the Proceedings Book

Transcription

ES 2708 EJC EACR-23 Abstract Cover_EJC EACR-23 Abstract Cover 28/05/2014 11:50 Page 1
50
S5
Volume 50, Supplement 5, July 2014 ISSN 0959-8049
EUROPEAN JOURNAL OF CANCER
J
EC
Vol. 50/S5 (2014)
THE OFFICIAL
JOURNAL OF
EORTC
European Organisation for
Research and Treatment of Cancer
ECCO
European CanCer Organisation
EACR
23rd Biennial Congress of the
European Association for Cancer Research
EUROPEAN ASSOCIATION
FOR CANCER RESEARCH
From Basic Research to
Personalised Cancer Treatment
PROCEEDINGS
BOOK
EAS 2015 Advert-210x280.indd 1
30/05/14 17:36
European Association
for Cancer Research
EUSOMA
European Society of
Breast Cancer Specialists
Vol. 50 Supplement 5
July 2014
ISSN 0959-8049
European Journal of Cancer
European Association for
Cancer Research
5–8 July 2014
Munich, Germany
Proceedings Book
Amsterdam • Boston • London • New York • Oxford • Paris • Philadelphia • San Diego • St Louis
Editor-in-Chief:
Editors:
Basic and Preclinical Research:
Drug Development:
Early Breast Cancer:
Advanced Breast Cancer:
Gastrointestinal Cancers:
Genitourinary Cancers:
Lung Cancer:
Gynaecological Cancers:
Head and Neck Cancer:
Sarcomas:
Melanoma:
Neuro-oncology:
Epidemiology and Prevention:
Paediatric Oncology:
Founding Editor:
Past Editors:
Editorial Office:
Alexander M.M. Eggermont
Institut Gustave Roussy
Villejuif, France
Richard Marais, Manchester, UK
Ulrich Keilholz, Berlin, Germany
Jordi Rodon, Barcelona, Spain
Kathleen I. Pritchard, Toronto, Canada
David Cameron, Edinburgh, UK
Volker Heinemann, Munich, Germany
Michel Ducreux, Villejuif, France
Karim Fizazi, Villejuif, France
Mary O’Brien, London, UK
Ignace Vergote, Leuven, Belgium
Kevin Harrington, London, UK
Jean-Yves Blay, Lyon, France
Dirk Schadendorf, Essen, Germany
Roger Stupp, Zurich, Switzerland
Jan Willem Coebergh, Rotterdam, The Netherlands
Rob Pieters, Rotterdam, The Netherlands
Henri Tagnon
Michael Peckham, London, UK; Hans-Jörg Senn, St Gallen, Switzerland; John Smyth, Edinburgh, UK
Elsevier, The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK
Tel: +44 (0) 1865 843590, Email: ejcancer@elsevier.com
EDITORIAL BOARD
CLINICAL ONCOLOGY
J.-P. Armand (France)
A. Ayhan (Japan)
R. Blamey (UK)
M. Bolla (France)
J. Boyages (Australia)
N. Brünner (Denmark)
F. Cardoso (Portugal)
J. Cassidy (UK)
M. Castiglione (Switzerland)
L. Cataliotti (Italy)
L. Cheng (USA)
H. Cody (USA)
R. Coleman (UK)
A. Costa (Italy)
J. De Bono (UK)
M.J.A. De Jong (The Netherlands)
E. de Vries (The Netherlands)
A. Dicker (USA)
R. Dummer (Switzerland)
F. Eisinger (France)
S. Erridge (UK)
G. Ferrandina (Italy)
H. Gabra (UK)
H. Gelderblom (The Netherlands)
B. Hasan (Belgium)
J.C. Horiot (Switzerland)
C. Huber (Germany)
R. Jakesz (Austria)
J. Jassem (Poland)
D. Jodrell (UK)
V.C. Jordan (USA)
A. Katz (Brazil)
M. Kaufmann (Germany)
I. Kunkler (UK)
L. Lindner (Germany)
P.E. Lønning (Norway)
P. Lorigan (UK)
K. McDonald (Australia)
R. Mertelsmann (UK)
F. Meunier (Belgium)
T. Mok (Hong Kong)
D. Nam (Korea)
P. O’Dwyer (USA)
J. Overgaard (Denmark)
N. Pavlidis (Greece)
J. Perry (Canada)
P. Price (UK)
D. Raghavan (USA)
J. Ringash (Canada)
J. Robert (France)
A. Rody (Germany)
D. Sargent (USA)
M. Schmidinger (Austria)
S. Sleijfer (The Netherlands)
P. Sonneveld (The Netherlands)
A. Sparreboom (USA)
M. van den Bent (The Netherlands)
M. Van Glabbeke (Belgium)
G. Velikova (UK)
U. Veronesi (Italy)
A. Vincent-Salomon (France)
A. Voogd (The Netherlands)
E. Winquist (Canada)
BASIC, PRECLINICAL AND TRANSLATIONAL RESEARCH
A. Albini (Italy)
P. Allavena (Italy)
F. Balkwill (UK)
M. Barbacid (Spain)
M. Broggini (Italy)
C. Catapano (Switzerland)
J. Collard (The Netherlands)
E. Garattini (Italy)
A. Gescher (UK)
R. Giavazzi (Italy)
I. Hart (UK)
W. Keith (UK)
J. Lunec (UK)
D.R. Newell (UK)
G.J. Peters (The Netherlands)
A. Puisieux (France)
V. Rotter (Israel)
M. Schmitt (Germany)
C.G.J. Sweep (The Netherlands)
G. Taraboletti (Italy)
P. Vineis (UK)
N. Zaffaroni (Italy)
D. Forman (France)
A. Green (Australia)
K. Hemminki (Germany)
C. Johansen (Denmark)
L.A. Kiemeney (The Netherlands)
E. Lynge (Denmark)
M. Maynadié (France)
H. Møller (UK)
P. Peeters (The Netherlands)
A.G. Renehan (UK)
S. Sanjose (Spain)
M.K. Schmidt (The Netherlands)
I. Soerjomataram (France)
H. Storm (Denmark)
L.V. van de Poll-Franse (The Netherlands)
H.M. Verkooijen (The Netherlands)
E. de Vries (The Netherlands)
R. Zanetti (Italy)
G. Chantada (Argentina)
F. Doz (France)
A. Ferrari (Italy)
M.A. Grootenhuis (The Netherlands)
K. Pritchard-Jones (UK)
L. Sung (Canada)
M. van den Heuvel-Eibrink (The Netherlands)
M. van Noesel (The Netherlands)
EPIDEMIOLOGY AND PREVENTION
B. Armstrong (Australia)
P. Autier (France)
J.M. Borras (Spain)
C. Bosetti (Italy)
H. Brenner (Germany)
L.E.M. Duijm (The Netherlands)
J. Faivre (France)
S. Franceschi (France)
PAEDIATRIC ONCOLOGY
C. Bergeron (France)
A. Biondi (Italy)
E. Bouffet (Canada)
M. Cairo (USA)
H. Caron (The Netherlands)
Aims and Scope
The European Journal of Cancer (EJC) is an international multidisciplinary oncology journal, which publishes original research, reviews, and editorial
comments on basic and preclinical cancer research, translational oncology, clinical oncology – including medical oncology, paediatric oncology, radiation
oncology, and surgical oncology, and cancer epidemiology and prevention. The EJC is the official journal of the European Organisation for Research and
Treatment of Cancer (EORTC), the European CanCer Organisation (ECCO), European Association for Cancer Research (EACR) and the European Society of
Breast Cancer Specialists (EUSOMA).
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9 – 12 July 2016 | Manchester, UK
24th Biennial Congress of the
European Association
for Cancer Research
From Basic Research to Precision Medicine
Organised by
SAVE THE DATE
www.ecco-org.eu/EACR
#EACR24
Volume 50
Suppl. 5
2014
Contents
Acknowledgements
Official Media Partners
Letter of Welcome
Congress Committees
Accreditation Information
General Information
Awards
About AEK
About EACR
Susan G. Komen
Floorplans
List of Exhibitors
Exhibitor Profiles
Satellite Symposia
Innovation Launch Special Session
Programme at a Glance
Scientific Programme
Saturday 5 July 2014
Sunday 6 July 2014
Monday 7 July 2014
Tuesday 8 July 2014
Poster Sessions, Sunday 6 July 2014
Poster Sessions, Monday 7 July 2014
Abstracts
Sunday 6 July 2014
Monday 7 July 2014
Tuesday 8 July 2014
Poster Sessions (Sunday 6 July & Monday 7 July 2014)
Index of presenting authors
Indexed/Abstracted in:
Current Contents;
EMBASE/Excerpta Medica;
Index Medicus; MEDLINE;
CABS, BIOSIS Database;
PASCAL-CNRS Database; CINAHLs®
VI
VI
VII
VII
IX
X
XV
XVIII
XIX
XX
XXI
XXIII
XXIV
XXIX
XXXI
XXXII
XXXIV
XXXIV
XXXV
XXXVIII
XL
XLI
LXI
S1
S1
S3
S12
S21
S23
S243
ISSN 0959-8049
vi
Acknowledgements
The European Association for Cancer Research (EACR) would like to acknowledge the generous ongoing support
of its Sustaining Members:
And expresses sincere thanks for the generous support of the organisations sponsoring Symposia, Keynote and
Award Lectures.
EACR is also grateful for the support from the following sponsors:
EACR also wishes to thank the following companies and organisations for their support of the Congress by taking
part in the exhibition:
Abcam
Advanced Cell Diagnostics
Affymetrix
Agilent Technologies
American Association for Cancer
Research (AACR)
Amsbio
Automated Lab Solutions (ALS)
Beckman Coulter Genomics
BD Biosciences
Cell Signaling Technology
Charles River
CYTOO
Essen BioScience
Illumina
Indivumed
iThera Medical
IUL Instruments
Jackson Immuno Research
LGC
LI-COR Biosciences
Life Technologies
Lonza
Miltenyi Biotec
NanoString Technologies
Novus Biologicals
OMNI Life Science
Oxford Optronix
PerkinElmer
Polyplus-transfection
Proteintech
QIAGEN
R&D Systems
SEQUENOM
Sirion Biotech
Spandidos Publications
SYNkinase
Takara Clontech
The Company of Biologists
Official Media Partners
On behalf of the EACR-23 Congress Committees, we would like to acknowledge the collaboration and support
of our official media partners:
vii
Letter of Welcome
On behalf of the Executive Scientific Committee, we are pleased to welcome you to Munich for the 23rd Biennial
EACR Congress.
We are very pleased that the EACR-23 Congress is actively supported by the German Cancer Society,
Experimental Cancer Research (AEK).
EACR congresses are well known for providing a rich and diverse programme. During the next four days EACR-23
will provide a unique opportunity for participants to learn from and meet with leaders in the field of basic and
translational research, apply new data to inform practice and ultimately improve patient outcomes.
The Congress will focus on the latest developments in basic and discovery driven translational research, through
to personalised precision cancer treatment. Keynote and Award Lectures are complemented with focussed
symposia across eighteen topics. The programme will offer exceptional Plenary Lectures, Plenary Symposia with
ample time for discussions, Scientific Symposia, Workshops and Oral and Poster presentations.
We are also pleased that the Congress will be held in one of Europe’s most lively cities; a city with a leading
position in oncology and a remarkable reputation for cancer research and cancer care.
We trust that you will return from the Congress inspired by colleagues from around the world and that you will
have made new friends and scientific contacts that will support you in your essential work.
We are delighted to be welcoming you at what promises to be another highly educational, collaborative and
successful EACR Congress.
Kind regards,
Professor Moshe Oren
Congress & Scientific Chair
Congress Committees
Organising & Scientific Programme Committee
Moshe Oren (IL), Congress & Scientific Chair
Anne-Lise Børresen-Dale (NO)
Julio Celis (DK)
Donatella Del Bufalo (IT)
Ivan Dikic (DE)
Rainer Engers (DE)
Clare Isacke (UK)
Robert Kenney (UK)
National & Local Organising Committee
Axel Ullrich (DE)
Horst Domdey (DE)
Rainer Engers (DE)
Christof von Kalle (DE)
Richard Marais (UK)
Daniel Peeper (NL)
Joan Seoane (ES)
Axel Ullrich (DE)
Emmy Verschuren (FI)
Christof von Kalle (DE)
Karen Vousden (UK)
Moshe Yaniv (FR)
18
18th ECCO - 40th ESMO
European Cancer Congress
Reinforcing multidisciplinarity
VIENNA, AUSTRIA, 25 - 29 SEPTEMBER 2015
35
www.ecco-org.eu
SIOP
SIOP Europe
the European Society for Paediatric Oncology
ix
Accreditation Information
The ‘23rd Biennial Congress of the European Association for Cancer Research’ is accredited by the European
Accreditation Council for Continuing Medical Education (EACCME) to provide the following CME activity
for medical specialists. The EACCME is an institution of the European Union of Medical Specialists (UEMS),
www.uems.net
EACR-23 is designated for a maximum of 18 hours of European external CME credits. Each medical
specialist should claim only those hours of credit that he/she actually spent in the educational activity. The
EACCME credit system is based on 1 ECMEC per hour with a maximum of 3 ECMECs for half a day and
6 ECMECs for a full-day event.
European Accreditation is granted by the EACCME in order to allow participants who attend the above-mentioned
activity to validate their credits in their own country.
Through an agreement between the European Union of Medical Specialists and the American Medical
Association, physicians may convert EACCME credits to an equivalent number of AMA PRA Category 1
Credits™. Information on the process to convert EACCME credit to AMA credit can be found at:
www.ama-assn.org/go/internationalcme
Live educational activities, occurring outside of Canada, recognised by the UEMS-EACCME for ECMEC credits
are deemed to be Accredited Group Learning Activities (Section 1) as defined by the Maintenance of Certification
Program of The Royal College of Physicians and Surgeons of Canada.
x
General Information
Congress Secretariat
c/o ECCO − the European CanCer Organisation
eacr23@ecco-org.eu
www.ecco-org.eu/eacr23
During the congress, the Secretariat can be reached at +49 89 949 79401
Congress Venue
International Congress Centre Munich (ICM)
Messegelände
DE-81823 München, Germany
Phone: +49 89 949-23410
www.icm-muenchen.de
Airport Check-in
As of Monday 7 July, delegates can check in online for their flight using the terminals and printers located in the
registration area.
App
All attendees can download the free ECCO App on iPhone, iPad, or Android supported devices.
Features include EACR-23 related information and news. The App contains the complete list of scientific
sessions, session types, speakers, exhibitors, and satellite symposia. Users can save their selected sessions,
notes, favourites, as well as exporting sessions to their smartphone calendar. To download the App, search for
ECCO cancer on iTunes or Google Play.
Learn more at www.ecco-org.eu/app or use the QR code below for direct download.
Badges
For security reasons, delegates are requested to wear their badge at all times during the congress. Delegates
having lost their badge can obtain a new badge at the registration desk. A replacement fee of 75 EUR per
participant will be charged.
Catering
Coffee Breaks: Coffee breaks, courtesy of the organisers, have been scheduled as follows:
Saturday 5 July:
Sunday 6 July:
15:00–15:30
Monday 7 July:
10:15−10:45
10:15−10:45
15:45−16:15
15:45−16:15
Tuesday 8 July:
10:15−10:45
Coffee breaks will take place in the catering areas of the exhibition, except on Tuesday, when refreshments will
be served in the foyer areas on the first floor.
Lunches: Delegates can purchase food and beverages from the cash bars at the Congress Centre.
Certificate of Attendance
Certificates of Attendance recording CME credits will be available online immediately after the Congress. You
will receive an email link to a short questionnaire which also provides the link for you to print your Certificate of
Attendance.
EACR-23, General Information / European Journal of Cancer 50, Suppl. 5 (2014) x–xiii
xi
We kindly ask you to keep your Congress badge as you will need the unique badge code to print your Certificate
of Attendance.
The Congress Secretariat will not mail Certificates of Attendance to participants after the Congress.
For information on CME accreditation see page IX.
City Information
München Tourismus/Munich Convention Bureau will operate a city information desk on Saturday 5 and Sunday 6
July, located in the entrance hall.
Cloakroom
A cloakroom is located on the mezzanine level.
Cloakroom Opening Hours:
Saturday 5 July:
11:00−20:30
Sunday 6 July:
07:30−21:30
Monday 7 July:
07:30−19:00
Tuesday 8 July:
08:00−13:30
The rate for use of the cloakroom is 2 EUR per coat or bag.
Congress Dinner: Monday 7 July 20:00
The EACR-23 Congress Dinner will take place at the Munich Old Town Hall. Ticket sales were limited owing to
the capacity of the venue and sold out prior to the start of the congress. Ticket holders will be asked to present
their ticket upon arrival at the venue.
EACR General Assembly and Awards Ceremony: Sunday 6 July 19:00
The General Assembly and Awards Ceremony of the European Association for Cancer Research will be held in
room 14A of the Congress Centre. This event is open to EACR members and award winners.
Exhibition
The EACR-23 exhibition is held in Hall B0 on the ground floor of the Congress Centre. Entrance is free for
registered delegates but limited to researchers, oncology professionals, press and exhibitors.
Exhibition Opening Hours:
Saturday 5 July:
15:00−20:00
Sunday 6 July:
10:15−17:15
Monday 7 July:
10:15−17:15
For the exhibition floorplan and list of exhibitors, please see the exhibition section (p.
Book.
XXII)
of this Proceedings
First Aid
A first aid room is located on the ground floor, close to the main entrance. In case of emergency, please inform
the nurse on duty via +49 89 949 28103.
Insurance
The organisers of EACR-23 do not accept liability for individual medical, travel or personal insurance. Participants
are strongly recommended to obtain their own personal insurance policies. The organisers of EACR-23 accept
no responsibility for loss due to theft or negligence.
Internet Wi-Fi Access
General Wi-Fi access is available throughout the Congress Centre. For access, activate the Wi-Fi network on
your laptop or device, select the network listed as EACR23, and enter the user name and password EACR23.
Internet Zone
The official EACR-23 Internet Zone is available free of charge during the Congress. The terminals provide you
with the following services: internet browsing, access to web-based mail, the congress searchable programme
and exhibitor information.
xii
Language & Translation
The official language of the congress is English. Translation is not provided.
Lost & Found
All enquiries should be directed to the registration helpdesk in the entrance hall. The organisers accept no
responsibility for loss due to theft or negligence.
Opening Lecture and Ceremony: Saturday 5 July 17:00
The Opening Lecture and Ceremony will be held at the Congress Centre in room 14B. Access is free for all
registered participants. Please refer to the Scientific Programme for further details.
The Opening Lecture and Ceremony will be followed by an Exhibitors’ Reception taking place in the Exhibition
Hall B0 where light refreshments will be served.
During the reception, a World Cup quarter final match will be broadcast live in the Exhibition Hall.
Poster Sessions
Posters will be on display for one day in the dedicated poster area (Sunday or Monday, across the various topics.
For details please refer to the Scientific Programme). Poster presenters will be allowed access to the Exhibition
Hall as of 08:00 AM to mount their poster on the day their poster is to be presented.
Posters must be removed by 17:15 on the day the poster was presented. Any posters remaining after this time
will be removed by the organisers and cannot be reclaimed.
Registration
EACR-23 is open to all registered participants. Your official name badge is required for admission to the Congress
Centre and all Congress events. For security reasons, participants are requested to wear their badge at all times.
Registration Opening Hours:
Saturday 5 July:
08:00−19:00
Sunday 6 July:
07:30−18:00
Monday 7 July:
07:30−17:30
Tuesday 8 July:
08:00−12:00
Registration Package
The full congress registration package includes:
− Entry to all scientific sessions and to the Opening Lecture and Ceremony on Saturday 5 July;
− Entry to the exhibition (restricted to researchers, oncology professionals and media);
− EACR-23 Proceedings Book;
− EACR-23 coffee breaks;
− Internet access via the Internet Zone and Wi-Fi access in the Congress Centre;
− Congress bag including a city map.
The day registration package includes:
− Access to all scientific sessions on that day;
− Entry to the exhibition (restricted to researchers, oncology professionals and media);
− Proceedings Book (depending on availability);
− EACR-23 coffee breaks on that day;
− Internet access via the Internet Zone and Wi-Fi access in the Congress Centre;
− Congress bag including a city map (depending on availability).
Satellite Symposia
Several satellite symposia are taking place during EACR-23. For the detailed programme, please see the satellite
symposia section (page XXIX) of this Proceedings Book.
Social Media
Twitter is available during the congress − tweet, network and follow updates using hashtag #EACR23.
Find links, tutorials and tips: www.ecco-org.eu/social
xiii
Speaker Preview Room
The Speaker Preview Room is located in room 12A (first floor). Speakers are requested to bring their PowerPoint
presentations to the Speaker Preview Room at least 4 hours before their session starts or one day in advance if
the session starts early in the morning. Provision for presentations using laptops is not available in the session
rooms.
Speaker Preview Room Opening Hours:
Saturday 5 July:
09:30−18:00
Sunday 6 July:
07:30−18:00
Monday 7 July:
07:30−17:30
Tuesday 8 July:
08:00−12:30
xv
Awards
Mike Price Gold Medal Award
The Mike Price Gold Medal is presented biennially by EACR to a senior researcher who has made an exceptional
contribution to cancer research in Europe.
Award winner: Harald zur Hausen
Harald zur Hausen was born on March 11, 1936 in Gelsenkirchen-Buer, Germany. He
studied Medicine at the Universities of Bonn, Hamburg and Düsseldorf and received
his M.D. in 1960. After his internship he worked as a postdoctoral fellow at the Institute of
Microbiology in Düsseldorf, and subsequently in the Virus Laboratories of the Children’s
Hospital in Philadelphia where he was later appointed as Assistant Professor. After a
period of 3 years as a senior scientist at the Institute of Virology of the University of
Würzburg, he was appointed in 1972 as Chairman and Professor of Virology at the
University of Erlangen-Nürnberg. In 1977 he moved to a similar position at the University
of Freiburg. From 1983 until 2003 he held the post of Scientific Director of the Deutsches
Krebsforschungszentrum (German Cancer Research Centre) in Heidelberg. He retired
from this position in 2003.
He has received a number of national and international awards, among them the Robert-Koch-Prize, the
Charles S. Mott Prize of the General Motors Cancer Research Foundation, the Federation of the European Cancer
Societies Clinical Research Award, the Paul-Ehrlich-Ludwig Darmstädter-Prize, the Jung-Prize, Hamburg, the
Charles Rodolphe Brupbacher Prize, Zürich, the Prince Mahidol Award, Bangkok, the Raymond Bourgine Award,
Paris, the Coley-Award, New York, the Life Science Achievement Award of the American Association for Cancer
Research, San Diego, the German Special Order of Merit with Star, the Tsungming Tu Award and the Nobel Prize
for Medicine, 2008.
From January 2000 to December 2009 zur Hausen was Editor-in-Chief of the International Journal of Cancer , and
from 2006 to 2010 he was a member of the Board of Directors of the International Union against Cancer (UICC).
From 2003 to 2010 he was vice-president of the German National Academy for Natural Sciences and Medicine
LEOPOLDINA in Halle. Since 2006 he has been a member of the Scientific Council of the National Science and
Technology Development Agency in Bangkok, Thailand and from 2010 to 2012 Chairman of this Council.
The Pezcoller Foundation − EACR Cancer Researcher Award
The Pezcoller Foundation − EACR Cancer Researcher Award celebrates academic excellence and achievements
in the field of cancer research. The award is presented biennially to a researcher of excellence with no more
than 15 years post doctoral experience.
Award Winner: Eduard Batlle
Eduard Batlle (Barcelona, 1970) received a Doctorate in Biology from the University of
Barcelona. His studies on the mechanisms responsible for tumour cell invasion led him
to discover originally the role of the transcription factor Snail as repressor of E-cadherin
expression in tumour cells. This crucial discovery founded the field of research on Epithelial
to Mesenchymal Transition (EMT) in Cancer. He then moved to The Netherlands to work
on WNT signalling under the mentorship of the renowned scientist Hans Clevers. His
postdoctoral work at the Clevers lab revealed the first connection between WNT signalling,
intestinal stem cells and colorectal cancer and resulted in a series of papers that have
become classics. In 2004, Eduard Batlle joined the Institute for Research in Biomedicine
(IRB Barcelona) as Head of the Oncology Programme and ICREA Research Professor.
xvi
EACR-23, Awards / European Journal of Cancer 50, Suppl. 5 (2014) xv–xvii
The research activity in his lab is focused towards the characterisation of the mechanisms that drive colorectal
cancer initiation and progression. In the last ten years and amongst other findings, the Batlle laboratory has made
key contributions to understand how stem cell gene programs control the behaviour of tumour cells and impose a
hierarchical organisation on colorectal cancer. More recently, he has investigated the phenomenon of metastasis
and particularly how disseminated tumour cells depend upon signals from the tumour microenvironment for
survival during colonisation of foreign organs. His track record has been recognised by several awards and
honours such as the Debiopharm Life Science Award (2006), European Research Council (ERC) Starting Grant
(2007), Banc de Sabadell Award for Biomedical Research (2010), Josef Steiner Cancer Research Foundation
Award (2013), Diz Pintado Award (2013) and by an ERC Advanced Grant (2013).
Anthony Dipple Carcinogenesis Award
The Anthony Dipple Carcinogenesis Award is for major contributions to research in the field of carcinogenesis.
Award Winner: Varda Rotter, Weizmann Institute for Science, Rehovot, Israel
Varda Rotter is among the pioneers who discovered the p53 protein, and provided
original insights for the understanding of the molecular and cellular biology of its function
as a cancer hallmark. In addition to the identification of important structural functional
domains of the p53 protein, she identified and molecularly characterised several mouse
and human p53 non-producer cell lines that was seminal for the establishment of the
well-accepted concept that wild type p53 functions as a tumour suppressor while the
mutant p53 exhibits an oncogenic activity.
Most significant is her seminal work in showing that mutant p53 has a gain-offunction activity in carcinogenesis. Her major contributions included the realisation that
mutant p53 promotes cancer through its transcriptional activity that renders tumour cells
resistant to apoptosis. Varda Rotter’s research focused on the establishment of in-vitro models to permit the
extensive gene expression analysis and adoption of system biology approaches, which led to successfully identify
gene networks that are associated with p53 gain-of-function in cancer progression. In an effort to resolve the role
of p53 in stem cells she observed that expression of mutant p53 in stem cells led to the development of cancer
stem cells that initiate cancer development.
Carcinogenesis Young Investigator Award
The Carcinogenesis Young Investigator Award is for a recent, significant contribution to carcinogenesis research
by an investigator under the age of 40.
Award Winner: Lin He
Lin He is Assistant Professor of Cell and Developmental Biology at the Department
of Molecular & Cell Biology, University of California, Berkeley, USA. She received her
doctoral degree at Stanford Medical School with Dr. Greg Barsh, before her postdoctoral
training with Dr. Greg Hannon at Cold Spring Harbor Laboratory. She joined the faculty
of UC Berkeley in 2008 to explore the roles of microRNAs in development and cancer
using mouse tumour models.
EACR-23, Awards / European Journal of Cancer 50, Suppl. 5 (2014) xv–xvii
xvii
EACR Meeting Bursary Award Winners
This year 43 awards have been made to support EACR members’ attendance at EACR-23
Margarida Abrantes, Portugal; Sinead Aherne, Ireland; Narmen Azazmeh, Israel; Jorge Barbazan, Spain; Elisa
Caiola, Italy; David Cerezo Fernández, Spain; Gina Chan Tse, UK; Elvira D’Ippolito, Italy; Saikat Das, India;
Oriol de Barrios, Spain; Alessandra Decio, Italy; Evgeny Denisov, Russia; Camila Henrique de Sousa, UK;
Alexia Hervieu, UK; Thomas Hopkins, UK; Theodoros Karampelas, Greece; Terézia Kiskova, Slovakia; Sebastian
Kurscheid, Switzerland; Stephania Libreros, USA; Ka Ian Lio, UK; Carrie Lovitt, Australia; Paulo Luraghi, Italy;
Diana Marouco, UK; Nerea Matamala, Spain; Ivana Matic, Serbia; Tanja Matijevic Glavan, Croatia; Emanuela
Minna, Italy; Tashfina Mirza, UK; Andrea Morandi, Italy; Brianna Morten, Australia; Gabor Pajor, Hungary;
Pasquale Pellegrini, Spain; Pedro Pinheiro, Portugal; Salomé Pires, Portugal; Carmit Strauss, Israel; Roula
Tahtouh, Lebanon; Victoria Thompson, UK; Pedro Torres, Spain; Dana Tsui, UK; David Vetvicka, Czech Republic;
Claudia Vollbrecht, Germany; Jennifer Wymant, UK
Proffered Paper Awards
The following have been selected to present a proffered paper in the symposia at EACR-23
Mohamed Bentires-Alj, Switzerland; Christian Breunig, Germany; Hani Choudhry, UK; Ross D. Hannan, Australia;
Claus Jorgensen, UK; Neige Journy, France; Valery Krizhanovsky, Israel; Niamh Lynam-Lennon, Ireland; James
Mansfield, USA; Philipp Mertins, USA; Nuria Lopez-Bigas, Spain; Ohad Tarcic, Israel; Victoria Thompson, UK;
Tushar Tomar, Netherlands; Dana Tsui, UK; Anne Kathrin Volkmer, Germany; David Wedge, UK; Lisa Wiesmüller,
Germany
Susan G. Komen Scholarship Awards
The Komen awards reflect the highest scoring abstracts submitted in the field of breast cancer research
Mohamed Bentires-Alj, Switzerland; Jason Carroll, UK; Hani Choudhry, UK; Philipp Mertins, USA; Manasa
Ramakrishna, UK; Anne Kathrin Volkmer, Germany; Lisa Wiesmüller, Germany
xviii
About AEK
AEK is the Division of Experimental Cancer Research of the German Cancer Society and was founded in 1979.
With around 900 members, AEK is one of the biggest divisions of the German Cancer Society and represents
the majority of experimental cancer scientists in Germany.
Major aims of the AEK:
(1) Increase visibility and underscore impact of experimental cancer research
(2) Support of scientific exchange in cancer research not only amongst German cancer researchers but also on
an international level as well as between basic cancer scientists, translational cancer researchers and clinical
oncologists
(3) Support of young scientists on the field of experimental cancer research
(4) Representation of experimental cancer research in national and international institutions, organisations and
societies in accordance with the board and presidency of the German Cancer Society
(5) Organisation of scientific meetings, in particular the biennial International AEK Cancer Congress
The biennial International AEK Cancer Congress is the central congress for basic and translational cancer
researchers from Germany and neighbouring countries. The congress format with renowned international and
national speakers stimulates scientific exchange on a very high level and provides ample opportunity for young
scientists to present and discuss their work.
The forthcoming 18th International AEK Cancer Congress will be held in Heidelberg, Germany, from March
19−21, 2015 (Congress Chair: Lisa Wiesmueller, Ulm, Germany; Congress Co-Chair: Petra Boukamp, Heidelberg,
Germany).
Further information will be available through www.aek-congress.org
Looking forward to welcoming you in Heidelberg in 2015
and with my best regards,
Rainer Engers
(AEK president)
Contact address: Prof. Dr. Rainer Engers
Zentrum für Pathologie, Zytologie und Molekularpathologie
Am Hasenberg 44
D-41462 Neuss
phone: +49 2131 6659 1350
fax:
+49 2131 6659 1353
E-mail: engers@pathologiezentrum-neuss.de
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xx
Susan G. Komen
Susan G. Komen has supported the EACR-23 Congress by providing seven Susan G. Komen Scholarship
Awards, awarded to the presenters of the very best abstracts in the field of breast cancer research.
Susan G. Komen’s Sustained Commitment to Young Investigators Worldwide
As with all biomedical research, successful research for breast cancer relies on the talent and dedication of the
scientific workforce, as well as a continued supply of highly trained researchers who can bring new insights to
our understanding of breast cancer biology and advance the translation of these insights into improved health.
Supporting opportunities for these young investigators has long been a key component of Susan G. Komen’s
research investment.
Susan G. Komen is the leading non-profit funder of breast cancer research, worldwide. Essential to their global
mission of ending breast cancer forever, Komen has awarded over 30 international young investigators with
Post-Doctoral Fellowship, Dissertation Research and Career Catalyst Awards. These awards help researchers
gain a foothold in the profession and help fortify their paths to independence in breast cancer research. In fact,
preliminary results from a recent survey reveal that 88% of Komen Post-Doctoral Fellows award recipients (both
international and national) have stayed in the field of breast cancer.
Awardees have studied with distinguished mentors such as Dr. Ramon Colomer, Head of the Medical Oncology
Division at the Hospital Universitario La Princesa in Madrid, Spain and Dr. Nancy Hynes of the Friedrich Miescher
Institute for Biomedical Research in Switzerland. Dr. Albana Gatelli received a Komen Post-Doctoral Fellowship
award in 2010 under the mentorship of Dr. Hynes to study the role of Ret receptor in endocrine resistant breast
cancer. Based on the findings generated during the period of her fellowship, Dr. Gatelli plans to continue this
research as an independent investigator in her own country of Argentina. In 2013, Susan G. Komen awarded two
Career Catalyst grants to Spanish investigators Dr. Aleix Prat, of Vall d’Hebron Institute of Oncology and Dr. Eva
Gonzalez Suarez, of the Bellvitge Institute for Biomedical Research.
Susan G. Komen also provides conference support, enabling young investigators to travel to scientific conferences
to present their work, interact with some of the most influential breast cancer researchers in the world, meet with
peers and develop their enthusiasm for breast cancer research. Since 2009, Komen has funded the American
Association for Cancer Research’s (AACR) Scholar in Training Awards to enhance the education and training of
early career scientists by providing financial support for their attendance at AACR conferences and meetings.
To date, over 35 young investigators from a dozen European countries have received these awards. Komen has
also provided funding in multiple years for the European Society for Medical Oncology’s IMPAKT Breast Cancer
Conference in Brussels, Belgium, awarding over 65 travel scholarships including many awards to young scientists
from European countries including Austria, Italy, Poland, Romania and the United Kingdom.
Essential to their global mission of ending breast cancer forever, Susan G. Komen has designated 2014 as
the Year of The Young Investigator and redoubled its commitment to supporting young investigators worldwide.
Research funding opportunities are released in late spring and applications are awarded, based on merit, across
the globe.
xxi
Floorplans
Venue Floorplan
First Floor
Room 11A
Speaker
Preview Room
Room 13
Room 14A
Room 14B
Room 14C
Hall 1
Ground Floor
Exhibition Hall B0
Entrance
xxii
EACR-23, Floorplans / European Journal of Cancer 50, Suppl. 5 (2014) xxi–xxii
Catering Area
Catering Area
Exhibition Floorplan
Poster Area
C7
Catering Area
C1
C5
C6
B3
B4
B7
B8
B1
B2
B5
B6
A9
A10
A13
A14
A7
A8
A11
A12
C12
C13
C11
C15
C9
B9
A18
B11
A19
A20
A15
Entrance
C18
C21
C22
C16
C19
C20
B21
B22
B19
B20
A24
A27
A28
A22
A25
A26
B15
A21
Catering Area
Seating Area
xxiii
List of Exhibitors
Exhibitor Name
Booth number
Abcam
C21
A27
Affymetrix
B11
C9
American Association for Cancer Research (AACR)
A12
Amsbio
A11
B2
BD Biosciences
C1
A22
Cell Signaling Technology
C11
Charles River
A20
CYTOO
B6
ECCO − the European CanCer Organisation
A8
European Association for Cancer Research (EACR)
A15
Essen BioScience
C12
Illumina
C15
Indivumed
C18
iThera Medical
A24
IUL Instruments
C19
Jackson Immuno Research
C16
LGC
B4
LI-COR Biosciences
B9
Life Technologies
C7
Lonza
B22
Miltenyi Biotec
C5
B21
Novus Biologicals
B5
OMNI Life Science
B1
Oxford Optronix
C6
PerkinElmer
A21
A18
Proteintech
C22
QIAGEN
B15
R&D Systems
B7
SEQUENOM
C13
Sirion Biotech
B8
Spandidos Publications
B3
SYNkinase
A13
Takara Clontech
B19
A14
This list reflects confirmed exhibitors as of 26 May 2014
xxiv
Exhibitor Profiles
This list reflects confirmed exhibitors as of 13 May 2014
Abcam
Booth C21
www.abcam.com
Abcam is a provider of protein research tools and services, with an unrivalled range of products and expert
technical support, enabling scientists to analyse living cells at the molecular level and improving the understanding
of health and disease.
Booth A27
www.acdbio.com
Advanced Cell Diagnostics, Inc. (ACD) is a leader in the field of molecular pathology, developing
cell-and-tissue based diagnostic tests for personalised medicine. ACD’s products and services are based on its
proprietary RNAscope® Technology capable of detecting RNA biomarkers in situ at single molecule sensitivity.
Affymetrix
Booth B11
www.affymetrix.com
Affymetrix’ tools for cancer research enable whole-genome analysis to single-gene
validation across diverse sample types including FFPE tissue and blood. Our solutions for expression, miRNA,
genotyping, copy number, flow cytometry, and immunophenotyping assays provide an integrated view of tumour
samples at the gene, protein and cellular level, for faster translation of discoveries to treatments.
Booth C9
www.agilent.com
Agilent Technologies is a leading supplier of life science research systems that
enable scientists to study complex biological processes and disease mechanisms. By integrating multiple
comprehensive analyses—genomics, transcriptomics, proteomics, and metabolomics—Agilent is your partner to
help you generate a better pathway-level understanding of your biological system and to be at the forefront of
oncology research.
American Association for Cancer Research (AACR)
Booth A12
www.aacr.org
The mission of the AACR is to prevent and cure cancer through research, education, communication, and
collaboration. Through its programs and services, the AACR fosters research in cancer and related biomedical
science; accelerates the dissemination of new research findings among scientists and others dedicated to the
conquest of cancer; promotes science education and training; and advances the understanding of cancer etiology,
prevention, diagnosis, and treatment throughout the world.
Booth B2
www.als-jena.com
We manufacture and market automated laboratory systems with a focus on automation of cell
culture analysis, rare cell isolation, and cell picking. ALS CellCelector™ is a flexible platform for
automated screening and recovery of single cells (including CTCs), clusters or cell colonies for
downstream molecular characterisation or cell culturing.
Booth A22
www.beckmangenomics.com
Beckman Coulter Genomics specialises in next generation and Sanger sequencing services
across the Illumina HiSeq 2000/2500, Roche 454 FLX and ABI 3730XL sequencing platforms supporting life
science, healthcare and the biopharma industries worldwide. Applications supported include exome and targeted
resequencing, RNA-Seq, genotyping and expression analysis.
EACR-23, Exhibitor Profiles / European Journal of Cancer 50, Suppl. 5 (2014) xxiv–xxviii
xxv
BD Biosciences
Booth C1
www.bdbiosciences.com/eu
BD Biosciences, a segment of Becton, Dickinson and Company, is one of the world’s leading businesses focused
on bringing innovative tools to life science researchers and clinicians. Its product lines include: flow cytometers
and cell sorters, monoclonal antibodies, research reagents, diagnostic assays, and tools to support cell analysis.
Cell Signaling Technology (CST)
Booth C11
www.cellsignal.com
Founded by research scientists, Cell Signaling Technology (CST) is active in applied systems biology research,
particularly as it relates to cancer. Understanding the importance of using antibodies with high levels of specificity
and consistency, CST scientists produce, validate, and support all our antibodies in-house.
Charles River
Booth A20
www.criver.com
Charles River provides essential products and services to help pharmaceutical, biotechnology
companies and leading academic institutions accelerate their research and drug development efforts. We are
focused on providing clients with what they need to improve the discovery, development and safe manufacture
of new therapies.
CYTOO
Booth B6
www.cytoo.com
CYTOO has pioneered the ability to control cells microenvironment and has developed a platform
of micropattern technologies to enable significantly greater physiologically relevant cellular models in
comparison to standard techniques used by most labs and CRO’s. CYTOO solutions allow for simple conversion
of poorly robust and low throughput assays into highly reliable and data rich HCS efforts.
ECCO − the European CanCer Organisation
Booth A8
www.ecco-org.eu
The European CanCer Organisation (ECCO) represents the interests of over 60,000
oncology professionals. It drives multidisciplinarity in the field and connects the
European cancer community by leveraging knowledge, promoting education and building awareness. ECCO also
proactively engages with policymakers to promote the interests of cancer research, cancer patients, and all other
oncocommunity members.
Essen BioScience
Booth C12
www.essenbioscience.com
IncuCyte − 300 publications − kinetic real-time non-invasive assays − perfect for Cancer
biology. Whether it’s label free (proliferation (confluence), migration or invasion), or quantification in green and
red channels, for example, in cell counting, apoptosis, angiogenesis and cytotoxicity. The IncuCyte is simple and
productive for any cancer laboratory.
European Association for Cancer Research (EACR)
Booth A15
www.eacr.org
The European Association for Cancer Research (EACR) is the largest member society for cancer
research in Europe and has a membership of over 9,500. In seeking to advance cancer research,
EACR supports its members through a wide range of activities, scientific meetings and other opportunities for
communication and interaction.
Illumina
Booth C15
www.illumina.com
At Illumina, our goal is to apply innovative sequencing and array technologies to the analysis of genetic variation
and function, making studies possible that were not even imaginable just a few years ago. Our solutions are not
only innovative, but flexible, scalable, and complete with industry-leading support and service.
xxvi
Indivumed
Booth C18
www.indivumed.com
Indivumed runs the world’s leading high-quality tumour biobank and database to support development of cancer
companion diagnostics and therapies. The company offers large expertise in target and biomarker validation for
companion diagnostic development, human tumour precision-cut tissue slices for protein pathway analyses and
protocol validation.
iThera Medical
Booth A24
www.ithera-medical.com
iThera Medical develops and markets biomedical imaging systems based on a novel technology called
Multispectral Optoacoustic Tomography (MSOT). MSOT utilises the photoacoustic effect to visualise and quantify
anatomical, functional and molecular information, in vivo, in deep tissue and in real time.
Jackson Immunoresearch Europe Ltd
Booth C16
www.jireurope.com
Jackson ImmunoResearch Laboratories Inc. (JIR) specialises in Secondary Antibodies
and Conjugates. Our products are used in all immunological applications, including Western Blot, Immunohistochemistry, Flow Cytometry and ELISA.
Our European subsidiary, Jackson ImmunoResearch Europe Ltd, provides direct support, including fast delivery,
to the European scientific community.
LGC
Booth B4
www.lgcstandards.com
LGC partnership with ATCC provides efficient access to ATCC’s biological resources to scientists
throughout Europe. As the exclusive European distributor for ATCC’s unique collections, you can be
assured that the ATCC products purchased through LGC are the original materials supported by our in-house
renowned expertise.
LI-COR® Biosciences
Booth B9
www.licor.com
LI-COR® Biosciences is a leading manufacturer of near-infrared and chemiluminescent imaging platforms,
analysis software, and IRDye® infrared dye reagents for quantitative Western Blotting, small animal imaging,
and DNA fragment analysis.
Life Technologies™
Booth C7
www.lifetechnologies.com
The Life Technologies™ genetic analysis portfolio includes instruments, software, reagents and
support for every lab, every application. At this year’s EACR Congress, we are showcasing our solutions that
enable scientists to understand cancer genomics and perform complex cancer cell analysis.
Lonza
Booth B22
www.lonza.com
Lonza provides the pharma market with tools life science researchers use to develop and test therapeutics.
Lonza Pharma − Bioscience Solutions products and services range from cell culture and discovery research
technologies, to quality control tests and software to ensure product quality. Lonza serves customers from
companies and research institutions worldwide.
Miltenyi Biotec
Booth C5
www.miltenyibiotec.com
Miltenyi Biotec provides products and services that advance biomedical research and cellular
therapy. Our innovative tools support research at every level, from basic research to translational
research to clinical application. Our more than 25 years of expertise includes immunology, stem cell biology,
neuroscience, and cancer. Miltenyi Biotec has more than 1,400 employees in 25 countries.
xxvii
Booth B21
www.nanostring.com
NanoString Technologies provides innovative products that unlock valuable and clinically actionable genomic
information from small amounts of tissue. The company is committed to offering tools that enable scientists and
clinicians to translate today’s leading genomic research into clinically actionable tests that improve patient care.
Novus Biologicals
Booth B5
www.novusbio.com
Novus Biologicals is a global proteomics company. We commercialise more than 200,000
guaranteed research products, and specialise in antibodies, proteins and peptides. All of our products are
100% guaranteed. Novus Biologicals recently purchased Imgenex, broadening its product range and expertise
to Innate Immunity, Toll Like Receptors and reporter cell lines.
Oxford Optronix
Booth C6
www.oxford-optronix.com
Oxford Optronix specialises in offering the Cancer Biology community world-renowned
solutions for Automated Colony Counting of adherent/non-adherent colony assays. We show a new generation
system for direct Dissolved Oxygen (pO2) monitoring and offer this in combination laser Doppler measurements.
Oxford Optronix launches a new bench-top, barometric compensated hypoxia workstation.
PerkinElmer
Booth A21
www.perkinelmer.com/lifesciences
PerkinElmer translational imaging. Learn more about pathway characterisation, therapeutic
effect, and treatment at the cellular, whole-body, and tissue levels with PerkinElmer’s complete
offering of translational imaging and analysis solutions. Our intuitive, high-performance software, broad portfolio
of reagents, and leading imaging systems enable you to see and understand more in every area of research.
Booth A18
www.polyplus-transfection.com
Polyplus-transfection is a biotechnology company that develops and manufactures innovative
solutions for transfection. The company is focused on synthetic reagents for delivery of biomolecules (DNA, siRNA
and proteins) to Eukaryotic cells or in vivo models. We also offer reagents for Large scale Bioproduction.
Proteintech
Booth C22
www.ptglab.com
Proteintech Group currently has over 10,000 antibodies in its catalog, all of which have been
by a specialist in-house team in multiple species and multiple applications. Over 90% of our antibodies are raised
against the whole recombinant protein which gives them superior protein recognition capabilities and versatility.
QIAGEN
Booth B15
www.QIAGEN.com
QIAGEN, the leading global provider of Sample & Assay Technologies, enables access to content from
any biological sample. QIAGEN markets more than 500 products worldwide, both consumable kits and automation
systems to customer in Molecular Diagnostics, Applied Testing, Pharma and Academia.
R&D Systems
Booth B7
www.RnDSystems.com
R&D Systems has over 25 years experience developing innovative, high quality cancer research
reagents. We offer over 25,000 products, developed, manufactured and controlled in our own laboratories. These
include a wide range of high performance antibodies, and the most referenced collection of bioactive proteins
and immunoassays in the industry.
xxviii
SEQUENOM
Booth C13
www.sequenom.com/bioscience
SEQUENOM designs, develops, manufactures and markets innovative instrumentation and tests that serve
oncology research and clinical molecular diagnostics markets.
SEQUENOM’s MassARRAY® technology is ideally suited for rapidly validating genetic discoveries and translating
them into cost effective genetic tests. The technology is applied by the leading genome centres worldwide.
SIRION Biotech
Booth B8
www.sirion-biotech.com
State of the art service provider for preclinical target research.
SIRION Biotech is a technology leader in the field of functional gene analysis and sophisticated cell modeling
for basic research, the drug, food and cosmetic industries. Combining its proprietary viral vector platform, RNAi
technology and outstanding expertise in cells, SIRION Biotech enables much improved target research and
compound screening.
SYNkinase
Booth A13
www.synkinase.com
SYNkinase manufactures kinase inhibitors, and other small molecules, inhibitor arrays and
KiNet-1 for drug-discovery researchers. KiNet-1 (CTx-0294885) is a novel, proprietary pan-kinase binder,
immobilised on Sepharose beads which binds over 250 protein kinases.
Focusing on quality, ensures we consistently supply high purity, high reactivity material time and time again.
Takara Clontech
Booth B19
www.takara-bio.eu, www.clontech.com
Clontech offers a range of products under the Takara and Clontech brands including PCR/qPCR reagents;
RT enzymes; library construction kits; the In-Fusion Cloning system; and nucleic acid purification products.
These products support applications including gene discovery, regulation, and function; protein expression and
purification; plant and food research; and RNAi studies.
Booth A14
www.biologists.com
The Company of Biologists is the not-for-profit publisher of the three distinguished journals
Development, Journal of Cell Science and The Journal of Experimental Biology. The Company also publishes
two open access journals, Disease Models & Mechanisms and Biology Open.
The Company also supports innovation in all aspects of biological research, providing grants in the areas in which
it publishes.
xxix
Satellite Symposia
Sunday 6 July 2014
Satellite Symposium: BD Biosciences
Understanding Tumour Heterogeneity through Phenotypic Analysis
Room 14A, 12:30−14:00
12:30 Phenotype and genotype of single cells dissociated from solid tumours
Speaker: S. Ghanekar (Belgium)
13:15 Epithelial to mesenchymal transition controls tumour heterogeneity and stemness in skin squamous cell
carcinoma
Speaker: D. Nassar (Belgium)
Satellite Symposium: Illumina
Transform the Future of Oncology with Genomics
Room 14C, 12:30−14:00
12:35 Explore the future with Cancer Genomics
Speaker: C. Attwooll (United Kingdom)
12:55 The genomic landscape of SCLC single CTCs and CTC derived mouse models
Speaker: C. Dive (United Kingdom)
13:25 Translating the Cancer Genome: Therapeutic Options
Speaker: E. Mardis (USA)
13:55 Close
xxx
EACR-23, Satellite Symposia / European Journal of Cancer 50, Suppl. 5 (2014) xxix–xxx
Monday 7 July 2014
Satellite Symposium: Affymetrix
Finding and Implementing the Right Clinically Actionable
Cancer Biomarker: 360º Tumour Profiling
Room 14A, 12:30−14:00
12:30 Phenotypic identification of subclones in multiple myeloma with different genomic profile, clonogenic
potential and drug sensitivity
B. Paiva (Spain)
13:00 Use of Affymetrix Arrays (Human Transcriptome Array − HTA and Cytoscan HD Array) in haematological
malignancy studies
G. Martinelli (Italy)
13:30 From trials evaluating drugs to trials evaluating treatment algorithms − Focus on the SHIVA trial
C. Le Tourneau (France)
Satellite Symposium: Life Technologies
New Approaches for the Genetic Analysis of Solid Tumours
Room 14C, 12:30−14:00
12:30 Welcome and Introduction
12:40 A single targeted re-sequencing workflow developed for routine TP53 analysis using an Ion AmpliSeq™
gene panel and validated in a breast cancer cohort
Speaker: A. Langerød (Norway)
13:00 Development and validation of an RNA fusion transcript panel for targeted analysis of lung tumours
Speaker: P. Laurent-Puig (France)
13:20 Profiling of miRNA in testicular cancer using qPCR
Speaker: L. Looijenga (The Netherlands)
13:40 HER-2 Copy Number Variation assessment in breast cancer translational research
Speaker: B. Ping (United Kingdom)
xxxi
Innovation Launch Special Session
Sunday 6 July 2014
Revolutionise your NGS Workflow with QIAGEN’s
Platform-Agnostic Sample to Insight Solution
Room 14C, 19:00−21:00
19:00 Welcome with typical Bavarian food and refreshments
19:15 Improving NGS results through removal of sequence artifacts in FFPE samples
Speaker: D. O’Neil (Germany)
19:40 NGS based genome and transcriptome analysis of single cells
Speaker: C. Korfhage (Germany)
20:05 Comparison of variant calling from whole exome and transcriptome sequencing using CLC Cancer
Research Workbench
Speaker: A. Arens (Germany)
20:30 Comprehensive network and pathway analysis of RNA sequencing of triple negative breast cancers
Speaker: J-N. Billaud (Germany)
xxxii
Programme at a Glance
Saturday5July2014
Room 14B
Sunday6July2014
Room 14B
Room 1
Room 13
Symposium
08:30Ͳ10:15
GenomeStability
Symposium
08:30Ͳ10:15
TumourHeterogeneity
Symposium
08:30Ͳ10:15
TumourMetabolism
CoffeeBreak/PosterViewing/Exhibition
10:15Ͳ10:45
OpeningAddress
13:00Ͳ13:15
MühlbockLecture
13:15Ͳ14:15
DevelopmentofTargeted
CancerTherapies
PosterViewing/Exhibition
12:30Ͳ14:00
TheAICRLecture
14:15Ͳ15:00
UsingSwitchableMouseGeneticsto
ModelCancerTherapies
Symposium
14:00Ͳ15:45
TheTumourMicroenvironment
YoungCancer
ResearcherWS
13:00Ͳ14:00
WomeninScience
Symposium
14:00Ͳ15:45
CancerBiomarkers
Symposium
10:45Ͳ12:30
CancerPrevention
SatelliteSymposium
(Room 14A)
12:30Ͳ14:00
SatelliteSymposium
(Room 14C)
12:30Ͳ14:00
Symposium
14:00Ͳ15:45
CellDeath/Autophagy
PlenarySymposium
15:30Ͳ16:45
Epigenetics
OpeningCeremony/OpeningLecture
16:45Ͳ17:50
FollowedbyExhibitors'Reception
18:00Ͳ20:00
Exhibition15:00Ͳ20:00
CoffeeBreak(ExhibitionHall)
15:00Ͳ15:30
Coffeebreak
15:45Ͳ16:15
PosterDefence/Exhibition
15:45Ͳ17:15
AnthonyDipple
CarcinogenesisAward
17:15Ͳ18:00
CarcinogenesisYoung
Investigator'sAward
18:15Ͳ19:00
GeneralAssembly
(Room 14A)
19:00Ͳ21:00
InnovationLaunch
SpecialSession
(Room 14C)
19:00Ͳ21:00
Symposium
10:45Ͳ12:30
MechanismsofDrugResistance
PosterViewing10:15Ͳ17:15
Symposium
10:45Ͳ12:30
SenescenceandAgeing
EACR-23, Programme at a Glance / European Journal of Cancer 50, Suppl. 5 (2014) xxxii–xxxiii
Monday7July2014
Room 14B
Room 1
Tuesday8July2014
Room 13
Room 14B
EMBOLecture
08:45Ͳ09:30
HallmarksofCancer
OECISymposium
08:30Ͳ10:15
PersonalisedCancerMedicine
Symposium
08:30Ͳ10:15
CancerImmunology
Symposium
08:30Ͳ10:15
NonͲcodingRNAsinCancer
ThePezcollerFoundationͲEACR
CancerResearcherAwardLecture
09:30Ͳ10:15
CoffeeBreak
10:15Ͳ10:45
CoffeeBreak/PosterViewing/Exhibition
10:15Ͳ10:45
Symposium
10:45Ͳ12:30
MechanismsofTumour
Suppression
SatelliteSymposium
(Room 14A)
12:30Ͳ14:00
PosterViewing/Exhibition
12:30Ͳ14:00
SatelliteSymposium
(Room 14C)
12:30Ͳ14:00
Symposium
14:00Ͳ15:45
NovelCancerTherapies
Symposium
14:00Ͳ15:45
InflammationandCancer
Coffeebreak
15:45Ͳ16:15
PosterDefence/Exhibition
15:45Ͳ17:15
PlenarySymposium
17:15Ͳ18:45
CancerGenomics
CongressDinner
20:00Ͳ22:30
Symposium
14:00Ͳ15:45
Imaging
PlenarySymposium
10:45Ͳ12:15
StemCells
AEKsupportedSymposium
10:45Ͳ12:30
InvasionandMetastasis
PosterViewing10:15Ͳ17:15
Symposium
10:45Ͳ12:30
NewStrategiesinEarlyDetection
ClosingRemarks,PosterAwards
andEACRͲ23Highlights
12:30
MikePriceGoldMedal
AwardLecture
12:50Ͳ13:30
xxxiii
xxxiv
Scientific Programme
13:00–13:15
Opening Address (Room 14B)
13:00 Welcome to EACR-23
M. Oren, EACR President and Congress Chair
13:07 Welcome Message from the National Committee Chair
A. Ullrich (Germany)
13:15–14:15
Mühlbock Lecture: Development of Targeted Cancer Therapies
(Room 14B)
Abstract number
Chair: R. Marais (United Kingdom)
13:15 Development of targeted cancer therapies
A. Ullrich (Germany)
14:15–15:00
AICR Lecture: Using Switchable Mouse Genetics to Model
Cancer Therapies (Room 14B)
1
Abstract number
Chair: E. Verschuren (Finland)
14:15 Using switchable mouse genetics to model cancer therapies
G. Evan (United Kingdom)
15:30–16:45
Plenary Symposium: Epigenetics (Room 14B)
2
Abstract number
Chair: M. Yaniv (France)
15:30 The cancer epigenome
P. Jones (USA)
16:00 Epigenetic targets in cancer
K. Helin (Denmark)
16:30 Discussion
16:45–17:50
Opening Ceremony / Opening Lecture (Room 14B)
3
4
Abstract number
Chair: M. Oren (Israel)
16:50 Prevention is the cure
C. Maar, Felix Burda Foundation (Germany)
17:05 Cancer research from the bench to the bedside
O. Wiestler (Germany)
17:35 My life for football
H. Herrlich, Coach of Bayern Munich U17 and brain cancer survivor (Germany)
18:00–20:00
Exhibitors Reception (Exhibition Hall)
5
EACR-23, Scientific Programme / European Journal of Cancer 50, Suppl. 5 (2014) xxxiv–lxxxiv
xxxv
Sunday 6 July 2014
08:30–10:15
Symposium: Genome Stability (Room 14B)
Abstract number
Chair: Y. Shiloh (Israel)
08:30 Targeting MTH1 prevents sanitation of oxidised dNTP pools and causes cancer-selective DNA
damage and cell death
T. Helleday (Sweden)
09:00 Chromatin dynamics and cell cycle: From histone variants to heterochromatin
G. Almouzni (France)
09:30 Proffered Paper: Mono- and polygenic breast cancer susceptibility associates with error-prone
homologous DNA double-strand break repair in mouse and man
L. Wiesmüller , M. Deniz, K.J. Obermeier, M. Böhringer, M. Keimling, N. Griner,
D.J. Jerry (Germany)
09:45 The ATM-mediated DNA damage response: Implications for cancer development and treatment
Y. Shiloh (Israel)
08:30–10:15
Symposium: Tumour Heterogeneity (Room 1)
6
7
8
9
Abstract number
Chair: A. Berns (Netherlands)
08:30 Intra-tumour heterogeneity in early-stage lung cancer inferred by multi-region sequencing
E. De Bruin (United Kingdom)
09:00 Functional heterogeneity of tumour-initiating cells in gastrointestinal cancers
H. Glimm (Germany)
09:30 Proffered Paper: The life history of lethal metastatic prostate cancer (The UK prostate cancer
working group of the International Cancer Genome Consortium)
D.C. Wedge, G. Gundem, P. Van Loo, D. Brewer, K. Leinonen, R. Eeles, C. Cooper, T. Visakorpi,
U. McDermott, G.S. Bova (United Kingdom)
09:45 Tumor heterogeneity and cell-of-origin of mouse small cell and non-small cell lung cancer
A. Berns (Netherlands)
08:30–10:15
Symposium: Tumour Metabolism (Room 13)
10
11
12
13
Abstract number
Chair: K. Vousden (United Kingdom)
08:30 Balancing energetic and anabolic needs of cancer cells
K. Vousden (United Kingdom)
09:00 Role of autophagy in the metabolism and growth of lung cancer
E. White (USA)
09:30 Proffered Paper: Altered mitochondrial function and energy metabolism is associated with a
radioresistant phenotype in oesophageal adenocarcinoma
N. Lynam-Lennon, S.G. Maher, A. Maguire, J. Phelan, C. Muldoon, J.V. Reynolds,
J.N. O’Sullivan (Ireland)
09:45 Magnetic resonance imaging of tumour metabolism
K.M. Brindle (United Kingdom)
10:45–12:30
Symposium: Senescence and Ageing (Room 14B)
14
15
16
17
Abstract number
Chair: J. Campisi (USA)
10:45 Epigenetics of cellular senescence, insights into cancer and aging
P. Adams (United Kingdom)
11:15 Aneuploidy and telomerase in stem cell derived cancer
K. Rudolph (Germany)
18
19
xxxvi
11:45 Proffered Paper: Senescent cells impact premalignant microenvironment by direct protein transfer
A. Biran, M. Perelmutter, D. Burton, Y. Ovadya, T. Geiger, V. Krizhanovsky (Israel)
12:00 Cancer and ageing: Rival demons?
J. Campisi (USA)
10:45–12:30
Symposium: Mechanisms of Drug Resistance (Room 1)
20
21
Abstract number
Chair: D. Peeper (Netherlands)
10:45 Mechanisms and biomarkers of acquired resistance to targeted cancer therapies
R. Marais (United Kingdom)
11:15 Mutations and acquired resistance in colorectal cancer
A. Bardelli (Italy)
11:45 Proffered Paper: JAK2/STAT5 inhibition circumvents resistance to PI3K/mTOR blockade:
A rationale for co-targeting these pathways in metastatic breast cancer
A. Britschgi, R. Andraos, H. Brinkhaus, I. Klebba, V. Romanet, U. Mueller, M. Murakami,
T. Radimerski, M. Bentires-Alj (Switzerland)
12:00 In vivo RNAi screening for novel therapeutic cancer targets
D. Peeper (Netherlands)
10:45–12:30
Symposium: Cancer Prevention (Room 13)
22
23
24
25
Abstract number
Chair: J. Cuzick (United Kingdom)
10:45 Progress in breast cancer prevention
J. Cuzick (United Kingdom)
11:15 Worldwide prevention of cervical cancer
J. Peto (United Kingdom)
11:45 Proffered Paper: Cancer risk induced by computed tomography scans in pediatrics: first results
from a national cohort study in France
N. Journy, J.L. Rehel, J.F. Chateil, H. Ducou Le Pointe, M. Mezzarobba, S. Caer-Lorho,
D. Laurier, M.O. Bernier (France)
12:00 Alcohol and cancer risk, with focus on low doses
C. La Vecchia (Italy)
13:00–14:00
26
27
28
29
Young Cancer Researcher Workshop: Women in Science (Room 1)
Coordinator: D. Del Bufalo (Italy)
13:00 A. Buzyn (France)
13:25 Questions & Answers
14:00–15:45
Symposium: The Tumour Microenvironment (Room 14B)
Abstract number
Chair: F. Balkwill (United Kingdom)
14:00 From the immune contexture to the Immunoscore in cancer
J. Galon (France)
14:30 Understanding transcriptional regulation of myeloid cells in the tumor microenvironment
J. Schultze (Germany)
15:00 Proffered Paper: Vessel co-option in colorectal cancer liver metastases mediates resistance to
conventional anti-angiogenic therapy
V. Thompson, S. Frentzas, P. Vermeulen, S. Foo, Z. Eltahir, G. Brown, D. Cunningham,
A.R. Reynolds (United Kingdom)
15:15 Targeting cancer-related inflammation
F. Balkwill (United Kingdom)
30
31
32
33
14:00–15:45
Symposium: Cancer Biomarkers (Room 1)
xxxvii
Abstract number
Chair: M. Mann (Germany)
14:00 Progress toward a biomarker discovery-to-development pipeline in clinical proteomics
S.A. Carr (USA)
14:30 Personalised proteotypes and their association with disease
R. Aebersold (Switzerland)
15:00 Proffered Paper: Proteogenomic analysis of human breast cancer connects genetic alterations
to phosphorylation networks
P. Mertins, K.R. Clauser, D.R. Mani, M. Gillette, D. Fenyo, P. Wang, A. Paulovich, M. Ellis,
S.A. Carr, Clinical Proteomic Tumor Analysis Consortium (USA)
15:15 Recent developments in the technology of mass spectrometry-based clinical proteomics
M. Mann (Germany)
14:00–15:45
Symposium: Cell Death/Autophagy (Room 13)
34
35
36
37
Abstract number
Chair: A. Kimchi (Israel)
14:00 Points of interface between autophagy and apoptosis
A. Kimchi (Israel)
14:30 Autophagy and cell death
S. Fulda (Germany)
15:00 Proffered Paper: miRNA induces escape from NK cell-mediated cytotoxicity and resistance
towards apoptosis in breast cancer
C. Breunig, M. Küblbeck, M. Miller, D. Antonelli, C. Wirth, N. Erdem, Y. Yarden, A. Cerwenka,
S. Wiemann (Germany)
15:15 Sphingomyelin metabolism as a target for cancer therapy
M. Jäättelä (Denmark)
17:15–18:00
Award Lecture: Anthony Dipple Carcinogenesis Award
(Room 14B)
38
39
40
41
Abstract number
Chair: C.C. Harris (USA)
17:15 The mutant p53-cancer stem cells and drug resistance paradigm
V. Rotter (Israel)
18:15–19:00
Award Lecture: Carcinogenesis Young Investigator’s Award
(Room 14B)
42
Abstract number
Chair: C.C. Harris (USA)
18:15 Outside the coding genome: microRNAs make a big difference
L. He (USA)
19:00–21:00
EACR General Assembly (Room 14A)
43
xxxviii
Monday 7 July 2014
08:30–10:15
OECI Symposium: Personalised Cancer Medicine (Room 14B)
Abstract number
Chair: O. Kallioniemi (Finland)
08:30 The benefits of precision medicine will require us to examine both what we do and how we do it
S. Friend (USA)
09:00 OECI Lecture: Cancer genomics and plasma DNA: Towards new classification of breast cancer
and monitoring therapy response
C. Caldas (United Kingdom)
09:30 Proffered Paper: Therapeutical landscape of cancer drivers
A. Gonzalez-Perez, D. Tamborero, C. Rubio, M. Schroeder, C. Perez-Llamas, J. Deu-Pons,
N. Lopez-Bigas (Spain)
09:45 Individualised systems medicine for cancer care
O. Kallioniemi (Finland)
08:30–10:15
Symposium: Cancer Immunology (Room 1)
44
45
46
47
Abstract number
Chair: A. Hayday (United Kingdom)
08:30 Peptide-based immunotherapy of cancer
H. Rammensee (Germany)
09:00 Blocking the PD-1/PD-L1 axis for cancer therapy
C. Drake (USA)
09:30 Proffered Paper: Overcoming immune evasion in ovarian and breast cancer with anti-CD47
antibody blockade: A novel class of immune therapy
A.K. Volkmer , S.B. Willingham, S.R. Tseng, P.Y. Ho, J.P. Volkmer, B.I. Sikic, R. Majeti,
I.L. Weissman (Germany)
09:45 How T cells may distinguish stress from normality in an epithelium
A. Hayday (United Kingdom)
08:30–10:15
Symposium: Non-coding RNAs in Cancer (Room 13)
48
49
50
51
Abstract number
Chair: A. Harel-Bellan (France)
08:30 Long non-coding RNA and cancer
S. Diederichs (Germany)
09:00 miRNA−protein interaction networks in cancer
S. Wiemann (Germany)
09:30 Proffered Paper: Hypoxic regulation of the long non-coding transcriptome in breast cancer
H. Choudhry, A. Albukhari, J. Schodel, S. Oikonomopoulos, S. Haider, P. Ratcliffe, I. Ragousis,
D. Mole, A. Harris (United Kingdom)
09:45 Nuclear RNAi and cell fate
A. Harel-Bellan (France)
10:45–12:30
Symposium: New Strategies in Early Detection (Room 14B)
52
53
54
55
Abstract number
Chair: C. Dive (United Kingdom)
10:45 The NCI early detection research network: Charting the course of biomarker research
S. Srivastava (USA)
11:15 Early detection biomarkers for oesophageal cancer
P. Lao-Sirieix (United Kingdom)
56
57
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11:45 Proffered Paper: Noninvasive monitoring of tumour mutations and dynamics in circulating DNA
of non-small cell lung cancer patients treated with EGFR inhibitors
D. Tsui, A.S.C. Wong, M. Murtaza, T. Forshew, R. Soo, H.L. Lim, B.C. Goh, D. Gale, T.M. Chin,
N. Rosenfeld (United Kingdom)
12:00 CTCs and cfDNA, will they be useful for early detection of cancer?
C. Dive (United Kingdom)
10:45–12:30
AEK Supported Symposium: Invasion and Metastasis (Room 1)
58
59
Abstract number
Chair: C. Isacke (United Kingdom)
10:45 Selection and adaptation during metastatic cancer progression
C. Klein (Germany)
11:15 Role of the Hippo transducers YAP and TAZ in mechanotransduction and cancer stem cells
S. Piccolo (Italy)
11:45 Proffered Paper: Cell-specific proteomic analysis of contact-initiated tumour-endothelial signalling
identifies novel regulators of transendothelial migration
M. Locard-Paulet, L. Lim, J. Sinclair, C. Jorgensen (United Kingdom)
12:00 Tumour–stroma interactions in breast cancer progression
C. Isacke (United Kingdom)
10:45–12:30
Symposium: Mechanisms of Tumour Suppression (Room 13)
60
61
62
63
Abstract number
Chair: M. Oren (Israel)
10:45 Role of TP53 and other tumour suppressors in breast cancer development and progression
A. Børresen-Dale (Norway)
11:15 Functional interplay between the DNA damage response and ARF pathways in human cancer
V.G. Gorgoulis (Greece)
11:45 Proffered Paper: Regulation of NF-kB, inflammation and cancer by the E3 ligase RNF20
O. Tarcic, M. Oren (Israel)
12:00 Unexpected cancer-related properties of BRCA1
D. Livingston (USA)
14:00–15:45
Symposium: Novel Cancer Therapies (Room 14B)
64
65
66
67
Abstract number
Chair: C. Von Kalle (Germany)
14:00 Novel therapeutic approaches to lung cancer
C. Rudin (USA)
14:30 The TGF-beta pathway as a therapeutic target
J. Seoane (Spain)
15:00 Proffered Paper: Inhibition of RNA Polymerase I transcription by CX-5461 as a completely new
approach to treat highly refractory haematological malignancies
R. Hannan, N. Hein, S.E. O’Brien, D. Drygin, C. Cullinane, G. Matthews, R.W. Johnstone,
R.B. Pearson, G. McArthur, S. Harrison (Australia)
15:15 Targeted cancer genetics
C. Von Kalle (Germany)
14:00–15:45
Symposium: Inflammation and Cancer (Room 1)
68
69
70
71
Abstract number
Chair: F.M. Watt (United Kingdom)
14:00 Inflammation in colorectal and liver tumorigenesis
M. Karin (USA)
14:30 Parainflammation in cancer
Y. Ben-Neriah (Israel)
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15:00 Proffered Paper: Investigation of the spatial heterogeneity of specific immune cell phenotypes
in the tumor microenvironment of follicular lymphoma
J. Mansfield, L. Nelson, B. Wendik, C. Van der Loos, C. Rose, R. Byers (USA)
15:15 Role of inflammation in epidermal carcinogenesis
F.M. Watt (United Kingdom)
14:00–15:45
Symposium: Imaging (Room 13)
74
75
Abstract number
Chair: M. Schwaiger (Germany)
14:00 Multiparametric imaging in cancer research
A.K. Buck (Germany)
14:30 Molecular imaging for personalised treatment of cancer
W.J.G. Oyen (Netherlands)
15:00 Proffered Paper: Dual wavelength near-infrared fluorescence imaging of VEGF and IGF-1R in
ovarian cancer patient-derived xenografts
T. Tomar , N.G. Alkema, A.G. Terwisscha van Scheltinga, J.A.L. Visser, G.J. Meersma,
E.W. Duiker, E.G.E. De Vries, A.G.J. Van der Zee, G.B.A. Wisman, S. De Jong (Netherlands)
15:15 Imaging as tool for translational research in oncology
M. Schwaiger (Germany)
17:15–18:45
Plenary Symposium: Cancer Genomics (Room 14B)
76
77
78
79
Abstract number
Chair: C. Von Kalle (Germany)
17:15 Recent advances in cancer genomics
E. Mardis (USA)
17:50 Oncogenomics of brain tumors: Integrated approaches reveal novel pathomechanisms
P. Lichter (Germany)
18:25 Discussion
80
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Tuesday 8 July 2014
08:45–09:30
The EMBO Lecture: Hallmarks of Cancer: Applications to Cancer Abstract number
Medicine? (Room 14B)
Chair: A. Børresen-Dale (Norway)
08:45 Hallmarks of cancer: applications to cancer medicine?
D. Hanahan (Switzerland)
09:30–10:15
The Pezcoller Foundation − EACR Cancer Researcher Award
Lecture (Room 14B)
82
Abstract number
Chair: D. Del Bufalo (Italy)
09:30 Introduction of the Award Lecturer
D. Bassi (Italy)
09:35 Mechanisms of metastasis by colorectal cancer stem cells
E. Batlle (Spain)
10:45–12:15
Plenary Symposium: Stem Cells (Room 14B)
83
Abstract number
Chair: J. Seoane (Spain)
10:45 Mesenchymal and MDS stem cells shape an interactive disease unit in the bone marrow
A. Trumpp (Germany)
84
11:20 Stem cells in homeostasis and cancer
E. Fuchs (USA)
11:55 Discussion
12:30–12:50
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85
Closing Ceremony (Room 14B)
EACR-23 Highlights and Closing Remarks
M. Oren (Israel)
Poster Awards
Announcement of EACR24
R. Marais (United Kingdom)
12:50–13:30
Mike Price Gold Medal Award Lecture (Room 14B)
Abstract number
Chair: J.E. Celis (Denmark)
12:50 Infections causing human cancers − mechanisms and perspectives
H. zur Hausen (Germany)
86
Poster Sessions, Sunday 6 July 2014
10:15–17:15
Cell and Tumour Biology I
Abstract/poster number
100
IKKb dependent NF-kB activation suppresses progression of lung metastases during breast
carcinogenesis
H.Y. Fang, O.C. Olson, J.A. Joyce, M. Quante, F.R. Greten (Germany)
SWAP-70 is required for spontaneous transformation of mouse embryo fibroblasts
101
Y.T. Chang, C.L. Shu, J.Y. Lai, C.Y. Lin, C.P. Chuu, T. Ichikawa, K. Morishita, R. Jessberger,
Y. Fukui (Taiwan)
102
Growth-inhibitory effect of conjugated linolenic acid on human eosinophilic leukemia EoL-1 cells
W.N. Liu, K.N. Leung (Hong Kong)
103
The tumour–stroma niche of ovarian cancer in 3D
D. Loessner , B.M. Holzapfel, J. Baldwin, A. Rockstroh, V. Magdolen, D.W. Hutmacher,
J.A. Clements (Australia)
ZEB1 modulates expression of CDX1 and CDX2 caudal homeobox genes in colorectal carcinoma cell
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lines
O. De Barrios, C.A. Orozco, E. Sánchez-Tilló, L. Fanlo, A. Castells, A. Postigo (Spain)
Anoikis of colon carcinoma cells triggered by beta-catenin loss can be enhanced by Tumor Necrosis
105
Factor Receptor 1 antagonists
K. Rosen, O. Masson, Y. Li, B. Yoo (Canada)
The positive feedback between interleukin-8 (IL-8) and matrix metalloproteinase-9 (MMP-9) in hormone
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dependent breast cancer
J. Milovanovic, N. Todorovic-Rakovic, Z. Abu Rabi (Serbia)
108
Potential effects of telomerase activity and Bcl-2 expression on the apoptosis of the human brain tumors
C. Kim, J.H. Cheong, J.M. Kim (Korea)
Biological activity of novel platinum(II)–iodido complexes
110
L. Filipovic, A. Savic, S. Arandjelovic, T. Sabo, S. Grguric-Sipka, S. Radulovic (Serbia)
111
RANK pathway as a new therapeutic target in primary breast cancer
P. Pellegrini, A. Cordero, E. González-Suarez (Spain)
xlii
Expression of voltage-gated sodium channel beta1 subunits in breast cancer: Promotion of tumor
growth and metastasis
M. Nelson, R. Millican-Slater, L.C. Forrest, W. Brackenbury (United Kingdom)
An aqueous extract of Limoniastrum guyonianum gall induces anti-tumor effects in melanoma-injected
mice via modulation of the immune response
M. Krifa, I. Skandrani, A. Pizzi, N. Nasr, K. Ghedira, L. Chekir (Tunisia)
The role of sumoylation in the regulation of p53 response
D. Marouco, N.A. Barlev (United Kingdom)
The mRNA-binding protein LARP1 is a pro-survival factor that promotes tumourigenicity and
chemotherapy resistance in ovarian cancer
T.G. Hopkins, J. Weir, M. Mura, N. Abd-Latip, K. Sweeney, S. Ghaem-Maghami, G. Gabra,
S.P. Blagden (United Kingdom)
Interaction of C-FABP and PPARS in prostate cancer
F. Seyed Forootan, S. Seyed Forootan Shiva, Y. Ke (United Kingdom)
Autocrine human growth hormone promotes the oncogenic behaviour of hepatocellular carcinoma cells
Y.J. Chen, X.J. Kong, Z.S. Wu, Y.C. Lau, J.J. Wang, T. Zhu, P.E. Lobie (Singapore)
Poly(I:C) and chemotherapeutics synergistically induce cell death in head and neck cancer cell lines
T. Matijevic Glavan, B. Verillaud, P. Busson, J. Pavelic (Croatia)
M30 assay may not be an accurate method for apoptosis in the cancer cells expressing low level of
cytokeratin
B. Cevatemre, F. Ari, M. Sarimahmut, A. Yilmaztepe Oral, E. Ulukaya (Turkey)
Vincristine induces autophagy-mediated HMGB1 release via transcriptional regulation of Mcl-1
antagonizes apoptosis in human oral cancer cells
S. Yang, C. Lin, M. Hsieh (Taiwan)
Autophagy effects of pterostilbene on human oral cancer cells through modulation of Akt and
mitogen-activated protein kinase pathway
C. Lin, S. Yang, M. Hsieh (Taiwan)
Metabolic and motile reprogramming of ER positive breast cancer cells following long-term estrogen
deprivation
A. Morandi, M. Bacci, L.A. Martin, M.L. Taddei, E. Giannoni, P. Chiarugi (Italy)
Loss of far upstream element (FUSE) binding protein (FBP)-interacting repressor (FIR) function
supports HCC growth
M. Malz, M. Bovet, J. Samarin, D.F. Calvisi, S. Rössler, S. Singer, M. Weber, M. Zörnig, P. Schirmacher,
K. Breuhahn (Germany)
Semaphorin7A increases growth and metastasis of mammary tumours by promoting tumour cell
survival and motility
R. Garcia-Areas, S. Libreros, S. Amat, C. Castro-Silva, P. Robinson, E. Wojcikiewicz, K. Schilling,
V. Iragavarapu-Charyulu (USA)
Eight microRNAs as biomarkers for metastatic spread in triple negative breast cancer
A. Mathe, K.A. Avery-Kiejda, M. Wong-Brown, J.F. Forbes, S.G. Braye, R.J. Scott (Australia)
Diacylglycerol kinase alpha regulates Src and promotes 3D growth in breast cancer
P. Torres-Ayuso, M. Daza-Martin, A. Ávila-Flores, I. Mérida (Spain)
Autocrine human growth hormone suppression of E-cadherin via p44/42 MAPK promotes
epithelial-to-mesenchymal transition of colorectal carcinoma cells
J. Wang, Z. Wu, V. Pandey, Y. Chen, T. Zhu, P. Lobie (Singapore)
Oral cavity changes in lymphoma patients
I. Besu Zizak, S. Jelic, S. Matkovic, B. Mihaljevic, L.J. Jankovic (Serbia)
The role of Delta-40p53 and p53 in Estrogen Receptor-a signalling pathways in breast cancer
B. Morten, R.J. Scott, K.A. Avery-Kiejda (Australia)
A novel monoclonal antibody allowing for the isolation and analysis of Lgr5 positive cells from cell lines
and primary human tissue
D. Agorku, N. Chelius, A. Bosio, O. Hardt (Germany)
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Antitumor activities of some macrofungi extracts
Z. Zizak, A. Klaus, M. Kozarski, J. Vunduk, M. Niksic, Z. Juranic (Serbia)
Induction of cellular senescence and hair follicle stem cell dysfunction upon p14ARF and p16Ink4a
expression in the skin
N. Azazmeh, R. Tokarsky-Amiel, I. Ben-Porath (Israel)
MET signaling in colorectal cancer-initiating cells blunts response to EGFR inhibition: Implications for
targeted therapy
P. Luraghi, G. Reato, E. Cipriano, E. Menietti, F. Sassi, V. Bigatto, F. Orzan, A. Bertotti, L. Trusolino,
C. Boccaccio (Italy)
RET/PTC1 in vitro models unveil a novel tumor suppressor miRNA in papillary thyroid carcinoma
E. Minna, P. Romeo, L. De Cecco, M. Dugo, G. Cassinelli, C. Lanzi, S. Pilotti, M.A. Pierotti, A. Greco,
M.G. Borrello (Italy)
Proteolytic regulation of the EphB4 receptor in prostate cancer
J. Lisle, I. Mertens-Walker, C. Stephens, S. Stansfield, A. Herington, J. Clements,
S. Stephenson (Australia)
Zoledronate suppresses human osteosarcoma (U2OS) cells invasion and migration by affecting cell
morphology and cytoskeletal organization via FAK, RhoA, Ras, vimentin and E-cadherin
K. Lu, S. Yang (Taiwan)
Breast cancer tumor secreted factors induce both endothelial activation and increment of vascular
permeability in vitro
M.A. Gallardo Vera, J.L. Ventura Gallegos, A. Zentella Dehesa (Mexico)
Tumor microenvironment influencing 18 F-FDG uptake − an in vitro study in three different colorectal
carcinoma cell lines
A. Abrantes, A.S. Pires, M. Laranjo, A.C. Mamede, A.F. Brito, J. Casalta-Lopes, A.C. Gonçalves,
A.B. Sarmento-Ribeiro, M.F. Botelho (Portugal)
Exploring the metabolic profile of colorectal cancer cells from different origin
A. Abrantes, L. Tavares, A.S. Pires, M. Laranjo, J. Casalta-Lopes, R.A. Carvalho, M.F. Botelho (Portugal)
Is 18 F-FDG uptake in three different colon cancer cell lines affected by incubation with sodium butyrate?
T.J. Gonçalves, A.S. Pires, J.C. Encarnação, R. Teixo, A.F. Brito, J.E. Casalta-Lopes, A.M. Abrantes,
M.F. Botelho (Portugal)
High selective cytotoxic activity of sesamol induced mitochondrial-mediated apoptosis pathway in lung
adenocarcinoma cells
B. Siriwarin, N. Weerapreeyakul, S. Barusrux (Thailand)
Impact of the microenvironment on therapeutics in breast cancer
C.J. Lovitt, V.M. Avery (Australia)
Antithetic effect of PDGFRbeta-induced miR-9 and ZEB-1-repressed miR-200cin vasculogenic mimicry
of triple negative breast cancer
E. D’Ippolito, I. Plantamura, P. Casalini, S. Baroni, C. Piovan, R. Orlandi, F. De Braud, E. Tagliabue,
M.V. Iorio (Italy)
Novel CSF-1 receptor ligand IL-34 modulates macrophage-breast cancer cell crosstalk
S. Gunawardhana, K. Zins, T. Lukas, D. Abraham (Austria)
Reduced expression of GRHL genes in human non-melanoma skin cancers
A. Kikulska, B. Wilczynski, T. Rausch, V. Benes, P. Rutkowski, T. Wilanowski (Poland)
Unravelling the role of Caveolin-1 in metabolic reprogramming during hepatocellular carcinoma
Z. Nwosu, S. Dooley, C. Meyer (Germany)
11beta-hydroxysteroid dehydrogenase in human colon and colonic tumors
J. Pácha, P. Ergang, M. Moravec, J. Bryndová, S. Zbánková, M. Kment, V. Mandys (Czech Republic)
Pterostilbene inhibit hepatocellular carcinoma cells proliferation by induce autophagy and apoptosis
in vitro
H. Chiou, S. Yang, M. Hsieh (Taiwan)
Role of Id1–IGF2–VEGF–VEGFR relay in esophageal tumorigenesis
A.L.M. Cheung, W.W. Xu, B. Li, S.W. Tsao, K.W. Chan (Hong Kong)
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Specific EMT-inducers signature associates with oncogenic events in breast tumour progression
C. Moyret-Lalle, E. Ruiz, S. Courtois-Cox, C. Bardel, A. Veron (France)
Metabolic activity of tumor cells in a variable microenvironment: combinations of glucose, glutamine,
lactate and pH
A.M. Otto, C. Janzon, J. Hutterer (Germany)
Interplay between EMT-inducers and miRNAs during breast tumorigenesis
C. Moyret-Lalle, B. Vitton-Méa, E. Ruiz, C. Hermann, E.W. Lam, A. Puisieux (France)
The role of the transcription factor ecotropic viral integration site 1 in prostate cancer
A. Queisser , S. Hagedorn, W. Vogel, D. Böhm, A. Von Mässenhaussen, C. Lengerke,
S. Perner (Germany)
FoxF1 is a potential oncogene in prostate cancer
M. Nowak, R. Menon, F.A. Kunze, M.A. Svensson, J. Carlsson, N. Wernert, G. Kristiansen, O. Andrén,
S. Perner (Germany)
Role of trefoil factor-3 peptide (TFF3) in prostate cancer progression
M. Nowak, C. Merz, A. Von Maessenhausen, W. Vogel, D. Boehm, N. Wernert, G. Kristiansen,
S. Perner (Germany)
Regulation of S-adenosylmethionine synthesis in a sequential model of cirrhosis-hepatocellular cancer
by adenosine derivative
L.R. Marı́a Guadalupe, V.L. Nora Gabriela, D.L. Mariana, C.S. Victoria (Mexico)
Autoschizis: a cell death induced by the anticancer, pro-oxidant stress of ascorbate:menadione
combination
J. Gilloteaux, J.M. Jamison, J.L. Summers (United Kingdom)
A novel Zn(II)-compound reactivates mutant p53 protein in cancer cells: molecular mechanisms and
therapeutical implications
A. Garufi, D. Pucci, M.L. Avantaggiati, G. D’Orazi (Italy)
Cycling hypoxia amplifies tumor microenvironment inflammation
C. Michiels, C. Tellier, C. Graux, L. Finet, M. Raes, O. Feron (Belgium)
Serum endostatin levels are elevated in colorectal cancer and correlate with invasion and systemic
inflammatory markers
A. Tuomisto, T. Kantola, J.P. Väyrynen, K. Klintrup, J. Mäkelä, S.M. Karppinen, T. Pihlajaniemi,
H. Autio-Harmainen, T. Karttunen, M. Mäkinen (Finland)
Role of NANOS family members in tumor progression
E. De Keuckelaere, V. Andries, K. Staes, S. Grelet, B. Nawrocki-Raby, P. Birembaut, F. Van Roy (Belgium)
Vasoactive intestinal peptide decreases MYCN expression and AKT activity via a PKA-dependent
pathway in neuroblastoma cells
M. De Boisvilliers, A. Garnier, A.C. Meunier, S. Bensalma, J.M. Muller, C. Chadeneau (France)
miR-27a is a functional biomarker for tamoxifen treatment of luminal A/B breast tumors
B. Ljepoja, J. Garcia-Roman, F. Kopp, E. Wagner, A. Roidl (Germany)
Characterization and analysis of deregulation of signaling pathways of cancer stem cells derived from
oral squamous cell carcinoma
N. Andrade, M.F.S.D. Rodrigues, C.O. Rodini, F.D. Nunes (Brazil)
ADAM9 coordinates genes in anoikis resistance for lung cancer metastases
Y. Sher , C. Lin, C. Huang, L. Lai, T. Kuo, G. Tseng, M. Hung (Taiwan)
COX-2 independent induction of apoptosis by two synthetic COX-2 inhibitors in breast cancer cell line
M. Salimi, S. Norouzi, M. Norouzi, M. Amini, A. Amanzadeh (Iran)
Antiproliferative effects of different fractions obtained from Anthemis nobilis leaves on human oral
cancer cell
M. Salimi, N. Gheisarzadeh, K. Azadmnaesh, N. Rastkari, M. Salimi (Iran)
Cellular and molecular regulations of head and neck carcinogenesis under diabetic environment
W.J. Chang, C.Y. Chen, T.Y. Chen, P.L. Lee, W.C. Li (Taiwan)
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IGF family genes expression in clear cell renal cell cancer and their corresponding adjacent
non-cancerous kidney tissues
R.S. Braczkowski, M. Bialozyt, B. Braczkowska (Poland)
Class III beta-tubulin is a target gene of HIF-2alpha in glioblastoma cells exposed to hypoxia
K. Bordji, A. Grandval, L. Cunha-Alves, E. Lechapt-Zalcman, M. Bernaudin (France)
From ERa66 to ERa36: a new predictive marker for cancer progression and therapeutic response in
breast tumors?
C. Chamard, A. Jung, A. Chesnel, J. Abecassis, S. Flament, C. Macabre, S. Ledrappier, T. Boukhobza,
H. Dumond (France)
Roles of digoxin and digitoxin in hepatocellular carcinoma
W. Huang, Y. Jeng, I. Fong, H. Hsu (Taiwan)
Regulation of Panax notoginseng extract on metastasis in human colon cancer cells
C.C. Wu, Y.H. Kuo, C.Y. Lee, C.C. Tsai, S.L. Hsieh (Taiwan)
The anti-cancer effects of clioquinol on oral cancer
M.W. Lee, P.C. Lin, W.C. Tsai (Taiwan)
The expression of the human Sprouty protein-1 (hSpry1) inversely correlates with proliferation, migration
and invasion of the SKOV-3 and 1A9 human ovarian cancer cells
S. Masoumi-Moghaddam, A. Amini, D.L. Morris (Australia)
Bromelain and N-acetylcysteine induce cytotoxic effects and reduce the expression of mucin in
mucin-producing carcinoma cell lines of gastrointestinal origin
A. Amini, S. Masoumi-Moghaddam, D.L. Morris (Australia)
Characterization of ovarian carcinoma-associated fibroblasts: The capability in predicting tumor
aggressiveness
C. Liu, T. Mao (Taiwan)
Involvement of CHK2 kinase in pre-mRNA splicing
H.H. Choi, H.K. Choi, Y. Bae, S.T. Kim, T. Kim (South Korea)
Heterogeneity among early-stage K-Ras driven lung adenocarcinoma predicts tumor aggressiveness
and identifies Ddr1 as a therapeutic target
C. Ambrogio, G. Gomez, D. Santamaria, M. Barbacid (Spain)
Pinus morrisonicola Hayata extracts inhibit cell proliferation and promote apoptosis of human
promyelocytic HL-60 leukemia cells
G.Y. Liu, Z.W. Wang (Taiwan)
Tetraindole suppresses breast cancer growth and metastasis by reversing epithelial–mesenchymal
transition state associated with up-regulation of the miR-200 family
W. Li, C. Wang, C. Chen, S. Jao (Taiwan)
Platycodi Radix facilitates autophagy through increasing ROS formation in NCI-H460 human non-small
lung carcinoma cells
S.H. Hong, C. Park, M.H. Han, Y. Choi (South Korea)
System-wide characterization of dynamics of cytosolic macromolecular protein complexes during
oncogene-induced cell transformation
B. Diedrich, K.G. Rigbolt, S. Bestmann, T. Brummer, J. Dengjel (Germany)
Tetraarsenic hexaoxide induces apoptosis in human bladder cancer 5637 cells via reactive oxygen
species generation and DNA damage
M.H. Han, Y. Choi, S.H. Hong, W.J. Kim, C. Park (South Korea)
Effects of pro-apoptotic Bcl-2 on morin-induced apoptosis in human leukemia U937 cells
C. Park, Y. Choi, M.H. Han, W.J. Kim, S.H. Hong (South Korea)
ATM kinase expression regulates breast cancer stem-like phenotype
M. Antonelli, M. Sambucci, R. Brandi, I. Arisi, M. D’Onofrio, D. Barilà, V. Stagni (Italy)
Potential therapeutic targets for the hypoxic niche in glioblastoma
A. Dirkse, M. Sanzey, J. Bohler, R. Bjerkvig, A. Golebiewska, S. Niclou (Luxembourg)
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The aging suppressor hormone klotho: A novel tumor suppressor and regulator of estrogen signaling
in ovarian cancer
I. Lojkin, O. Schwarzman, T. Rubinek, B.Y. Karlan, I. Wolf (Israel)
Simultaneous measurements of multiple injected contrast agents using multispectral optoacoustic
tomography (MSOT) in phantoms and in vivo
T. Sardella, N.C. Burton, W.H.P. Driessen, J. Claussen, S. Morscher, D. Razansky,
V. Ntziachristos (Germany)
The ethanolic extract of Phellinus linteus inhibits breast cancer cell growth through autophagy-related
cell death
W. Lee, T. Chiang (Taiwan)
Pre-mRNA alternative splicing controls VEGF-A autocrine functions and response to bevacizumab in
non small cell lung carcinoma
A. Boudria, S. Gout, M. Keramidas, J.L. Coll, S. Lantuejoul, E. Brambilla, S. Gazzeri, B. Eymin (France)
GDF15 knockdown induces resistance to temozolomide treatment in glioblastoma cell line
M. Baroni, P.F. Fedatto, A.F. Andrade, V.K. Suazo, R.G.P. Queiroz, L.G. Tone, C.A. Scrideli (Brazil)
A tea polyphenol epigallocatechin gallate (EGCG) displays a superior effect on enzyme inhibition of
human ornithine decarboxylase
H.C. Hung, L. Pan (Taiwan)
GALNT1 knockdown suppresses hepatocellular carcinoma cell malignant behaviours and decreases
EGFR activation and recycling
M. Huang, Y.M. Wu, M.C. Huang (Taiwan)
K06, a novel CD43 epitope on human thymocytes, is involved in caspase-independent cell death
T.J. Kim, Y. Bae (Korea)
Effect of sesamin, sesamolin and sesamol on P-glycoprotein mediated efflux
C. Junhom, B. Siriwarin, N. Weerapreeyakul, S. Barusrux (Thailand)
Co-evolution of stroma and neoplastic cells in prostate cancer is sustained by reprogramming energy
metabolism
M. Bologna, P. Sanita’, A. Angelucci, P. Muzi, C. Vicentini (Italy)
WIP, a novel negative regulator in WBP2-mediated Wnt pathway activation
S. Lim, Y.P. Lim (Singapore)
The direct cytotoxic and P-glycoprotein modulating effects of Thai indigenous plant extracts in drug
resistant HepG2 cells
C. Junhom, N. Weerapreeyakul, S. Barusrux, T. Titimatharoj (Thailand)
The deubiquitinating enzyme USP15 deubiquitinates the E3 ligase SMURF2
P. Iyengar, P. Jaynes, P. Eichhorn (Singapore)
Activated B cell receptor signaling pathway contributes to bortezomib resistance in mantle cell lymphoma
A. Kim, S. Park, D. Shin, W. Jang, S. Lee, S. Lee (Korea)
Role of extracellular S100A4 in stimulation of melanoma cells crossing the blood–brain barrier in vitro
and in vivo
N. Herwig, S. Wolf, C. Haase-Kohn, J. Steinbach, J. Pietzsch (Germany)
The identification of novel targets in b-catenin-driven acute myeloid leukemic stem cells
H. Yi, J. Wang, M. Kavallaris, J.Y. Wang (Australia)
The role of WWOX gene in EMT process in endometrial cancer
E. Pluciennik, M. Nowakowska, K. Pospiech, K. Kosla, I. Baryla, K. Wojcik-Krowiranda, A. Bienkiewicz,
M. Galdyszynska, M. Popeda, A.K. Bednarek (Poland)
Inactivation of nucleoside-derived anticancer drugs by catabolic prokaryotic enzymes
J. Vande Voorde, S. Sabuncuoglu, J. Balzarini, S. Liekens (Belgium)
Up-regulation of C1GALT1 promotes breast cancer cell growth through MUC1-C signaling pathway
C. Chou, M.C. Huang (Taiwan)
The T-box transcription factor, TBX3, promotes tumourigenesis in soft tissue and bone sarcomas: a
possible therapeutic target
T. Willmer , S. Prince (South Africa)
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Chronic intermittent hypoxia triggers adaptive changes that promote protection against cell death
J. Matschke, H. Riffkin, R. Handrick, L. Klein-Hitpass, V. Jendrossek (Germany)
HIF prolyl hydroxylase PHD3 maintains hypoxic cell cycle through cyclin-dependent kinase inhibitor P27
H. Högel, P. Miikkulainen, L. Bino, P.M. Jaakkola (Finland)
Effect of HuIFN-a and Royal jelly on the proliferation of human colon cancer (CaCo-2) cells in vitro
K. Rihar , D. Gregoric Exel, S. Sladoljev, B. Filipic (Slovenia)
Patient-derived tissue culture and xenograft models for studying prostate tumor responses to therapy
L. Yu, T.E. Kähkönen, P. Taimen, P. Boström, J. Tuomela, P. Härkönen (Finland)
Role of EGFR-regulated microRNAs in malignant processes of non-small cell lung cancer
S. Langsch, S. Schäfer, M.P. Tschan, E. Vassella (Switzerland)
The WWOX gene modulates the adhesion of neural cells to ECM
K. Kosla, E. Styczen-Binkowska, M. Nowakowska, K. Pospiech, E. Pluciennik, I. Baryla,
A.K. Bednarek (Poland)
A novel animal model of phaeochromocytoma for preclinical therapy evaluation
M. Ullrich, R. Bergmann, J. Pietzsch, M. Cartellieri, M. Peitzsch, G. Eisenhofer, S.R. Bornstein,
C.G. Ziegler (Germany)
The LIM domain protein cysteine-rich protein 2 (CRP2) promotes breast cancer progression
C. Hoffmann, X. Mao, M. Dieterle, F. Moreau, A. Steinmetz, C. Thomas (Luxembourg)
BRAF V600E-mutated cells in a papillary thyroid carcinoma do not form a compact ball, but a mesh
of cells sparsely embedded within the stroma
A. Antoniou, M. Tarabichi, S. Le Pennec, V. Detours, C. Maenhaut (Belgium)
Murine tumors microenvironment after irradiation characterized by diffusion-weighted, dynamic contrastenhanced MRI and immunohistology
A. Drzal, M. Gonet, T. Skorka, M. Elas (Poland)
The glycolytic enzyme GPI/AMF is a stimulator of glioma cell migration and directly contributes to the
pro-migratory phenotype under hypoxic conditions
A. Kathagen, M.H. Holz, A.S. Schulte, M.W. Westphal, K.L. Lamszus (Germany)
Clinical and prognostic values of the pretreatment PSA doubling time in patients with prostate cancer
O. Bogomolov, G. Zharinov, M. Shkolnik (Russian Federation)
MMP2 as a molecular biomarker of proficient tumor–stroma cross-talk in lung cancer
E. Baldoli, T. Caputo, G. Bertolini, M. Moro, F. Facchinetti, R. Caserini, U. Pastorino, G. Sozzi,
L. Roz (Italy)
Identification and characterization of novel cancer-associated molecules
A. Chernenko, P.R. Karhemo, M. Reinman, Y. Chen, S. Goodison, K. Takkinen, P. Panula,
P. Laakkonen (Finland)
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Cancer Genomics, Epigenetics and Genome Instability I
Understanding estrogen receptor transcription in breast cancer
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J. Carroll (United Kingdom)
Cancer stem cells, a fuzzy evolving concept: A cell population or a cell property?
376
J. Dumont, A. Antoniou, A. Hebrant, C. Maenhaut (Belgium)
DNA2 is highly mutated in estrogen-dependent cancers; from a bioinformatics screen to the effect of
378
clinical mutations on cellular growth
C. Strauss, A. Benvenisty, T. Ravid, A. Arbel, I. Ben-Porath, M. Goldberg (Israel)
379
POLE and POLD1 screening in Portuguese patients with hereditary colorectal cancer
M. Viegas, S. Saraiva, M. Areias, D. Brito, R. Carvalho, S. Alves, M. Lopes, A. Cadime,
T.C. Martins (Portugal)
Characterization of CpG islands and methylation status of Claspin gene (CLSPN) promoter
380
D. Azenha, C. Lopes, T.C. Martins (Portugal)
The ApcMin/+ mouse to identify “driver” epigenetic lesions in colorectal cancer: promoter
381
hypermethylation of the protocadherins
M. Williams, K. Malik, A.R. Dallosso, M. Szemes, H.G. Linhart (United Kingdom)
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Identification of pathogenic virus sequences in pancreatic cancer
M. Amaravadi, A.S. Bauer, A. Hotz-Wagenblatt, S.K. Botla, M. Löchelt, M. Pawlita, M.W. Büchler,
N. Giese, J.D. Hoheisel (Germany)
Effects of different additives for detection of single nucleotide polymorphisms in promoter sequence
EGFR gene in NSCLC
V. Jurisic, J. Obradovic (Serbia)
The role of ODZ4 in epithelial cancers
T. Mirza, K.D. Howarth, E. Turro, R.C. Fitzgerald, P.A.W. Edwards (United Kingdom)
The role of miR-375 in prostate carcinogenesis
P. Costa-Pinheiro, F. Quintela Vieira, J. Ramalho-Carvalho, J. Torres-Ferreira, J. Oliveira, R. Henrique,
C. Jerónimo (Portugal)
Profiling of plasma microRNAs in a group of healthy smokers and non-smokers to evaluate their
potential as biomarkers of effect following tobacco exposure
A. Banerjee, D. Waters, E. Minet (United Kingdom)
Quantitative analysis of CDKN2A methylation, mRNA, and p16INK4a protein expression in children and
adolescents with Burkitt lymphoma: Biological and clinical implications
M.C. Robaina, V.O. Arruda, L.M.M. Rezende, G.M. Vasconcelos, R.S. Faccion, A.G. Apa, C. Bacchi,
C.E. Klumb (Brazil)
A building-block lentiviral vector system that facilitates combinatorial genetics and tumor modelling in
primary cells and in mouse tissues
J. Albers, S. Brandt, P.K. Bode, B. Bode-Lesniewska, P. Wild, I.J. Frew (Switzerland)
Process-automation of next generation sequencing for high-throughput mutation analyses in cancer
C. Vollbrecht, U. Koitzsch, K. Koenig, M. Kloth, R. Buettner, M. Odenthal (Germany)
Downregulation of the DLEC1 gene is not associated with promoter methylation in head and neck cancer
N. Buyru, E. Baltaci, E. Yavuz, D. Seven, E. Kilic, E. Karaman (Turkey)
Copy number alterations in lung cancer
N. Dalay, O. Baykara, B. Bakir, A. Demirkaya, N. Buyru (Turkey)
Rab25 is downregulated and is not associated with invasion in head and neck squamous cell carcinoma
D. Seven, S. Dogan, E. Kilic, E. Karaman, H. Koseoglu, N. Buyru (Turkey)
Molecular characterization through massive parallel sequencing unveils clues regarding origin of
undifferentiated endometrial carcinomas
J.M. Rosa-Rosa, E. Cristobal-Lana, G. Muñoz, M.A. Lopez-Garcia, L. Romero-Perez, A. Pascual,
J. Prat, R.A. Soslow, X. Matias-Guiu, J. Palacios-Calvo (Spain)
Epigenetic deregulation of HOXA genes associated with aberrant expression in glioblastoma
S. Kurscheid, D. Sciuscio, P. Bady, R. Stupp, M. Delorenzi, M. Hegi (Switzerland)
Malignant transformation in high hyperdiploid pediatric acute lymphoblastic leukemia (HHDpALL)
is characteristically featured with sequential and hierarchical numerical chromosomal changes, and
chromosomal instability (CIN)
D.R. Alpar, D.R. Varga, D.R. Pótó, D.R. Mátics, D.R. Vojcek, D. Ottoffy, P.R.O.F. Szuhai,
G. Pajor (Hungary)
miR-141 and miR-375 expression in plasma samples from Bulgarian prostate cancer patients and
controls
D. Kachakova, E. Popov, A. Mitkova, I. Popov, A. Vlahova, T. Dikov, S. Christova, C. Slavov, V. Mitev,
R. Kaneva (Bulgaria)
Evaluation of microRNA expression profiles in triple-negative (ER, PR and Her2/neu) breast cancer
patients with and without germ-line BRCA mutations
E. Erturk, G. Cecener, U. Egeli, B. Tunca, G. Tezcan, S. Gokgoz, S. Tolunay, I. Tasdelen (Turkey)
Dependence of malignancy grade of gastrointestinal stromal tumor from a genetic profile
I. Gagarin, A. Shveikin, A. Barinov, E. Tsepenshchikova, N. Savelov, V. Grinevitch, I. Demidova (Russian
Federation)
382
383
384
385
386
387
388
389
391
392
393
394
395
396
397
398
399
Changes in colorectal carcinoma genomes under anti-EGFR therapy identified by whole-genome
plasma DNA sequencing
E. Heitzer , S. Mohan, P. Ulz, I. Lafer, M. Auer, S. Lax, G. Hoefler, T. Bauernhofer, J.B. Geigl,
M.R. Speicher (Austria)
Chromatin remodelling by the p400 ATPase influences DNA double-strand breaks repair and genetic
instability independently of the H2AZ histone variant incorporation
Y. Canitrot, C. Courilleau, C. Chailleux, G.C. Taty-Taty, M. Quaranta, A. Jauneau, E. Boutet-Robinet,
D. Trouche (France)
Mutational landscape of the mediator complex across human cancers
A. Offermann, Z. Shaikhibrahim, D. Böhm, M. Deng, S. Perner (Germany)
Comparative evaluation of EGFR mutation testing of tissue and cytology using direct sequencing,
pyrosequencing and peptide nucleic acid clamping in lung adenocarcinoma
W. Kim, K.W. Min, K.Y. Lee (South Korea)
Tolerance to mitotic defects contributes to chromosomal instability in post-tetraploid progeny
A. Kuznetsova, S. Müller, M. Dürrbaum, Z. Storchova (Germany)
Constitutional and somatic rearrangement of chromosome 21 in acute lymphoblastic leukaemia
Y. Li, C. Schwab, S. Ryan, E. Papaemmanuil, H.M. Robinson, P. Jacobs, P. Van Loo, M.R. Stratton,
P.J. Campbell, C.J. Harrison (United Kingdom)
AML mesenchymal stem cells signature
N. Oliveira, E. Abdelhay, R. Binato (Brazil)
Transcription factors with differential transcription start sites during B-cell differentiation in normal
tissues and diffuse large B-cell lymphoma
J. Bødker , M.K. Kjeldsen, M.B. Kloster, M. Rodrigo-Domingo, A.E. Bilgrau, P. Johansen, A. Schmitz,
H.E. Johnsen, M. Bøgsted, K. Dybkær (Denmark)
HER2 binds to the genome at specific regulatory sites of key genes in breast cancer
A. Redmond, J.S. Carroll (United Kingdom)
Epigenetic mechanisms of cadmium urothelial carcinogenesis
L. Cowling, C. Varley, T. Minshull, M. Dickman, J. Catto, J. Southgate (United Kingdom)
DNA methylation levels of OPCML and SFRP1 as diagnostic biomarkers in cholangiocarcinoma
T. Limpaiboon, R. Amornpisutt, S. Proungvitaya, P. Jearanaikoon (Thailand)
TET1 was an important factor for DNA demethylation to regulate the expression of MUC1 and MUC4
in lung cancer
S. Yokoyama, S. Kitamoto, M. Higashi, H. Tsutsumida, J. Wakimoto, S. Yonezawa (Japan)
Epigenetic modifications of androgen receptor signaling in castration resistant prostate cancer (CRPC)
H. Saraç, O.D. Toparlak, A. Kaplan, A. Ebrahimi, T. Bagci-Önder, T. Onder, N.A. Lack (Turkey)
Telomerase reverse transcriptase promoter mutations in primary cutaneous melanoma
B. Heidenreich, E. Nagore, P.S. Rachakonda, Z. Garcia-Casado, C. Requena, V. Traves, K. Hemminki,
R. Kumar (Germany)
DNA methylation profiles in invasive breast tumours associate with methylation in lymph node
metastases and not in plasma samples
I. Fridrichova, I. Zmetakova, V. Kajabova, M. Mego, Z. Cierna, T. Krivulcik, B. Smolkova (Slovak
Republic)
Visualization of tumor formation in the WAP-TGFa/FGFR4Arg385 KI breast cancer mouse model
B. Sperl, R. Abraham, J. Bussemer, C. Wallasch, M. Schwaiger, A. Ullrich (Germany)
Microsatellite unstable neuroendocrine carcinomas are a distinct clinico-pathologic entity in the
gastroenteropancreatic system
D. Furlan, N. Sahnane, M. Monti, E. Vicari, A. Vanoli, C. Capella, E. Solcia, F. Sessa, S. La Rosa (Italy)
Next-generation sequencing to dissect transcriptome complexity of childhood ALL and of different
therapy response
I. Cifola, M. Severgnini, G. Fazio, S. Bungaro, A. Biondi, G. Cazzaniga, G. De Bellis (Italy)
Functional role of the long noncoding RNA ANRIL in silencing of the INK4/ARF tumor suppressor locus
in urothelial carcinoma
M. Hoffmann, G. Niegisch, W. Schulz (Germany)
xlix
400
401
402
403
405
407
408
409
410
411
412
413
414
415
416
417
418
419
420
l
Class-I histone deacetylases are deregulated in urothelial cancer but may differ in suitability as
therapeutic targets
M. Lehmann, M.J. Hoffmann, W.A. Schulz, G. Niegisch (Germany)
Genomic structural instability and homologous recombination deficiency in breast and ovarian cancers
T. Popova, E. Manié, A. Battistella, X. Sastre-Garau, O. Goundiam, A. Vincent-Salomon,
D. Stoppa-Lyonnet, M.H. Stern (France)
421
422
Signalling Pathways I
Dissecting the 5 untranslated region of the PTCH1b tumor suppressor gene
471
P. Ozretic, A. Bisio, V. Musani, D. Trnski, M. Sabol, S. Levanat, A. Inga (Croatia)
Canonical Notch signalling is inactive in urothelial carcinoma
473
A. Koch, S. Jankowiak, G. Niegisch, M.J. Hoffmann, W.A. Schulz (Germany)
475
The role of ZEB2-induced epithelial–mesenchymal transition in DNA repair
G.C. Tse, H. Abbas, A.E. Sayan, M.D. Evans, E. Tulchinsky (United Kingdom)
476
Understanding and targeting PI3K pathway downstream of Met oncogenic mutant
A. Hervieu, C. Joffre, Z. Leung, S. Kermorgant (United Kingdom)
The role of Breast Cancer Associated gene 2 in EGFR endocytosis, downregulation and breast cancer
479
survival
J. Wymant, S. Hiscox, A. Westwell, S. Urbé, M. Clague, A.T. Jones (United Kingdom)
Calcium sensing receptor mediated downstream signalling in colonocytes
480
S. Tennakoon, A. Aggarwal, L. Hegedus, M. Wohlgenannt, E. Kallay (Austria)
482
The p53-Rbm38 loop and its role in tumor suppression and premature aging
X. Chen, M. Zhang, J. Zhang (USA)
Assessment of fibroblast growth factor receptor 2 amplification in gastric cancer using quantitative
483
real-time polymerase chain reaction, fluorescent in situ hybridization and immunohistochemistry
Y.S. Park, H.R. Kim, M.H. Ryu, Y.S. Na, S.R. Park, B.Y. Ryoo, Y.G. Kang (South Korea)
Leukocyte cell-derived chemotaxin 2 antagonizes MET receptor activation and results in the suppression
484
of hepatocellular carcinoma vascular invasion
K.T. Hua, C.K. Chen, M.L. Kuo (Taiwan)
Characterization of the Src-regulated kinome in breast cancer by chemical proteomics and functional
485
genomics
L. Zhang, J. Wu, I. Holmes, R.J. Daly (Australia)
HER-2 overexpression is closely related with genes that belong to the Shh pathway in medulloblastoma
486
G.A.V. Cruzeiro, V.S. Silveira, K.B. Salomão, D.A. Moreno, V.K. Suazo, J.A. Yunes, S.R. Brandalise,
S.S. Aguiar, C.A. Scrideli, L.G. Tone (Brazil)
Liaison between signaling and metabolism in ovarian cancer: ErbB/PI3K and fatty acid synthase
487
T. Grunt (Austria)
PDGFRa-driving mutations found in gastro-intestinal stromal tumors induce receptor mislocalisation
488
and alter the PDGFR-a conventional signalling characteristics
R. Eulenfeld, C. Bahlawane, M.Y. Wiesinger, J. Wang, A. Müller, L. Vallar, T. Sauter, V. Satagopam,
S. Haan (Luxembourg)
Mechanisms linking obesity to altered metabolism in colon carcinogenesis
489
L. Nimri, E. Yehuda-Shnaidman, B. Schwartz (Israel)
Long chain alkylphenol mixture promotes mammary epithelial cell metaplastic phenotype through an
490
estrogen receptor alpha 36 mediated mechanism
C. Chamard, E. Bresso, T. Boukhobza, M.D. Devignes, M. Smaı̈l-Tabbone, A. Chesnel,
H. Dumond (France)
491
Interaction of Hedgehog-Gli and estrogen receptor signaling pathways in breast cancer cells
S. Levanat, D. Trnski, Z. Uzarevic, P. Ozretic, V. Musani, M. Sabol (Croatia)
Development of a 3D culture system to study the deterioration of oral cancer
492
S.J. Chang, Y.M. Chen, M.W. Lee (Taiwan)
ATM kinase sustains HER2 tumorigenicity in breast cancer
V. Stagni, V. Oropallo, M. Mottolese, A. Di Benedetto, I. Manni, G. Piaggio, R. Falcioni, S. Di Carlo,
M.T. Cencioni, D. Barilà (Italy)
Prostaglandin E2 trans-activates the Colony-Stimulating Factor-1 (CSF-1) receptor and synergizes with
CSF-1 in the induction of cell migration in macrophages via the mitogen-activated protein kinase ERK1/2
G. Digiacomo, M. Ziche, P. Dello Sbarba, S. Donnini, E. Rovida (Italy)
AKT and gefitinib resistance in mutant KRAS non-small cell lung cancers through mechanisms
dependent of acetylation
V. Jeannot, B. Busser, E. Brambilla, M. Wislez, B. Robin, J. Cadranel, J.L. Coll, A. Hurbin (France)
Nuclear EGFR controls the expression of the ARF tumor suppressor
D. Dayde, P. Ozenne, P. Perron, C. Barrial, B. Eymin, S. Gazzeri (France)
Activation of TRKA receptor by nerve growth factor induces shedding of P75 receptor related with
progression of epithelial ovarian cancer
C. Romero, C. Vallejos, F. Gabler, A. Selman, M. Vega (Chile)
Two distinct antitumor pathways activated by transfected poly(I:C) in androgen-independent prostate
cancer cells
S. Palchetti, D. Starace, P. De Cesaris, A. Filippini, E. Ziparo, A. Riccioli (Italy)
The role of Hedgehog signaling pathway in the regulation of SOX18 gene expression in cervical
carcinoma cell line
I. Petrovic, M. Milivojevic, M. Mojsin, D. Drakulic, N. Kovacevic Grujicic, V. Topalovic, S. Davidovic,
M. Stevanovic (Serbia)
Analysis of the metastasis/tumor-suppressing effects of SASH1
U. Nitsche, E. Lichtenegger, S. Leis, B. Heckl, H. Weidmann, E. Nino, K.P. Janssen (Germany)
The prognostic value of b-catenin in anal cancer
H. Jacob, O. Dahl, M. Myklebust (Norway)
AKT inhibition induces a prosurvival autophagy response that limits combination effects with gefitinib
S.M. Bokobza, Y. Jiang, A.M. Devery, A.M. Weber, A.J. Ryan (United Kingdom)
Novel FLT3 mutation shows dominant negative effects on the wild-type FLT3 receptor
J. Bauer , N. Sandhöfer, W. Hiddemann, K. Spiekermann (Germany)
Functional proteomic analysis of leukaemogenic protein tyrosine kinase targets
N. Che Azmi, A. Pierce, A. Williamson, A. Whetton (United Kingdom)
Reverse phase protein array profiling of lobular and triple negative breast cancers within the RATHER
consortium
L. De Koning, B. He, A. Barbet, F. Bard, C. Lecerf, C. Baldeyron, T. Dubois (France)
Relevance of BRAFG615E and BRAFG474R mutations in undifferentiated thyroid carcinomas
R. Muñoz Martinez, G. Fernández Ballester, A.M. Costa, T. Alvarez Gago, G. Garcia-Rostan (Spain)
The tumour-promoting receptor tyrosine kinase, EphB4, regulates expression of integrin b8 in prostate
cancer cells
A. Herington, I. Mertens-Walker, B. Fernandini, A. Rockstroh, C. Nelson, S. Stephenson (Australia)
Fibroblast growth factor receptor 1 controls cancer cell survival via ERK signaling
L. Ling, T.H. Goh, R.J.E. Chua, V. Nurcombe, A. Van Wijnen, S.M. Cool (Singapore)
Nuclear PI3K signalling in colorectal cancer
M. Palmieri, B. Catimel, A. Holmes, O. Sieber (Australia)
Urokinase modulates EGF-dependent signalling, proliferation and motility of the two breast cancer cell
lines MCF-7 and MDA-MB-231
N. Kozlova, A. Samoylenko, L. Drobot, T. Kietzmann (Finland)
GPR84 is a positive regulator of beta-catenin signalling and is required for the maintenance of
MLLAF9-rearranged acute myeloid leukaemia
P.A. Dietrich, M.D. Norris, J.Y. Wang (Australia)
li
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
509
511
512
513
lii
Carcinogenesis I
Telomerase inhibition decreases alpha-fetoprotein expression in hepatocellular carcinoma cells in vitro
557
and in vivo: Possible involvement of interleukin-6 induced PI3K/Akt/mTor pathway
R. Tahtouh, A.S. Azzi, S. Chammat, H. Bou Haroun, N. Alaaeddine, L. Wardi, G. Hilal (Lebanon)
p53 and other components of the DNA damage response as determinants of cell sensitivity to
558
ATR inhibition
F.K. Middleton, T. Chen, J.R. Pollard, N.J. Curtin (United Kingdom)
560
Artemin promotes metastasis through p44/42 MAPK pathway in colorectal carcinoma
Q. Zhuang, X.J. Kong, T. Zhu, Z.S. Wu, P.E. Lobie (Singapore)
Characterising the role of Heterochromatin Protein 1 gamma in normal intestinal homeostasis and
563
tumourigenesis
K. Lio, A. Clarke, A. Reed (United Kingdom)
564
The expression level of MACC1 in early stage CRC patients of Turkish population
S. Ceylan, S. Ak, B. Tunca, E. Ozturk, G. Tezcan, G. Cecener, U. Egeli, T. Yilmazlar, O. Yerci (Turkey)
Expression of annexin A2 controls cell motility in renal cell carcinoma
565
C.W. Cheng, C.J. Hsiao, H.L. Hsu, T.K. Chao, Y.H. Wu, Y.H. Sung, Y.F. Lin (Taiwan)
566
Investigating PI3Kinase and K-ras pathway interactions in mouse model of colorectal cancer
V. Meniel, I. Martin-Berenjeno, B. Vanhaesebroeck, A.R. Clarke (United Kingdom)
567
The impact of chronic cisplatin treatment on DNA repair in head and neck squamous cell carcinoma
W. Su, W.C. Su, J.Y. Chang, H.J. Liaw (Taiwan)
The role of Slit1 in angiogenesis
568
K.F. Pan, T.Y. Yeh, F.C. Peng (Taiwan)
Chronic and low doses exposure of environmental pollutants induces phenotypic alterations in a tumour
569
progression model of breast cancer
C.F. Donini, E. Grisard, V. Maguer-Satta, P. Balaguer, A. Escande, M.L. Bayle, C. Casellas, V. Cavailles,
B. Fervers, P.A. Cohen (France)
Global DNA methylation profiling identifies epigenetic differences between spontaneous and
570
radiation-induced rat mammary carcinomas
M. Takabatake, K. Daino, T. Imaoka, M. Nishimura, M. Fukushi, Y. Shimada (Japan)
The Hippo pathway gene, YAP, is an oncogene in ovarian cancer
571
X. Zhang, J. George, S. Deb, J.L. Degoutin, E.A. Takano, S.B. Fox, A.O.C.S. Study group,
D.D.L. Bowtell, K.F. Harvey (Australia)
Epigenetic silencing of tumor suppressor miR-3151 in Chinese chronic lymphocytic leukemia patients
572
L.Q. Wang, K.Y. Wong, C.S. Chim (China)
Epigenetic inactivation of miR-9 family microRNAs in chronic lymphocytic leukemia − implications on
573
constitutive activation of NF-úB pathway
L.Q. Wang, Y.L. Kwong, C.S.B. Kho, K.F. Wong, K.Y. Wong, M. Ferracin, G.A. Calin, C.S. Chim (China)
Role of prostate apoptosis response-4 in ovarian cancer cells
574
S. Meynier , M. Kramer, P. Ribaux, J.C. Tille, F. Delie, P. Petignat, M. Cohen (Switzerland)
Translational Research I
High incidence of EGFR gene mutations in Serbian female lung adenocarcinoma patients
593
M. Cavic, A. Krivokuca, K. Brotto, E. Malisic, S. Radulovic, R. Jankovic (Serbia)
595
The miR21/10b ratio as a prognostic marker in metastasis-free clear cell renal cell carcinoma patients
H. Fritz, D. Lindgren, B. Ljungberg, H. Axelson, B. Dahlbäck (Sweden)
Tumor miRNA expression profiling to identify circulating miRNAs for breast cancer detection
596
N. Matamala, M.T. Vargas, R. González-Cámpora, J.I. Arias, P. Menéndez, E. Andrés, K. Yanowsky,
R. Miñambres, B. Martı́nez-Delgado, J. Benı́tez (Spain)
Design, synthesis and evaluation of COX-2 PET imaging probes
598
A. Pacelli, G. Smith, J. Greenman, C. Cawthorne (United Kingdom)
GLUT-1 inhibition: A new therapeutic approach against hepatocellular carcinoma?
A.F. Brito, A.M. Abrantes, M. Laranjo, A.C. Ribeiro, A.C. Gonçalves, A.B. Sarmento-Ribeiro,
F. Castro-Sousa, J.G. Tralhão, M.F. Botelho (Portugal)
Targeting nucleolin in lung cancer − an emerging strategy to overcome stroma-mediated anti-VEGF
resistance
A. Valério-Fernandes, N. Fonseca, V. Moura, A. Ladeirinha, T. Ferreira, A. Alarcão, L. Carvalho,
S. Simões, J.N. Moreira (Portugal)
Cisplatin response in a panel of patient-derived ovarian carcinoma xenografts: roles of epithelial
mesenchymal transition and DNA repair
F. Ricci, F. Guffanti, M. Fratelli, P. Perego, R. Fruscio, R. Baldo, S. Magni, M. Broggini, G. Damia (Italy)
Detection of hot spot and pathway related variants in chronic lymphocytic leukemia by multiplexed
amplicon sequencing
C. Vollbrecht, K. Koenig, L.C. Heukamp, M. Peifer, R. Buettner, M. Odenthal,
C.D. Schweighofer (Germany)
Testing cancer related gene panels for high throughput parallel sequencing
C. Vollbrecht, F.D. Mairinger, C.D. Schweighofer, L.C. Heukamp, S. Merkelbach-Bruse, R. Buettner,
M. Odenthal (Germany)
JK-1(FAM134B) gene implications in colorectal carcinoma: A gene expression and functional study
K. Kasem, V. Gopalan, A. Salajegheh, A. Lam (Australia)
Chick embryo chorioallantoic membrane model for testing novel anticancer substances
K. Zabielska, I. Dolka, M. Król, K.M. Pawlowski, A. Zbikowski, M. Wójcik, W. Lewandowski,
J. Mieczkowski, R. Lechowski (Poland)
A systematic approach to the metastatically relevant microRNA landscape
G. Mudduluru, M. Abba, J. Batliner, N. Patil, J.H. Leupold, W. Thiele, M. Rothley, A. Benner, J. Sleeman,
H. Allgayer (Germany)
The new orthotopic locally advanced animal model of prostate cancer
E. Tavares-Silva, A.C. Mamede, S. Guerra, P. Simoes, A. Abrantes, A. Mota, M.F. Botelho (Portugal)
Cell-based biopharmaceuticals in a Phase I/II trial (TREAT-ME 1) for targeted drug- and gene delivery
as an innovative treatment modality in advanced cancer
R. Huss, J. Von Einem, F. Hermann, H. Niess, M. Michl, V. Scherhammer, C. Bruns, V. Heinemann,
C. Guenther (Germany)
Overexpression of BMI-1 immortalises normal human urothelial cells
E.J. Chapman, L.E. De Faveri, J. Roulson, M. Sanchez-Carbayo, M.A. Knowles (United Kingdom)
Fluorescence analysis of urine: Role in differential diagnostics between benign and malignant ovarian
tumours
D. Martinicky, M. Zvarik, L. Sikurova, L. Hunakova (Slovak Republic)
Prognostic significance of neuroendocrine components in gastric carcinomas
H.R. Kim, C.M. Choi, J.C. Lee, M.H. Ryu, J.H. Yook, B.S. Kim, Y.S. Park (South Korea)
Immunohistochemical evaluation of the mammalian target of rapamycin pathway in stage I non-small
cell lung carcinoma
H.R. Kim, C.M. Choi, J.C. Lee, E. Shin, Y.S. Park (South Korea)
Efficacy of temsirolimus for advanced hepatocellular carcinoma (HCC) − assessment of pS6 as
potential prognostic biomarker of response
W. Yeo, J. Tong, A.W. Chan, S. Chan, F.K. Mo, E.P. Hui, J. Koh, L. Li, S.C. Yu, K.F. To (Hong Kong)
In vivo models of pancreatic cancer for translational medicine
D. Behrens, C. Hallas, D. Anders, I. Fichtner (Germany)
Tumor–stroma cross talk and cancer progression in breast cancer is related to tenascin-C expression
C. Kronberger , K. Reiter, B. Lauss, M. Knollhofer, H. Jaksch-Bogensperger, G. Hutarew, O. Dietze,
R. Reitsamer (Austria)
Autophagosome-mediated EGFR down-regulation induced by CK2 inhibitor enhances the efficacy of
EGFR-TKI on EGFR-mutant lung cancer cells with T790M
J. Lee, C.M. Choi, H.R. Kim, Y.S. Park, G.S. So, J.K. Rho, S.H. Jang (South Korea)
liii
599
600
601
604
605
606
607
608
611
615
619
620
621
622
623
624
625
626
liv
MED12 overexpression is a frequent event in castration-resistant prostate cancer
A. Offermann, Z. Shaikhibrahim, M. Braun, R. Menon, C. Ruiz, T. Zellweger, C. Rentsch, O. Andren,
L. Bubendorf, S. Perner (Germany)
MED15 in head and neck squamous cell carcinoma: Clinical and molecular implications
A. Offermann, Z. Shaikhibrahim, R. Halbach, M. Braun, G. Kristiansen, F. Bootz, R. Mikut, M. Reischl,
A. Schröck, S. Perner (Germany)
Expression and role of the embryonic protein SOX2 in head and neck squamous cell carcinoma
A. Schröck, M. Bode, A. Franzen, L. Heasley, C. Lengerke, S. Perner, A. Queisser (Germany)
Receptor tyrosine kinases as novel therapeutic targets in head and neck squamous cell carcinoma
A. Von Mässenhausen, M. Deng, W. Vogel, A. Queisser, S. Perner (Germany)
Impact of TP53 accumulation on ovarian cancer prognosis in BRCA1 mutation carriers
I.K. Rzepecka, L. Szafron, A. Felisiak-Golabek, A. Podgorska, J. Kupryjanczyk (Poland)
Tamoxifen resistance can be overcome by salinomycin treatment
A. Sommer, F. Mickler, A. Herrmann, A. Hermawan, C. Bräuchle, E. Wagner, P. Knyazev, A. Ullrich,
A. Roidl (Germany)
Real-time RT-PCR analysis of galectin-3 expression in fine-needle aspirates of thyroid papillary
carcinoma and thyroid non-neoplastic lesions
I. Samija, N. Matesa, Z. Kusic (Croatia)
CA IX inhibitor 4-(3 -(3 ,5 -dimethylphenyl)ureido)phenyl sulfamate (S4) as an adjuvant to doxorubicin
chemotherapy reduces metastatic dissemination
R.G. Gieling, M. Babur, B.A. Telfer, K.J. Williams (United Kingdom)
The PATH biobank: German breast cancer patients approve of genome wide association studies
C. Mayer , U. Ohlms, C. Waldner, D.C. Schmitt, H. Bodenmüller, T. Anzeneder (Germany)
Clinical relevance of telomere length and telomerase activity in colorectal cancer
C. De Juan Chocano, T. Fernández-Marcelo, I. Pascua, J. Head, A. Sánchez-Pernaute, A.J. Torres,
M. Benito, P. Iniesta (Spain)
DAXX/ATRX loss and chromosomal instability in pancreatic neuroendocrine tumors (pNETs)
I. Marinoni, A. Schmitt-Kurrer, E.J. Speel, A. Perren (Switzerland)
Telomere length and DAPK1 expression: prognosis implication in non-small cell lung cancer
T. Fernández-Marcelo, I. Pascua, C. De Juan, J. Head, A. Gómez, F. Hernando, J.R. Jarabo, A.J. Torres,
M. Benito, P. Iniesta (Spain)
Whole-genome sequencing of plasma DNA reveals frequently occurring copy number changes in
patients with metastatic breast cancer
M. Auer , E. Heitzer, P. Ulz, G. Pristauz, E. Petru, S. Jahn, M.R. Speicher, J.B. Geigl (Austria)
11
C-MET-PET for monitoring of early treatment response in multiple myeloma
K. Lückerath, C. Lapa, S. Samnick, H. Einsele, S. Knop, A.K. Buck (Germany)
Transglutaminase 2, a potential biomarker and therapeutic target for meningioma by microarray analysis
Y.C. Huang, K.C. Wei, C.N. Chang, P.Y. Chen, C.P. Chen, C.S. Lu, H.L. Wang, D.H. Gutmann,
T.H. Yeh (Taiwan)
miRNA-mRNA integrated analysis in breast cancer: Let-7 cluster as a meta-signature for grade
classification
Y. Oztemur, T. Bekmez, A. Aydos, I. Yulug, B. Gur Dedeoglu (Turkey)
Predicting colon cancer occurrence from transcriptomic, splicing and genomic data in colon adenomas
L. Corcos, M. Pesson, A. Uguen, K. Trillet, S. Redon, P. De La Grange, M. Aubry, M. Robaszkiewicz,
G. Le Gac, B. Simon (France)
Partial wave spectroscopic nanocytology to personalize management of early stage prostate cancer
H. Roy, C. Brendler, H. Subramanian, D. Zhang, C. Maneval, K. Kaul, B. Helfand, C. Wang,
M. Paterakos, V. Backman (USA)
Regulation of norepinephrine transporter expression in pheochromocytoma by dual PI3K/mTOR inhibition
M. Lee, F.C. Gaertner, N.S. Pellegata (Germany)
ZIRA: A new prognostic biomarker of estrogen receptor-positive (ER+) breast cancers
J. Vendrell, N.T. Nguyen, B. Gyorffy, S. Léon, E. Grisard, T. Bachelot, I. Treilleux, P.A. Cohen (France)
627
628
629
630
631
632
634
635
637
638
639
640
641
642
643
644
646
647
648
649
Atypical nuclear localization of VIP receptors in glioma cell lines and patients
A. Barbarin, P. Séité, S. Bensalma, J.M. Muller, C. Chadeneau (France)
An array-based pharmacogenetic study on elderly patients with advanced breast cancer treated with
aromatase inhibitors
E. Rumiato, A. Brunello, S. Ahcene-Djaballah, L. Borgato, M. Gusella, F. Pasini, P. Fiduccia, A. Amadori,
V. Zagonel, D. Saggioro (Italy)
Patient-derived xenograft models of acute leukemias for translational research
A. Siegert, B. Brzezicha, A. Wulf-Goldenberg, I. Fichtner, J. Hoffmann (Germany)
Comparison of direct sequencing and peptide nucleic acid clamping of EGFR gene in patients with
non-small cell lung cancer
Y. Kim, S.H. Yoon, I.J. Oh, K.S. Kim, H.J. Cho, Y.D. Choi (South Korea)
lv
650
651
652
653
Experimental/Molecular Therapeutics, Pharmacogenesis I
Plaunotol inhibits doxorubicin-induced renal cell death
716
W. De-Eknamkul, C. Chaotham, P. Chanvorachote (Thailand)
PCL-grafted chondroitin sulfate copolymers to promote dual-medicated endocytosis for enhanced
717
anti-cancer drug delivery
L. Wang, Y. Liu (Taiwan)
720
Augmentation of NAD+ by NQO1 activation attenuates cisplatin-mediated hearing impairment
H. So, H. Kim, G. Oh, S. Yang, S. Lee, S. Moon, K. Kwon, R. Park (South Korea)
Aprepitant combinations (AC) for chemotherapy-induced nausea and vomiting (CINV) in adults:
721
A meta-analysis of randomized controlled trials (RCTs)
N. Gupta, H. Hatoum, O. Al Ustwani, P. Danchaivijitr, K. Wang, R. Pili (USA)
722
Iron chelation by biopolymers for an anti-cancer therapy; binding up the ‘ferrotoxicity’ in the colon
R. Horniblow, S. Radulescu, M. Schneider, M. Dowle, T. Iqbal, R. Palmer, G. Latunde-Dada,
Z. Pikramenou, C. Tselepis (United Kingdom)
A sorafenib derivative and novel SHP-1 agonist, SC-59, acts synergistically with radiotherapy in
723
hepatocellular carcinoma cells through inhibition of STAT3
C.Y. Huang, W.T. Tai, C.Y. Hsieh, Y.J. Lai, W.M. Hsu, L.J. Chen, C.W. Shiau, K.F. Chen (Taiwan)
Biodegradable amphiphilic gelatin/calcium phosphate co-delivery systems with simultaneously
725
encapsulating hydrophilic and hydrophobic drugs for enhanced tumor therapy
S. Chen, W. Li, C. Su, Y. Chen (Taiwan)
Drug resistance acquisition is associated to expression changes in caspases and Bcl-2 proteins in a
726
human lymphoblastic cell line
D. Cerezo Fernández, M. Cánovas, P. Garcı́a-Peñarrubia, E. Martı́n-Orozco (Spain)
727
Novel human drug-resistant lymphoblastic cell line exhibits collateral sensitivity to cold stress
D. Cerezo Fernández, P. Garcı́a-Peñarrubia, M. Cánovas, E. Martı́n-Orozco (Spain)
DNA-PKcs inhibition enhances response to DNA damaging agents and is associated with Rad51
728
downregulation
C. Sousa, E. Maginn, H. Gabra, H. Wasan, E. Stronach (United Kingdom)
Nupharidine inhibits NF-kappa B activity, has synergistic cytotoxic activity with cisplatin and etoposide
729
and induces apoptosis
J. Gopas, J. Ozer, N. Eisner, D. Benharroch, A. Golan-Goldhirsh (Israel)
ACK1 is a potential target for anti-metastasis therapeutic development
730
B.T. Chua, Y.D. Cheng, S.C. Tham (Singapore)
731
Studies on the modulation of cisplatin activity in NSCLC by KRAS: Role of specific mutations at codon 12
E. Caiola, R. Frapolli, M. Broggini, M.C. Garassino, G. Farina, M. Marabese (Italy)
Heterogeneous response to bevacizumab combined with chemotherapy in patient-derived ovarian
732
cancer xenografts
A. Decio, M. Cesca, F. Bizzaro, M.R. Bani, R. Giavazzi (Italy)
Novel photodynamic therapy with glucose conjugated chlorin for GIST
735
M. Tanaka, H. Kataoka, N. Hayashi, S. Yano, T. Joh (Japan)
lvi
Sorafenib and 18 F-fluorocholine: New options in cholangiocarcinoma therapy and diagnosis?
A. Brito, A.M. Abrantes, A.C. Gonçalves, A.B. Sarmento-Ribeiro, F. Castro-Sousa, J.G. Tralhão,
ICP-133 controls in vitro cell proliferation of multiple myeloma cancer stem cells
M. Issa, M. Cuendet (Switzerland)
Circulating miRNAs associated with progression of colorectal cancer
S. Aherne, S.F. Madden, D.J. Hughes, B. Pardini, A. Naccarati, M. Levy, P. Vodicka, P. Neary,
P. Dowling, M. Clynes (Ireland)
Can photodynamic therapy make a difference in retinoblastoma? In vitro studies
R. Teixo, M. Laranjo, A.C. Serra, M. Piñeiro, A.M. Abrantes, A.C. Gonçalves, J. Casalta-Lopes,
A.B. Sarmento-Ribeiro, A.M. Rocha-Gonsalves, M.F. Botelho (Portugal)
Can photodynamic therapy make a difference in retinoblastoma? In vivo studies
G. Brites, M. Laranjo, R. Teixo, A.C. Serra, M. Piñeiro, A.M. Abrantes, J. Casalta-Lopes,
A.M. Rocha-Gonsalves, M.F. Botelho (Portugal)
PARP-inhibitor PJ34 potentiates the anticancer effect of chiral 6,7-bis(hydroxymethyl)-1H,3Hpyrrolo[1,2-c]thiazoles against breast cancer cell lines
K. Santos, M. Laranjo, M.I. Soares, A.S. Oliveira, A.M. Abrantes, A. Brito, T. Pinho e Melo,
Chiral 6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles as anticancer agents against breast cancer
MCF7 and HCC1806 cell lines
K. Santos, M. Laranjo, M.I. Soares, A.S. Oliveira, A.M. Abrantes, A. Brito, A.C. Gonçalves,
A.B. Sarmento-Ribeiro, T. Pinho e Melo, M.F. Botelho (Portugal)
Gold nanoparticle conjugated lignan derivatives inhibited the proliferation of MCF-7 human breast
cancer cells
F. Bakar , G. Caglayan, F. Onur, S. Nebioglu, I.M. Palabiyik (Turkey)
Clinical and histopathological characteristics of HER2-positive breast cancer in patients treated with
adjuvant trastuzumab
P. Jurcic (Croatia)
Metabolic radiotherapy in cholangiocarcinoma: An option in the future?
A.I. Fernandes, A.C. Ribeiro, A.F. Brito, A.M. Abrantes, K. Santos, A.C. Gonçalves, A.B. SarmentoRibeiro, J.G. Tralhão, M.F. Botelho (Portugal)
Novel anthraquinone-thiosemicarbazones with tautomerizable methylene group as anti-metastatic and
anti-angiogenic agents
B. Kolundzija, T. Stanojkovic, M.D. Joksovic (Serbia)
Targeting cell-surface nucleolin in metastatic breast cancer
A. Gregório, N. Fonseca, V. Moura, G. Domingues, M. Lacerda, P. Figueireido, S. Simões, S. Dias,
J.N. Moreira (Portugal)
Human amniotic membrane secreted factors plus chemotherapy: A mishmash of effects?
S. Guerra, A.C. Mamede, M. Laranjo, A.S. Pires, M.J. Carvalho, A.F. Brito, P. Moura, A.M. Abrantes,
C.J. Maia, M.F. Botelho (Portugal)
Butyrate and irinotecan combination as a new therapeutic approach for colon cancer
J.C. Encarnação, A.S. Pires, T.J. Gonçalves, J.E. Casalta-Lopes, A.C. Gonçalves, A.M. Abrantes,
A.B. Sarmento-Ribeiro, M.F. Botelho (Portugal)
Anti-cancer proteins found in amniotic membrane: extraction, identification and cellular effects
A.C. Mamede, S. Guerra, M. Laranjo, A.F. Brito, A.S. Pires, M.J. Carvalho, P. Moura, A.M. Abrantes,
C.J. Maia, M.F. Botelho (Portugal)
Disulfiram-induced cytotoxicity and endolysosomal sequestration of zinc in breast cancer cell models
H. Wiggins, S. Hiscox, A. Westwell, K. Taylor, A.T. Jones (United Kingdom)
Experimental topical photodynamic therapy of various carcinomas
D. Vetvicka, M. Zadinova, M. Nekvasil, P. Jezek, J. Rakusan, M. Karaskova, J. Kralova, V. Kral,
P. Pouckova (Czech Republic)
736
737
738
739
740
741
742
743
744
746
747
749
750
751
752
753
754
A gemcitabine prodrug for the treatment of castration-resistant prostate cancer: Reduced metabolic
inactivation combined with targeted drug delivery
T. Karampelas, O. Argyros, N. Sayyad, A.G. Tzakos, D. Fokas, C. Tamvakopoulos (Greece)
Ascorbic acid-induced cytotoxicity in colon cancer cell lines
A.S. Pires, C.R. Marques, J.C. Encarnação, T.J. Gonçalves, A.C. Mamede, J.E. Casalta-Lopes,
A.C. Gonçalves, A.M. Abrantes, A.B. Sarmento-Ribeiro, M.F. Botelho (Portugal)
The effects on apoptotic and autophagic cell death pathways of endemic Colchicum baytopiorum
C.D. Brickell extract in HeLa cells
O. Dagdeviren, G. Ozcan Arican, N. Sütlüpinar, S. Kayacan, M. Oztürk (Turkey)
Ficus carica latex inhibits GBM cell proliferation by modulating let-7d expression
G. Tezcan, B. Tunca, A. Bekar, G. Cecener, U. Egeli, H. Malyer (Turkey)
Enhanced antitumor effects of oncolytic reovirus and trastuzumab combination therapy in human
HER2-positive gastric cancer
H. Kataoka, S. Hamano, M. Aoyama, Y. Mori, M. Tanaka, N. Hayashi, E. Kubota, R. Johnston,
T. Joh (Japan)
Curcumin induces the apoptosis of human monocytic leukemia THP-1 cells via the activation of
JNK/ERK Pathways
W. Yang, C. Yang (Taiwan)
Novel therapeutic compounds against human pancreatic cancer cells from wasabi component
6-(methylsulfinyl) hexyl isothiocyanate and derivatives
H.F. Liao, Y.J. Chen, T.H. Tsai, Y.C. Huang (Taiwan)
A new survival model for hyperthermic intraperitoneal chemoperfusion (HIPEC) in tumor-bearing rats
in the treatment of advanced ovarian cancer
G.S. Kireeva, O.A. Belyaeva, V.G. Bespalov, K.Y. Senchik, A.N. Stukov, A.M. Belyaev (Russian
Federation)
Human recombinant arginase I (Co)-PEG5000 [HuArgI (Co)-PEG5000]-induced arginine depletion is
selectively cytotoxic to human glioblastoma cells
O. Khoury, A. Bekdash, E. Stone, G. Georgiou, A. Frankel, R. Abi-Habib (Lebanon)
Ubiquitinated protein accumulation: A novel approach to treating bladder cancer
A. Sato, T. Asano, M. Isono, K. Ito, T. Asano (Japan)
MDR1 confers the acquired resistance to 17-DMAG in lung cancer with ALK rearrangement
C. Choi, J.C. Lee, H.R. Kim, Y.S. Park, Y.J. Choi, J.K. Rho, K.Y. Lee (South Korea)
A novel photodynamic therapy using mannose conjugated chlorin targeting cancer cells and
tumor-associated macrophages
N. Hayashi, H. Kataoka, S. Yano, M. Tanaka, T. Joh (Japan)
Ritonavir interacts with belinostat to cause endoplasmic reticulum stress and histone acetylation
synergistically in renal cancer cells
M. Isono, A. Sato, T. Asano, K. Ito, T. Asano (Japan)
Targeting MYC-driven growth and proliferation in prostate cancer
R. Rebello, D. Drygin, D. Cameron, G.P. Risbridger, R.B. Pearson, R.D. Hannan, L. Furic (Australia)
Docosahexaenoic acid may indirectly increase proteasome activity through reactive oxygen species in
human cervical cancer HeLa cells
K. Lim, K. Jing, S. Shin, S. Jeong, S. Kim, J. Heo, G. Kweon, S. Park, J. Park (Korea)
A novel pharmacological approach to inhibit Myc in cancer
M.E. Beaulieu, J.R. Whitfield, T. Jauset, D. Massó-Vallés, E. Serrano, F. Canals, P. Lavigne,
M. Montagne, L. Maltais, L. Soucek (Spain)
A potent betulenic acid analogue induces apoptosis in HT29 cells
D. Dutta, B. Chakraborty, C. Chowdhury, P. Das (India)
Synergistic targeting to HER2-positive breast cancer via nanocapsules with tunable ligand density and
magnetic targeting
C. Chiang, S.Y. Chen (Taiwan)
Inhibition of the IRE-1/XBP-1 pathway synergizes with ibrutinib for the treatment of B cell cancer
C.H. Tang, S. Ranatunga, C. Kriss, C. Cubitt, J. Tao, J. Pinilla-Ibarz, J. Del Valle, C.C. Hu (USA)
lvii
755
756
758
759
760
762
763
764
765
767
768
769
770
771
772
773
774
775
776
lviii
Quercetin induces mitochondrial-derived apoptosis via reactive oxygen species-mediated ERK
activation in acute myeloid leukemia (AML) cells and AML xenograft model
M. Chien, W. Lee, M. Hsiao, J. Chang, S. Yang, J. Chow, L. Lee (Taiwan)
Anti-tumor activity of a novel monoclonal antibody recognizing claudin-3 and -4
X. Li, Y. Kimura, M. Iida, H. Kuniyasu, M. Fukasawa, M. Tada, A. Ishii, A. Watari, K. Yagi,
M. Kondoh (Japan)
Induction of cavitation by a bubble-generating liposomal system for physical cancer therapy
M.F. Chung, Z.X. Liao, H.W. Sung, W.T. Chia (Taiwan)
Hyperthermia-mediated local drug delivery by a bubble-generating liposomal system for tumor specific
chemotherapy
H.W. Sung, K.J. Chen, E.Y. Chaung, C.C. Lin (Taiwan)
Transfection of chitosan/KillerRed/gPGA complex to intrinsically generate photosensitizer for
photodynamic therapy
C.C. Huang, Z.X. Liao, Y.C. Li, H.M. Lu, H.W. Sung (Taiwan)
M-Trap: Exosome-based capture of tumor cells as a new therapeutic technology in peritoneal metastasis
M. Abal, A. De la Fuente, L. Alonso-Alconada, J. Cueva, R. Lopez-Lopez (Spain)
Modification of topoisomerases in cancer stem cells derived from MCF7 breast cancer cell line − clinical
implications for cancer treatment
E. Priel, R. Peleg (Israel)
A strategy to screen and subsequently identify therapeutically valuable microRNAs that target a
clinically established KITENIN oncogene in colorectal cancer
S.M. Yoon, S.Y. Park, J.A. Bae, Y.S. Ko, H.G. Kim, K.K. Kim (South Korea)
Multicellular tumor spheroid 3D models to decipher cancer cell biology and to evaluate anticancer drugs
V. Lobjois, B. Ducommun (France)
The mitogen-activated protein kinase ERK5 regulates the development and growth of hepatocellular
carcinoma
E. Rovida, G. Di Maira, S. Cannito, I. Tusa, C. Paternostro, X. Deng, P. Dello Sbarba, N.S. Gray,
M. Parola, F. Marra (Italy)
Transferrin-conjugated self assembled nanoparticles incorporating ZOL as a tool for the targeting of
glioblastoma
M. Caraglia, S. Zappavigna, M. Porru, L. Amalia, S. Lusa, G. Salzano, S. Artuso, A. Stoppacciaro,
C. Leonetti, G. De Rosa (Italy)
Expression of Annexin A4 regulates cisplatin-susceptibility in malignant mesothelioma
K. Nagano, T. Yamashita, M. Inoue, K. Higashisaka, Y. Yoshioka, Y. Abe, Y. Mukai, H. Kamada,
Y. Tsutsumi, S. Tsunoda (Japan)
Combinatorial treatment of lung cancer cell lines and their spheroids-cancer stem cells with standard
therapeutic drug and salinomycin
Z. Xiao, B. Sperl, A. Ullrich, P. Knyazev (Germany)
Netropsin inhibits the growth of Burkitt’s lymphoma cells by forcing the loss of Epstein−Barr virus (EBV)
genomes from those cells
A. Chakravorty, B. Sugden (USA)
777
778
779
780
781
782
783
784
785
786
787
788
789
790
Tumour Immunology I
Immuno-modulatory effect of the photosensitizer BAM-SiPc in cancer treatment
867
H.Y. Yeung, P.C. Lo, D.K.P. Ng, W.P. Fong (Hong Kong)
CD70 methylation and expression in early breast cancer
868
C. Petrau, M. Cornic, P. Bertrand, C. Maingonnat, V. Marchand, J.M. Picquenot, F. Jardin,
F. Clatot (France)
Transcriptome and pathway analysis identifies IRF1 as a predictor of progression free and overall
869
survival in ovarian carcinoma
J. Billaud, S. Cohen, R. Mosig, R. Halpert, P. Dottino, J. Martignetti (USA)
A new bispecific T cell recruiting antibody enhances anti-tumor activity of adoptive T cell transfer
S. Kobold, J. Steffen, C. Lampert, R. Castoldi, J.C. Schmollinger, C. Sustmann, G. Niederfellner,
C. Klein, C. Bourquin, S. Endres (Germany)
A new PD1-CD28 chimeric receptor overcomes PD-1-mediated immunosuppression in adoptive T cell
therapy
S. Kobold, S. Grassmann, M. Chaloupka, C. Lampert, J.C. Schmollinger, S. Endres (Germany)
Activation of systemic anti-tumor immunity by in situ ablation of breast carcinoma by intratumoral
224
Ra-loaded wires
H. Confino, I. Hochman, M. Efrati, M. Schmidt, V. Umansky, I. Kelson, Y. Keisari (Israel)
Macrophage migration and polarization is triggered by oxygen gradient in glioblastoma
M.M. Leblond, A.N. Gérault, A. Corroyer-Dulmont, E. Petit, M. Bernaudin, S. Valable (France)
Allergen induced pulmonary inflammation enhances mammary tumor growth and metastasis: Role of
CHI3L1
S. Libreros, R. Garcia-Areas, P. Robinson, A. Humbles, V. Iragavarapu-Charyulu (USA)
Changes of the white blood cells subsets in HER2 positive breast cancer patients treated with
trastuzumab
I. Matic, M. Grujic, B. Kolundzija, A. Damjanovic Velickovic, Z. Tomasevic, I. Bozovic Spasojevic,
R. Dzodic, Z. Zdrale, Z. Juranic (Serbia)
Cryotherapy enhances the antigen-specific T cell immune responses and therapeutic antitumor effects
generated by therapeutic HPV DNA vaccine
S.Y. Lee, J.K. Sim, K.H. Min, G.Y. Hur, J.J. Shim, K.H. In, K.H. Kang, C.F. Hung, T.C. Wu (Korea)
Interactions of immunoglobulin-like cell adhesion molecule hepaCAM with CD137 in cell–matrix
interactions, cell motility and escape from immune surveillance in Hodgkin Lymphoma
J. Seah (Singapore)
Depletion of tumor-associated macrophages enhances peptide-based cancer immunotherapy
S. Liu, K. Shen, Y. Song, I. Chen, P. Chong (Taiwan)
Early development of CD49d-high Th1 memory phenotype CD4+ T cells in the murine peritoneal cavity
Y. Bae, H. Moon, J.G. Lee, S.H. Shin, C.H. Park, J.H. Lee, K.J. Kang, T.J. Kim (South Korea)
Serum-dependent processing of late apoptotic cells for enhanced efferocytosis
Y. Liang, T. Arnold, A. Michlmayr, D. Rainprecht, B. Perticevic, A. Spittler, R. Oehler (Austria)
lix
870
871
874
875
876
877
879
880
881
882
883
Radiobiology/Radiation Oncology I
Psammaplin A (PsA) derivatives inhibit DNA methyltransferase (DNMT) and radiosensitize A549 lung
901
cancer cells
H.J. Kim, E.S. Ma, B.S. Shin, J.H. Kim, I.H. Kim (South Korea)
902
Mesenchymal stem cell therapy antagonizes radiation-induced endothelial cell damage and metastasis
D. Klein, A. Schmetter, V. Kleff, H. Jastrow, M. Stuschke, V. Jendrossek (Germany)
903
Single-fraction of gamma radiation induces apoptosis in cultured HeLa cervical cancer cells
W. Khalilia, G. Ozcan Arican (Turkey)
Investigation on the role of low dose radiation as chemo-potentiator in locally advanced carcinoma
904
cervix: A new treatment paradigm based on radiobiological advantage
S. Das, T.S. Vijaykumar, R.R. Singh, A. Chandramohan, J. Subhashini (India)
A novel role of kaempferol: Enhancing the radiosensitivity on lung cancer cells
905
C. Yao, Y. Tsai, W. Kuo, J. Lian, Y. Kuo, Y. Chen, S. Kuo (Taiwan)
Investigating the cellular and molecular mechanisms of tumour and host immune response to ionizing
906
irradiation in zebrafish
I.N. Aylott, A.C. Williams, C. Paraskeva, P. Martin (United Kingdom)
Effects of ionizing radiation on H69 cell line
908
F. Mendes, S. Schugk, T. Sales, A. Abrantes, A.C. Goncalves, P. Cesar, P. Soares, A.B. SarmentoRibeiro, M.F. Botelho, M.S. Rosa (Portugal)
Drug delivery evaluation with cell viability: A potential misinterpretation
909
H. Tseng, S.H. Hung, P.N. Chen (Taiwan)
lx
External beam radiotherapy synergizes rhenium 188-liposome against human esophageal cancer
xenograft and modulates rhenium 188-liposome pharmacokinetics
Y.J. Chen, C.H. Chang, S.Y. Liu, C.W. Chi, H.L. Yu, T.J. Chang, T.W. Lee (Taiwan)
Molecular mechanisms of the antineoplastic action of erufosine in hypoxic tumor cells
H. Riffkin, S. Oeck, G. Renzelman, R. Handrick, V. Jendrossek (Germany)
Analysing the effects of radiotherapy on the metastatic phenotype: A role for combined therapeutic
approaches incorporating Src and PI3K targeting
E.J. Rowling, K.J. Williams, P. Elvin (United Kingdom)
Whole thorax irradiation initiates a local and systemic accumulation of immunosuppressive FoxP3+
regulatory T cells
F. Wirsdörfer , M. Niazman, F. Cappuccini, S. De Leve, A.M. Westendorf, L. Lüdemann, M. Stuschke,
V. Jendrossek (Germany)
Radiobiological and clinical evaluation results of radiotherapy cancer cervix
V. Turkevich (Russian Federation)
Claudin-3 overexpression confers radioresistance of colorectal cancer cells
N. Fortunato-Miranda, W.F. Souza, J.A. Morgado-Dı́az (Brazil)
Importance of stromal caveolin-1 for tumor growth and radiosensitivity of epithelial tumors
V. Verhelst, D. Klein, T. Schmitz, A. Sak, V. Jendrossek (Germany)
910
911
912
913
914
915
916
Molecular and Genetic Epidemiology I
XRCC1 Arg399Gln polymorphism and susceptibility for hereditary BRCA1/2 negative breast cancer in
934
Serbian women
A. Krivokuca, E. Malisic, J. Rakobradovic, R. Jankovic, M. Brankovic-Magic (Serbia)
938
Deleterious RAD51C germ line mutations rarely predispose to breast and ovarian cancer in Pakistan
M. Rashid, N. Muhammad, S. Faisal, A. Amin, U. Hamann (Pakistan)
A highly polymorphic AG repeat in the upstream regulatory region of the estrogen-induced gene
939
EIG121 is a modifier of disease risk in endometrial cancer
K.A. Bolton, E.G. Holliday, N.A. Bowden, K.A. Avery-Kiejda, R.J. Scott (Australia)
Role of BRCA2 in the susceptibility to familial colorectal cancer type X
944
P. Garre, L. Martı́n, P. Diaque, A. Tosar, J. Sanz, E. Dı́az-Rubio, M. De la Hoya, T. Caldés (Spain)
945
Increased overall risk of cancer in patients with type 2 diabetes mellitus, but not their siblings or spouses
X. Liu, K. Hemminki, K. Sundquist, J. Sundquist, J. Ji (Sweden)
946
Incidence of second malignancies for prostate cancer in the canton of Zurich, 1980−2010
M. Van Hemelrijck, A. Feller, H. Gormo, D. Korol, F. Valeri, S. Dehler, S. Rohrmann (Switzerland)
Italianity is associated with lower risk of prostate cancer mortality in Switzerland
947
A. Richard, D. Faeh, S. Rohrmann, J. Braun, S. Tarnutzer, M. Bopp (Switzerland)
948
Genetic variants of susceptibility in second primary esophageal cancer patients
E. Boldrin, E. Rumiato, M. Fassan, M. Rugge, M. Cagol, V. Chiarion-Sileni, A. Ruol, M. Gusella,
A. Amadori, D. Saggioro (Italy)
Melatonin secretion in patients with breast cancer associated with primary hypertension
949
A. Tavartkiladze, G. Simonia (Georgia)
Intake and potential cancer risk of polycyclic aromatic hydrocarbons associated with traditional grilled
950
meat consumption in Iran
R. Ahmadkhaniha, N. Rastkari, M. Zare Jeddi, M. Yunesian, M. Es’haghi Gorji, M. Moazzen (Iran)
The polymorphism in C677T methylenetetrahydrofolate reductase, and the risk of colorectal cancer in
951
the Moroccan population
F. Azzam, A. Laraqui, F. El Boukhrissi, H. El Rhaffouli, Y. Bakri, R. Aboukhalid, I. Lahlou-amine,
S. Amzazi (Morocco)
Frequency of KRAS and NRAS mutations in Bulgarian patients with colorectal cancer
952
S. Bichev, S. Andonova, L. Hristova, A. Savov (Bulgaria)
lxi
Prevention and Early Detection I
Epidemiology of oral and pharyngeal cancer at the National Cancer Institute, Cairo Egypt
972
N. Labib (Egypt)
976
Nocturnal administration of resveratrol prevents breast cancer initiation in rats
T. Kisková, V. Demeckova, S. Gancarcikova, Z. Jendzelovska, M. Mekkova (Slovak Republic)
Circulating free DNA: a ‘liquid biopsy’ for the early detection of cancer?
977
R.M. Trigg, S.M. Giblett, C.A. Pritchard, J.A. Shaw (United Kingdom)
978
A multimarker panel for circulating tumor cells detection predicts patient outcome and therapy response
in metastatic colorectal cancer
J. Barbazan, L. Muinelo-Romay, M. Vieito, S. Candamio, A. Dı́az-López, A. Cano, A. Gómez-Tato,
M.A. Casares de Cal, M. Abal, R. López-López (Spain)
The immunomodulating and anti-inflammatory effects of garlic organosulfur compounds in cancer
979
prevention
J. Hitchcock, G. Schäfer, A. Katz, C.H. Kaschula (South Africa)
Identification of potential biomarkers in pancreatic ductal adenocarcinoma associated to tumor–stroma
980
interaction
A. Resovi, G. Taraboletti, A. Scarpa, R.T. Lawlor, R. Giavazzi, D. Belotti (Italy)
Proteomic analysis of urinary exosomes in patients with non-small cell lung cancer and tuberculosis
981
pleurisy using SWATH technique
K. Lee, H. Kim, D. Choi, K. Kim (Korea)
982
Prooxidant activity of resveratrol is associated with higher efficacy in breast cancer prevention
V. Demeckova, T. Kiskova, D. Mudronova, S. Gancarcikova, Z. Jendzelovska, L. Culka (Slovak Republic)
Hypermethylation biomarkers associated with inorganic arsenic metabolism are predictors to the
983
occurrence of internal organ cancers among arseniasis residents
P. Liao, H. Chiou, C. Chen, K. Hsu (Taiwan)
984
Tartrate-resistant acid phosphatase role in the diagnosis of metastatic bone lesions
V. Protsenko, A. Ilnitsky, B. Duda (Ukraine)
Poster Sessions, Monday 7 July 2014
10:15–17:15
Cell and Tumour Biology II
236
Novel approaches for dynamic biomarker imaging by multispectral optoacoustic tomography (MSOT)
W. Driessen, S. Morscher, N.C. Burton, T. Sardella, D. Razansky, V. Ntziachristos (Germany)
Sequential application of targeted therapies guided by biomarkers overcomes therapy resistance in
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rapidly evolving highly aggressive mammary tumors
O. Sahin, Q. Wang, S.W. Brady, H. Wang, C. Chang, S.T. Wong, W.J. Muller, F.J. Esteva, J. Chang,
D. Yu (Turkey)
Vascular endothelial growth factor-C modulates proliferation and chemoresistance in acute myeloid
238
leukemic cells through an endothelin-1-dependent induction of cyclooxygenase-2
Y. Yang, K. Hua, M. Chien, M. Kuo (Taiwan)
SOX14 downregulates SOX1 expression in HeLa cells
240
I. Petrovic, J. Popovic, D. Stanisavljevic, M. Schwirtlich, A. Klajn, J. Marjanovic, N. Kovacevic Grujicic,
V. Topalovic, M. Mojsin, M. Stevanovic (Serbia)
Bcl-xL protein overexpression enhances tumor progression of human melanoma cells in zebrafish
241
xenograft model: involvement of interleukin 8
C. Gabellini, E. Gómez-Abenza, S. De Oliveira, D. Del Bufalo, V. Mulero (Spain)
Characterisation of retinoic acid effect on breast cancer cell plasticity
242
G. Paroni, A. Zanetti, R. Affatato, E. Garattini (Italy)
lxii
p27 is a haploinsufficient tumor suppressor in MENX rats and this associates with the development of
invasive medullary thyroid carcinoma
N. Pellegata, S. Molatore, F. Neff, M. Irmler, E. Pulz, F. Roncaroli (Germany)
Aromatase expression contributes to the survival and metastasis of estrogen receptor positive breast
cancer cells
L.Z. Sun, K. Mukhopadhyay, Z. Liu, A. Bandyopadhyay (USA)
CD9 is required for stromal cell invasion of breast cancer cells
G. Rappa, T. Green, A. Lorico (USA)
Effects of the potential energy restriction mimetic agent delta2-troglitazone in breast cancer cells
I. Grillier-Vuissoz, C. Colin-Cassin, X. Yao, C. Cerella, A. Berthe, S. Mazerbourg, M. Boisbrun, S. Kuntz,
M. Diederich, S. Flament (France)
Ovarian cancer cells treated with leptin contribute with a pro-tumoral phenotype in macrophages in vitro
L. Abarzua-Catalan, S. Kato, A.M. Delpiano, G.I. Owen, M.A. Cuello (Chile)
Radiotherapy response of breast cancer stem cells
P. Cordeiro, T. Costa, M.J. Carvalho, M. Laranjo, P. Rachinhas, J.E. Casalta-Lopes, A.M. Abrantes,
M. Botelho (Portugal)
Radiosensitive and radioresistant colorectal cancer cells
A. Ferreira, J.E. Casalta-Lopes, M. Laranjo, A.M. Abrantes, A. Cavaco, M. Borrego, P. Soares,
M. Botelho (Portugal)
Apoptosis and proliferation in micropapillary structures of colorectal polyps and carcinomas
M. Patankar , S. Sajanti, A. Tuomisto, M. Mäkinen, T. Karttunen (Finland)
Polo-like kinase 4 (plk4) modulates cancer cell polarity and invasion
K. Kazazian, R. Xu, O. Brashavitskaya, F. Zih, C.J. Swallow (Canada)
RhoGTPase-based regulation of cell spreading by Plk4
V. Brashavitskaya, K. Kazazian, R. Bagshaw, C.O. Rosario, F.S.W. Zih, Y. Haffani, J.W. Dennis,
T. Pawson, C.J. Swallow (Canada)
Expression of TRF2 and its prognostic relevance in advanced stage cervical cancer patients
O. Orun, S. Ozden, P. Mega Tiber, N. Serakinci, Z. Ozgen, H. Ozyurt (Turkey)
Role of prominin-1 (CD133)-exosomes released by melanoma cells in intercellular communication
A. Lorico, T. Green, F. Anzanello, G. Rappa (USA)
Apoptosis-inducing effect of Usnea filipendula Stirt. in breast cancer cells in vitro
M. Sarimahmut, S. Celikler, F. Ari, S. Oran, N. Aztopal, S. Ozturk, E. Ulukaya (Turkey)
TXNRD2 and DCBLD2 are novel targets of osteosarcoma metastasis
E. Topkas, N. Cai, A. Cumming, N. Saunders, L. Endo-Munoz (Australia)
Stimulated prostatic angiogenesis in senescence resembles the microenvironment of neoplastic lesions
and can be reversed by antiangiogenic therapy
F. Montico, L.A. Kido, A.C. Hetzl, V.H.A. Cagnon (Brazil)
Procathepsin B and endogenous inhibitors of cysteine proteases as possible biomarkers of ovarian
cancer
E. Gashenko, V. Lebedeva, E. Tsykalenko, G. Russkikh, K. Loktev, I. Brak, T. Korolenko (Russian
Federation)
A novel MYC directed apoptosis pathway controls NOXA and BIM transcription
M. Wirth, N. Stojanovic, R. Schmid, O.H. Krämer, D. Saur, G. Schneider (Germany)
Cancer-associated fibroblast (CAF)-derived exosome may mediate breast cancer progression by
reducing exosomal microRNAs
J.E. Kim, N.H. Cho (Korea)
STAT3-induced WDR1 expression is associated with breast cancer cell migration
J.H. Lee, N.H. Cho (Korea)
Mimicking the tumour microenvironment (TME): Angiogenesis in tumour progression
U.H. Bonda, L.J. Bray, N. Lister, S. Ellem, G. Risbridger, U. Freudenberg, C.C. Werner, D.W. Hutmacher,
E.M. De-Juan-Pardo, D. Loessner (Australia)
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Histone deacetylase inhibitors resensitize glioblastoma cells to EGFR-directed therapy with tyrosine
kinase inhibitors after primary treatment failure
K. Liffers, K. Kolbe, M. Westphal, K. Lamszus, A. Schulte (Germany)
The effects of INhibitor of Growth 3 (ING3) on prostate cancer cell survival and invasion
A. Nabbi, A. Almami, T. Bismar, K. Riabowol (Canada)
EphB4 negatively regulates blood vessel network formation and perfusion in human A375 melanoma
xenografts
C. Neuber , F. Hofheinz, R. Bergmann, S. Meister, J. Steinbach, B. Mosch, J. Pietzsch (Germany)
Mesenchymal cells regulate growth of intestinal crypts by a Wnt independent mechanism in 3D culture
system
A. Pastula, S. Hauck, K.P. Janssen, R.M. Schmid, M. Quante (Germany)
Comparison of membrane fatty acid compositions of mesenchymal stem cells and mature mesenchymal
cell lines using GC-FID method
T. Ozgurtas, A. Tas, F. Yesildal, F. Avcu (Turkey)
Multidrug resistance gene MDR1 is transactivated by Oct4 to increase chemotherapy resistance
C.L. Wu, C.S. Lu, A.L. Shiau (Taiwan)
TPRG1L is a novel microRNA-21 target: a possible linkage between miR-21 and cellular senescence
in liver fluke-associated cholangiocarcinoma
P. Chusorn, N. Namwat, W. Loilome, A. Techasen, C. Pairojkul, N. Khuntikeo, B. Tean Teh, I. Lee,
P. Yongvanit (Thailand)
AR-associated p21 induces apoptosis by neutralizing anti-apoptotic Bcl-2 protein in androgen-dependent
prostate cancer LNCaP cells
K. Choi, H. Suh, C.H. Lee (Korea)
TWIST1 and ZEB1 EMT inducers contribute to melanoma development through regulating MITF
G. Richard, M. Houang, A. De la Fouchardière, R. Marais, L. Larue, S. Dalle, E. Tulchinsky, S. Ansieau,
A. Puisieux, J. Caramel (France)
Androgen receptor interacting protein HSPBAP1 facilitates growth of prostate cancer cells in
androgen-deficient conditions
P. Ostling, K. Saeed, M. Björkman, T. Mirtti, T. Vesterinen, J. Lundin, A. Rannikko, J.P. Mpindi,
O. Kallioniemi, J. Rantala (Finland)
Tumor metabolism and docetaxel resistance in prostate cancer
M. Taddei, A. Marini, L. Ippolito, A. Morandi, E. Giannoni, P. Chiarugi (Italy)
MiR-136 targeting Notch3 is involved in chemoresistance and angiogenesis in ovarian cancer cells
H. An, J. Jeong, M. Lee, J. Song, Y. Jung, Y. Kim, A. Kwon, J. Huh, K. Kim, H. Kang (Korea)
COX-2/PGE2 promotes lung cancer invasion/metastasis via MIG-7 and phosphorylated prohibitin
S.M. Liang, M.Y. Ho, C.M. Liang (Taiwan)
The kallikrein-related serine peptidase, KLK4, regulates the TGFb1 pathway in the tumour–stroma
microenvironment in prostate cancer
J. Clements, R. Fuhrman-Luck, S. Stansfield, M. Hastie, T. Stoll, C. Stephens, D. Loessner, M. Lehman,
C. Nelson, J. Gorman (Australia)
Alterations of hepatocyte metabolic identity are triggered by the hepatitis C virus through systemic
wnt/b-catenin signalling
M. Moreau, B. Rivière, S. Vegna, J. Ramos, E. Assenat, U. Hibner (France)
Silencing of mitochondrial Lon protease deeply alters mitochondrial proteome and functionality in RKO
colorectal carcinoma cells
L. Gibellini, M. Pinti, F. Boraldi, V. Giorgio, P. Bernardi, M. Nasi, S. De Biasi, P. Pinton, D. Quaglino,
A. Cossarizza (Italy)
A panel of 20 genes involved in cellular adhesion and ECM remodelling distinguishes renal cancer and
control samples
J. Boguslawska, H. Kedzierska, B. Rybicka, P. Poplawski, Z. Tanski, A. Nauman,
A. Piekielko-Witkowska (Poland)
p53-directed translational control can shape and expand the universe of p53 target genes
S. Zaccara, C. Martinez Bolado, C. Pederiva, T. Tebaldi, Y. Ciribilli, A. Bisio, A. Inga (Italy)
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Disruption of cortical tension patterning drives the outgrowth of oncogenic cells
S. Wu, G.A. Gomez, M. Michael, S. Verma, H. Cox, J.G. Lefevre, R.G. Parton, N.A. Hamilton,
A.S. Yap (Australia)
Evidence of a correlation between bcl-2 protein and miR-211 expression in melanoma cell lines
T. De Luca, A. Pelosi, D. Trisciuoglio, S. D’Aguanno, A. Felsani, A. Urbani, M.G. Rizzo,
D. Del Bufalo (Italy)
MicroRNA expression in triple-negative versus other subtypes of breast cancer
D. Kalniete, M. Nakazawa-Miklasevica, I. Strumfa, A. Abolins, A. Irmejs, G. Trofimovics, J. Gardovskis,
E. Miklasevics (Latvia)
Expression of markers of epithelial–mesenchymal transition E-cadherin and vimentin in different
immunohistochemical subtypes of breast cancer
Y.M. Zasadkevich, S.V. Sazonov, A.A. Brilliant (Russian Federation)
Improved robustness for fully automated 3D spheroid HCA screening
S. Degot, J. Young, E. Duchemin-Pelletier, F. Monjaret (France)
Effect of boric acid on head and neck cancer cell lines
M. Gunduz, M. Acar, K. Fakioglu, B. Dogan, M. Oznur, E. Gunduz (Turkey)
Examination of role of ING1 splicing variant (p33ING1) in carcinogenesis and metastasis of head and
neck carcinomas
E. Gunduz, O.F. Hatipoglu, K.O. Yaykasli, K. Erdogan, E.N. Cetin, G. Nas, M. Gunduz (Turkey)
The 5 -untranslated region of p16INK4a acts as a cellular IRES, controls mRNA translation during
hypoxic and energetic stresses, and is a target of YBX1
A. Bisio, E. Latorre, V. Andreotti, P. Ghiorzo, B. Bressac-de Paillerets, R.C. Spitale, A. Provenzani,
A. Inga (Italy)
Cytotoxic effect and apoptosis induction by phytohemagglutinin erythroagglutinating on lung cancer cells
K.Y. Chen, W.T. Kuo, Y.J. Ho, C.H. Yao (Taiwan)
MNT roles and expression in the absence of MAX
M.C. Lafita, A. Quintanilla, J. Rodrı́guez, I. Varela, A. Von Kriegsheim, J. León (Spain)
In vitro modulation of CITED4 gene expression in a colorectal cancer cell line
M.A. Rogers, V. Kalter, G. Marcias, M. Zapatka, S. Barbus, B. Radlwimmer, P. Lichter (Germany)
Twist oncoproteins are modulators of cellular stress
J. Kolodziejski, U. Hibner, P. Lassus (France)
Preclinical validation of the therapeutic potential of neuropilin-1 targeting transmembrane peptide in
glioblastoma
J. Fritz, L. Jacob, A. Fernandez, D. Bagnard (France)
The metabolic cooperation between prostate carcinoma cells and cancer associated fibroblasts:
pyruvate kinase M2 at the crossroads
E. Giannoni, M.L. Taddei, A. Morandi, G. Comito, P. Chiarugi (Italy)
Induction of HIF1a influences estrogen receptor expression in ex-vivo culture of tumour tissue
E. Davies, A. Rahi, M. Cumberbatch, C. Eberlein, E. Anderson, S. Wedge, M. Smalley, S. Barry (United
Kingdom)
Metformin induces apoptosis and dowregulates pyruvate kinase M2 in MCF7 breast cancer cells only
when grown in nutrient-poor conditions
A. Silvestri, I. Rasi, F. Palumbo, D. Posca, L. Castagnoli, G. Cesareni (Italy)
Loss of PI3K-C2a promotes tumorigenesis and aneuploidy in breast cancer
M. Martini, F. Gulluni, M.C. De Santis, A. Ghigo, J.P. Margaria, E. Ciraolo, U. Ala, F. Cavallo, R. Chiarle,
E. Hirsch (Italy)
Normal and oncogenic proliferation under control of microRNAs: A functional high content screening
D. Sastre, I.M.S. Lima, J.F. Guerreiro, D.T. Covas, M.A. Zago, R.A. Panepucci (Brazil)
A role for senescent cell-derived IL6 in HER2+ breast cancer progression
M. Zacarias Fluck, B. Morancho, P.D. Angelini, R. Vicario, L. Villarreal, C. Aura, P. Nuciforo,
J. Villanueva, I.T. Rubio, J. Arribas (Spain)
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Myc mediates the phosphorylation and degradation of p27 through activation of Cyclin A/CDK1
L. Garcı́a-Gutiérrez, G. Bretones, I. Arechaga, D. Santamarı́a, M. Barbacid, J. León (Spain)
Natural and synthetic inhibitors of mTOR blunt the p53 response to low concentrations of actinomycin
D but not nutlin-3
K.M. Goudarzi, M. Nistér, M.S. Lindström (Sweden)
Chemotherapy sensitizes p95HER2-positive breast cancers to trastuzumab
B. Morancho, J.L. Parra-Palau, V. Peg, R. Vicario, M. Zacarias-Fluck, K. Pedersen, C.M. Perou, A. Prat,
I.T. Rubio, J. Arribas (Spain)
Gelsolin promotes the survival of cancer cells under stress by modulating autophagy
S. Deng, L. Tochhawng, T.D. Dinh, H.M. Shen, C.T. Yap (Singapore)
Hybrid peptide tat-Ram13-induced necrosis-like cell death depends on expression of PTEN in human
leukemia cell lines
A. Kuniyasu, M. Kurogi, M. Setoguchi, M. Makise (Japan)
Clonal succession of transiently active TIC clones in human pancreatic cancer
C.R. Ball, F. Oppel, R. Ehrenberg, J. Weitz, J. Werner, F. Bergmann, N. Ishaque, B. Brors, C. Von Kalle,
H. Glimm (Germany)
The novel use of BAMLET in the treatment of oral squamous cell carcinoma
N. Sinevici, N. Harte, K. Hun Mok, Y. Xie, J. O’Sullivan (Ireland)
A CXCR4-positive subpopulation exhibits cancer stem cell properties in renal cell carcinoma
W. Zimmermann, M. Gassenmaier, D. Chen, A. Buchner, H. Pohla (Germany)
Zoledronic acid impairs stromal reactivity by inhibiting M2-macrophages polarization and prostate
cancer-associated fibroblasts
G. Comito, C. Pons Segura, K. Sobierajska, L. Ippolito, M.L. Taddei, E. Giannoni, P. Chiarugi (Italy)
Breast cancer stem cells are more resistant than parental cells to a novel palladium (II) saccharinate
complex
D. Karakas, N. Aztopal, B. Cevatemre, F. Ari, A. Yilmaztepe Oral, V. Turan Yilmaz, E. Ulukaya (Turkey)
BCLAF1; a multi-faceted protein involved DNA repair, apoptosis and autophagy
E. Barros, K.S. Savage, D.P. Harkin (United Kingdom)
Elovl6 overexpression promotes liver carcinogenesis
A.L. Shiau, Y.C. Su, Y.H. Feng, H.T. Wu, Y.S. Huang, P. Wu, C.J. Chang, C.L. Wu (Taiwan)
Influence of cancer stem cells on drug resistance in prostate cancer
E.A. Castellón, V. Castillo, R. Valenzuela, H.R. Contreras (Chile)
Analysis of HER2 amplification and IGF-IR expression in CETCs and its possible association with
resistance to trastuzumab in HER2 positive breast cancers
D. Zimon, M. Pizon, U. Pachmann, K. Pachmann (Germany)
Chemosensitivity differs between CETCs and spheroids grown from the CETCs in cancer patients with
solid tumors
M. Pizon, D. Zimon, U. Pachmann, K. Pachmann (Germany)
Expression profile of fibroblasts from tumor bearing prostate exhibits significant differences compared
to BPH-derived fibroblasts
V. Jung, K. Schmitt, M. Saar, K. Junker, M. Stöckle, G. Unteregger (Germany)
Expression and cytogenetic profile of intratumor morphological heterogeneity in breast cancer
E. Denisov, T. Gerashchenko, N. Skryabin, S. Vasilyev, M. Zavyalova, N. Litviakov, V. Perelmuter,
N. Cherdyntseva (Russian Federation)
Identification of GREB1 as a potential mediator of estrogen effects on ovarian cancer progression in
a mouse model
K.M. Hodgkinson, L.A. Laviolette, B.C. Vanderhyden (Canada)
Simplifying high throughput 3D tumour spheroid growth and shrinkage assays using live content imaging
T. Dale, K. Patel, B. O’Clair, T. O’Callaghan, D. Appledorn, D. Trezise (United Kingdom)
BRCA1 as a regulator of global cell metabolism − identifying functional targets through integromic
analysis
A. Powell, K.I. Savage, D.P. Harkin (United Kingdom)
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Expression analyses of long non-coding RNAs in breast cancer
E. Knutsen, T. Fiskaa, S.L. Figenschau, E.S. Brun, S. Fismen, O.M. Seternes, E.S. Mortensen,
S.D. Johansen, M. Perander (Norway)
Paraffin embedded tissue as valuable and challenging resource for studying intratumoral genetic
heterogeneity of clear cell renal cell carcinoma
P. Ferronika, J. Bergsma, J. Li, M.M. Terpstra, A.M. Lelieveld-Kors, R. Danarto, R.H. Sijmons,
G. Kats-Ugurlu, A. Moeljono, K. Kok (Netherlands)
Identification and quantification of BRCA1 splicing variants
F. Lhota, J. Hojny, P. Kleiblova, J. Sevcik, J. Soukupova, M. Janatova, P. Boudova, M. Borecka,
P. Pohlreich, Z. Kleibl (Czech Republic)
Inhibition of thioredoxin-1 by small interfering RNA reduces chemoresistance for doxorubicine in two
diffuse large B-cell lymphoma cell lines
M.E.L. Kuusisto, P. Honkavaara, A. Hakalahti, P. Karihtala, T. Turpeenniemi-Hujanen,
O. Kuittinen (Finland)
Only an EpCAM–claudin-7 complex acts as a cancer initiating cell biomarker
F. Thuma, S. Heiler, R. Philip, M. Zöller (Germany)
Integrative analysis reveals extensive association between microRNA expression and mRNA−protein
translation
M.R. Aure, S. Jernström, M. Krohn, E. Due, Oslo Breast Cancer Consortium, G.B. Mills,
A.L. Børresen-Dale, K.K. Sahlberg, O.C. Lingjærde, V.N. Kristensen (Norway)
Stem cell fractions of extra- and intrahepatic cholangiocellular carcinoma cell lines differ in size and
phenotype
R. Schmuck, C.V. Fischer, A. Schirmeier, P. Neuhaus, M. Bahra (Germany)
Long-term inhibition of miR-200c in pancreatic adenocarcinoma cells changes CDH1 expression and
cell migration
M. Stölting, J. Schwarze, M. Starke, J. Haier, N. Senninger, S.T. Mees (Germany)
EDI3, a glycerophosphodiesterase linking metabolism to cellular migration and attachment
R. Marchan, M.S. Lesjak, B. Buettner, J.D. Stewart, J. Lambert, H. Keun, J. Rahnenfuehrer,
J.G. Hengstler (Germany)
STAT3, IL-17 and COX-2 features in the transgenic adenocarcinoma of mouse prostate (TRAMP) model
and in the aging mice (FVB) submitted to goniothalamin therapy
L.A. Kido, F. Montico, D.B. Vendramini-Costa, J.E. Carvalho, R.A. Pilli, V.H.A. Cagnon (Brazil)
Smad ubiquitination regulatory factor 2 (Smurf2) regulates the gap junction protein connexin43 during
mitosis
T.A. Fykerud, S.D. Koirala, M.Z. Totland, L.M. Knudsen, Z. Yohannes, Y. Omori, A. Brech,
E. Leithe (Norway)
MALDI-MS peptide profiling of thyroid cancer cell lines to detect peptide signatures changes with the
PI3-K inhibitor GDC-0941, in hypoxia and normoxia
F. Henderson, K. Williams (United Kingdom)
Resistin induces a disintegrin and metalloproteinase with thrombospondin motifs-4 gene expression in
human chondrosarcoma cell line
O.F. Hatipoglu, K.O. Yaykasli, E. Yaykasli, E. Kaya, M. Ozsahin, M. Uslu, K. Yildirim, H.E. Gurses,
E. Gunduz (Turkey)
SLC22A18, a solute carrier transporter, acts on a tumor suppressor in colorectal cancer via KRAS
Y. Jung, H.Y. Lee, J. Keum, Y. Jung, S. Kim, H.K. Chun, W.Y. Lee, S. Lee, J. Kim (South Korea)
Modulated electrohyperthermia induced immunogenic cell death specific signals in colorectal
adenocarcinoma model
N. Meggyeshazi, G. Andocs, C. Kovago, L. Balogh, T. Krenacs (Hungary)
Deciphering the role of ubiquitin specific peptidase 15 (USP15) in human glioblastoma
M. Oikonomaki, M.E. Hegi (Switzerland)
Characterisation of RNA content of cancer-derived exosomes and microvesicles
A. Line, D. Andrejeva, A. Cirulis, A. Abols, P. Zayakin (Latvia)
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In vivo model of dormancy in ER breast cancer to bone metastasis
S. Gawrzak, M. Guiu, J. Urosevic, E. Fernandez, M. Pavlovic, R.R. Gomis (Spain)
Fibroblast crosstalk with anti-Her2 therapies breast cancer resistant clones
P. Fernandez, G. Fuster, M. Mancino, A. Zubeldia, E. Ametller, E. Enreig, P. Gascón, H. Slovang,
H. Russnes, V. Almendro (Spain)
BCAT1 promotes cell proliferation through amino acid catabolism in gliomas carrying wild-type IDH1
B. Radlwimmer , M. Tönjes, S. Barbus, Y.J. Park, W. Wang, I. Weibrecht, S.M. Hutson, C. Plass,
G. Reifenberger, P. Lichter (Germany)
Potential interconnection between alternative splicing and microRNA regulation of SRSF1 oncogene
in renal cancer
J. Boguslawska, E. Sokol, H. Kedzierska, B. Rybicka, P. Poplawski, Z. Tanski, A. Nauman,
A. Piekielko-Witkowska (Poland)
Recapitulation of non-small-cell lung carcinoma microenvironment in perfusion bioreactor cultures: the
impact of hypoxia on tumour−stroma crosstalk
V. Espı́rito Santo, M. Estrada, S. Veloso, M.F.Q. Sousa, H. Van der Kuip, M. Oren, E.R. Boghaert,
P.M. Alves, C. Brito (Portugal)
The TALE homeodomain transcription factor MEIS1 activates the pro-metastatic melanoma cell
adhesion molecule Mcam to promote migration of pancreatic cancer cells
J. Von Burstin, F. Bachhuber, O.P. Da Costa, T. Buch, A.K. Rustgi, R.M. Schmid (Germany)
Modulation of canonical WNT signaling in combination with temozolomide inhibits growth of glioblastoma
cancer stem cells in vitro
F. Schneider , A. Flieger, J. Gempt, F. Ringel, J. Schlegel (Germany)
microRNA-449 acts as a barrier to stemness
C. Gallinas Suazo, A. Klimke, M. Lizé, M. Kessel, M. Dobbelstein (Germany)
Role of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase in lung cancer
K. Leithner , A. Hrzenjak, M. Trötzmüller, T. Moustafa, H.C. Köfeler, C. Wohlkoenig, E. Stacher,
A.L. Harris, A. Olschewski, H. Olschewski (Austria)
Effects of anti-proliferative lichen metabolite, protolichesterinic acid on fatty acid synthase and lipid
composition in breast cancer cells
M. Bessadottir , F.F. Eiriksson, S. Gowan, S. Eccles, S. Omarsdottir, M. Thorsteinsdottir,
H.M. Ogmundsdottir (Iceland)
Effect of SNAIL1 expression on the epithelial–mesenchymal transition in prostate cancer
H. Contreras, L. Osorio, N. Farfán, E.A. Castellón (Chile)
Targeted therapy makes EGFR promiscuous: EGFR and c-Met interaction in lung cancer
E. Ortiz-Zapater , R.W. Lee, G. Fruhwirth, G. Weitsman, W. Owen, T. Ng, G. Santis (United Kingdom)
Role of the polarity protein Par3 in promotion and suppression of skin tumors
D. Kleefisch, S. Vorhagen, C. Niessen, S. Iden (Germany)
Reduced promoter methylation and increased expression of CSPG4 negatively influences survival of
HNSCC patients
R.W. Warta, J.C. Chaisaingmongkol, A.M. Mock, O.P. Popanda, E.H. Herpel, C.P. Plass, P.S. Schmezer,
V.E. Eckstein, G.D. Dyckhoff, C.H.M. Herold-Mende (Germany)
The putative tumor suppressor gene Dickkopf-3 (DKK3) and its role in the carcinogenesis of breast cancer
E. Lorsy, J. Veeck, R. Knüchel, E. Dahl (Germany)
BMP4 suppresses breast cancer metastasis through down-regulation of G-CSF
R.L. Anderson, Y. Cao, A. Swierczak, B.L. Eckhardt, J.A. Hamilton (Australia)
The ING1 tumour suppressor induces senescence via altering endocytosis
K. Riabowol, U.K. Rajarajacholan, S. Thaalippilly (Canada)
VHL-dependent changes in global kinome expression in renal cell carcinoma
K. Rantanen, P. Kouvonen, G.L. Corthals, P.M. Jaakkola (Finland)
The interplay between the redox environment, MTH1 and cancer
L. Braeutigam, R. Fiskesund, U. Warpman-Berglund, T. Helleday (Sweden)
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The role of tumour derived extracellular matrices on macrophage polarization
M.L. Pinto, E. Rios, A. Silva, A.T. Pinto, A.P. Cardoso, D. Nascimento, P. Pinto do Ó, F. Carneiro,
M.B. Barbosa, M.J. Oliveira (Portugal)
Elucidating the synthetic lethal effect of CDK inhibition in triple negative breast cancer
J. Rohrberg, A. Corella, L. Starnes, A. Goga (USA)
ErbB3 in the nucleus − mechanisms of nuclear entry
R. Reif , A. Adawy, G. Günther (Germany)
An in vivo shRNA screen to identify novel drivers and therapeutic targets of breast cancer metastasis
A. Van Weverwijk, M. Ashenden, N. Murugaesu, M. Iravani, C.M. Isacke (United Kingdom)
Lymphotoxin signalling alters the vasculature to increase tumour cell metastasis
N. Simonavicius, B. Seubert, L. Borsig, D. Wohlleber, J. Browning, A. Krüger, M. Heikenwalder (Germany)
Esophageal squamous cell carcinoma cells stimulate the formation of a hyaluronan-rich
microenvironment: implications for fibroblast phenotype and sensitivity to tyrosine kinase inhibitors
I. Kretschmer , T. Freudenberger, S. Twarock, J.W. Fischer (Germany)
c-Abl antagonizes the YAP oncogenic function
R. Keshet, J. Adler, Y. Shaul (Israel)
c-Abl tyrosine kinase regulates recovery from the G2-M DNA damage induced checkpoint
V. Meltser , N. Reuven, Y. Shaul (Israel)
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370
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Cancer Genomics, Epigenetics and Genome Instability II
423
Epigenetic effects of radiation on hTERT-immortalized mesenchymal stem cells
O. Orun, P. Mega Tiber, N. Serakinci (Turkey)
TET2 gene expression and 5-hydroxymethylcytosine levels are decreased in pediatric patients with
424
myelodysplastic syndrome
D. Coutinho, B.C.R. Monte-Mor, D. Vianna, S. Rouxinol, A.B. Batalha, A.P. Bueno, M.S. Pombo-deOliveira, E. Abdelhay, I. Zalcberg (Brazil)
RUNX1 structural variants in oesophageal adenocarcinoma
425
R.C.J. Hsu, R.C. Fitzgerald, P.A.W. Edwards (United Kingdom)
426
MicroRNA 196b regulates radioresistance of gastric cancer cells by targeting HR23B
S. Kim, Y.N. Shen (Korea)
NuRD complex co-operates with PRC2 to suppress gene expression
427
W. Zhu, F. Wei, X.I. Wang, H. Wang (China)
428
Detecting and quantifying low level variants in Sanger sequencing traces
E. Schreiber , S. Berosik, M. Wenz (USA)
429
Targeted next-generation sequencing − Identification of Lynch syndrome cases
B.A. Talseth-Palmer, T.J. Evans, A. Spigelman, R.J. Scott (Australia)
Identification of deregulated genes in colorectal cancer metastasis through whole genome and
431
transcriptome sequencing
C. Hauser , B. Hutter, I. Buchhalter, A. Shavinskaya, M. Abba, H. Yazdanparast, R. Eils, B. Brors,
H. Allgayer (Germany)
Comparison of two library construction strategies for targeted resequencing of BRCA1/2 genes in
432
Bulgarian breast cancer patients on NGS platform
D. Dacheva, I. Popov, R. Dodova, T. Goranova, A. Mitkova, R. Kaneva, V. Mitev (Bulgaria)
An integrated multi level ‘-omics’ approach to decipher disease recurrence in epithelial ovarian cancer
433
P. Orsini, L. De Cecco, E. Cecchin, M.L. Carcangiu, F. Raspagliesi, D. Lo Russo, G. Toffoli,
D. Mezzanzanica, S. Canevari, M. Bagnoli (Italy)
434
MicroRNAs at the human 14q32 locus have significance in malignant phyllodes tumour
D.C. Kim, M.R. Lee, J.W. Lee, S.E. Cho (Korea)
435
Identification of hypoxia-induced miRNAs in colon cancer stem cells
P. Ullmann, K. Baig, W. Ammerlaan, S. Haan, E. Lettelier (Luxembourg)
Epigenetic regulation of AREG and EREG expression by ZBTB33/KAISO in colorectal cancer
436
S. Ispasanie, F. Bormann, N. Kuhn, S. Tierling, J. Walter, C. Sers (Germany)
Identification of a specific and sensitive urinary DNA hypermethylation signature for bladder cancer
diagnosis
C.U. Köhler , M. Ahrens, T. Behrens, M. Eisenacher, K. Braun, K.H. Jöckel, R. Erbel, A. Tannapfel,
T. Brüning, H.U. Käfferlein (Germany)
Comprehensive mutational landscape of human stable colorectal tumors in stage II
R. Sanz-Pamplona, A. López-Dóriga, L. Paré-Brunet, K. Lázaro, H. Alonso, S. Beltran, F. Castro,
M. Gut, L. Agueda, V. Moreno (Spain)
Effects of the histone deacetylase inhibitor romidepsin in lymphoma B-cells and BCL6 regulation
M.G. Cortiguera, L. Garcı́a-Gaipo, A. Batlle-López, M.D. Delgado (Spain)
Genomic copy number changes and the prognostic impact of the encoded genes PTEN and BIRC5 in
malignant peripheral nerve sheath tumours
M. Høland, M. Kolberg, B. Davidson, K. Sundby Hall, F. Mertens, E. Van den Berg, S. Smeland,
P. Picci, O.C. Lingjærde, R.A. Lothe (Norway)
Expression profiling of microRNAs in neuroblastoma tumors in different stages and histology
K. Ergin, S. Aktas, Z. Altun, G. Diniz, N. Olgun (Turkey)
Combined microRNA and ER expression: a new classifier for familial and sporadic breast cancer patients
S. De Summa, K. Danza, R. Pinto, B. Pilato, M. Carella, O. Palumbo, G. Simone, D. Sambiasi,
E. Savino, S. Tommasi (Italy)
The DNA demethylating agent zebularine inhibits medulloblastoma growth in vitro in association with
the regulation of cell cycle and apoptosis-related genes
A.F. Andrade, K.S. Borges, V.K. Suazo, R.G.P. Queiroz, C.A. Scrideli, L.G. Tone (Brazil)
A gain of function by the cancer-associated FGFR4 c.1162G>A (p.Gly388Arg) variant
V.K. Ulaganathan, B. Sperl, T. Mayr, R. Hornberger, U.R. Rapp, A. Ullrich (Germany)
Classification of human cutaneous metastatic melanoma cell lines and clinical samples by receptor
tyrosine kinases- and phenotype-driven gene expression signatures
M. Dugo, S. Canevari, A. Anichini, M.L. Sensi (Italy)
Promoter hypermethylation and overexpression of putative oncomiRs may contribute to silencing of the
tumor suppressor gene PCDH17 in laryngeal squamous cell carcinoma
E. Byzia, M. Szaumkessel, K. Kiwerska, M. Kostrzewska-Poczekaj, M. Jarmuz-Szymczak, N. Zemke,
R. Grenman, K. Szyfter, M. Giefing (Poland)
Gene-specific methylation profiles in male breast cancer
P. Rizzolo, V. Silvestri, A.S. Navazio, V. Valentini, V. Zelli, M. Falchetti, I. Zanna, S. Bianchi, D. Palli,
L. Ottini (Italy)
The consequence of SETD2 mutation in clear cell renal cell carcinoma progenitor cells
J. Li, J. Osinga, P. Ferronika, M.B. Van Werkhoven, M.M. Terpstra, P. Van der Vlies, G. Duns,
H. Westers, R.H. Sijmons, K. Kok (Netherlands)
Epigenetic changes and dietary factors in human and experimental breast cancer
E. Escrich, C. Rodrı́guez-Miguel, N. Braviz, A. Modolell, T. Checa, R. Moral (Spain)
Modifications in gene expression profile of mammary gland and experimental tumors by effect of high
fat diets
R. Moral, R. Escrich, M. Solanas, E. Vela, M.C. Ruı́z de Villa, E. Escrich (Spain)
Enhancer of Zeste Homolog 2 (EZH2) modulation in either embryonal or PAX3-FOXO1 alveolar
rhabdomyosarcoma shows different anti-tumoral effects
R. Rota, E. Carcarino, M. De Salvo, L. Adesso, R. Ciarapica, V.E. Marquez, A. Mai, P.L. Puri,
D. Palacios, F. Locatelli (Italy)
Modelling mutational landscapes of primary cancers using an in vitro cell immortalization assay
M. Olivier , A. Weninger, M. Ardin, H. Huskova, G. Team, T. Nedelko, K.R. Muehlbauer, G. Byrnes,
M. Hollstein, J. Zavadil (France)
The role of new cancer hallmarks in tumorigenesis
A. Gonzalez-Perez, A. Jene-Sanz, D. Tamborero, N. Lopez-Bigas (Spain)
Improving cell based models through viral vector technology − chances for functional genomics and
target research
K. Schmitt, M. Salomon, V. Jagusch, S. Schrödel, C. Thirion (Germany)
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442
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446
447
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449
450
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455
456
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Diversity of methylation patterns in HPV16 LCR in cervical cancer
S. Amaro-Filho, A.C. Brant, J.P. Vidal, S.P. Felix, C.R. Bonvincino, M.A.M. Moreira (Brazil)
Identification of novel epigenetic modulators of acquired chemoresistance in colon cancer
E. Enreig, M. Mancino, P. Fernandez, F.J. Casado, P. Gascón, N. Carbó, E. Ametller, V. Almendro (Spain)
COSMIC: Exploring novel cancer biomarkers
S. Forbes, D. Beare, K. Leung, N. Bindal, S. Bamford, S. Ward, C. Dunham, U. McDermott,
M.R. Stratton, P. Campbell (United Kingdom)
Genomic copy number and microRNA profiles of 27 colon cancer cell lines
K.G. Berg, P.W. Eide, I.A. Eilertsen, L. Cekaite, A. Sveen, R.A. Lothe (Norway)
Mutational characterisation of 400 breast cancers genomes
M. Ramakrishna, H.R. Davies, S. Nik-Zainal, S. Martin, P.J. Campbell, M.R. Stratton (United Kingdom)
A molecular study of breast cancer progression stages from normal breast tissue to invasive cancer
H. Bergholtz, R. Lesurf, S. Myhre, V.D. Haakensen, A.L. Børresen-Dale, T. Bathen, F. Wärnberg,
V.N. Kristensen, A. Helland, T. Sørlie (Norway)
Epigenetic pattern analysis for sub-classification of cancer and immune cell infiltration
R.P. Loewe, M. Hippich, J. Mellad, M. Schneider (Germany)
A possible genetic signature of DNA repair genes in triple negative breast cancers by a NGS approach
L. Spugnesi, M. Gabriele, M. Tancredi, G. Gaetana, L. Maresca, B. Salvadori, I. Bertolini,
M.A. Caligo (Italy)
Potential oncogenic activity of CDK1 gene in laryngeal squamous cell carcinoma
K. Szyfter, K. Bednarek, M. Bodnar, M. Kostrzewska-Poczekaj, R. Grenman, M. Jarmuz-Szymczak,
A. Marszalek, M. Giefing (Poland)
Whole-genome sequencing of Asian lung cancers: Second-hand smoke unlikely to be responsible for
higher incidence of lung cancer among Asian never-smokers
A. Hillmer , V.G. Krishnan, P.J. Ebert, J.C. Ting, E. Lim, S.S. Wong, G.B. Nilsen, T.D. Barber, P. Tan,
P.C. Ng (Singapore)
CLIP2 as radiation biomarker in papillary thyroid carcinoma
M. Selmansberger , A. Feuchtinger, L. Zurnadzhy, I. Höfig, T. Bogdanova, A. Walch, K. Unger,
H. Zitzelsberger, J. Hess (Germany)
Mechanism of tumourigenesis caused by loss of SMARCB1 in malignant rhabdoid tumors
M. Finetti, A. Del Carpio Pons, J. Wood, B. Skalkoyannis, M. Selby, A. Smith, S. Crosier, S. Bailey,
S. Clifford, D. Williamson (United Kingdom)
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460
461
462
463
464
465
466
468
469
470
Signalling Pathways II
Assessing functional ER pathway activity using a computational pathway model
514
P. Van de Wiel, W. Verhaegh, M. Alves de Inda, H. Van Ooijen, E. Den Biezen, A. Van Brussel,
M. Smid, J. Martens, J. Foekens, A. Van de Stolpe (Netherlands)
P53 protein evolutionary functional divergence through the lens of a yeast-based transactivation assay
515
I. Raimondi, M. Lion, S. Donati, O. Jousson, Y. Ciribilli, A. Inga (Italy)
516
Role of LPAR3, PKC and EGFR in LPA-induced cell migration in oral squamous carcinoma cells
I. Brusevold, I.H. Tveteraas, M. Aasrum, J. Odegård, D.L. Sandnes, T. Christoffersen (Norway)
miR-125b confers NF-úB activity and temozolomide resistance by targeting TNFAIP3 and NKIRAS2 in
518
glioblastomas
S. Haemmig, U. Baumgartner, S. Zbinden, A. Glück, M. Tschan, A. Kappeler, I. Vajtai,
E. Vassella (Switzerland)
Fibroblasts isolated from the cancer resistant blind mole rat Spalax exert an extraordinary anticancer
519
activity towards human cancer cells by triggering mitochondrial-dependent apoptosis
I. Manov, V. Domankevich, A. Avivi, I. Shams (Israel)
Investigation of the expression and the role of avb3, avb5 and a5b1 integrins in head and neck cancers
520
H. Ahmedah, L. Patterson, S. Shnyder, H. Sheldrake (United Kingdom)
Role of hypoxia and Hypoxia Inducible Factor 1a (HIF1a) in regulation of the HSPA2 gene expression
521
in human keratinocytes
A. Habryka, A. Gogler-Piglowska, M. Kryj, D. Sojka, Z. Krawczyk, D. Scieglinska (Poland)
Her-receptor ligands and their role in cetuximab sensitivity in gastric cancer
J. Kneissl, A. Hartmann, S. Keller, T. Lorber, H. Hoefler, B. Luber (Germany)
Identification of kallikrein-related peptidase 7 degradome and transcriptome targets in ovarian cancer
L. Munasinghage Silva, T. Stoll, S. Stansfield, C. Stephens, M. Hastie, H. Irving-Rodgers, Y. Dong,
J. Gorman, J.A. Clements (Australia)
A c-Rel-NFATc2-COX2 pathway is critical for TRAIL resistance of pancreatic cancer cells
C. Geismann, F. Grohmann, G. Wirths, A. Dreher, R. Häsler, P. Rosenstiel, G. Schneider, S. Schreiber,
H. Schäfer, A. Arlt (Germany)
Reactive oxygen species and nitric oxide in irradiated and irradiation-induced bystander cells
M. Skonieczna, K. Gajda, K. Biernacki, D. Hudy, A. Krzywon, S. Student, I. Slezak-Prochazka,
M. Widel, J. Rzeszowska-Wolny (Poland)
TRAP1 is responsible for the co-translational regulation of BRAF and the downstream attenuation of
ERK phosphorylation and cell cycle progression: A novel molecular target for human BRAF-mutated
colorectal carcinomas
L. Sisinni, V. Condelli, A. Piscazzi, D.S. Matassa, F. Maddalena, G. Lettini, G. Palladino, M.R. Amoroso,
F. Esposito, M. Landriscina (Italy)
A hormone-dependent feedback-loop controls androgen receptor levels by limiting Midline1, a novel
translation enhancer and promoter of oncogenic signaling
U. Demir, A. Koehler, E. Kickstein, B. Aranda-Orgillés, H. Bu, M.R. Schweiger, G. Schaefer,
S. Schweiger, H. Klocker , R. Schneider (Austria)
Cooperation between Rho-ROCK and STAT 3 signaling modulates colon cancer cell proliferation
R. Peres-Moreira, F. Leve, R. Binato, E. Abdelhay, J.A. Morgado-Dı́az (Brazil)
Identification of novel determinants of cetuximab-resistance in triple negative breast cancer
A. Albukhari, H. Choudhry, S. Haider, F. Buffa, A. Ahmed, A. Kong (United Kingdom)
Induction of survival pathways in human cholangiocarcinoma cells following photodynamic therapy
R. Weijer , M. Broekgaarden, A. Jongejan, P.D. Moerland, A.H.C. Van Kampen, T.M. Van Gulik,
M. Heger (Netherlands)
MYCN induced miR-18a interferes with neuroblastoma cell differentiation by inhibition of estrogen and
NGF signaling
J. Dzieran, U.K. Westermark, M. Arsenian Henriksson (Sweden)
A combined transcriptional and proteomic approach to identification of SOX11 regulated pathways in
mantle cell lymphoma
V. Kuci, S. Ek (Sweden)
Parthenolide, a NF-úB pathway inhibitor, reduces tumorigenic potential in human colorectal cancer cells
A.S. Gehren, W.F. De Souza, J.A. Morgado-Dı́az (Brazil)
The role of the cell adhesion molecule E-cadherin in cetuximab sensitivity of gastric cancer cell lines
S. Keller, B. Mühlthaler, M. Krummhaar, J. Kneißl, H. Höfler, B. Luber (Germany)
Small RNA sequencing identifies microRNAs involved in B-cell receptor signaling in chronic lymphocytic
leukemia
C.J. Blume, T. Hielscher, A. Hotz-Wagenblatt, L. Sellner, J. Hüllein, T. Stolz, C.C. Oakes, O. Merkel,
T. Zenz (Germany)
Immunofluorescent analysis and estimation of clinical significance of ERCC1 expression in ovarian
cancer tissue
A.V. Semakov, T.A. Bogush, E.A. Dudko, A.N. Grishanina, A.S. Tjulandina, S.A. Tjulandin,
V.T. Zarkua (Russian Federation)
Nup155 is linked to the p53 pathway in hepatocellular carcinoma
K. Holzer , A. Ori, J. Winkler, A. Cooke, E. Eiteneuer, M. Beck, P. Schirmacher, S. Singer (Germany)
Wnt secretion is required to maintain Wnt activity in colon cancer
O. Voloshanenko, G. Erdmann, T.D. Dubash, I. Augustin, M. Metzig, G. Kerr, C.R. Ball, H. Glimm,
R. Spang, M. Boutros (Germany)
mTORC2, but not mTORC1, regulates chemotherapy resistance in A549 lung cancer cells
M. Chawsheen, P.R. Dash (United Kingdom)
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527
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531
532
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534
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536
537
538
540
lxxii
Protein complex composition distinguishes different cancer types
A. Ori, S. Singer, M. Beck (Germany)
Quantitative analysis of NFúB transactivation abilities using a yeast-based in vivo functional assay
V. Sharma, A. Bisio, Y. Ciribilli, J. Jordan, M.A. Resnick, A. Inga (Italy)
XIAP E3 ligase activity signals intra-mitochondrial processing of depolarized mitochondria to pre-empt
intrinsic apoptosis
A. Hamacher-Brady, S.C. Choe, N.R. Brady (Germany)
2-Hydroxyethyl methacrylate-dependent cytotoxicity: A gene expression study
C. Di Nisio, S. Zara, M. De Colli, V. Di Valerio, A. Cataldi, V. Di Giacomo (Italy)
Lithium downregulates PTEN by activating Wnt/b-catenin signaling but prevents the tumorigenic
potential of colorectal cancer cells
A.W. Wallace, P.D. Pedro Daniel, B.F. Bruno, J.V. Joao Viola, J.M. Jose Morgado (Brazil)
Quantitative analysis of autophagic flux in pancreatic ductal adenocarcinoma (PDAC) by cellular
heterogeneity measurements
N.R. Brady, A. Hamacher-Brady (Germany)
Different micro-RNA expression profiles distinguish subtypes of neuroendocrine tumours of the lung:
results of a profiling study
F. Mairinger , S. Ting, R. Werner, R.F.H. Walter, C. Vollbrecht, D.C. Christoph, T. Mairinger,
D. Theegarten, K.W. Schmid, J. Wohlschlaeger (Germany)
mRNA biomarker screening in pulmonary tumors showing neuroendocrine differentiation via NanoString
nCounter
F. Mairinger , R.F.H. Walter, R. Werner, C. Vollbrecht, S. Ting, D. Theegarten, D.C. Christoph,
K.W. Schmid, J. Wohlschlaeger (Germany)
A biochemical, immunocytochemical, and bioinformatics analysis of the functional association between
TBC domain-containing proteins and Rab G proteins
S. Faitar , J. Davie (USA)
A functional Cav1-Fas interplay as a new mechanism of chemoresistance in colon cancer
N. Carbó, M. Fontcuberta, C. Rodrı́guez, E. Enreig, G. Fuster, M. Camps, P. Gascón, F.J. Casado,
E. Ametller, V. Almendro (Spain)
CDH3/P-cadherin is negatively regulated by TAp63 in a p53-dependent manner in breast cancer cells:
Effects on P-cadherin-mediated invasion and self-renewal
A. Albergaria, A.R. Nobre, A.S. Ribeiro, A.F. Vieira, R. Seruca, F. Schmitt, J. Paredes (Portugal)
Proteomic profiling of N-Myc-associated proteins in neuroblastoma
M. Halasz, T. Santra, J. Rodriguez, B. Kholodenko, W. Kolch (Ireland)
Identification of molecular targets and signalling networks that influence hypersensitivity to ionizing
radiation
A. Michna, H. Braselmann, A. Gürtler, M. Gomolka, S. Hornhardt, N. Blüthgen, A. Sieber,
H. Zitzelsberger, K. Unger (Germany)
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543
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546
547
548
549
550
552
553
554
Carcinogenesis II
The oncogenic potential of TWIST1 is specifically assigned to the TWIST1-E2A complex
575
L. Jacqueroud, C. Bouard, A. Tissier, G. Richard, L. Payen-Gay, J. Caramel, A. Puisieux,
S. Ansieau (France)
Modulating the Wnt-pathway in colorectal adenoma cells by 1,25-dihydroxyvitamin D3
576
C. Gröschel, A. Aggarwal, M. Wohlgenannt, T. Manhardt, B. Marian, E. Kállay (Austria)
577
Lats1 knockout mouse model recapitulating human dedifferentiated liposarcoma
S. Kim, D. Lim, N. Cho, W. Yang (South Korea)
Critical age windows of radiation exposure for cancer risk in experimental animal models
578
Y. Shimada, M. Nishimura, T. Imaoka, K. Daino, Y. Yamada, K. Ariyoshi, C. Tsuruoka,
S. Kakinuma (Japan)
Subtype specific expression of HRH1 contributes to increased chemoresistance of breast cancer
579
M. Mancino, P. Fernandez-Nogueira, E. Enreig, E. Ametller, P. Gascon, V. Almendro (Spain)
Sensitive detection of NRAS mutations using mutant-enriched PCR and reverse-hybridization teststrips
M. Novy, B. Rauscher, N. Fakhreddin, R. Mahfouz, C. Oberkanins (Austria)
The influence of pre- and postharvest treatments on selected biological and epigenetic activities of
Brassica sprouts
D. Kolodziejski, J. Cyprys, M. Groszewska, A. Piekarska, A. Bartoszek (Poland)
Dietary lipids modify the composition of tumour cell membrane microdomains in experimental mammary
cancer
M. Solanas, M. Pelicano-Esqueta, I. Costa, E. Escrich (Spain)
Recurrent BRCA1/2 mutations in Bulgarian patients with hereditary breast and ovarian cancer
R. Dodova, A. Mitkova, D. Dacheva, M. Taushanova, S. Valev, A. Vlahova, T. Dikov, C. Timcheva,
S. Christova, R. Kaneva (Bulgaria)
Dietary lipids may modulate the xenobiotic metabolism in a chemically-induced breast cancer model
M.A. Manzanares, C. De Miguel, E. Escrich, M. Solanas (Spain)
The synthetic Tryptanthrin analogue suppresses STATs signalling and induces caspase dependent
apoptosis via ERK up regulation in human leukemia HL-60 cells
A. Pathania, S. Kumar, S. Guru, F. Malik, S. Bhushan, A. Kumar (India)
Metallothionein up-regulation in response to cadmium exposure in a normal human urothelial cell system
R. McNeill, J. Southgate (United Kingdom)
Are molecular findings of papillary thyroid microcarcinoma associated with its morphology?
C. Cacchi, E. Maldi, R. Boldorini, C.J. Haas, H. Gabbert, S. Allegrini (Germany)
Modulation of CXCR4/CXL12 axis by hypoxia and pharmacological drug combination in colon cancer
D. Guenot, B.R. Romain, M.H.H. Hachet-Haas, J.L.G. Galzi, E.P. Pencreach (France)
New molecular markers associated with clinical outcome in locally advanced head and neck carcinoma
M.A. Pavon, M. Téllez-Gabriel, I. Arroyo-Solera, X. Leon, M. Lopez, M.V. Céspedes, I. Casanova,
A. Barnadas, R. Mangues, M. Parreño (Spain)
Dickkopf-3 (DKK3), biomarker in head and neck cancer and its implication in tumor progression
S. Cisa, A. Martinez-Limon, T. Dediulia, X. Leon, J. Ubeda, J.M. Nomdedu, A. Villanueva,
M. Parreño (Spain)
S100P, a calcium-binding protein, is associated with polypoid tumour growth in colorectal carcinogenesis
J. Chiang, J. Chen (Taiwan)
Relationship between oxidative DNA damage and p53 mutation in colorectal adenoma and
adenocarcinoma
D.G. Priolli, J.R. Scalise, J.C. Valdivia, N.P. Martinez, D.Y. Kanno, M.G. Marques, I.A. Cardinalli (Brazil)
lxxiii
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592
Translational Research II
Exome sequencing of normal DNA from young non-smokers with lung cancer
654
A.G. Iyevleva, A.P. Sokolenko, E.S. Kuligina, N.V. Mitiushkina, E.V. Preobrazhenskaya, V.I. Tyurin,
E.N. Imyanitov (Russian Federation)
Identification of new synergistic drug combinations on basis of cell panel profiling
655
J.C.M. Uitdehaag, J.A.D.M. De Roos, J.A.P. Spijkers-Hagelstein, A.M. Van Doornmalen, M.B.W. Prinsen,
J. De Man, R.C. Buijsman, G.J.R. Zaman (Netherlands)
656
Effects of chemotherapy of small cell lung cancer on pH-regulating proteins
L. Klameth, M. Redl, T. Thalhammer, G. Hamilton (Austria)
Real time noninvasive 2D and 3D multispectral optoacoustic tomography (MSOT) for clinical imaging
657
of vessel oxygenation and melanin distribution
N. Burton, A. Ulrich, W. Driessen, S. Morscher, T. Sardella, E. Nasanova, D. Razansky,
V. Ntziachristos (Germany)
High-grade serous ovarian cancer subtyping identifies pathways for targeted therapy
658
K. Huhtinen, P. Chen, P. Mikkonen, K. Kaipio, V. Aittomäki, R. Lindell, A. Auranen, R. Lehtonen,
S. Hautaniemi, O. Carpén (Finland)
Somatic mutation profiles in primary colorectal cancers and matching ovarian metastases: Identification
659
of driver and passenger mutations
S. Crobach, D. Ruano, R. van Eijk, M. Schrumpf, G. Fleuren, T. Wezel, H. Morreau (Netherlands)
lxxiv
In vitro model for DNA double-strand break (DSB) repair analysis in cells derived from breast cancer
specimens identifies new prognostic markers
M. Deniz, T. Gundelach, J. Kaufmann, M. Keimling, W. Janni, L. Wiesmüller (Germany)
Wnt/b-catenin signalling inhibition decreases cell proliferation in Wnt autocrine-soft tissue sarcoma cells
A. Obrador-Hevia, S. Calabuig-Fariñas, E. Martı́nez, A. Ochoa, I. Felipe-Abrio, J. Martı́n,
R. Alemany (Spain)
HER2 isoforms in mammary carcinogenesis and targeted therapy susceptibility
A. Palladini, M. Dall’Ora, V. Grosso, M.L. Ianzano, D. Ranieri, T. Balboni, M. Iezzi, C. De Giovanni,
P.L. Lollini, P. Nanni (Italy)
Inter- and intra-tumoral heterogeneity in oncogenic activation of PI3K and MAPK pathways in aggressive
thyroid carcinomas (PDC and ATC) - Cohabitation rate
R. Muñoz Martı́nez, A.M. Costa, J. Cameselle Teijeiro, T. Alvarez Gago, G. Garcia-Rostan (Spain)
Expression of w3-desaturase gene inhibits tumorigenicity and metastasis of prostate cancer cells
K. Lim, S. Shin, K. Jing, S. Jeong, S. Kim, J. Heo, Y. Dai, S. Park, G. Kweon, J. Park (Korea)
Cullin-RING ligases inhibition is a potential new therapeutic strategy in soft tissue sarcomas
A. Martı́n, I. Felipe-Abrio, A. Obrador-Hevia, S. Calabuig-Fariñas, R. Alemany, J. Martı́n (Germany)
A novel zebrafish model of glioma reveals cell fate alteration through expression of oncogenic RAS
M. Spitzner , M. Reischl, M. Mione (Germany)
Shorter multimarker signatures: a new tool to facilitate cancer diagnosis
M. Guergova-Kuras, M.P. Schneider , N. Jullian, M. Afshar (France)
Predictive value of Notch associated genes for response and survival in neoadjuvant treated gastric
cancer
A. Munzig, L. Bauer, J. Slotta-Huspenina, A. Novotny, K. Becker, A. Hapfelmeier, H. Höfler,
G. Keller (Germany)
The transcription factor Phox2a plays a pro-tumorigenic role in pheochromocytoma
I. Leinhäuser , M.J. Atkinson, N.S. Pellegata (Germany)
Pharmacokinetic modelling in dynamic contrast enhanced multispectral optoacoustic tomography
(DCE-MSOT)
S. Morscher, W.H.P. Driessen, N.C.B. Burton, T. Sardella, D. Razansky, V. Ntziachristos (Germany)
Inhibition of angiogenesis promotes a homogeneous intra-tumor distribution of chemotherapy associated
with better antitumor response
M. Cesca, L. Morosi, M. Zucchetti, R. Frapolli, S. Giordano, P. Richter, O. Dirsch, A. Bernd,
R. Giavazzi (Italy)
ALK fusion in a cohort of 469 Finnish patients with non-small cell lung cancer
K. Merkkiniemi, M. Kero, S. Mäki-Nevala, V.K. Sarhadi, M. Tikkanen, T. Wirtanen, M. Rönty, A. Knuuttila,
S. Knuutila (Finland)
uPAR and its interaction partners: potential new therapy targets in triple negative breast cancer
M. Huber , N. Falkenberg, M. Schmitt, H. Braselmann, R. Mall, M. Jakovac, A. Walch, H. Höfler,
M. Aubele (Germany)
Oxidized macrophage migration inhibitory factor (oxMIF) occurs specifically in malignant tissue and is
a potential new drug target and biomarker in cancer
A. Schinagl, M. Thiele, P. Douillard, T. Hagemann, F. Scheiflinger, R. Kerschbaumer (Austria)
Therapeutic and diagnostic targeting of gastrointestinal tumors with Shiga Toxin B subunit
M. Maak, P. Geyer, D. Saur, D. Wilhelm, T. Müller, C. Späth, J. Kleeff, A. Schnieke, L. Johannes,
K. Janssen (Germany)
Pretreatment telomere length of peripheral blood neutrophil as a prognostic factor in metastatic
S.H. Jang, J.H. Kim, Y.I. Hwang, K.S. Jung (South Korea)
Photodynamic therapy effects on tumor vasculature and oxygenation in vivo
M. Krzykawska-Serda, K. Jasinska, J. Dabrowski, G. Stochel, L.G. Arnaut, K. Urbanska,
M. Elas (Poland)
Comprehensive meta-analysis of publicly available microarray data in head and neck cancer
L. De Cecco, P. Bossi, M. Nicolau, L. Locati, L. Licitra, M.G. Daidone, S. Canevari (Italy)
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662
663
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665
666
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669
670
671
672
673
674
675
676
678
679
miR-187 targets the androgen-regulated gene ALDH1A3 in prostate cancer
I. Casanova-Salas, E. Masiá, A. Armiñán, A. Calatrava, C. Mancarella, J. Rubio-Briones, K. Scotlandi,
M.J. Vicent, J.A. López-Guerrero (Spain)
Mutations and expression of genes in PI3K-AKT pathway in radiation-induced mammary carcinoma of
(Sprague-Dawley × Copenhagen) F1 hybrid rats
M. Nishimura, K. Showler, K. Daino, T. Takabakake, M. Takabatake, T. Imaoka, S. Kakinuma,
Y. Shimada (Japan)
Synergistic combination of valproic acid and oncolytic parvovirus H-1PV as a potential therapy against
cervical and pancreatic carcinomas
J. Li, S. Bonifati, G. Hristov, T. Marttila, J. Rommelaere, A. Marchini (Germany)
Ex vivo modelling of Kras-driven murine non-small cell lung cancer
K. Närhi, E. Parri, A. Nagaraj, P. Kovanen, R. Turkki, A. Schoonenberg, O. Brenner, M. Kaustio,
S. Blom, E. Verschuren (Finland)
Helsinki Urological Biobank (HUB): A new-generation integrated biobank for facilitating precision
medicine and translational research in urological cancers
T. Af Hällström, T. Mirtti, K. Saeed, V. Rahkama, M. Puhka, P. Ostling, T. Vesterinen, K. Pitkänen,
O. Kallioniemi, A. Rannikko (Finland)
Linking tumor evolution and therapy response using diagnostic targeted next generation sequencing in
colorectal cancer
S. Mamlouk, L. Childs, T. Redmer, H. Bläker, D. Aust, R. Schäfer, C. Sers (Germany)
microRNAs in hepatocellular carcinoma
A. Balakrishnan, A. Goga, M.P. Manns, M. Ott (Germany)
Tumor-infiltrating lymphocytes in human gastric cancer and disseminated tumor cells
S. Osinsky, A. Kovelskaya, L. Bubnovskaya, D. Osinsky, I. Ganusevich, L. Gumenyuk, S. Merentsev,
L. Mamontova (Ukraine)
CXCL12/CXCR4 ratio in colorectal cancer patients
L. Stanisavljevic, J. Aßmus, O. Dahl, M.P. Myklebust (Norway)
Bellini duct carcinomas have a different genetic signature compared to urothelial carcinomas of the
upper urinary tract
V. Jung, F. Becker, A. Hartmann, R. Grobholz, B. Wullich, S. Füssel, A. Strauss, W. Otto, M. Stöckle,
K. Junker (Germany)
Precision cut cancer tissue slices as a preclinical drug testing platform
F.T. Unger , J. Krueger, J. Schaller, P. Uhlig, H. Juhl, K.A. David (Germany)
SERPINE1 immunostaining is associated with clinical outcome in head and neck squamous cell
carcinoma
M. Téllez-Gabriel, A. Gallardo, I. Arroyo-Solera, L.C. Navas, X. León, M. Quer, A. Barnadas,
R. Mangues, M. Pavon (Spain)
Effect of specific PI3K/mTOR inhibitors in squamous lung cancer cells carrying PI3K alterations
M. Bonelli, A. Cavazzoni, S. La Monica, M. Galetti, C. Caffarra, D. Cretella, F. Saccani, C. Fumarola,
R. Alfieri, P.G. Petronini (Italy)
Intrinsic resistance to sunitinib in clear cell renal cell carcinoma: A gene expression analysis
O. Reig, M. Marı́n-Aguilera, J.J. Lozano, B. Gonzalez, C. Mallofré, M. Campayo, P. Gascon,
B. Mellado (Spain)
MicroRNAs for detection of pancreatic neoplasia: biomarker discovery by next generation sequencing
and validation in EUS FNA and plasma samples
M. Gironella, E. Vila, M. Vila, S. Durán, L. Moreira, A. Ginés, R. Miquel, J.J. Lozano, A. Castells (Spain)
Glycoprofiling of serous ovarian tumours is a promising strategy for developing new diagnostic tools
S. Ricardo, L. Silva, D. Pereira, N. Lunet, L. David (Portugal)
Significance of NF-kB2 genetic variations rs7897947, rs11574852 and rs12769316 in non-small-cell
lung cancer: a case−control study
F. Dimitrakopoulos, A.G. Antonacopoulou, A.E. Kottorou, S. Maroussi, I. Koukourikou, C. Scopa,
D. Dougenis, H. Papadaki, H. Kalofonos (Greece)
lxxv
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681
682
683
684
685
686
687
688
689
690
691
692
693
695
696
697
lxxvi
Gene expression of SOX2, SOX6, SOX8 and SOX9 and their prognostic role in patients with gliomas
G. Stancheva, T. Goranova, M. Laleva, M. Kamenova, A. Mitkova, N. Velinov, G. Poptodorov, R. Kaneva,
V. Mitev, N. Gabrovsky (Bulgaria)
MicroRNA profiling in serum samples from donors with germ cell cancer
K. Lee, S. Patel, K. Hayashibara, S. Chu, A.D.J. Gillis, M. Rijlaarsdam, L.C.J. Dorssers,
L.H.J. Looijenga (Medical and Applied Sciences)
Confocal fluorescence endomicroscopy in vivo for the detection of tumour microenvironment and
healthy tissue margin
S.P. Johnson, C. Schneider, A. Desjardins, S. Walker-Samuel, D. Hawkes (United Kingdom)
The alternative pathway of NF-kB is deregulated in NSCLC?
F.I. Dimitrakopoulos, A.E. Kottorou, A.G. Antonacopoulou, C. Scopa, D. Dougenis, H. Papadaki,
H. Kalofonos (Greece)
Pathway analysis of ovarian cancer
S.L. O’Kane, M.A. Murphy, F. O’Hannigan, S. O’Toole, J.J. O’Leary, D.J. Cahill (Ireland)
Whole transcriptome analysis of testicular germ cell tumors
L. Degoricija, K.Y. Lee, S. Patel, S. Chu, A.J.M. Gillis, M.A. Rijlaarsdam, L.C.J. Dorssers,
L.H.J. Looijenga (USA)
Phenotype based discovery of novel small molecule anti-angiogenic drugs
C. Butler (Ireland)
Shifts in mitochondrial energy metabolism are correlated with disease progression in Barrett’s
oesophagus
J. Phelan, F. McCarthy, R. Feighery, N.J. O’Farrell, N. Lynam-Lennon, B. Doyle, D. O’Toole, B. Kennedy,
J.V. Reynolds, J.N. O’Sullivan (Ireland)
The relevance of Epstein–Barr virus (EBV) DNA load and the oncogenic protein BARF1, as response
and therapeutic monitoring predictive factors in undifferentiated nasopharyngeal carcinoma (UNPC) −
The VEBINASO study
D. Costa, M. Cunha, S. Esteves, C. Ornelas, M. Silveira, M. Magalhaes, V. Leite, M. Ferreira,
I. Sargento, A. Moreira (Portugal)
Multi-point targeting of the synthetic lethal interactions between Myc, ribosome biogenesis and ribosome
function cooperates to treat B-cell lymphoma
R. Pearson, J.R. Devlin, K.M. Hannan, N. Hein, M.J. Bywater, D. Drygin, S. O’Brien, C. Cullinane,
G. McArthur, R.D. Hannan (Australia)
A Phase Ib study of BKM120 combined with abiraterone acetate for castrate-resistant, metastatic
prostate cancer
A. Patnaik, J. Kung, J. Wu, M. Loda, P. Kantoff, L.C. Cantley, S. Balk, G. Bubley (USA)
Clinical implication of UGT1A1 promoter polymorphism for irinotecan dose escalation in metastatic
colorectal cancer patients treated with bevacizumab combined with FOLFIRI in the first-line setting
J. Wang, C.Y. Lu (Taiwan)
Live imaging of cellular signaling in patient-derived tumor organoids
C. Verissimo, B. Ponsioen, M. Van de Wetering, R.M. Overmeer, I. Verlaan, H. Clevers, J.L. Bos,
H.J. Snippert (Netherlands)
The pig as a model for human cancer
A. Saalfrank, T. Flisikowska, K. Flisikowski, S. Eser, E. Wolf, D. Saur, A. Schnieke (Germany)
BCL-2 dependence in T-cell leukemia is defined by the maturation stage of the clone of origin
T. Ni Chonghaile, J. Roderick, G. Gutierrez, M. Kelliher, A. Letai (Ireland)
698
699
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702
703
704
705
706
707
708
710
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715
Experimental/Molecular Therapeutics, Pharmacogenesis II
Investigation of the mode of action of sunitinib kinase inhibitor profile analogues in insulin release
791
Z. Orfi, M. Falcenberg, D. Eros, L. Orfi, G. Keri, A. Ullrich (Germany)
792
Polysaccharide-based nanocarriers targeting CD44 for lung cancer treatment
V. Jeannot, S. Mazzaferro, J. Lavaud, V. Josserand, M. Henry, C. Schatz, S. Lecommandoux, A. Hurbin,
J.L. Coll (France)
Effective inhibition of glioblastoma growth with dacomitinib: an irreversible EGFR inhibitor
C. Zahonero, P. Aguilera, A. Pérez, A. Hernández-Laı́n, M.V. Bolós, J.M. Sepúlveda,
P. Sánchez Gómez (Spain)
Crizotinib is an allosteric ABL-inhibitor targeting both native BCR/ABL and BCR/ABL-T315I in vitro and
in vivo models of PH+ leukemia
J. Mahajna, H. Khamaisie, N. Ruimi, A. Agbarya, E. Eshel, N. Dally, O. Ottmann, R. Biondi, A.A. Mian,
M. Ruthardt (Israel)
Plausible extraction technique for cancer chemopreventive isothiocyanate compounds from the
Raphanus sativus L. var. caudatus Alef
S. Sangthong, N. Weerapreeyakul, S. Barusrux (Thailand)
The G-quadruplex ligand EMICORON has antitumoral activity against orthotopic and patient-derived
human colon cancer xenografts
M. Porru, S. Artuso, A. Bianco, M. Franceschin, M.G. Diodoro, D. Passeri, A. Orlandi, A. Biroccio,
C. Leonetti (Italy)
Enhancement of the cytotoxic effect of doxorubicin through gap junctional intercellular communication
in esophageal cancer cells
T. Tanaka, S. Matono, N. Mori, H. Hino, K. Okada, K. Shirouzu (Japan)
Reovirus infection dysregulates the Ras signaling pathway in canine mast cell tumour cells
C.C. Hwang, M. Coffey, S. Noguchi, M. Okuda, T. Mizuno (Japan)
Role of cyclosporine A as chemomodulator of cisplatin cytotoxicity in ovarian cancer cell lines
G. Hamilton, L. Klameth, B. Rath, E. Obermayr, R. Zeillinger, T. Thalhammer (Austria)
The effect of 50% hydroethanolic extract of Pinus kesiya on apoptosis in hepatocellular
carcinoma (HepG2) cells
A. Nonpanya, N. Weerapreeyakul, S. Barusrux (Thailand)
Activation of HER3 interferes with antitumor effects of Axl receptor tyrosine kinase inhibitors −
suggestion of combination therapy
R. Torka, K. Pénzes, C. Baumann, S. Gusenbauer, I. Szabadkai, L. Orfi, G. Kéri, A. Ullrich (Germany)
Drug delivery systems (DDS) for anticancer drugs originated from medicinal plant
X. Siwe Noundou, R.W.M. Krause, S. Moeno (South Africa)
Polymeric nanogel delivery system with pH/thermo-responsive doxorubicin release for intracellular drug
delivery
H.C. Chiu, W.H. Chiang, W.C. Huang, Y.J. Chang (Taiwan)
Meripilus giganteus in prevention of cancer: Phenolic profile, biological potential and antitumor effect
via upregulation of p53 and SOX1 expression in HeLa cells
I. Petrovic, N. Kovacevic Grujicic, D. Stojkovic, S. Davidovic, J. Glamoclija, A. Ciric, M. Sokovic,
M. Stevanovic (Serbia)
Cancer stem cell-like cells mediate adaptive resistance to targeted cancer therapies
E. Jokinen, N. Laurila, P. Koivunen, J.P. Koivunen (Finland)
Concentration dependent effect of sesamol on cell cycle arrest
N. Weerapreeyakul, M. Srisayam, S. Barusrux (Thailand)
Combined treatment of pancreatic tumour cells with bioactive food components reduces tumour growth
and potentiates gemcitabine toxicity in a preclinical mouse model
S. Dabernat, E. Peuchant, I. Moranvillier, B. Rousseau, A. Bedel, F. Beliveau, H. De Verneuil,
F. Moreau-Gaudry (France)
The PI3K/mTOR inhibitor NVP-BEZ235 is able to revert selumetinib resistance in colorectal cancer
cellular models
I. Martinez-Lacaci, M. Carballo-Santana, E. Tristante, L. Mayor-López, S. Grasso, P. Carbonell,
C. De Torre, J. Luján, J.L. Alonso, F. Carballo (Spain)
Salivary exosomes of oral cancer patients may serve as a diagnostic tool since they differ from those
found in the saliva of healthy individuals
A. Zlotogorski-Hurvitz, D. Dayan, G. Chaushu, T. Salo, M. Vered (Israel)
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802
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Photodynamic therapy in non-small cell lung cancer cell line
M. Laranjo, K. Santos, R. Teixo, M. Piñeiro, A.C. Gonçalves, A.M. Abrantes, A.B. Sarmento-Ribeiro,
A.M. Rocha-Gonsalves, A.C. Serra, M. Botelho (Portugal)
Fully human antibodies specific for oxidized macrophage migration inhibitory factor (oxMIF) exhibit
anti-cancer activity in animal models
M. Thiele, T. Hagemann, M. Freissmuth, P. Douillard, D. Völkel, F. Scheiflinger,
R.J. Kerschbaumer (Austria)
Systematic investigation of drug resistance factors in colorectal cancer cells using pooled CRISPR/Cas9
knockout screens
T. Zhan, M. Breinig, F. Heigwer, S. Leible, M.P. Ebert, M. Boutros (Germany)
Development of self-assembling lecithin-based polymeric mixed micellar systems for quercetin in cancer
treatment
Y.C. Chen, T.W. Chung, M.T. Sheu (Taiwan)
Preparation of micellar oral dosage form of amphotericin B for synergistic enhancement of anti-cancer
effects with the pretreatment of ethanolic extract of Taiwanofungus camphoratus
L.C. Chen, M.T. Sheu, T.W. Chung (Taiwan)
Inhibitory effect of xanthohumol on STAT3 activation and cholangiocarcinoma development
H. Dokduang, W. Loilome, N. Namwat, C. Pairojkul, M. Sakurai-Yageta, Y. Murakami,
P. Yongvanit (Thailand)
Investigations into the anti-cancer mechanism of action of garlic related disulfides
C.H. Kaschula, M. Smith, R. Hunter, M.I. Parker (South Africa)
Human antibodies specific for oxidized macrophage migration inhibitory factor (oxMIF) synergize with
chemotherapeutic agents in animal models of cancer
D. Voelkel, T. Hagemann, M. Freissmuth, M. Thiele, A. Schinagl, P. Douillard, F. Scheiflinger,
R.J. Kerschbaumer (Austria)
Anti-migratory and cytotoxic properties of medicinal plants against melanoma
A. Alqathama, J.M. Prieto (United Kingdom)
Chemo-resistant gastric cancer: changes in Notch signalling
L. Bauer , A. Munzig, E. Müller, J. Slotta-Huspenina, K. Becker, A. Hapfelmeier, A. Novotny, H. Höfler,
G. Keller (Germany)
The histone acetyltransferases inhibitor CPTH6 preferentially inhibits proliferation of patient-derived
lung cancer stem cells in vitro and in vivo
M. Di Martile, M. Desideri, C. Gabellini, A. Eramo, S. Carradori, D. Secci, M. Milella, D. Del Bufalo,
D. Trisciuoglio (Italy)
3-deazaneplanocin A (DZNep), an inhibitor of histone methyltransferase EZH2, induces apoptosis and
reduces cell migration in chondrosarcoma cells
N. Girard, C. Bazille, E. Lhuissier, H. Benateau, A. Llombart-bosch, K. Boumediene, C. Bauge (France)
Re-engineering vesicular stomatitis virus to abrogate neurotoxicity, circumvent humoral immunity and
enhance oncolytic potency
D. Von Laer , A. Muik, L.J. Stubbert, R.Z. Jahedi, Y. Geiß, C. Dold, R. Tober, U. Dietrich, H. Miletic,
J.C. Bell (Austria)
Improved cytotoxic therapy by dual targeting of a5b1 integrin and p53 pathway in glioblastoma
M. Dontenwill, H. Janouskova, G. Renner, A.M. Ray, F. Noulet, L. Choulier, N. Etienne-Selloum,
M. Lehmann, I. Lelong-Rebel, S. Martin (France)
Primary T-prolymphocytic leukemia (T-PLL) cells are sensitive to BCL-2 and HDAC inhibitors: Results
from high-throughput ex vivo drug testing
E. Andersson, T. Pemovska, A. Lauhio, P. Pietarinen, C. Mateos, E. Faber, T. Brümmendorf,
O. Kallioniemi, K. Wennerberg, S. Mustjoki (Finland)
Ex vivo combinatorial screen to identify drug sensitivity and rational treatment of chronic lymphocytic
leukemia
M. Lukas, L. Sellner, M. Oles, J. Huellein, S. Anders, T. Stolz, C. Muley, C. Blume, W. Huber,
T. Zenz (Germany)
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819
820
822
823
824
825
826
827
The anti-tumor activity of simvastatin encapsulated in long circulating liposomes is dependent on the
intratumoral macrophages
E. Licarete, M.C. Alupei, L.I. Patras, M. Banciu (Romania)
Quality of life, efficacy and tolerability of long term treatment with BP-C1, in patients with stage IV
breast cancer
S. Larsen, S.L.J. Lindkær-Jensen (Norway)
Phytochemical extract from Senecio graveolens (Chachacoma): Searching new candidates for
anti-cancer drugs
C. Echiburú-Chau, C. Salas, M. Cuellar, J. Santander, J.P. Ogalde, N. Brown, S. Alfaro-Lira, N. López,
F. Rothhammer (Chile)
Induction of apoptosis by pyrrolo-1,5-benzoxazepines in oral squamous carcinoma cells
K. O’Callaghan, E. Palagano, S. Butini, G. Campiani, D.C. Williams, D.M. Zisterer, J. O’Sullivan (Ireland)
MicroRNA-139-5p: A tool for metastasis and chemotherapeutic resistance control in pancreatic
adenocarcinoma
M. Passadouro, C. Nóbrega, L. Almeida, M. Pedroso de Lima, H. Faneca (Portugal)
The effect of fibroblast growth factor receptor inhibition on the growth of breast cancer cells
T.E. Kähkönen, M. Toriseva, K.K. Ivaska, P. Härkönen (Finland)
Chemotherapeutic-loaded biodegradable nanoparticles in the treatment of chronic lymphocytic leukemia
S. Capolla, N. Mezzaroba, L. Nunez, S. Biffi, G. Baj, S. Zorzet, M. Granzotto, G. Pozzatto,
P. Macor (Italy)
Inhibiting MTH1 kills cancer cells by preventing sanitisation of oxidised dNTP pool
U. Warpman Berglund, H. Gad, A.S. Jemth, T. Koolmeister, C. Gokturk, T. Pham, J. Nilsson, S. Eshtad,
M. Scobie, T. Helleday (Sweden)
Hyperthermia potentiates cisplatin and doxorubicin effects by inhibition of PARP-dependent DNA repair
L. Schaaf , H. Van der Kuip, T.E. Mürdter, W.E. Aulitzky, C. Ulmer (Germany)
The combination of retinoic acid and protein kinase C modulators as a novel therapy for the growth
inhibition of cancer stem cells in a triple negative murine mammary cancer model
D. Berardi, C. Flumian, M.I. Dı́az Bessone, S. Cirigliano, E. Bal de Kier Joffé, A. Urtreger,
L. Todaro (Argentina)
Panobinostat reduces hypoxia-related cisplatin resistance of non-small cell lung carcinoma cells via
HIF-1alpha destabilization
A. Hrzenjak, C. Fischer, K. Leithner , C. Wohlkoenig, F. Quehenberger, A. Olschewski,
H. Olschewski (Austria)
Development of a novel anti-angiogenic therapy based on multi-target destabilization of short-lived
mRNAs
F. Rataj, S. Planel, J.J. Feige, N. Cherradi (France)
Development of substrate-based radiotracers for imaging of tumour-associated lysyl oxidase activity by
positron emission tomography
M. Kuchar, R. Bergmann, R. Wodtke, B. Mosch, J. Lenk, J. Steinbach, J. Pietzsch, R. Löser (Germany)
Colorectal cancer cells with acquired resistance to anti-EGFR mAb ICR62 remain highly sensitive to
treatment with small molecule tyrosine kinase inhibitors
S. Khelwatty, S. Essapen, A. Seddon, Z. Fan, H. Modjtahedi (United Kingdom)
Systematic phenotypic profiling of chemical-genetic interactions in human cancer cells
M. Breinig, F. Klein, W. Huber, M. Boutros (Germany)
Increasing the efficacy of photodynamic therapy of solid cancers by inhibition of hypoxic signaling with
acriflavine
M. Broekgaarden, R. Weijer, M. Krekorian, B. Van den IJssel, R. Van Vught, M. Kos, L.K. Alles,
T.M. Van Gulik, M. Heger (Netherlands)
A novel strategy for the treatment of Hodgkin lymphoma
D. Stolfa, L. Porcelli, A.E. Quatrale, A. Stefanachi, R.M. Iacobazzi, L. Sidella, S. Cellamare,
A. Azzariti (Italy)
lxxix
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
lxxx
The ROS/SUMO axis is involved in acute myeloid leukemia (AML) cells response to chemotherapeutic
drugs and constitutes a potential target to overcome chemoresistance in AML
M. Piechaczyk, J.E. Sarry, C. Kifagi, M. Ristic, E. Saland, T. Salem, H. Baik, C. Récher, S. Manenti,
G. Bossis (France)
Antitumor activity of the synthetic retinoid ST1926 in colorectal cancer cells: involvement of DNA damage
N. Darwiche, R. Abdel-Samad, R. Hmadi, M. El-Sabban, H. Muhtasib, C. Pisano (Lebanon)
Hsp90 inhibitor–drug conjugates (HDCs): concept and preclinical activity
W. Ying, D. Chimmanamada, T. Przewloka, J. Zhang, J. Jiang, D.A. Proia, J. Sang, J. Chu, N. Tatsuta,
T. Inoue (USA)
The characterization of ascorbic acid loaded solid lipid nanoparticles
G. Dikmen, G. Guney (Turkey)
PI3K/mTOR dual inhibition modulates Bcl-2 family proteins expression and sensitizes ovarian carcinoma
cells to ABT-737
A. Jebahi, M. Villedieu, C. Pétigny, E. Brotin, M.H. Louis, E. Abeilard, F. Giffard, P. Gauduchon,
S. Lheureux, L. Poulain (France)
The antitumor agent obatoclax displays antimetastatic properties on melanoma cells in vitro and in vivo
V. Soto-Cerrato, M. Espona-Fiedler, P. Manuel-Manresa, C. Garcı́a-Martı́nez, A.M. Rodilla, R. Quesada,
R. Pérez-Tomás (Spain)
Pre-clinical efficacy of the synthetic retinoid ST1926 for treating chronic myeloid leukemia
N. Darwiche, R. Hmadi, R. El-Eit, A. Iskandarani, C. Pisano, R. Nasr (Lebanon)
Branched cell-penetrating peptide drug conjugates for overcoming drug resistance
S. Kaloyanova, M. Lelle, K. Müllen, K. Peneva (Germany)
Development of a novel tumor homing compound targeting glioblastoma cells
E. Giannopoulou, K. Siatis, A. Lampropoulou, C. Papadopoulos, S. Christoforidis, S. Papas,
E. Briasoulis, A. Tzakos, V. Tsikaris, H.P. Kalofonos (Greece)
Interplay between mTOR pathway and Unfolded Protein Response in stress response of neuroendocrine
tumors: a possible key to enhance tumors response to targeted therapy?
P. Freis, J. Lebeau, J. Bollard, C. Vercherat, P. Massoma, T. Walter, S. Manie, C. Roche, J.Y. Scoazec,
C. Ferraro-Peyret (France)
Perspectives of hormonal therapy in non-small cell lung cancer (NSCLC) based on estrogen
receptor beta expression in tumor tissue
T.A. Bogush, E.A. Dudko, A.S. Shaturova, R.J. Ramanauskaite, B.E. Polotsky, S.A. Tjulandin,
M.I. Davydov (Russian Federation)
Molecular mechanisms of sorafenib-induced apoptosis in cancer cells
C. Kuznia, B. Klinger, J. Keil, B. Seliger, C. Falk, N. Rohwer, T. Cramer, R. Schäfer, N. Blüthgen,
C. Sers (Germany)
Aggravation of ER stress by combination of proteasome inhibitors and HIV protease inhibitors results
in preferencial killing of triple-negative breast cancer cells in vitro
J. Gibbons Marsico, T. Heger, M. Kraus, J. Bader, M. Jörger, C.H. Driessen (Switzerland)
Simvastatin modifies the expression of stemness and epithelial–mesenchymal transition (EMT) markers
in ovarian cancer-initiating cells resulting in decreased cell migration
S. Kato, L. Abarzua, K. Hormazabal, A. Delpiano, M.L. Bravo, R. Orellana, J. Branes, E. Castellon,
M. Cuello-Fredes, G.I. Owen (Chile)
Survey of activated kinase proteins reveals PI3K/Akt and Wnt/b-catenin signaling pathways as potential
targets for cholangiocarcinoma treatment
W. Loilome, H. Dokduang, S. Yothaisong, P. Bungkanjana, S. Juntana, A. Techasen, N. Namwat,
P. Yongvanit, N. Khuntikeo, A. Puapairoj (Thailand)
Effects of novel anticancer tambjamine analogs on lung cancer cell lines and human lung primary cultures
P. Manuel-Manresa, V. Soto-Cerrato, A.M. Rodilla, R. Quesada, R. Pérez-Tomás (Spain)
Targeting the HSF1/HSP70/BAG3 pathway for reactivation of apoptosis in glioma
D. Koegel, P. Antonietti, S. Rakel, V. Seifert, F. Gessler (Germany)
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
Inhibition of carbonic anhydrases 9/12 decreases proliferation leading to cell death in vitro and in vivo
and enhances chemosensitivity in glioblastoma cells
S. Teixeira, J.A. Pezuk, A.F. Andrade, V.K. Suazo, L.G. Tone, R.P.Q. Gomes, C.G. Carlotti,
C.A. Scrideli (Brazil)
Chemosensitization and radiosensitization effects of glioblastoma cells by the histone deacetylase
inhibitor, givinostat (ITF2357) in glioblastoma cell models
G.L. Gravina, F. Leoni, E. Benedetti, S. Delle Monache, A. Mancini, A. Angelucci, E. Di Cesare,
A.M. Cimini, G. Porro, C. Festuccia (Italy)
CXCR4 inhibition reduces bone metastases by affecting tumour growth and tumorigenic potential in
prostate cancer preclinical models
G.L. Gravina, A. Mancini, L. Ventura, L. Scarsella, A. Jitariuc, A. Colapietro, E. Ricevuto, E. Di Cesare,
C. Festuccia (Italy)
Therapeutic response of modified rutin (Q3G) in animal model of human adenocarcinoma colon
D. Castilho, N.P. Martinez, R. Colenci, J.C. Valdivia, D.F. Gimenez, V. Bonadio, A.C. Duarte,
G.C. De Miguel, D.G. Priolli (Brazil)
Screening of Lactobacillus reuteri strains for their short chain fatty acids production, stability and
potential in colorectal cancer: In-vitro analysis
I. Kahouli, M. Meenakshi, C. Tomaro-Duchesneau, S. Saha, D. Marinescu, L. Sonia Rodes,
M.M. Aloui-Jamali, S. Prakash (Canada)
lxxxi
862
863
864
865
866
Tumour Immunology II
Do serum components play a role in the elimination of mitochondria from necrotic cells by phagocytes?
884
A. Hessenberger, T. Arnold, A. Sousek, K. Nowikovsky, R. Oehler (Austria)
Cell-type specific functions for the immune adapter protein MyD88 in colorectal carcinogenesis
885
A. Holtorf , A. Conrad, B. Holzmann, K.P. Janssen (Germany)
886
Prognostic relevance of tertiary lymphoid organs in oral squamous cell carcinoma
A. Wirsing, O. Rikardsen, S.E. Steigen, L. Uhlin-Hansen, E. Hadler-Olsen (Norway)
Expression and turnover of proteins govern their sampling for HLA class I presentation
887
M. Bassani-Sternberg, S. Pletscher-Frankild, L. Juhl Jensen, M. Mann (Germany)
Activation and homing profile of dendritic cells in human colorectal cancer − effect of tumour derived
888
factors or leaky lymph nodes?
G. Lee, G. Malietzis, D. Bernardo, S.K. Clark, H.O. Al-Hassi, S.C. Knight (United Kingdom)
889
Circulating dendritic cells are gut homing in colorectal cancers − A role in diagnosis and prognosis in
colorectal cancer?
G. Lee, G. Malietzis, D. Bernardo, S.K. Clark, S.C. Knight, H.O. Al-Hassi (United Kingdom)
Plasma levels of cell death biomarker HMGB1 during the first cycle of epirubicin/docetaxel or
890
5-FU/oxaliplatin
M. Sachet, T. Arnold, S. Baumann, R. Bartsch, M. Gnant, T. Bachleitner-Hofmann, M. Bergmann,
R. Oehler (Austria)
Cytotoxic T lymphocytes functionally active against tumor microenvironment-derived specific antigen
891
M. Bojin, O.I. Gavriliuc, C.S. Tatu, G. Tanasie, C. Panaitescu, C.A. Tatu, V. Paunescu (Romania)
892
Peripheral T cell responses in glioblastoma patients are associated with an improved survival
C. Herold-Mende, J. Mossemann, C. Jungk, R. Ahmadi, D. Capper, A. Von Deimling, A. Unterberg,
P. Beckhove (Germany)
In situ tumor ablation by intratumoral pulsed electric currents and activation of systemic anti-tumor
893
immunity
Y. Keisari, I. Hochman, H. Confino, M. Efrati, V. Umansky, R. Korenstein (Israel)
894
Muscle mass of the host and dendritic cell phenotype in patients with colorectal cancer
G. Malietzis, G.H. Lee, D. Bernardo, N. Johns, K.C.H. Fearon, A. Blakemore, J.T. Jenkins, H. Al-Hassi,
S. Knight (United Kingdom)
lxxxii
Dasatinib-induced reduction of tumor growth is accompanied by the changes in the immune profile in
melanoma B16.OVA mouse model
M. Ilander , C. Hekim, M. Vähä-Koskela, P. Savola, S. Tähtinen, A. Hemminki, K. Porkka,
S. Mustjoki (Finland)
Differentially expressed functions and genes between serrated adenocarcinoma and sporadic colorectal
carcinoma showing histological and molecular features of high level of microsatellite instability
M. Turpı́n Sevilla, R. Carbonell-Muñoz, J. Garcia-Solano, D. Torres-Moreno, F. Garcı́a-Garcı́a,
A. Conesa, M. Perez-Guillermo, P. Conesa-Zamora (Spain)
Co-expression of chimeric antigen receptor (CAR) and miRNAs to T cell therapy
M. Carneiro, L. Chicaybam, M.H. Bonamino (Brazil)
Impact of inflammation in a spontaneous model of melanoma in zebrafish
E. Gómez-Abenza, C. Gabellini, D. Garcı́a-Moreno, M. Mione, V. Mulero (Spain)
Identifying genes involved in the colon cancer initiating cells (CICs) survivability against montelukast
treatment in xenograft model
K. Bellamkonda, S. Savari, N. Chandrashekar, A. Sjolander (Sweden)
Changes in NK cell effector function, subset distribution and receptor repertoire following in vitro NK cell
and K562 tumor cell contact in melanoma patients
G. Konjevic, A. Vuletic, K. Mirjacic Martinovic (Serbia)
895
896
897
898
899
900
Radiobiology/Radiation Oncology II
PI3K/Akt and ERK1/2 signaling pathways are involved with the aggressive potential of the progeny
917
derived from radioresistant colorectal cancer cells
P.G. Marcondes, L.G. Bastos, J.A. Morgado-Dı́az (Brazil)
918
Is single amino acid arginine deprivation a strategy to treat non-auxotrophic glioblastoma?
M. Ingargiola, L. Radon, N. Hinrichs, A. Köhn Luque, R. Wiedemuth, A. Temme, A. Deutsch,
L. Kunz-Schughart (Germany)
Arginine deprivation and radiation as combination therapy: A systematic study in HNSCC 2-D vs. 3-D
919
culture models
F. Manig, L. Lehmann, M. Huether, M. Baumann, O. Stasyk, L.A. Kunz-Schughart (Germany)
DNA damage and oxidative stress after low doses of X and proton beam irradiation
920
K. Jasinska, A. Cierniak, A. Borkowska, J. Jura, P. Olko, B. Romanowska-Dixon, M. Elas,
K. Urbanska (Poland)
Importance of Mcl-1 and the Mcl-1-modulating enzyme USP9x for radiosensitivity in prostate cancer cells
921
J. Rudner , S.A. Hogh-Binder, F. Wolfsperger, S. Huber, V. Jendrossek (Germany)
Relevance of 3D-microtissues to investigate the therapeutic oncogenes HER2 and PTK6 in breast cancer
922
I. Hoefig, N. Falkenberg, M. Rosemann, J. Szumielewski, S. Richter, M.J. Atkinson, M. Aubele,
N. Anastasov (Germany)
Cellular motility properties after X and proton beam irradiation
923
K. Jasinska, A. Borkowska, P. Koczurkiewicz, M. Michalik, Z. Madeja, P. Olko, B. Romanowska-Dixon,
M. Elas, K. Urbanska (Poland)
Comparative study of chondrosarcomas response to DNA damage: Impact of HIF2 expression
924
E. Lhuissier, N. Girard, O. Cauvard, C. Bazille, H. Benateau, A. Batalla, A. Llombart-bosch, C. Bauge,
K. Boumediene (France)
Multiparametric MRI evaluation of radiotherapy effects on the vascular compartment and white matter
925
for glioblastoma treatment
A. Gerault, A. Corroyer-Dulmont, A. Chakhoyan, D. Divoux, J. Toutain, M. Bernaudin, S. Valable,
E. Petit (France)
Application of texture analysis to SPECT images of 125I-A5B7 anti-CEA antibody localisation in
926
metastatic colorectal cancer models: Correlation with histological microarchitecture and response to
antivascular therapy
V. Rajkumar , V. Goh, M. Siddique, M. Robson, G. Boxer, B. Pedley, G. Cook (United Kingdom)
Immunohistochemical analysis reveals frequent tumoural loss of ATM protein expression in lung cancer
927
A.M. Weber , A.M. Devery, S.M. Bokobza, A.J. Ryan (United Kingdom)
Met signaling sustains glioblastoma stem cells radioresistance
F. De Bacco, E. Casanova, A. D’Ambrosio, F. Orzan, S. Pellegatta, R. Neggia, R. Albano,
G. Finocchiaro, P.M. Comoglio, C. Boccaccio (Italy)
Early NFKB signalling pathway activation in Ramos B cells in response to ionizing radiation
C. Di Nisio, S. Sancilio, V. Di Giacomo, M. Rapino, L. Sancillo, R.A. Rana, R. Di Pietro (Italy)
Effect of radiotherapy on macrophages
A.T. Pinto, H. Osório, M.L. Pinto, A.P. Cardoso, C. Monteiro, M. Marques, R. Seruca, M.B. Barbosa,
S. Rocha, M.J. Oliveira (Portugal)
Photodynamic therapy as an option for osteosarcoma
G. Chohfi de Miguel, M. Laranjo, A.M. Abrantes, R. Teixo, T. Rocha, A.C. Serra, M. Piñeiro,
A.M. Rocha-Gonsalves, M.F. Botelho, D.G. Priolli (Brazil)
Comparative global characterisation of microRNA-expression in radiation-associated and sporadic
breast carcinomas
C. Wilke, J. Heß, A. Pitea, D. Schmidl, S. Klymenko, H. Zitzelsberger, K. Unger (Germany)
FancA overexpression confers radioresistance to cells of head and neck squamous cell carcinoma
I. Gimenez-Aznar , A. Michna, L. Hieber, H. Braselmann, V. Jendrossek, V. Zangen, K. Unger,
H. Zitzelsberger, K. Lauber, J. Heß (Germany)
lxxxiii
928
929
930
931
932
933
Molecular and Genetic Epidemiology II
HDAC9 inhibition decreases cell proliferation in acute lymphoblastic leukemia
953
D. Moreno, K.D. Salomão, G.A. Cruzeiro, V.K. Suazo, R.G.P. Queiroz, R.C.P. Lira, C.A. Scrideli,
L.G. Tone (Brazil)
Cervical cancer incidence in the Kyrgyz Republic
954
E. Makimbetov, Z.M. Izmailova (Kyrgyzstan)
Functional germline variants in driver genes of breast cancer
955
S. Göhler , M.I. Da Silva Filho, R. Johansson, K. Enquist-Olsson, R. Henriksson, K. Hemminki,
P. Lenner, A. Försti (Germany)
Double heterozygotes among breast cancer patients analyzed for BRCA1, CHEK2, ATM, NBN/NBS1,
956
and BLM germ-line mutations
A. Sokolenko, N. Bogdanova, W. Kluzniak, E. Preobrazhenskaya, A. Iyevleva, S. Aleksakhina,
E. Kuligina, A. Jakubowska, T. Dörk, E. Imyanitov (Russian Federation)
Inherited variants in genes somatically mutated in thyroid cancer
957
C. Campo, A. Köhler, G. Figlioli, R. Elisei, C. Romei, M. Cipollini, F. Bambi, F. Gemignani, S. Landi,
A. Försti (Italy)
Effect of Survivin gene −1547 A>G (rs3764383) polymorphism in Turkish breast cancer patients
958
M. Acar , H. Sahin, M. Oznur, O. Bender, O. Surgit, E. Gunduz, M. Gunduz (Turkey)
Identification of pathogenic mutations in RAD51 paralogs in BRCA1/2-negative ovarian cancer patients
959
in the Czech Republic
M. Janatova, J. Soukupova, Z. Kleibl, P. Kleiblova, F. Lhota, J. Hojny, M. Borecka, P. Boudova,
P. Pohlreich (Czech Republic)
Effect of asthma on cancer incidence and survival: A population based study in Sweden
960
J. Ji (Sweden)
961
Low-penetrance alleles and male breast cancer risk: Results from a multicenter study in Italy
V. Silvestri, P. Rizzolo, G. Giannini, S. Tommasi, A. Viel, L. Cortesi, M. Montagna, P. Radice, D. Palli,
L. Ottini (Italy)
Association of SULT1A1 Arg213His polymorphism with male breast cancer risk: a case−control study
962
in Italy
P. Rizzolo, V. Silvestri, G. Giannini, L. Varesco, A. Viel, L. Cortesi, M. Montagna, P. Radice, D. Palli,
L. Ottini (Italy)
Potential of tumour-associated autoantibodies as biomarkers for gastric cancer detection
963
Z. Kalnina, I. Meistere, P. Zayakin, K. Silina, A. Pismennaja, M. Leja, A. Line (Latvia)
FokI and TakI vitamin D receptor polymorphism in patients with prostate cancer
964
R. Kwiatkowski, R. Braczkowski, A. Danikiewicz, B. Braczkowska (Poland)
lxxxiv
Characterization of breast cancer (BC) predisposition variants in high risk BRCA1- and BRCA2-negative
BC patients using panel next-gen sequencing
F. Lhota, P. Boudova, V. Stranecky, J. Soukupova, P. Kleiblova, J. Hojny, M. Janatova, M. Borecka,
BRCA1 VUS: an approach to assess functional impact using transcription activation and PALB2
interaction
G. De Gregoris, A.N. Monteiro, M.A. Carvalho (Brazil)
Hereditary variants of genes coding for TP53 and its regulators (CHK2 and WIP1) in high-risk breast
cancer patients
P. Kleiblova, J. Hojny, F. Lhota, L. Macurek, J. Soukupova, M. Janatova, P. Boudova, M. Borecka,
Avoided cost in chemotherapy drugs attributable to patients inclusion in clinical trials
M. Sanchez, J.I. Chacon, A.R. Rubio, A. Gonzalez, M.A. Cruz, P. Moya (Spain)
RAF1 c.*266C>T polymorphism as a protective factor in bladder cancer
N. Ersoy Tunali, F. Koksalar, N.O. Tiryakioglu (Turkey)
Epigenetic gender differences in colorectal cancer
R. Toth, N. Habermann, D. Scherer, B. Gigic, P. Schrotz-King, J. Staffa, A. Ulrich, E. Herpel, H. Brenner,
C.M. Ulrich (Germany)
Carcinogenic and non-carcinogenic risk assessment of bis(2-ethylhexyl) phthalate in bottled water
N. Rastkari, M. Zare Jeddi, M. Yunesian, R. Ahmadkhaniha (Iran)
965
966
967
969
970
971
971A
Prevention and Early Detection II
A novel sampling device for collecting mucocellular material from the unprepared rectum
985
J. Booth, J. Lacy-Colson, M. Norwood, C. Murray (United Kingdom)
Urolithins A and B, walnut polyphenol metabolites, modulate androgen receptor expression in a prostate
986
cancer cell model
C. Sanchez-Gonzalez, C.J. Ciudad, V. Noé, M. Izquierdo-Pulido (Spain)
987
High intensity of cytoplasmic peroxiredoxin VI predicts poor outcome in diffuse large B-cell lymphoma
M.E.L. Kuusisto, K.M. Haapasaari, E. Jantunen, O. Kuittinen, Y. Soini, P. Peroja, T. TurpeenniemiHujanen, P. Karihtala (Finland)
Hair dyes in breast cancer risk
990
S. Heikkinen, N. Malila, M. Koskenvuo, T. Sarkeala, J. Pitkäniemi (Finland)
Highly sensitive pancreatic cancer diagnosis by serum exosome stem cell and miRNA markers
991
M. Zöller , S. Yue, B. Madhavan, U. Galli, W. Gross, N.A. Giese, H. Kalthoff, M.W. Büchler (Germany)
993
Quantification of donor/recipient chimerism in leukemia by digital PCR
K. Hayashibara, A. Jiménez-Velasco (USA)
996
Prevalence of germline MUTYH mutations among Lynch-like syndrome patients
G. Vargas, M. Navarro, S. González, J. Brunet, T. Ramón y Cajal, J. Balmaña, C. Lázaro, I. Blanco,
M. Pineda, G. Capellá (Spain)
Non-small cell lung cancer (NSCLC) cancer biomarkers are present in sputum and can be used for
997
diagnosis and therapeutic intervention
D. Jones (United Kingdom)
European Journal of Cancer (2014) 50(S5), S1–S2
Available at www.sciencedirect.com
ScienceDirect
journal homepage: www.ejcancer.com
13:15−14:15
Mühlbock Lecture: Development of Targeted
Cancer Therapies
14:15−15:00
AICR Lecture: Using Switchable Mouse Genetics
to Model Cancer Therapies
1 Development of targeted cancer therapies
2 Using switchable mouse genetics to model cancer therapies
A. Ullrich1 . 1 Max-Planck Institute for Biochemistry, Department of Molecular
Biology, Martinsried, Germany
No abstract received.
No conflict of interest information specified.
Cancer represents a disease prototype that is connected to defects in the
cellular signaling network that controls proliferation, motility, invasivity, survival
and recognition by the immune surveillance system. We obtained the first
insights into the genetic basis of cancer in the early 1980ies by comparing
the sequences of retroviral oncogenes of animal origin with human protooncogenes that encoded components of the cellular signal transduction
network. Currently the spectrum of known genetic alterations in cancer
cells includes mutations in a variety of genes leading to structural and
functional dysfunctions in cellular signal transmission and -definition as well
as over- or under expression of positive or negative signal regulatory proteins
respectively.
For the past years we have investigated various aspects of signaling
systems in tumor cells in order to identify critical switch points in the pathophysiological process that results in malignancy. These efforts aim at the
selective blockade of abnormal, disease-promoting signaling mechanisms by
monoclonal antibodies, or small molecule kinase inhibitors. This strategic
approach began with the cloning of the EGF receptor cDNA (1984) and
the related receptor HER-2/neu (1985) and translated the animal oncogene
concept into target-directed therapy of human cancer. This work yielded the
first specific oncogene target-based personalized, FDA-approved therapeutic
agent, “Herceptin”, for the treatment of metastatic breast cancer (1998).
Earlier and subsequent “target-driven drug development” efforts that employed
various genomic analysis strategies led to cancer therapies that are based
on EGFR, HER3, FGFR4, Axl/Ufo and Flk-1/VEGFR2 as critical signaling
elements in tumor progression. The latter served, in cooperation with
SUGEN Inc./Pharmacia/Pfizer, as basis for the development of SU11248.
The drug discovery process that led to SU11248 represents a prototypical
example for the adaption of cancer therapeutics from highly specific to multitargeted drugs. In 2006 the FDA approved SU11248/SUTENT/Sunitinib for
the treatment of Gleevec-resistant GIST and Renal Cell Carcinoma (Pfizer)
and the European Agency EMEA followed suit. Current research efforts aim
at the elucidation of the mechanistic relevance of the Sunitinib target profile,
which may aid in the prediction of patient response to this multi-specific cancer
therapeutic.
While all novel cancer therapies target genetic alterations in tumor tissues
innovative strategies are aimed at investigating the contribution of germ line
determinants of the patient to disease progression and therapy response. One
example is the common (50% in the Caucasian population) polymorphism at
codon position 388 in the human FGFR4 gene of which the Arg388 allele
represents a target for the development of individual genotype-dependent
cancer therapy development. Current findings and their consequences for
patient-specific cancer therapy and therapy response prediction will be
discussed.
No conflict of interest.
15:30−16:45
Plenary Symposium: Epigenetics
3 The cancer epigenome
P. Jones1 . 1 Van Andel Research Institute, Michigan, USA
Epigenetic processes are reinforced by interactions between covalent
chromatin marks such as DNA methylation, histone modifications and variants.
These marks ultimately specify the locations of nucleosomes particularly
with respect to transcriptional start sites and other regulatory regions. We
have developed a new methodology to simultaneously map nucleosomal
positioning and DNA methylation on individual molecules of DNA and show
that the methylation of CpG islands at the transcriptional start sites of key
tumor suppressor genes results in the stable placement of nucleosomes at
the transcription start site. Inhibition of DNA methylation by 5-azanucleoside
treatment results in an immediate inhibition of DNA methylation and a
sequence of downstream events ultimately resulting in the eviction of the
nucleosomes from the transcription start site and the activation of gene
expression.
4 Epigenetic targets in cancer
K. Helin1 . 1 University of Copenhagen, Centre for Epigenetics Biotech
Research & Innovation Centre (BRIC), Copenhagen, Denmark
Cell fate decisions are regulated by an intricate interplay between the
cellular environment, growth factors and intracellular signaling pathways. The
balance of stability versus plasticity in stem cells is a regulatory challenge,
and extensive studies in recent years have focused on understanding the
contribution of transcription factors and epigenetic enzymes in the regulation of
cellular identity and differentiation pathways. Disruption of epigenetic control
is a frequent event in disease, and the first epigenetic-based therapies for
cancer treatment have been approved. A generation of new classes of potent
and specific inhibitors for several chromatin-associated proteins have shown
promise in pre-clinical trials. Although the biology of epigenetic regulation is
complex, these and other new inhibitors will hopefully be of clinical use in the
coming years.
Our research is focused on understanding the role of epigenetic regulators in
maintaining cellular identity and how their deregulation leads to cancer. At the
meeting, I will review the progress that has been made on the development
of new epigenetic inhibitors and discuss some of our recent data on the
understanding of the biological function of chromatin-associated proteins and
how they contribute to cancer.
Conflict of interest: Ownership: EpiTherapeutics Aps. Advisory board:
EpiTherapeutics Aps. Board of directors: EpiTherapeutics Aps.
0959-8049/$ – see front matter © 2014 Elsevier Ltd. All rights reserved.
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EACR-23 Oral Presentations, Saturday 5 July 2014 / European Journal of Cancer 50, Suppl. 5 (2014) S1–S2
16:45−17:50
Opening Ceremony: Opening Ceremony / Opening
Lecture
5 Cancer research from the bench to the bedside
O. Wiestler1 . 1 Deutsches Krebsforschungszentrum, Heidelberg, Germany
Our field has recently encountered striking progress in unraveling basic
mechanisms of cancer development and this work has significant impact
on translational medicine, i.e. the fast and efficient transfer of research
findings into clinical applications. Important insights have been gained in
areas such as the molecular and cellular basis of malignant transformation,
basic principles of tumor progression, metastasis formation, cancer initiating
cells, tumor immunology, infection and cancer, genomics and epigenetics
as well as bioinfomatics. At the same time, significant technical and
methodological advances have been made, as for example in the field of next
generation sequencing, sophisticated disease models as well as the increased
accessibility of primary human tumor material. Based on these achievements it
is now possible to speed up the delivery of new innovative drugs and therapies
for cancer patients by developing targeted intervention strategies. In addition,
since cancer is a highly individual genetic disease, treatment and care can be
approached in an individualized manner. Finally, highly interesting prospects in
preventive oncology, i.e. risk assessment in healthy individuals, early detection
and prevention have come up.
To highlight this exciting era of bench to bedside research, a number of
prominent examples from the German Cancer Research Center (DKFZ)
together with collaborators from university medicine and pharmaceutical
industry partners will be presented. The focus will particularly be on the
translational potential of cancer initiating cells, cancer stem cells, the progress
made by exploiting high throughput genome sequencing tools, novel strategies
in cancer immunotherapy, imaging and radiation therapy, as well as new
approaches for individualizing cancer treatment and cancer care.
ScienceDirect
Sunday 6 July 2014
Sunday 6 July 2014
08:30−10:15
Symposium
Genome Stability
6 Targeting MTH1 prevents sanitation of oxidised dNTP pools and
causes cancer-selective DNA damage and cell death
T. Helleday1 . 1 Karolinska Institutet, Department of Medical Biochemistry and
Biophysics, Stockholm, Sweden
DNA damaging agents, i.e., radio- and chemotherapy, constitute the backbone
for treatment of a wide variety of cancers and may result in complete cure
from the disease. Emerging data demonstrate that cancer cells harbour high
level of endogenous DNA damage caused by replication, oxidative and other
DNA damage stress. Consequently, cancer cells may require a specific DNA
repair pathway to mediate survival to the high load of endogenous DNA
damage. Cancers have deregulated levels of reactive oxygen species (ROS),
damaging both DNA and free dNTPs. The MTH1 protein sanitises oxidized
dNTP pools, converting 8-oxo-dGTP to 8-oxo-dGMP, to prevent incorporation
of damaged bases during DNA replication. MTH1 overexpression reverses
the mutator phenotype caused by mismatch repair defects and prevents Rasinduced senescence by suppressing the overall level of DNA damage. These
data suggest that a majority of damage in cancer cells occur on the free dNTP
pool and that this need sanitation for cancer cell survival. Here we show that
cancer cells are dependent on MTH1 activity for survival, due to the effects of
MTH1 in preventing incorporation of oxidized dNTPs into DNA to avoid ATM
and p53 mediated apoptosis. As MTH1−/− mice are viable and MTH1 is not
required for survival of non-transformed cells, targeting MTH1 may selectively
cause DNA damage to cancer cells. We validate MTH1 as an anti-cancer
target in vivo and describe small molecules, TH287 and TH588 that potently
and selectively inhibit MTH1. Protein co-crystal structures demonstrate that the
compounds bind as inhibitors in the enzymatic pocket of MTH1. These first-inclass inhibitors of the Nudix hydrolase family cause increased incorporation of
oxidized dNTPs in cells subject to high ROS levels, causing DNA damage and
cytotoxicity to cancer cells. This study exemplifies a new general therapeutic
approach to convert oxidative stress to cytotoxic DNA damage and cancer cell
death.
Conflict of interest: Other substantive relationships: Named inventor on
submitted patent application.
7 Chromatin dynamics and cell cycle: From histone variants to
heterochromatin
G. Almouzni1 . 1 Institut Curie, Section de recherche UMR218, Paris, France
Chromatin organization in the nucleus provides a large repertoire of
information in addition to that encoded genetically. How histones, the major
protein components of chromatin, as particular variants or post-translationally
modified forms along with non coding RNA and other chromatin regulators,
can mark functional regions of the genome has been a major interest for my
group.
Understanding how chromatin organization is established, propagated, maintained, and changed during development and in response to environmental
cues has become a major theme in the field of Epigenetics. Errors in these
processes can lead to mis-regulation of genome functions and pathological
outcomes, such as cancer.
Our dissection of the mechanisms of chromatin assembly, from the basic
structural unit, the nucleosome, up to higher-order structures in the nucleus
has enabled to characterize key chaperones involved in nucleosome assembly
as well as to define the dynamics of new histone incorporation in chromatin.
We have examined specific nuclear domains: non-coding centromeric
heterochromatic regions, which are of major importance for chromosome
segregation. Our findings have shed light on the fundamental issues of the
dynamics, fate, and inheritance of histones, with their specific marks typical of
particular chromatin domains.
Our current hypothesis is that histone chaperones function in an ‘assembly
line’ with specificity for individual histone variants to mark defined regions of
the genome. Remarkably, we have found that misregulation of specific histone
chaperones is a common feature of aggressive breast cancers. Our recent
studies show that the depletion of the histone chaperone ASF1 leads to the
induction of the characteristics of Alternative Lengthening of Telomeres (ALT).
We will discuss our most recent advances in the regulatory pathways that
target histone chaperones and variants to control the assembly line and its
connecting network.
Reference(s)
Casanova M. et al. (2013) Heterochromatin reorganization during early mouse
development requires a single-strand non-coding transcript. Cell Rep., 4,
1156−67.
Abascal F. et al. (2013) Subfunctionalization via adaptive evolution influenced
by genomic context: the case of histone chaperones ASF1a and ASF1b.
Mol. Biol. Evol., 30, 1853−66.
Adam S. et al. (2013) Transcription recovery after DNA damage requires
chromatin priming by the H3.3 histone chaperone HIRA. Cell, 155, 94–
106.
Szenker E. et al. (2013) Properties and functions of histone variants. In
Fundamentals of Chromatin, pp. 375–427.
O’Sullivan R.J. et al. (2014) Rapid induction of the alternative lengthening of
telomeres by depletion of the histone chaperone ASF1. Nat. Struct. Mol.
Biol., 21, 167−74.
Lacoste N. et al. (2014) Mislocalization of the centromeric histone variant
CenH3/CENP-A in human cells depends on the chaperone DAXX. Mol.
Cell, 53, 631−44.
8 Proffered Paper: Mono- and polygenic breast cancer susceptibility
associates with error-prone homologous DNA double-strand break
repair in mouse and man
L. Wiesmüller1 , M. Deniz1 , K.J. Obermeier1 , M. Böhringer1 , M. Keimling1 ,
N. Griner2 , D.J. Jerry2 . 1 Ulm University, Department of Obstetrics and
Gynecology, Ulm, Germany, 2 University of Massachusetts, Department of
Veterinary & Animal Sciences, Amherst, USA
Background: High and moderate penetrance breast cancer susceptibility
genes have been linked to DNA double strand break (DSB) repair. However,
these genes explain only a small fraction of the familial risk and, low penetrance
modifier genes were demonstrated to have a dramatic impact on the individual
risk of high penetrance gene carriers. Modifier genes in BALB/c-Trp53 +/−
mice, resembling the hereditary Li-Fraumeni syndrome, were also proposed
to promote early-onset of spontaneous mammary tumor formation in an
unknown fashion. In search of novel comprehensive markers that may serve
as indicators for breast cancer risk, we analyzed DSB repair mechanisms and
genetic interactions in predisposed women as well as in BALB/c-Trp53 +/− mice
versus controls.
Material and Methods: Using a fluorescence-based test system for detection
of distinct mechanisms, we analyzed DSB repair in peripheral blood
lymphocytes (PBLs) from 35 female members of families with defined history
of familial breast and/or ovarian cancer, 175 female breast cancer patients
and 245 healthy women without previous cancer and without family history of
breast cancer. In parallel, we investigated mouse embryonic fibroblasts and
mammary epithelial cells from BALB/c-Trp53 +/− versus C57B/6-Trp53 +/− mice
regarding DSB repair pathway usage, lesion processing and molecular players
(immunofluorescence microscopy of nuclear signals, chromosome breakage,
G2 arrest, siRNA screen, gene expression).
Results and Discussion: Our study revealed association of an increase
of error-prone, particularly non-conservative homologous DSB repair
mechanisms in women with familial risk and young (<50), mostly predisposed
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EACR-23 Oral Presentations, Sunday 6 July 2014 / European Journal of Cancer 50, Suppl. 5 (2014) S3–S11
breast cancer patients (OR = 4.05–4.46), independently of single mutations
in BRCA1 or BRCA2. Strikingly, increased error-prone homologous DSB
repair was also found in BALB/c-Trp53 +/− mice. siRNA screening identified a
cluster of 25 genes causing DSB repair differences in the predisposed strain.
Interactome analysis revealed clustering of replication-related and Fanconi
anemia (FA)/breast cancer susceptibility (BRCA) genes. Dissection of the
functional and molecular changes in BALB/c-Trp53 +/− uncovered differences
in crosslink- and replication stress-associated repair and a FA pathway defect
downstream of FancD2 associated with reduced levels of BRCA2.
Conclusion: In conclusion, detection of error-prone homologous DSB
repair activities in patient cells could be a method to estimate breast
cancer susceptibility beyond the limitations of genotyping and to predict
responsiveness to therapeutics targeting DSB repair-dysfunctional tumors.
Consistent with polygenic models for breast cancer susceptibility, mammary
carcinogenesis in BALB/c-Trp53 +/− mice may be promoted by a modifier allele
in the FA pathway, again resulting in error-prone homologous DSB repair.
9 The ATM-mediated DNA damage response: Implications for cancer
development and treatment
Y. Shiloh1 . 1 Tel Aviv University, Department of Human Molecular Genetics
and Biochemistry, Tel Aviv, Israel
Genome stability is essential for the prevention of undue cellular death
and neoplasia. A central axis in maintaining genome stability is the DNA
damage response (DDR) − a complex signaling network that is vigorously
activated by DNA double strand breaks (DSBs). The primary transducer of the
DSB response is the serine-threonine kinase ATM, which mobilizes the DSB
response network by phosphorylating key players in its various branches. ATM
is missing or inactivated in patients with the genomic instability syndrome
ataxia–telangiectasia (A-T). A-T is characterized by neurodegeneration,
immunodeficiency, striking cancer predisposition and radiosensitivity. The
lymphoreticular malignancies in A-T patients reflect genomic aberrations
caused by defective rearrangement of the antigen receptor genes, a
physiological process which involves breakage and reunion of DNA. We are
exploring the DSB response network at both the transcriptional and posttranscriptional levels using systems biology tools, alongside proteomic and
genetic high-throughput screens. Subsequently, in-depth analysis of novel
pathways is carried out. Understanding this network is important not only
for understanding the early events in cancer formation but also for refining
radiation therapy and chemotherapy for cancer, since the cytotoxicity of these
agents results primarily from the damage they cause to the DNA molecule.
Importantly, loss of the ATM protein, as observed in A-T cells, confers the
highest degree of sensitivity to ionizing radiation and radiomimetic chemicals
known in humans. ATM is thus a preferred target for radio-sensitization of
cancer cells. Indeed, powerful inhibitors of ATM have recently been developed,
which can rapidly abolish ATM activity in a reversible manner. Targeting these
small molecules to cancer cells has the potential to radiosensitize these cells
differentially, allowing for the efficient treatment of patients with very low doses
of radiation or radiomimetic chemicals.
Sunday 6 July 2014
08:30−10:15
Symposium
Tumour Heterogeneity
10 Intra-tumour heterogeneity in early-stage lung cancer inferred
by multi-region sequencing
E. De Bruin1 , N. McGranahan2 , M. Salm3 , D. Wedge4 , R. Mitter3 , L. Yates4 ,
N. Matthews5 , A. Stewart3 , P. Campbell4 , C. Swanton1,2 . 1 University
College London Cancer Institute, Translational Cancer Therapeutics Lab,
London, United Kingdom, 2 Cancer Research UK London Research Institute,
Translational Cancer Therapeutics Lab, London, United Kingdom, 3 Cancer
Research UK London Research Institute, Bioinformatics, London, United
Kingdom, 4 Wellcome Trust Sanger Institute, Cancer Genetics and Genomics,
Cambridge, United Kingdom, 5 Cancer Research UK London Research
Institute, Advanced Sequencing Facility, London, United Kingdom
Introduction: Lung cancer is the most common cancer worldwide, and the
5-years overall survival remains poor. Understanding the genetic evolution of
the most common type of lung cancer, non-small lung cancer (NSCLC), may
improve therapeutic interventions.
Material and Methods: We performed multi-region exome and/or genome
sequencing to analyse a total of 25 tumour regions from seven early-stage
NSCLC samples.
Results and Discussion: All samples revealed branched tumour evolution
with potential driver mutations occurring both on the trunk and on the branches
of the phylogenetic trees. While most driver mutations and large-scale genomic
events such as genome doubling occurred predominantly early in the life
history of these tumours, on-going chromosomal instability enhanced the
intra-tumour heterogeneity at later stages of tumour development. Temporal
analyses of mutational patterns revealed regional differences in processes
that might drive further intra-tumour heterogeneity.
Conclusion: Our multi-region sequencing approach enables both spatial and
temporal dissection of mutations and of processes that drive these mutations,
revealing the persistent nature of genomic instability that occur in early-stage
NSCLCs.
11 Functional heterogeneity of tumour-initiating cells in
gastrointestinal cancers
12 Proffered Paper: The life history of lethal metastatic prostate
cancer (The UK prostate cancer working group of the International
Cancer Genome Consortium)
D.C. Wedge1 , G. Gundem1 , P. Van Loo1 , D. Brewer2 , K. Leinonen3 , R. Eeles4 ,
C. Cooper2 , T. Visakorpi3 , U. McDermott1 , G.S. Bova3 . 1 The Wellcome Trust
Sanger Institute, Cancer Genome Project, Cambridgeshire, United Kingdom,
2
University of East Anglia, Cancer Genetics School of Biological Sciences,
Norwich, United Kingdom, 3 University of Tampere, Institute of Biomedical
Technology, Tampere, Finland, 4 Institute of Cancer Research, Division of
Genetics and Epidemiology, Sutton, United Kingdom
Introduction: Tumour metastasis is the cause of 90% of cancer-related
deaths. Despite its clinical importance, little is known about the principles
governing the dissemination of cancer cells to distant organs. The analysis
of the cancer genomes of multiple metastases from a single patient yields
a wealth of information: allowing the separation of these processes across
time and space; revealing the effects of treatment and host tissue in driving
selection; and vastly increasing the power to identify competing subclones
within the tumour cell diaspora.
Materials and Methods: Fifty-two samples from the metastases and primary
prostate tumours of 10 men were whole genome sequenced to an average
60-fold coverage. Bioinformatic algorithms were developed to disaggregate
the population of tumour cells into competing subclones and identify the
mutational processes that have been operative on each subpopulation, using
an integrated analysis of point mutations, small insertions/deletions, copy
number changes and structural rearrangements.
Results: The phylogenetic trees for 10 tumours reveal a variety of
mutational processes operative at different timepoints. Loss of tumour
suppressor genes was commonly an early event, resulting from complex
structural rearrangement. In 2 patients, amplification of the AR locus resulted
from separate copy number events in different metastases, indicating the
occurrence of convergent evolution. The mutational processes operative in
different tumours could, in many cases, be linked to specific causes, such
as a tandem duplicator phenotype observed in 2 tumours with mutations
in genes associated with DNA repair. Mutational processes were ongoing
within all samples and indicated a temporal ordering of the appearance of the
founding cells of each metastasis. Metastases within the same tissue type were
more closely related than those in different tissue types. Integrated analyses
of subclonal architecture across the metastases of each patient produced
convincing evidence for the seeding of metastases from multiple cells derived
from different subclonal populations in up to 4 patients.
Conclusions: The application of bioinformatic analyses to whole genome
sequences of multiple metastases reveals the competing subclones within
the tumour population and the complexity of the mutational processes and
selection pressures acting thereon. These subclones are found to be spread
across time and space while maintaining their clonal integrity.
13 Tumor heterogeneity and cell-of-origin of mouse small cell and
A. Berns1 , K. Sutherland1 , M. Kwon1 , J.Y. Song2 , I. Huijbers1 . 1 The
Netherlands Cancer Institute, Division of Molecular Genetics, Amsterdam,
Netherlands, 2 The Netherlands Cancer Institute, Department of Pathology,
Amsterdam, Netherlands
Small cell lung cancer (SCLC) is one of the most lethal human malignancies,
due to its high metastatic potential and chemo-resistance upon relapse. Using
the Rbf/f;p53f/f mouse model for SCLC, we found that the tumors are often
composed of phenotypically different cells, characterized by mesenchymal and
neuroendocrine markers. These cells often share a common origin. Crosstalk
between these cells can endow the neuroendocrine component with metastatic
capacity, illustrating the potential relevance of tumor cell heterogeneity in
dictating functional tumor properties. Also specific genetic lesions appear to
S5
be associated with metastatic potential. We have studied the nature of this
crosstalk and found distinct signaling pathways to be critical.
We have also evaluated the relevance of additional lesions that were frequently
acquired in the mouse SCLC, such as amplification of Myc and Nfib. Therefore,
we have derived ES cells from Rbf/f;p53f/f, equipped these cells with an
exchange cassette in the ColA1 locus, and shuttled a conditional L-Myc and
Nfib under a strong promoter into this locus. This accelerated tumorigenesis
and resulted also in a shift in the metastatic phenotype.
To investigate the cell-of-origin of lung tumors, we have inactivated Trp53
and Rb1 in distinct cell types in the adult lung by targeting Cre-recombinase
expression to Clara, neuroendocrine, and alveolar type II cells using adenoviral
vectors. We could show that neuroendocrine cells serve as the predominant
although not the exclusive cell of origin of SCLC.
In contrast, activation of mutant Kras in the different cell types showed that
alveolar type II cells and Clara cells both can serve as the cell of origin of
adenocarcinomas (one of the NSCLC subtypes). Our data indicate that both
cell type specific features and the nature of the oncogenic lesion(s) are critical
factors in determining the tumor initiating capacity of lung (progenitor) cells.
Furthermore, the cell-of-origin appears to influence the malignant properties
of the resulting tumors.
function and lipid catabolism. Thus, autophagy is required for carcinoma
fate, and cancers require autophagy for distinct roles in metabolism that
are oncogene- and tumor suppressor gene-specific. To test the role of
autophagy in oncogenic signaling pathways downstream of RAS, Atg7
was deleted in a mouse model of BRAFV600E -induced lung cancer in the
presence or absence of the tumor suppressor Trp53. Atg7 deletion initially
induced oxidative stress and accelerated tumor cell proliferation in a manner
indistinguishable from Nrf2 ablation. Compound deletion of Atg7 and Nrf2
had no additive effect suggesting that both genes modulate tumorigenesis
by regulating oxidative stress, revealing a potential mechanism of autophagymediated tumor suppression. At later stages of tumorigenesis, Atg7 deficiency
resulted in an accumulation of defective mitochondria, proliferative defects,
reduced tumor burden, conversion of adenomas and adenocarcinomas to
oncocytomas, and increased mouse lifespan. Autophagy-defective tumorderived cell lines were defective in their ability to respire, survive starvation and
were glutamine-dependent, suggesting that autophagy-supplied substrates
from protein degradation sustains BrafV600E -tumor growth and metabolism.
Sunday 6 July 2014
N. Lynam-Lennon1 , S.G. Maher2 , A. Maguire3 , J. Phelan1 , C. Muldoon3 ,
J.V. Reynolds1 , J.N. O’Sullivan1 . 1 Trinity College Dublin, Department of
Surgery, Dublin, Ireland, 2 University of Hull, Cancer Biology and Therapeutics
Lab, Hull, United Kingdom, 3 St James’s Hospital, Department of Pathology,
Dublin, Ireland
08:30−10:15
Symposium
Tumour Metabolism
14 Balancing energetic and anabolic needs of cancer cells
K. Vousden1 , O. Maddocks1 , E. Cheung1 , P. Lee1 , C. Labuschagne1 , I. Mor1 .
1
Cancer Research UK Beatson Institute, Glasgow, United Kingdom
Cancer cells show changes in metabolism to help support their requirements
for growth and survival in abnormal environments. One challenge facing
cancer cells is fluctuating nutrient availability. We have recently found that
the tumour suppressor p53 can help to support the adaptation of cancer cells
to serine starvation by facilitating the switch to the de novo serine synthesis
pathway (SSP). The activation of p53 in response to serine depletion leads
to a transient cell cycle arrest, allowing the cells to channel metabolites into
glutathione synthesis and survive increased oxidative stress. Interestingly,
many transformed cells are highly dependent on the uptake of exogenous
serine and dietary depletion of serine in vivo reduces cancer growth without
impacting general health. We have also shown a synergy between serine
depletion and inhibitors of OXPHOS, such as oligomycin and Metformin.
Surprisingly, glycine cannot substitute for serine in supporting tumor cell growth
and our analyses indicate that this is due to a depletion of one-carbon pools
in glycine-fed cells.
p53 regulates the expression of various proteins that control glycolysis,
including TIGAR, an enzyme that regulates the level of fructose-2,6bisphosphate (F-2,6-BP) and redirects glucose from the glycolytic pathway to
alternative metabolic pathways. Our previous studies have demonstrated an
antioxidant function for TIGAR, resulting in part from increased flux through
the pentose phosphate pathway. We are now exploring the roles of TIGAR in
other metabolic pathways such as the hexosamine biosynthesis pathway. Our
work suggests that TIGAR expression may contribute to tumour development
and we are investigating the role of TIGAR in various in vivo cancer models.
Funded by CR-UK, ERC and MRC.
15 Role of autophagy in the metabolism and growth of lung cancer
E. White1 . 1 Rutgers University, Molecular Biology and Biochemistry, NJ
08901, USA
Autophagy degrades and recycles proteins and organelles to support
metabolism and survival starvation. Oncogenic RAS upregulates autophagy
required for mitochondrial function, stress survival, and engrafted tumor
growth. We deleted essential autophagy gene, autophagy-related-7 (Atg7)
concurrently with KrasG12D activation with or without intact Trp53 in two
mouse models for non-small-cell lung cancer (NSCLC). In both models, Atg7
deficiency caused tumor cells to accumulate dysfunctional mitochondria, and
acquire metabolic, growth and survival defects associated with reduction
in tumor burden. Importantly, Atg7 deficiency altered the fate of KrasG12D induced carcinomas to that of oncocytomas, rare, predominantly benign tumors
characterized by the accumulation of defective mitochondria. Surprisingly, lipid
accumulation was observed in Atg7-deficient tumors only when Trp53 was
deleted. Atg7-deficient tumor-derived cell lines (TDCLs) had compromised
starvation survival and formed lipidic cysts instead of tumors, suggesting
defective utilization of lipid stores. Atg7 deficiency reduced fatty acid
oxidation (FAO) and increased sensitivity to FAO inhibition, indicating that
with Trp53 loss, RAS-driven tumors require autophagy for mitochondrial
16 Proffered Paper: Altered mitochondrial function and energy
metabolism is associated with a radioresistant phenotype in
oesophageal adenocarcinoma
Introduction: Radiation therapy is fundamental to the treatment of
oesophageal cancer. However, radioresistance is a significant clinical problem.
We hypothesise that mitochondrial dysfunction may play a significant role in
driving radioresistance in oesophageal cancer.
Materials and Methods: An isogenic model of radioresistant oesophageal
adenocarcinoma (OAC) was established by chronically irradiating OE33
cells with clinically-relevant doses of 2 Gy X-ray radiation. Radiosensitivity
was assessed by clonogenic assay. Mitochondrial mutation frequency was
determined by random mutation capture assay. Mitochondrial function was
assessed by fluorimetry. Mitochondrial ultra-structure was assessed by
transmission electron microscopy (TEM). Gene expression was assessed by
qPCR arrays. Oxygen consumption rates (OCR) and extracellular acidification
rates (ECAR) were measured using the Seahorse XF Analyzer, with and
without treatment with oligomycin and antimycin. ATP was measured by
luminescence assay. ATP5B expression was assessed in 23 pre-treatment
OAC tumours by immunohistochemistry.
Results: Chronic exposure of OE33 cells to fractionated radiation produced
a radioresistant subline, OE33R. Characterisation of this model revealed
that, relative to the age- and passage-matched parent control (OE33P),
OE33R cells had increased basal frequencies of random mitochondrial
mutations (p < 0.01) and alterations in ROS and mitochondrial mass (p < 0.05).
TEM demonstrated altered mitochondrial morphology in OE33R cells, which
was coupled with altered expression of 15 mitochondrial-associated genes.
Energy metabolism was altered in radioresistant cells, with increased basal
OCR rates, a measure of oxidative phosphorylation, increased levels of
ATP (p < 0.05) and a higher proportion of oxygen consumption associated
with ATP synthesis (p < 0.05), when compared to OE33P cells. OE33R
cells also exhibited a significant increase (p < 0.05) in ECAR rates, a
marker of glycolysis, and clonogenic survival, upon inhibition of oxidative
phosphorylation. In pre-treatment OAC tumours, ATP5B was significantly
increased (p < 0.05) in tumours from patients who subsequently had a poor
response to chemoradiation therapy, when compared to good responders.
Conclusion: This study suggests for the first time, a role for specific
mitochondrial alterations and metabolic remodelling in the radioresistance of
OAC.
17 Magnetic resonance imaging of tumour metabolism
K.M. Brindle1 , D.E. Hu1 , T.B. Rodrigues2 , E.M. Serrao2 , K.N. Timm1 .
1
University of Cambridge, Department of Biochemistry, Cambridge, United
Kingdom, 2 University of Cambridge, CRUK Cambridge Institute, Cambridge,
United Kingdom
A better understanding of tumour biology has led to the development of
targeted therapies, in which a drug is designed to disrupt a specific biochemical
pathway important for tumour cell survival or proliferation. The introduction of
these drugs into the clinic has shown that patients can vary widely in their
responses. Molecular imaging is likely to play an increasingly important role
in predicting and detecting these responses and thus in guiding treatment
in individual patients [1]. We have been developing methods for detecting
the early responses of tumours to therapy, including magnetic resonance
S6
(MR) imaging of tumour cell metabolism using hyperpolarized 13 C-labelled
cell metabolites.
Magnetic resonance imaging of tissue water protons, which are present in
tissues at concentrations of ~80 M, can be used to produce relatively highresolution 3D images of tissue anatomy. However, it is also possible to detect
MR signals from tissue metabolites. The problem is that these are present at
~10,000× lower concentration than tissue water protons and therefore it is not
possible to image them at clinical magnetic field strengths, except at relatively
low spatial and temporal resolution. Moreover, single spectra or spectroscopic
images of tissue metabolites lack dynamic information about metabolic fluxes.
Hyperpolarisation of 13 C-labelled cell substrates increases their sensitivity to
detection in the MR experiment by more than 10,000×, making it possible
to not only image the location of a hyperpolarized 13 C-labelled cell substrate
in the body but, more importantly, the kinetics of its conversion into other cell
metabolites, with spatial resolutions of 2−5 mm and temporal resolutions in the
sub second range.
We have used metabolic imaging with hyperpolarized 13 C-labelled substrates
both to detect treatment response and to investigate the tumour microenvironment (reviewed in [2,3]). Exchange of hyperpolarized 13 C label between lactate
and pyruvate [4] and net flux of label between glucose and lactate [5] have
been shown to decrease post-treatment and hyperpolarized [1,4-13 C]fumarate
has been shown to detect subsequent cell necrosis [6,7]. Tumour pH
can be imaged using hyperpolarized H13 CO−3 [8] and redox state can be
determined by monitoring the oxidation and reduction of [1-13 C]ascorbate
and [1-13 C]dehydroascorbate respectively [9]. More recently we have shown
that we can follow the progression of pancreatic precursor lesions, in a
genetically engineered mouse model of the disease, which potentially could
be used clinically to guide earlier intervention. The technique has recently
transitioned to the clinic, with the completion of a clinical trial in prostate cancer
at UCSF [10], where it promises new opportunities for monitoring tumour
treatment response.
Reference(s)
[1] Brindle, K. Nature Rev Cancer 8, 94–107 (2008).
[2] Brindle, K.M., et al. Magn Reson Med 66, 505–519 (2011).
[3] Brindle, K. Brit J Radiol 85, 697–708 (2012).
[4] Day, S.E., et al. Nature Med 13, 1382–1387 (2007).
[5] Rodrigues, T.B., et al. Nature Med 20, 93−97 (2014).
[6] Gallagher, F.A., et al. Proc Natl Acad Sci U S A 106, 19801–19806
(2009).
[7] Clatworthy, M.R., et al. Proc Natl Acad Sci U S A 109, 13374–13379
(2012).
[8] Gallagher, F., et al. Nature 453, 940–943 (2008).
[9] Bohndiek, S.E., et al. J Am Chem Soc 133, 11795–11801 (2011).
[10] Nelson, S.J., et al. Science Translational Medicine 5, 198ra108 (2013).
Conflict of interest: Other substantive relationships: GE Healthcare.
Sunday 6 July 2014
10:45−12:30
Symposium
Senescence and Ageing
18 Epigenetics of cellular senescence, insights into cancer and aging
P. Adams1 . 1 University of Glasgow, Glasgow, United Kingdom
The incidence of many cancers increases with age, and age is the biggest
single risk factor for most cancers. However, the reason for this is not
well understood. While development and progression of cancer depends
on accumulation of multiple genetic and epigenetic alterations, the age
dependence of cancer is not well understood. Chromatin is an inherently
dynamic and plastic structure, as indicated by phenomena such as Position
Effect Variegation in flies and biochemical analyses from the ENCODE project.
Building on the work of a number of other labs, the Adams lab hypothesizes
that age-associated changes in this plastic chromatin structure predispose to
cancer and so contribute to the striking increased incidence of cancer with
age. Since most new targeted cancer therapies show only modest therapeutic
activity in the clinic, a better understanding of the age dependence of cancer
is likely to be critical to combat this disease, through risk assessment, early
detection and chemoprevention. We are investigating the relationship between
aging, epigenetics and cancer in senescent cells, as a model for some
molecular and cellular events associated with cell aging, and in mouse models
and human tissues.
19 Aneuploidy and telomerase in stem cell derived cancer
K. Rudolph1 . 1 University of Ulm, Institute of Molecular Medicine and
Max-Planck Research Group on Stem Cell Aging, Ulm, Germany
Aneuploidy represents a hallmark feature of carcinogenesis in humans but
molecular mechanisms that induce aneuploidy or survival of aneuploid human
cells remain to be defined. We conducted a genome-wide shRNA screen to
identify gene knockdowns leading to aneuploidy induction in human cancer
cells. A set of over 200 gene knockdowns was identified robustly inducing
aneuploidy. Based on bioinformatic analyses top candidate genes were
confirmed with single shRNAs. Transduction of top candidate shRNAs into
primary human fibroblast resulted in induction of aneuploidy. However, the
primary fibroblasts underwent premature senescence. Telomerase (TERT)
expression was sufficient to rescue induction of premature senescence
in primary human fibroblast transduced with aneuploidy inducing shRNAs.
Immortalized cultures exhibited aneuploidy and developed tumors in nude mice
in the absence of defective check-points.
20 Proffered Paper: Senescent cells impact premalignant
microenvironment by direct protein transfer
A. Biran1 , M. Perelmutter1 , D. Burton1 , Y. Ovadya1 , T. Geiger2 ,
V. Krizhanovsky1 . 1 Weizmann Institute of Science, Molecular Cell Biology,
Rehovot, Israel, 2 Tel Aviv University, Sackler School of Medicine, Tel-Aviv,
Israel
Introduction: Cellular senescence, a permanent cell-cycle arrest, prevents
both tumorigenesis and tissue damage. However, long-term presence of
senescent cells can paradoxically promote tumorigenesis and tissue damage
in their microenvironment. Soluble factors secreted from senescent cells
mediate these cell non-autonomous effects. Here we report for the first time
that senescent cells also use intercellular protein transfer to affect neighboring
cells.
Material and Methods: We studied the interaction between fibroblasts,
induced to senesce either with oncogenic Ras or with direct DNA damage, and
other cells including natural killer (NK) cells and epithelial cells. Intercellular
protein transfer was detected using FACS analysis of the transfer of fluorescent
proteins. To understand the mechanism of the transfer we identified all the
proteins that transfer from senescent cells to NK cells using SILAC (Stable
isotope labeling by/with amino acids)-mediated high-throughput proteomic
analysis. Based on the identified proteins we studied how protein transfer
is dependent on cell–cell contact and CDC42-regulated actin polymerization.
We then evaluated protein transfer in vivo in mice using co-expression of Red
Fluorescent Protein (RFP) and mutant K-ras in the pancreas.
Results and Discussion: Our results demonstrate that proteins are
preferentially transferred from senescent cells to NK cells as well as to
epithelial normal and cancer cells. The intercellular protein transfer leads
to increased activation of the NK cells. This transfer strictly depends on
cell contact and actin polymerization. CDC42, which regulates the actin
polymerization, is necessary for the protein transfer from senescent cells.
We propose that cytoplasmic bridges account for direct protein transfer from
senescent cells. Indeed, we detected such cytoplasmic bridges between
senescent cells and their neighboring cells, including NK cells. Moreover,
transfer of proteins to NK and T cells is increased in murine pre-neoplastic
pancreas, where senescent cells are present in vivo.
Conclusion: Our results present the first evidence for direct protein transfer
in vivo in mammals and suggest that it is a novel mode of inter-cellular
communication that may regulate immune surveillance of senescent cells,
tumourigenesis and tissue ageing.
21 Cancer and ageing: Rival demons?
J. Campisi1 . 1 Buck Institute for Age Research, Novato, USA
Aging is the largest risk factor for developing an array of diseases,
ranging from neurodegeneration to cancer. Several lines of evidence suggest
one or more basic aging process is a pivotal driver of the panoply of
age-related pathologies. Basic aging processes are attractive targets for
developing interventions that would revolutionize modern medicine − the
concurrent prevention, postponement or treatment of multiple age-related
diseases. Recent findings suggest one such basic aging process is cellular
senescence.
Cellular senescence is a potent anti-cancer mechanism that suppresses
the proliferation − essentially permanently − of cells at risk for malignant
transformation. Two important features of senescent are: (1) senescent cells
stably persist, certainly in culture and probably in vivo, and (2) senescent
cells create a pro-inflammatory milieu by secreting numerous inflammatory
cytokines, growth factors and proteases, collectively termed the senescenceassociated secretory phenotype (SASP). Several lines of evidence, including
new senescence-free mouse models, support the idea that senescent cells,
by virtue of the SASP, can disrupt normal tissue structure and function and,
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ironically, alter the tissue microenvironment to fuel the progression of late
life cancer. Mouse models have also uncovered a beneficial effect of cellular
senescence, distinct from its ability to suppress tumorigenesis early in life: the
transient presence of senescent cells promotes tissue repair in response to
injury. Their chronic presence, however, which occurs during aging, retards
tissue repair. Thus, cellular senescence is a complex response that can be
beneficial or deleterious, depending on the context and age of the organism.
Conflict of interest: Ownership: Cenexys, Inc. Corporate-sponsored
research: yes. Other substantive relationships: Scientific Founder.
Sunday 6 July 2014
10:45−12:30
Symposium
Mechanisms of Drug Resistance
22 Mechanisms and biomarkers of acquired resistance to targeted
cancer therapies
23 Mutations and acquired resistance in colorectal cancer
A. Bardelli1 . 1 University of Torino, Istituto di Candiolo − IRCCS, Candiolo
(Torino), Italy
It is now evident that colorectal cancers (CRC) indistinguishable by light
microscopy are actually distinct diseases requiring unique therapeutic
approaches. Tissue and liquid biopsies can be used to define CRC molecular
subtypes and to monitor response and resistance to therapy. Using these
approaches, CRC patients were found to respond selectively to targeted
agents interfering with oncogenic nodes of the EGFR signaling pathway.
Notably, the patient-specific responses can be recapitulated and paralleled in
cellular and mouse clinical proxies (CRC-avatars). The inevitable development
of acquired resistance to inhibitors of the EGFR signaling pathway presently
limits further clinical advances. Strategies to prevent or overcome resistance
are therefore essential to design the next generation of molecularly-driven
clinical trials for CRC patients.
24 Proffered Paper: JAK2/STAT5 inhibition circumvents resistance to
PI3K/mTOR blockade: A rationale for co-targeting these pathways
in metastatic breast cancer
A. Britschgi1 , R. Andraos2 , H. Brinkhaus1 , I. Klebba1 , V. Romanet2 ,
U. Mueller1 , M. Murakami2 , T. Radimerski2 , M. Bentires-Alj1 . 1 Friedrich
Miescher Institute, Mechanisms of Cancer, Basel, Switzerland, 2 NIBR, Onc,
Basel, Switzerland
Background: Hyperactive PI3K/mTOR signaling is prevalent in the majority
of human malignancies and its inhibition exhibits potent antitumor activity
in a wide spectrum of solid cancers. Unfortunately, single-agent targeted
cancer therapy is usually short-lived and thwarted by different resistance
mechanisms.
Material and Methods: We used a combination of in vitro and in vivo models
of luminal and triple-negative breast cancer and a battery of biochemical, cell
biological, tumorigenesis and metastasis assays.
Results: We discovered a JAK2/STAT5-evoked positive feedback loop that
causes adaptive resistance to dual PI3K/mTOR inhibition. Mechanistically,
PI3K/mTOR inhibition increased IRS1-dependent activation of JAK2/STAT5
and secretion of IL-8 in several cell lines and primary triple-negative breast
tumors. Genetic or pharmacological inhibition of JAK2 abrogated this vicious
feedback loop. Combined PI3K/mTOR and JAK2 inhibition synergistically
reduced cancer cell viability in vitro as well as tumor growth in vivo, and
decreased tumor seeding and metastasis due to its impact on the IL-8
receptor CXCR1+ tumor-initiating subpopulation of cells. We further found that
combined PI3K/mTOR and JAK2 inhibition increased event-free as a well as
overall survival of tumor bearing animals.
Conclusion: This study reveals a new link between growth factor signaling,
JAK/STAT activation, cytokine secretion and metastasis. Our results provide
a rationale for combined targeting of the PI3K/mTOR and JAK2/STAT5
pathways in triple-negative breast cancer, a particularly aggressive and
currently incurable disease.
Conflict of interest: Ownership: RA, VR, MM and TR are Novartis
employees.
25 In vivo RNAi screening for novel therapeutic cancer targets
D. Peeper1 . 1 Netherlands Cancer Institute − Antoni van Leeuwenhoek
Hospital, Division of Molecular Oncology, Amsterdam, Netherlands
Melanoma is the most aggressive type of skin cancer and its incidence is
steadily increasing. Melanomas tend to spread rapidly, which is associated
with a grim prognosis. Until recently, most advanced stage melanomas
were refractory to the available therapeutic options, but there are recent
developments offering better perspectives. For example, new therapeutic
approaches have become available, which target genetic vulnerabilities
within the melanomas. A primary example of such a dependency is
the common BRAFV600E mutation, which is essential for proliferation and
survival of melanoma cells. In the clinic, the mutant BRAF oncogene
product can be targeted by specific inhibitors, including vemurafenib, which
cause unprecedented melanoma regression. However, relapse eventually
occurs around six months due to a variety of resistance mechanisms, both
MAP kinase-dependent and -independent. Therefore, in spite of these new
perspectives, there is a dire need to identify additional targets amenable to
therapeutic intervention, to be used in combination with vemurafenib or other
specific inhibitors to overcome or prevent drug resistance and achieve more
durable responses. To achieve this, we set out to identify melanoma factors that
are required for proliferation and survival specifically in an in vivo setting. Thus,
we performed negative selection RNAi screens parallel in vitro and in vivo
and focused on the hits that were preferentially depleted in tumors relative
to the corresponding cells in culture. The results from these screens will be
discussed.
Conflict of interest: Other substantive relationships: CSO of MetaCurix.
Sunday 6 July 2014
10:45−12:30
Symposium
Cancer Prevention
26 Progress in breast cancer prevention
J. Cuzick1 . 1 Queen Mary University of London, Centre for Cancer Prevention,
London, United Kingdom
Two drugs, tamoxifen and raloxifene, are licensed for preventive therapy in the
United States. Both have been shown to reduce incidence by approximately
40%, but in a head-to-head comparison tamoxifen was about 25% more
effective. However as these drugs are now off patent there seems to be no
direct way for them to be licensed for prevention in Europe, although NICE
has now recommended their use for high risk women in the UK, and they can
be prescribed off label. More recently two other selective oestrogen receptor
modifiers (SERMs), lasofoxifene and arzoxifene have been investigated in large
prevention trials. Both appear to be at least as effective as tamoxifen in breast
cancer risk reduction, but lasofoxifene also shown large reductions in heart
disease and fracture rates, making it an ideal candidate for preventive therapy.
All of these drugs have recently been evaluated with extended follow up in
an individual patient overview where a 55% reduction in ER positive cancer
in the five years of active treatment was seen, but also a 42% reduction in
the next 5 years, as a result of “carryover” benefits after treatment cessation.
No reduction in ER negative tumours has occurred, and in fact a marginally
increased (14%, P = 0.09) incidence was seen. Newer approaches are looking
at the role of aromatase inhibitors, where substantial reductions in contralateral
tumours have been seen when they were used as adjuvant treatments for early
breast cancer. Two prevention trials in high risk women without breast cancer
have been conducted. The MAP.3 trial evaluated exemestane and a 65%
reduction in invasive tumours after a relatively short 30 months median follow
up was seen. More recently the IBIS 2 trial using anastrozole has completed
analysis of 3846 women with a median follow up of 5 years. Briefly a 53%
reduction in all breast cancer was seen, with a larger reduction in ER positive
invasive breast cancer. Fracture rates were not significantly increased, due
in part to bone mineral density scans at baseline, and monitoring of those
who were found to be osteoporotic or osteopenic with treatment as necessary.
Musculoskeletal and vasomotor symptoms were increased but only by about
10%, but rates were very high in the placebo arm, indicating that most of the
reported symptoms in uncontrolled situations are not drug related.
As both of these classes of drugs (SERMs and AIs) have important side effects,
it is important to focus their use among women most likely to benefit. Models
have been developed to aid this decision and the Tyrer-Cuzick model appears
to be one of the best at the moment. However newer results have shown
that mammographic breast density is an important predictor and a risk score
combining the 67 currently identified risk SNPs may also add to predictive
accuracy.
Conflict of interest: Corporate-sponsored research: AstraZeneca.
27 Worldwide prevention of cervical cancer
J. Peto1 . 1 London School of Hygiene & Tropical Medicine, Dept of
Non-Communicable Disease Epidemiology, London, United Kingdom
Most HPV vaccination programmes target adolescent girls and young women.
This will have little effect on overall cancer rates for several decades, as most
of the 8 million women who will develop cervical cancer over the next 20 years
have already been infected with HPV. The majority of HPV infections disappear
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within a year or two, and these confer little risk of progression to cancer. A
screen-and-treat policy based on a single HPV test therefore entails substantial
overtreatment, and does not protect against subsequent reinfection. Polyvalent
HPV vaccines that prevent the large majority of incident infections with highrisk HPV types and rapid HPV testing may soon be available at affordable
cost, and this will have important implications for vaccination and screening
policy, particularly in older women. Polyvalent HPV vaccination, followed 3
years later by a rapid HPV test and immediate ablative treatment of all
persisting HR-HPV infections (irrespective of cytology or colposcopy), would
greatly reduce cervical cancer incidence in women at all ages. This once in a
lifetime intervention might be a cost-effective alternative to regular screening
in developed countries, and in developing countries where regular cervical
screening is impractical it may be the only way to produce a large and rapid
reduction in cervical cancer incidence and mortality. The EUROGIN 2010
Roadmap on Cervical Cancer Prevention suggests that in developing countries
this might be achieved more easily by a programme of mass vaccination
followed a few years later by mass HPV testing than by attempting to follow
up individual women after vaccination.
28 Proffered Paper: Cancer risk induced by computed tomography
scans in pediatrics: first results from a national cohort study
in France
N. Journy1 , J.L. Rehel2 , J.F. Chateil3 , H. Ducou Le Pointe4 , M. Mezzarobba5 ,
S. Caer-Lorho5 , D. Laurier5 , M.O. Bernier5 . 1 Intitut de Radioprotection et de
Sûreté Nucléaire, Laboratory of Epidemiology, Fontenay-aux-Roses, France,
2
Institut de Radioprotection et de Sûreté Nucléaire, Medical Radiation
Protection Expertise Unit, Fontenay-aux-Roses, France, 3 CHU Pellegrin,
Department of Pediatric Radiology, Bordeaux, France, 4 CHU Trousseau,
Department of Pediatric Radiology, Paris, France, 5 Institut de Radioprotection
et de Sûreté Nucléaire, Laboratory of epidemiology, Paris, France
Background: The frequency of computed tomography (CT) examinations and
the magnitude of associated x-ray doses raise many concerns about potential
adverse effects, especially in children because of their high sensitivity to
radiation. Reasonable quantification of potential cancer risks should help to
identify situations in which the expected benefits do not outweigh the risks,
and clarify information to patients. Recent epidemiological studies in UK and
Australia have suggested an increased risk of cancer among children receiving
CT scans. The interpretation of such results is nevertheless questioned due
to the lack of information about the indication of examination.
Material and Methods: In France, a nationwide study is also ongoing to
assess cancer risks after CT scans in pediatrics. The included children
have received one or repeated CT scans before 10 years from 2000
in one of 21 university hospitals. Cumulative organ doses are estimated
from the protocols defined in the departments using a dedicated numerical
simulation software. Clinical information recorded during hospitalization was
used to identify children having medical conditions (such as Down syndrome,
neurofibromatosis, etc.) likely to increase both their risk of cancer and the
frequency of examination. Cancer incidence and mortality data are retrieved
through national registries.
Results: At the end of 2011, the cohort included more than 67,000 children,
30% of them have received a first CT scan before the age of 1 year. The
association between cancer risk and cumulative doses was consistent with
those from the two afore-mentioned studies. The first results suggest, however,
an indication bias in the estimation of excess risk of cerebral tumors, and
emphasize how some genetic defects and other conditions associated with
a high risk of cancer could affect the assessment of risk attributable to
CT scans.
Conclusions: This study takes part in a collaborative European project (EpiCT, International Agency for Cancer Research) which will gather nine national
cohorts to provide powerful results on cancer risks associated with CT.
29 Alcohol and cancer risk, with focus on low doses
C. La Vecchia1 . 1 IRCCS-Istituto di Ricerche Farmacologiche Mario Negri,
Department of Epidemiology, Milano, Italy
Alcohol increases the risk of head and neck and oesophageal cancers. The
risks are essentially due to total ethanol intake. Alcohol drinking has also been
associated with primary liver cancer, with cancers of the large bowel in both
sexes, of the female breast, and − at high doses only − of the pancreas [1].
To evaluate the strength of the evidence provided by the epidemiological
literature on the association between alcohol consumption and the risk of
18 known or potentially alcohol-related neoplasms, we performed a search
of the epidemiological literature from 1966 to 2012 using major bibliographic
data-bases. We fitted meta-regression models considering linear and nonlinear effects of alcohol intake. We also investigated the effects of selected
characteristics of the studies (e.g., allowance for tobacco) and of individuals
included in the studies (e.g., gender, age, etc.), as possible sources of
heterogeneity of the estimates. A total of approximately 600 studies including
200,000 cases were considered. Strong trends in risk were observed for
cancers of the oral cavity, oesophagus and larynx, the strongest one being
for oral cancer, with a relative risk (RR) around 5 for 50 g/day. Direct relations
were also observed for cancers of the colon and rectum, liver, breast, and (for
high doses only) pancreas and skin melanoma. For low doses (10 g/day)
some, direct association was observed for oral cancer in both sexes and for
female breast. No current association was observed for other sites. The risk of
kidney cancer, Hodgkin lymphoma and non Hodgkin lymphomas was reduced
in alcohol drinkers versus nondrinkers [2,3].
Approximately 400,000 cases of cancer are attributable to alcohol drinking
worldwide, representing 3.6% of all cancers (5.2% in men, 1.7% in women).
The corresponding figure for mortality is about 250,000 deaths (3.5% of all
cancer deaths). This proportion is particularly high among men in Central and
Eastern Europe. Among women, breast cancer comprised 60% of alcoholattributable cancers. Thus, the global burden of alcohol associated cancers
is substantial. Low alcohol drinking may account for about 12 to 15% of
alcohol-related cancers (50 to 60,000) and cancer deaths (30 to 40,000). Thus,
restricting alcohol drinking to up to 2 drinks per day in men and of one drink
in women would avoid over 85% of all alcohol-related cancers worldwide.
Reference(s)
[1] Pelucchi C, Tramacere I, Boffetta P, Negri E, La Vecchia C. Alcohol
consumption and cancer risk. Nutr Cancer 2011; 63: 983–990.
[2] Bagnardi V, Rota M, Botteri E, et al. Light alcohol drinking and cancer:
a meta-analysis. Ann Oncol 2013; 24: 301–308. 4
[3] Poli A, Marangoni F, Avogaro A, et al. Moderate alcohol use and health:
A consensus paper. Nutr Metab Cardiovasc Dis 2013; 23: 487–504.
Conflict of interest: Other substantive relationships: AIRC and Osservatorio
Permanente Giovani e Alcool.
Sunday 6 July 2014
14:00−15:45
Symposium
The Tumour Microenvironment
30 From the immune contexture to the Immunoscore in cancer
J. Galon1 . 1 INSERM UMRS1138, Cordeliers Research Center, Paris,
France
To date the anatomic extent of tumor (TNM classifications) has been by far the
most important factors to predict the prognosis of cancer patients. However,
this classification provides limited prognostic information in estimating the
outcome in cancer and does not predict response to therapy.
Materials and Methods: Using large-scale approaches and quantitative
measurements, we evaluated the importance of the host-immune response
within human tumors.
Results: We showed that tumors from human colorectal cancer with a high
density of infiltrating memory and effector memory T-cells (TEM) are less
likely to disseminate to lymphovascular and perineural structures and to
regional lymph-nodes. We showed that the combination of immune parameters
associating the nature, the density, the functional orientation and the location
of immune cells within the tumor was essential to accurately define the impact
of the local host immune reaction on patients prognosis. We defined these
parameters as the “immune contexture”, and identified factors modulating
the densities of intratumor immune subpopulations. Based on the immune
contexture, a standardized, simple and powerful immune stratification system,
termed the “Immunoscore”, was delineated that may bear a prognostic power
superior to that of the currently used cancer staging system. Tumor invasion
parameters were statistically dependent on the host-immune reaction. A
worldwide Immunoscore consortium is testing the prognostic value of the
Immunoscore, using a standardized assay to routinely measure the immune
status of a cancer patient.
Conclusions: The functional orientation of the immune contexture is
characterized by immune signatures qualitatively similar to those predicting
response to immunotherapy, Thus, a continuum of immune response is
existing, with similar predictive, prognostic, and mechanistic immune signature,
spanning a balance between tumor cell growth and elimination.
Conflict of interest: Advisory board: AstraZeneca, MedImmune, ImmunID.
Corporate-sponsored research: MedImmune. Other substantive relationships:
BMS, GSK, Janssen, Roche, Compugen.
31 Understanding transcriptional regulation of myeloid cells in the
tumor microenvironment
J. Schultze1 . 1 Universität Bonn, Bereich Genomforschung und
Immunregulation, Bonn, Germany
Tumor-associated macrophages (TAM) are a key component of the tumor
microenvironment linked to reduced survival in most tumor entities. Over
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the last decade, there have been numerous reports about their function
and polarization. It has been suggested that TAM follow a transcriptional
program termed M2 polarization that was initially attributed to IL4 stimulation of
macrophages. However, previous reports were often not comprehensive and
sometimes they were even contradictory. We have started to use genomewide transcriptome analysis of TAM derived from different murine syngeneic
and spontaneous tumor models. Already our initial analyses clearly revealed
that the previous interpretation of TAM activation being related to the socalled M2 polarization program is not apparent on the genome-wide level.
In contrast, we identify completely new biological processes that highlight
TAM reprogramming. Moreover, there seems to be a rather tumor type
specific alteration of TAM transcription and function. Since targeting the
tumor microenvironment therapeutically is a very attractive alternative or
complementary approach in cancer therapy, a better understanding of TAM
transcription and function on a global level will be a prerequisite for such
endeavors.
tumor angiogenesis and tumor macrophage infiltrate as well as reducing
paraneoplastic thrombocytosis. We are now investigating rational combinations
of anti-IL-6 antibodies with other treatments.
The chemokine receptor CCR4 is abnormally expressed on malignant cells as
well as on leukocytes in human renal cell cancer, RCC. Patient plasma levels
of the CCR4 ligands and their ratio reflect this abnormality and have prognostic
significance. Both a small molecule CCR4 inhibitor and an anti-CCR4 antibody
had anti-tumor activity in an RCC model with a novel mechanism of action on
tumor-associated macrophages.
Conflict of interest: Corporate-sponsored research: Affitech AS.
32 Proffered Paper: Vessel co-option in colorectal cancer liver
metastases mediates resistance to conventional anti-angiogenic
therapy
34 Progress toward a biomarker discovery-to-development pipeline
in clinical proteomics
1
1
2
1
3
3
V. Thompson , S. Frentzas , P. Vermeulen , S. Foo , Z. Eltahir , G. Brown ,
D. Cunningham3 , A.R. Reynolds1 . 1 The Institute of Cancer Research, Tumour
Biology Team, London, United Kingdom, 2 GZA Hospitals St. Augustinus,
Translational Cancer Research Unit, Antwerp, Belgium, 3 The Royal Marsden
Hospital, Oncology, London, United Kingdom
Introduction: The growth of metastatic tumors is considered to require
sprouting angiogenesis and this hypothesis has driven the development and
clinical application of vascular endothelial growth factor (VEGF) targeted
agents, such as bevacizumab. Responses to such agents in the clinic,
including in colorectal cancer (CRC) patients, are variable. The biology behind
the variable responses are not yet understood and we have no way of selecting
patients who may benefit. It is now evident that tumors can also utilize a
number of ‘VEGF-independent’ angiogenesis mechanisms, the existence of
which may limit the efficacy of conventional therapy. One such mechanism
is ‘vessel co-option’, whereby tumors hijack existing local blood vessels as
they invade into host tissue. Published studies reported this mechanism to
occur in ~28−30% of CRC liver metastases. The purpose of this study is:
(1) examine whether vessel co-option is associated with a lack of response
to Bevacizumab in advanced colorectal cancer patients, (2) identify whether
inhibition of pathways associated with vessel co-option sensitizes to antiangiogenic therapy in vivo.
Materials and Methods: Here we present data from a retrospective study of
patients with CRC liver metatases that were treated with bevacizumab whereby
responses to therapy were measured by radiological morphology (contrast
enhanced CT) or pathology (cell viability). The presence of vascular co-option
was quantified by histopathological analysis of resected specimens. Further to
this, we modeled this growth pattern in vivo by intrahepatic injection of HT29luc colorectal cancer cells into CB17-SCID mice. Candidate proteins believed
to be involved were stable knocked down using shRNA lentiviral technology.
Results: We demonstrate, in this patient cohort, the presence of vessel cooption in liver metastases was strongly associated with lack of response to
bevacizumab. We also show that a similar result is observed in a mouse
model of colorectal cancer liver metastasis. Interestingly, when we reduce
actin nucleation activity, via stable shRNA targeted knockdown of ArpC3 (p21ARC), in HT29 colorectal cancer cells, vessel co-option is compromised and
sensitivity to VEGF-targeted therapy is improved in vivo.
Conclusion: This data demonstrate a potential role for vessel co-option as
a negative predictive biomarker for anti-angiogenic therapy and may also
have consequences for the development of novel therapies for targeting tumor
angiogenesis.
33 Targeting cancer-related inflammation
F. Balkwill1 . 1 Barts and The London School of Medicine and Dentistry,
Cancers are driven by complex networks of cytokines and chemokines that
enable communication between the malignant cells and the many other cell
types of the tumor microenvironment.
Pre-clinical experiments have shown that targeting these networks with
therapeutic antibodies or small molecule inhibitors can decrease tumor growth
and spread. The challenge now is to translate these promising results to clinical
trial. This talk will focus on the cytokine IL-6 and the chemokine receptor
CCR4.
IL-6 is a key regulator of an inflammatory cytokine network of highgrade serous ovarian cancer, HGSC. Clinical, pre-clinical and in silico
experiments showed that antibodies to IL-6 can have multiple actions within
the tumor microenvironment including reductions in cytokine production,
Sunday 6 July 2014
14:00−15:45
Symposium
Cancer Biomarkers
S.A. Carr1 . 1 Broad Institute of MIT and Harvard, Proteomics, Cambridge,
USA
Better biomarkers are urgently needed to improve diagnosis, guide molecularly
targeted therapy, and monitor activity and therapeutic response across a
wide spectrum of disease. Proteomics methods based on mass spectrometry
hold special promise for the discovery of novel biomarkers that might
form the foundation for new clinical blood tests, but to date little has
been delivered. Proteomics-based biomarker discovery has been severely
hampered by inadequate number and quality of samples, poor study
design, and technological approaches lacking sufficient sensitivity, quantitative
precision, and capacity to analyze statistically relevant numbers of samples.
Another key problem has been the lack of robust quantitative methods to
credential candidate protein biomarkers in larger numbers of patient samples
prior to clinical evaluation. This problem extends into biology where the lack
of highly specific affinity reagents for novel candidate proteins and modified
peptides with sufficient sensitivity, specificity, reproducibility and throughput
has significantly hampered our ability to understand dynamic, protein-based
biological processes.
In our biomarker studies we are addressing both of these serious barriers.
In the discovery phase, we are employing multiplexed, quantitative MS
technologies that enable analysis of larger numbers of patient samples with
improved precision leading to better-qualified candidates. For verification of
candidate biomarkers, we are developing targeted mass spectrometry-based
technologies to screen and quantify low abundance proteins and modified
peptides in a variety of biological contexts including human tissue and
plasma. These assays are highly multiplexed, sensitive and specific and
do not suffer from interferences that plague immunoassays. Targeted MSbased approaches are helping to usher in a new era of quantitative biology
where proteins of interest and their modifications can be robustly measured
in any biological context. We are applying these quantitative approaches in
the context of a generalizable proteomics-based discovery-through-verification
pipeline to identify early biomarkers of cardiovascular injury, breast and
ovarian cancer. The same technologies are also being brought to bear to
understand response and resistance to therapy. These studies are beginning to
demonstrate that modern proteomic technologies when coherently integrated
can yield novel, credentialed protein and peptide biomarker candidates of
sufficient merit to warrant real clinical evaluation and to shed light on biological
function.
35 Personalised proteotypes and their association with disease
R. Aebersold1 . 1 ETH Zurich, Institute of Molecular Systems Biology, Zürich,
Switzerland
Powerful genomic technologies are now capable of determine genetic
variability at a genomic level and at unprecedented speed, accuracy and
(low) cost, and Genome Wide Association Studies (GWAS) have indicated the
(frequently weak) linkage of specific loci to disease phenotypes. The question
how genetic variability is translated into phenotypes is fundamental to biology
and of critical practical importance for medicine.
In this presentation we will discuss emerging computational and quantitative
proteomic technologies to relate genotypic variation to the proteotype, the
quantitative state of the proteome of a specific (disease) tissue. We will show
that precise and highly reproducible proteotype measurements can be now
generated at high throughput by the targeted proteomic methods selected
reaction monitoring (SRM) and, at higher throughput, by SWATH-MS.
We will discuss the principles of these mass spectrometric methods,
discuss the computational challenged they pose for data analysis and
demonstrate with selected applications their ability of proteotype data to
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classify clinically relevant samples (tissues/plasma) with respect to disease
relevant phenotypes, including clinical outcome or drug sensitivity.
36 Proffered Paper: Proteogenomic analysis of human breast cancer
connects genetic alterations to phosphorylation networks
P. Mertins1 , K.R. Clauser1 , D.R. Mani1 , M. Gillette1 , D. Fenyo2 , P. Wang3 ,
A. Paulovich4 , M. Ellis5 , S.A. Carr1 , Clinical Proteomic Tumor Analysis
Consortium6 . 1 The Broad Institute of MIT and Harvard, Proteomics Platform,
Cambridge, USA, 2 New York University, School of Medicine, New York, USA,
3
Icahn School of Medicine at Mount Sinai, Genetics and Genomic Sciences,
New York, USA, 4 Fred Hutchinson Cancer Research Center, Department
of Medicine/Division of Oncology, Seattle, USA, 5 Washington University
School of Medicine, Division of Medical Oncology, St. Louis, USA, 6 National
Institutes of Health, National Cancer Institute, Bethesda, USA
Background: The genetic landscape of human breast cancer has
been well defined in The Cancer Genome Atlas (TCGA) project. Mass
spectrometry (MS)-based global proteome and phosphoproteome analyses
may provide an orthogonal approach to genomic studies to further improve the
molecular taxonomy and our understanding of breast cancer. Central questions
in breast cancer biology that will be addressed in this study are: (1) Which
genomic characteristics are executed at the protein level? (2) How is the
molecular taxonomy of breast cancer reinforced and revised by protein and
phosphorylation data? and (3) What phosphorylation-driven signaling networks
emerge from genetic alterations?
Material and Methods: We analyzed 105 human breast cancer samples that
have been previously genetically characterized by the TCGA project. Tumor
samples were analyzed by global shotgun-proteomics and phosphoproteomics
using iTRAQ4-plex peptide labeling. All mass spectrometry data was acquired
using nearly 5,000 h of measurement time on a Q Exactive instrument and the
data was analyzed in Spectrum Mill using patient-specific RNA-sequencing
derived protein databases.
Results: In total we quantified >16,000 proteins and >70,000 phosphorylation
sites, with an average of >12,000 quantified proteins and >20,000
phosphorylation-sites for each tumor. Only 1−2% of all 9,600 genetically
encoded somatic single amino acid variants and 1−2% of 36,000 alternative
splice junctions were detected at the protein level despite the very
comprehensive proteome coverage obtained. While the global mRNA protein
abundance correlation was rather low (Spearman’s correlation of 0.25), we
found very good correlation for most protein kinase gene amplifications
for mRNA, protein and phosphoprotein abundance. Hierarchical clustering
analysis of both the proteome and the phosphoproteome data yielded an
overlapping set of three major clusters: a basal-like, a luminal and a new,
previously uncharacterized group. The two most recurrently mutated genes
in human breast cancer are PIK3CA and TP53 at frequencies of 30−40%.
Comparison of PIK3CA or TP53 mutated vs non-mutated tumors highlights
specific phosphorylation signaling events downstream of mutated PI3-kinase
and increased phosphorylation of cell cycle check point kinases in p53-mutated
tumors. Network and pathway analysis is being performed to comprehensively
integrate genetic and phospho-/proteomic alterations in one model. The effects
observed in human tumors will be compared to a set of 16 patient-derived
xenograft tumors that will allow drug efficacy studies in fully genetically
characterized tumors in the future.
Conclusions: Proteogenomic analysis of human breast cancers at
unprecedented proteome and phosphoproteome coverage further extends
the molecular annotation of breast cancer and reveals novel PI3K and p53
mutation specific phosphorylation events.
37 Recent developments in the technology of mass
spectrometry-based clinical proteomics
M. Mann1 , F. Cosica1 , N. Kulak1 , G. Pichler1 , S. Tyanova1 , K. Shaman1 ,
J. Wisniewski1 , J. Cox1 . 1 Max-Planck Institute for Biochemistry, Department
Proteomics and Signal Transduction, Martinsried, Germany
Mass spectrometry-based proteomics, particularly in a quantitative and high
resolution format, has become a very powerful technology to study gene
expression at its ‘end point’ − the level of proteins [1−3]. Here we describe
a state of the art workflow that now allows measuring accurate expression
changes for a very large fraction (more than 10,000 proteins) of mammalian
proteomes. This will be discussed using a recent project on 11 commonly
used mammalian cell lines. A current focus of our laboratory is rapid, ‘single
shot’ analysis of the proteome, which drastically increases sensitivity while
decreasing measuring time. Apart from ‘expression proteomics’, our generic
workflow can be applied to many other aspects of the dynamic proteomics,
including protein interaction, protein turnover, protein secretion [4] as well
as the detection and quantification of post-translational modifications. These
capabilities will be exemplified with applications from different areas of cell
biology and biomedicine.
Quantitative mass spectrometry is also a powerful technology for the study of
the cancer proteome. Our group has analyzed proteomics changes in breast
cancer, colon cancer, prostate cancer and many others. In contrast to mRNA
or genomic studies, proteomics yield a quantitative picture of the changes in
the entirety of the tumor proteins. We will show examples of how this can be
used to classify closely related tumor types and to study the tumor stromal
interface, amongst others.
Reference(s)
[1] Aebersold, R. & Mann, M. Mass spectrometry-based proteomics. Nature
422, 198–207 (2003).
[2] Cox, J. & Mann, M. Quantitative, high-resolution proteomics for data-driven
systems biology. Annual review of biochemistry 80, 273–299 (2011).
[3] Mann, M., Kulak, N.A., Nagaraj, N. & Cox, J. The coming age of complete,
accurate, and ubiquitous proteomes. Molecular cell 49, 583–590 (2013).
[4] Meissner, F., Scheltema, R.A., Mollenkopf, H.J. & Mann, M. Direct
proteomic quantification of the secretome of activated immune cells.
Science 340, 475–478 (2013).
Sunday 6 July 2014
14:00−15:45
Symposium
Cell Death/Autophagy
38 Points of interface between autophagy and apoptosis
A. Kimchi1 , Y. Gilad1 , A. Rubinstein1 , R. Shilioh1 , L. Wigdorovitz1 . 1 Weizmann
Institute of Science, Faculty of Biochemistry Department of Molecular
Genetics, Rehovot, Israel
Apoptosis and autophagy are two distinct biological processes, each driven by
a different set of protein–protein interactions. While many of the autophagic
genes have been identified so far (the Atg genes) and organized in multiprotein complexes, the mode of interactions between these complexes is
often unknown. In addition, there is a significant cross-talk between these two
biological processes driven by direct physical interactions between apoptotic
and autophagic proteins. In attempts to identify points of interface between
these two processes we performed siRNA screens with autophagic sublibraries searching for dual function proteins. This function-based screen
revealed that the autophagic protein Atg12 displays a pro-apoptotic function
by interacting with members of the Bcl-2 family and inducing mitochondrial
based caspase activation. Unlike its canonical function in autophagy, where
Atg12 conjugates to Atg5 and together with Atg16 functions as a E3 like
ligase driving LC3 lipidation, the pro-apoptotic function requires the free form
of Atg12. Phosphorylation by p38a strongly inhibited the effect of Atg12 on LC3
lipidation and the subsequent autophagic flux, whereas binding to Bcl-2 was
not interrupted, suggesting an efficient switch between the two processes by
this phosphorylation. We performed in our lab large scale unbiased screens
for protein–protein interactions using the protein fragment complementation
assay (PCA) in attempts to reveal novel protein interactions in autophagy and
in its cross talk with apoptosis. We constructed a library that encompasses 63
apoptotic and autophagic proteins, and applied it in a platform that enables
detection of protein–protein interactions in a high-throughput manner. By
running an unbiased screen of approximately 3800 interactions between all
the protein pair combinations we discovered new interactions among the
autophagic proteins, and established a global landscape of their connectivity to
apoptosis. Our work underscores the intricate interactions between these two
processes suggesting that their disruption has important patho-physiological
consequences including cancer development. These points of interface provide
alternative ways in killing tumor cells and may serve as potential targets for
drug treatment.
39 Autophagy and cell death
S. Fulda1 . 1 Goethe-University Frankfurt, Institute for Experimental Cancer
Research in Pediatrics, Frankfurt, Germany
Autophagy exerts dual functions in cancer, acting as both a tumor suppressor,
for example, by preventing the accumulation of damaged proteins and
organelles, and as a tumor promoter that supports tumor growth. Many
anticancer therapies engage autophagy as part of a cellular response.
However, the question of whether or not autophagic activity in cells undergoing
cell death is the cause of death or whether it is actually an attempt to support
survival in response to cellular stress conditions has been discussed with
great controversy. Recently, we identified Obatoclax (GX15-070)-stimulated
assembly of the necrosome on autophagosomal membranes as a key event
that connects GX15-070-stimulated autophagy to programmed cell death via
necroptosis. Since autophagy is commonly engaged as part of protective
antitumor response to cytotoxic therapies, further insights into the molecular
S11
basis of autophagic cell death will facilitate the development of new anticancer
drugs that interfere with autophagy signaling pathways to switch autophagy
into a cell death program.
T, Petersen NHT, Jäättelä M. Genetic and pharmacological inhibition of
acid sphingomyelinase inhibits autophagosome maturation. Manuscript in
preparation.
40 Proffered Paper: miRNA induces escape from NK cell-mediated
cytotoxicity and resistance towards apoptosis in breast cancer
Sunday 6 July 2014
C. Breunig1 , M. Küblbeck1 , M. Miller2 , D. Antonelli1 , C. Wirth1 , N. Erdem1 ,
Y. Yarden3 , A. Cerwenka2 , S. Wiemann1 . 1 Division of Molecular Genome
Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany,
2
Innate Immunity, Research Program Tumor Immunology, DKFZ,
3
Departments of Biological Regulation, Weizmann Institute of Science,
Rehovot, Israel
Introduction: Breast cancer is the most frequent type of cancer in women
(23% of all cancers), ranking first among cases of incidence, and is still the
second leading cause of cancer mortality in women worldwide. The escape
of tumor cells from the immune system by reducing immune cell recognition
and/or resistance towards the killing by immune cells correlate with poor patient
survival. The molecular mechanisms leading to resistance are diverse and
incompletely understood. Recent progress in cancer biology has revealed that
miRNAs are frequently deregulated in various human cancers, including breast
cancer.
Material and Methods: MCF10A and diverse breast cancer cell lines were
used to study the effect of the miRNA on NK cell-mediated cytotoxicity and
TRAIL and FasL-induced apoptosis. MCF10A cells were co-cultured with
primary human natural killer (NK) cells and activation and killing ability of
NK cells were measured. Diverse proliferation and apoptosis assays were
performed after overexpression of the miRNA in the presence of TRAIL and
FasL. MiRNA target genes were identified by luciferase assays and validated
with qRT-PCR and Western Blots. Clinical relevance of the miRNA and miRNA
target genes were analyzed using different breast cancer patient datasets.
Results and Discussion: We have identified a miRNA that is higher
expressed in aggressive types of breast cancer and to be associated with
decreased survival of breast cancer patients. In vitro experiments revealed
an inhibitory function of this miRNA for the recognition by and activation of
NK cells leading to a decreased killing of MCF10A cells. We identified NK
cell activation ligands as targets of the miRNA and to be responsible for
the decreased NK cell activation and killing. Moreover, we found the same
miRNA being involved in resistance mechanisms of TRAIL and FasL-induced
apoptosis, including the identification of the miRNA targets that are responsible
for the observed phenotype.
Conclusion: We have unraveled the mechanism of action of a miRNA and its
involvement in the aggressive phenotype of breast cancer by inducing escape
from NK cell-mediated cytotoxicity and resistance towards apoptosis.
17:15−18:00
Award Lecture: Anthony Dipple Carcinogenesis
Award
42 The mutant p53-cancer stem cells and drug resistance paradigm
V. Rotter1 . 1 Weizmann Institute of Science, Rehovot, Israel
Our working hypothesis is that drug resistance and cancer recurrence are
mediated by cancer stem cells that are expressing mutant p53. Based on
data accumulated, we suggest that wild type p53 plays a regulatory role in
cell development, cell differentiation and stem cell at large. Accordingly, and
in agreement with others, we observed that wild type p53 exerts a negative
effect of the rate of induced reprogrammed stem cells (iPSCs). However, we
found that iPSCs, as well as adult stem cells that are expressing mutant p53,
were found to exhibit a cancer stem cell phenotype. This is manifested in cells
exhibiting, among others, acquisition of a drug resistance that is mediated by
the specific induction of the well-characterized drug resistance genes, such as
MDR1, that are members of the ABC family and specific genes members of
ALDH family.
In all, the notion that p53 plays a regulatory role in the life of stem cells
coupled with the observations that p53 mutations may contribute to the
evolvement of cancer stem cells, makes it challenging to speculate that drug
resistance and cancer recurrence are mediated by cancer stem cells that are
expressing mutant p53. Accordingly, we would suggest that efficient cancer
therapy of mutant p53-expressing-tumors should be based on a combination
of chemotherapy and a p53-based therapy that target both tumor bulk and
cancer stem cells.
Reference(s)
Yoav Shetzer, Hilla Solomon, Gabriela Koifman, Alina Molchadsky, Stav
Horesh and Varda Rotter. The paradigm of mutant p53-cancer stem cells
and drug resistance (2014) Carcinogenesis, Rev in press 2014.
Sunday 6 July 2014
18:15−19:00
Award Lecture: Carcinogenesis Young Investigator
Award
41 Sphingomyelin metabolism as a target for cancer therapy
M. Jäättelä1 . 1 Danish Cancer Society, Apoptose Laboratoriet, Copenhagen,
Denmark
Transformation is associated with a decreased stability of lysosomal
membranes and an enhanced sensitivity to lysosomal cell death pathways
induced by various anti-cancer drugs. This sensitization is at least partially
brought about by the increased cysteine cathepsin expression and activity and
cathepsin-mediated degradation of lysosomal membrane stabilizing proteins
LAMP-1 and LAMP-2. On the other hand, lysosomal heat shock protein 70
(Hsp70) promotes the survival of cancer cells by enhancing the activity of
lysosomal acid sphingomyelinase (ASM) thereby stabilizing the lysosomal
membranes. These data prompted us to investigate whether lysosomal ASM
could serve as a direct target for cancer therapy. Data presented here show
that ASM activity is essential for the lysosomal stability, autophagosome
formation/closure and development of multidrug resistance in transformed
cells. Importantly, ASM can be inhibited by an experimental anti-cancer agent
siramesine as well as by several widely used and relatively safe cationic
amphiphilic drugs (tricyclic antidepressants, antihistamines, antipsychotics and
calcium channel blockers) that trigger lysosomal cancer cell death even in
apoptosis- and multidrug-resistant cells. The striking cancer selectivity of
ASM inhibitors is associated with significantly reduced expression of the
ASM encoding sphingomyelin phosphodiesterase 1 (Smpd1/SMPD1) gene in
transformed cells and various human cancers. Thus, ASM inhibitors should
prove efficacious in tumors with low sphingomyelinase activity, or when
combined with classic chemotherapy, even to treat tumors that have acquired
therapy resistance.
Reference(s)
Petersen et al. Transformation-associated changes in sphingolipid metabolism
sensitize cells to lysosomal cell death induced by inhibitors of acid
sphingomyelinase. Cancer Cell 24:379, 2013.
Vindeløv SD, Corcelle-Termeau E, Linkermann A, Mograbi B, Bräsen JH,
Szyniarowski P, Pedersen AK, Adam D, Hofman P, Krautwald S, Farkas
43 Outside the coding genome: microRNAs make a big difference
L. He. University of California, Department of Molecular and Cell Biology,
Berkeley, USA
In comparative genomic studies, the number of protein-coding genes within
a given genome does not seem to correlate well with the developmental and
pathological complexity of the organism. Emerging evidence has identified
numerous non-coding RNAs (ncRNAs), whose diversity scales with genomic
complexity and greatly exceeds that of protein-coding genes. It is increasingly
clear that ncRNAs have the potential to constitute integral components of
numerous molecular pathways, yielding unusual functionality and expression
regulation. The overall focus of my lab is to understand the unique biological
functions and molecular regulation of various ncRNAs in development and
disease, with a particular focus on microRNAs (miRNAs). miRNAs, a class
of small regulatory ncRNAs with pivotal roles in post-transcriptional gene
regulation, have distinct gene structures, expression regulation, and biogenesis
regulation. Our studies extend beyond the functional characterization of
miRNAs/ncRNAs, with a unifying theme of elucidating the distinct biological
functions and molecular regulation conferred by miRNAs/ncRNAs. Using
an interdisciplinary approach that includes mouse genetics and genomics,
Xenopus genetics, cell biology, and molecular biology, our studies have
elucidated the fundamental molecular mechanisms that govern the unique
functional complexity of ncRNAs.
ScienceDirect
Monday 7 July 2014
Monday 7 July 2014
08:30−10:15
OECI Symposium: Personalised Cancer Medicine
44 The benefits of precision medicine will require us to examine
both what we do and how we do it
S. Friend1 . 1 Sage Bionetworks EU, Seattle WA, USA
Scientific approaches used to solve biomedical problems that worked well for
hypothesis driven questions may not work for the new data driven approaches
required to define precision medicine. Similarly, the ways that data generators
have usually been the data analyzers may also not apply. Furthermore, the
communication and recognition systems enabling current University based
research may not be most useful as larger and more diverse teams tackle
complex problems. I will focus on the need to solve problems in different ways
that include the use of provenance, leaderboards, and efforts to set up open
Challenges with full transparency and accountability as an alternate to exiting
methods.
clonal architecture of tumor types − a key factor influencing the emergence of
resistances − varies widely between tumor types.
Conclusions: In this study we have surveyed all currently available drugs
and the most comprehensive catalog of cancer drivers to explore the present
therapeutical landscape of targeted drugs. We show that they can be
prescribed for a small fraction of patients of major cancer types, although
these figures may be improved by using drug repurposing strategies. Indirect
targeting, which currently is used by PARP-inhibitors, may also improve the
therapeutical opportunities in cancer, specially to act against genes bearing
loss-of-function driver mutations.
47 Individualised systems medicine for cancer care
Monday 7 July 2014
08:30−10:15
Symposium
Cancer Immunology
45 OECI Lecture: Cancer genomics and plasma DNA: Towards new
classification of breast cancer and monitoring therapy response
48 Peptide-based immunotherapy of cancer
H. Rammensee1 . 1 Eberhard-Karls-University Tübingen, Department of
Immunology, Tübingen, Germany
46 Proffered Paper: Therapeutical landscape of cancer drivers
A. Gonzalez-Perez1 , D. Tamborero1 , C. Rubio1 , M. Schroeder1 , C. PerezLlamas1 , J. Deu-Pons1 , N. Lopez-Bigas2 . 1 University Pompeu Fabra,
Barcelona, Spain, 2 ICREA and University Pompeu Fabra, Research Unit on
Biomedical Informatics, Barcelona, Spain
Introduction: Precision cancer medicine depends on the availability of drugs
targeting products of those mechanisms to which tumor cells are addicted.
Here, we have explored the therapeutical landscape that is currently offered
by targeted drugs available at several clinical stages taking into account their
primary indications as well as their repurposing opportunities for the cancer
drivers identified in 6,898 samples of 28 tumor types.
Methods: We comprehensively identified mutational cancer drivers by
detecting complementary signals of positive selection left by tumor evolution in
the mutation pattern of genes across tumors, Next, we detected which of these
driver genes contain activating mutations and which ones loose their function
upon mutation. Third, we systematically gathered all information currently
available on cancer drugs designed to directly target any of the identified
driver genes. We considered all pharmaceutical drugs approved by the FDA
for their use in any human tumor type, as well as drugs currently undergoing
clinical trials, and candidate drugs in pre-clinical stages. We finally combined
all this information to present the landscape of cancer targeted therapeutic
as it stands today, both in terms of the mutational drivers with targeted drugs
and of fraction of patients of major tumor types who could directly benefit from
these therapies.
Results: We identified 464 cancer drivers; 274 of them were predicted to
act via gain-of-function or unknown mechanisms and we extensively searched
therapeutic agents for their direct targeting. FDA approved drugs are available
for only 5 of these drivers which were mutated in 235 samples (3.4%).
Seventeen other drivers are the primary targets of cancer drugs currently
undergoing clinical trials, potentially benefiting to 777 (11.2%) samples of the
present cohort. Interestingly, 659 other samples (9.5%) could benefit of a FDAapproved drug via tumor type or off-target repurposing. Finally, products of 18
other driver genes could be targeted by molecules in pre-clinical trials, 44
are predicted to interact with existing small molecules and 32 are potentially
accessible by immunotherapy or protein therapy. We also found that the
Essentially, all of our cells express surface molecules (HLA) that perform a
kind of “real time analysis of the functional genome”: Samples of peptides
representing all gene products expressed by a cell are presented by HLA
molecules. This allows our T cells to sense the integrity of cells. Alterations,
including mutations intrinsic to tumor cells, can thus be sensed by T cells. Each
individual tumor expresses its own set of mutations (dozens to hundreds),
some being essential for tumor growth, others being passenger mutations.
Both categories can give rise to immunogenic peptides recognizable by T cells
that can kill the tumor cells. In addition, tumor cells show overexpression of
several gene products which can give rise to correspondingly higher density of
the respective HLA-presented peptides that again can be sensed by T cells.
Thus, we are confronted with two layers of individuality: Each human being
has his or her own set of HLA molecules, with individual peptide specificities,
and each tumor has its own individually accumulated set of mutations
and overexpression of genes. It is possible to find peptides representing
overexpressed gene products shared by cancer patients expressing a
particular HLA-allele, such as HLA-A2. Such peptides have been used for
cancer immunotherapy with some success.
The ultimate consequence for the design of immunotherapeutic applications,
however, is to take an individualized approach that involves identifying the
mutations/alterations in the tumor, identifying the new peptides presented on
the tumor’s HLA, synthesizing the peptides individually for each patient, and
vaccinating the same patient with these peptides using appropriate adjuvants
and conditions. The feasibility as well as the challenges of this strategy will be
discussed.
Conflict of interest: Ownership: Cofounder of immatics GmbH and CureVac
GmbH. Advisory board: immatics Biotechnologies GmbH, CureVac GmbH,
Synimmune GmbH, Bamomab GmbH. Other substantive relationships: Coowner of various patents related to immunotherapies.
49 Blocking the PD-1/PD-L1 axis for cancer therapy
C. Drake1 . 1 Johns Hopkins Sidney Kimmel Comp Cancer Center, Baltimore,
USA
Accumulating clinical data show that blocking the PD-1/PD-L1 axis using
monoclonal antibodies results in objective clinical responses in patients
with Non-Small Cell Lung Cancer (NSLC), Melanoma and Kidney Cancer.
EACR-23 Oral Presentations, Monday 7 July 2014 / European Journal of Cancer 50, Suppl. 5 (2014) S12–S20
Interestingly, some treated patients appear to enjoy long-term responses
off treatment. As these agents move toward regulatory approval, numerous
unanswered questions remain, including the determination of predictive
biomarkers, the precise nature of induced anti-tumor immune responses, and
the relative contribution of tumor heterogeneity to clinical outcome. In addition,
increasing the response rate to these agents through combinatorial regimens
involving radiation therapy, combination immune checkpoint blockade, or the
combination of anti-cancer vaccines with checkpoint blockade remains an
active area of research in both the pre-clinical and the clinical settings. This
session will discuss these concepts and provide an overview of ongoing
developments in the field.
Conflict of interest: Ownership: Compugen, NexImmune. Advisory board:
BMS, Compugen, Dendreon, ImmunExcite, NexImmune, Roche. Corporatesponsored research: Aduro Biotech, BMS.
50 Proffered Paper: Overcoming immune evasion in ovarian and
breast cancer with anti-CD47 antibody blockade: A novel class
of immune therapy
A.K. Volkmer1 , S.B. Willingham2 , S.R. Tseng2 , P.Y. Ho2 , J.P. Volkmer2 ,
B.I. Sikic3 , R. Majeti2 , I.L. Weissman2 . 1 University Hospital Düsseldorf,
Department of Obstetrics and Gynecology, Düsseldorf, Germany, 2 Stanford
University, Institute for Stem Cell Biology and Regenerative Medicine,
Stanford, USA, 3 Stanford Hospital and Clinics, Division of Oncology, Stanford,
USA
Background: CD47 is an anti-phagocytic ‘don’t eat me’ cell surface protein
mediating cancer cell evasion of phagocytosis by cells of the innate immune
system such as macrophages and dendritic cells. We have identified CD47
as a novel target for ovarian cancer (OC) and breast cancer (BC). We have
developed a humanized anti-CD47 monoclonal antibody (mAb), clone Hu5F9G4 that binds CD47 and blocks it from interacting with its ligand SIRP-alpha on
phagocytes. We investigated the effects of Hu5F9-G4 on OC cell growth and
compared its efficacy to the treatment with carboplatin and the VEGF-inhibitor
Bevacizumab. We also tested the efficacy of HuF9-G4 on the primary site and
established metastases in BC.
Material and Methods: In vitro phagocytosis assays of OC cells were
performed with human and mouse macrophages. Human OC cells transduced
with a GFP-luciferase encoding lentivirus were transplanted intraperitoneally
(ip) into NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ mice (NSG). After tumor engraftment
was confirmed by in vivo imaging, mice were randomized into treatment
cohorts. Mice were treated by systemic, ip, injections with Hu5F9-G4, PBS,
Bevacizumab, Carboplatin or combination of the former for up to 8 weeks.
Tumor growth was monitored by bioluminescent imaging.
Treatment of residual primary site disease and distant metastases of BC
with Hu5F9-G4 was assessed in an orthotopic, metastatic patient BC
xenotransplantation model. BC cells previously transduced with GFP-luciferase
encoding lentivirus were transplanted into the mammary fad pad of NSG mice.
After formation of primary site tumors and lung metastases primary tumors
were resected and mice were treated for 4 weeks with Hu5F9-G4 or PBS.
We have conducted studies in non-human primates to establish preclinical
pharmacokinetics and toxicology of Hu5F9-G4.
Result: CD47 blockade with Hu5F9-G4 enabled phagocytosis of OC cells
in vitro, inhibited tumor growth in vivo and prolonged survival significantly
compared to the treatment with Carboplatin and Bevacizumab.
Treatment of Hu5F9-G4 eliminated BC lung metastases and inhibited the
regrowth of resected primary BC tumors. Therapeutic serum levels of
Hu5F9-G4 determined in these studies have been established to be safely
administrable to non-human primates.
Conclusions: We have established blocking of CD47 with mAbs as a novel
immunotherapeutic approach for OC and BC. We have developed Hu5F9G4, a humanized anti-CD47 mAb that shows the desired therapeutic profile
in preclinical studies. Based on these data, and supported by the California
Institute for Regenerative Medicine (CIRM), Stanford has elected to move
Hu5F9-G4 into a full clinical development program.
51 How T cells may distinguish stress from normality in an epithelium
A. Hayday1 , F. Kyle1 , O. Nussbaumer1 , D. Enting1 , M.L. Iannitto1 . 1 King’s
College London, Division of Immunology Infection & Inflammatory Diseases,
Tumour infiltrating lymphocytes (TILs) characterise many solid tumours but
their efficacy is limited. Such immune-suppression has appropriately been
assigned to a spectrum of causes including tumour-associated myeloid
suppressor cells; regulatory T cells; and the prominence of negative costimulators (checkpoints). Moreover, clinical efficacy of checkpoint blockade
validates these perspectives. Nonetheless, our view is that rather than
modelling TILs on systemic T cells that infiltrate tissues, they should better be
viewed in relation to T cells that naturally associate with tissues. In that regard,
we shall present our current studies of how mouse and human intraepithelial
S13
T cells are regulated by epithelial cells at steady state and under conditions of
stress and shall consider the parallels that these forms of regulation have with
primary TILs studied from breast cancer. Based on these studies, we propose
a novel molecular axis for clinical targeting in efforts to re-activate TILs locally,
rather than suffering the consequences of systemic immune de-repression.
Monday 7 July 2014
08:30−10:15
Symposium
Non-coding RNAs in Cancer
52 Long non-coding RNA and cancer
S. Diederichs1 . 1 DKFZ − German Cancer Research Center, Molecular RNA
Biology & Cancer, Heidelberg, Germany
The majority of the human genome is transcribed into non-protein-coding RNA.
Hence, RNA is the primary product of the cancer genome. We have defined
the ncRNA expression landscape of lung, breast and liver cancer providing a
comprehensive expression map of over 17,000 long ncRNAs. We discovered
many new lncRNAs associated with cancer as well as tissue-, histology- and
prognosis-specific ncRNA signatures.
The long non-coding RNA MALAT1 was one of the first lncRNAs associated
with cancer: it is a highly conserved nuclear ncRNA and a predictive marker
for metastasis development in lung cancer. However, its high abundance and
nuclear localization have greatly hampered its functional analysis since it is
only inefficiently knocked down by RNA interference (RNAi).
To uncover its functional importance, we developed a MALAT1 knockout
model in human lung tumor cells by genomically integrating RNA destabilizing
elements site-specifically into the MALAT1 locus using Zinc Finger Nucleases
(ZFN) or CRISPR technology. This approach yielded a more than 1000-fold
silencing of MALAT1 providing a unique loss-of-function model.
Proposed mechanisms of action of MALAT1 include regulation of splicing or
gene expression. In lung cancer, MALAT1 does not alter alternative splicing
but actively regulates gene expression inducing a signature of metastasisassociated genes. Consequently, MALAT1-deficient cells are impaired in
migration and form fewer tumor nodules in a mouse xenograft model.
Encouraged by this discovery of the essential function of MALAT1 in lung
cancer metastasis, we wanted to analyze whether MALAT1 could also be
therapeutically targeted: We developed Antisense oligonucleotides (ASOs)
effectively blocking MALAT1 expression in the cell culture and in the animal.
Notably, MALAT1-ASO treatment prevents metastasis formation after tumor
implantation. Thus, targeting MALAT1 with antisense oligonucleotides provides
a potential therapeutic approach to prevent lung cancer metastasis with
MALAT1 serving as both, predictive marker and therapeutic target.
Lastly, regulating gene expression, but not alternative splicing is the critical
function of MALAT1 in lung cancer metastasis.
In summary, ten years after the discovery of the lncRNA MALAT1 as a
biomarker for lung cancer metastasis, our loss-of-function model unravels
the active function of MALAT1 as a regulator of gene expression governing
hallmarks of lung cancer metastasis.
53 miRNA−protein interaction networks in cancer
S. Wiemann1 , A. Bott2 , I. Keklikoglou2 , C. Giacomelli2 , A. Balwierz2 ,
S. Uhlmann2 , H. Mannsperger2 , U. Korf2 , C. Breunig2 . 1 German Cancer
Research Center, Heidelberg, Germany, 2 German Cancer Research Center,
Molecular Genome Analysis, Heidelberg, Germany
Cancer development and progression are mostly driven by alterations in
networks of signaling pathways. miRNAs are increasingly recognized as
regulators of signaling and, thus, as contributors to health and disease
phenotypes. We perform whole genome miRNA (miRome) screens to unravel
miRNA regulation of cellular pathways and signaling networks. In a large-scale
miRNA screening approach combined with quantitative targeted proteomics
readout we uncovered co-regulation patterns of miRNAs and their target
proteins in the EGFR pathway and the cell cycle control. Network-analysis
revealed redundancy in the activities of individual miRNAs on target genes
that act in the same functional modules. A screen investigating miRNAs
regulating the NF-úB pathway identified microRNA families impinging on
NF-úB activity. Deep analysis revealed miRNAs that directly target genes in
functionally connected pathways thus building a new regulation layer of cancer
phenotypes. Our current work embarks on the impact the miRNome has on
other, metastasis-relevant pathways as well as on tumor-interactions within
their microenvironment. This shall help to generate a comprehensive map of
the miRNA/pathway interaction network, and form the basis for experimental
testing of combinatorial perturbations in the network aimed at abrogating
cancer phenotypes.
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54 Proffered Paper: Hypoxic regulation of the long non-coding
transcriptome in breast cancer
H. Choudhry1 , A. Albukhari2 , J. Schodel3 , S. Oikonomopoulos1 , S. Haider2 ,
P. Ratcliffe4 , I. Ragousis5 , D. Mole4 , A. Harris2 . 1 University of Oxford,
Wellcome Trust Centre for Human Genetics, Oxford, United Kingdom,
2
University of Oxford, Department of Oncology, Oxford, United Kingdom,
3
Friedrich-Alexander-University, Department of Nephrology and Hypertension,
Erlangen, Germany, 4 University of Oxford, Henry Wellcome Building for
Molecular Physiology, Oxford, United Kingdom, 5 McGill University, Genome
Quebec Innovation Centre, Montréal, Canada
Introduction: Transcriptional responses to hypoxia are central to the
pathogenesis of many types of cancer. To date, pan-genomic analyses of
these transcriptional responses have focussed on protein-coding genes and
microRNAs. However, the role of other classes of non-coding RNAs, in
particular lncRNAs, in the hypoxia response is largely uncharacterised.
Material and Method: We undertook an integrated pan-genomic analysis of
normoxic and hypoxic MCF7 breast cancer cells, employing RNA-seq of polyA
selected and ribosomally depleted transcripts together with ChIP-seq for the
major hypoxia-inducible transcription factor HIF and for chromosomal markers
of active transcription (RNApol2 and histone H3K4 methylation).
Results and Discussion: Significant numbers of lncRNAs were up-regulated
in hypoxia and these were associated both with epigenetic marks of increased
transcription and with HIF binding. The most hypoxia upregulated lncRNA
was NEAT1, which is a direct transcriptional target of HIF-2 but not HIF-1.
However, the role of hypoxic NEAT1 in cancer has not been previously studied.
We demonstrate that hypoxic NEAT1 induction is common in breast cancer
cell lines and xenografts. NEAT1 directly induces the formation of nuclear
paraspeckles in hypoxia, contributes to tumourigenicity in cell proliferation and
colony forming assays and reduces rates of apoptosis. Finally, in a large cohort
of 2000 breast cancers, high levels of NEAT1 correlated with poor clinical
outcome.
Conclusion: Our findings extend the role of the hypoxic transcriptional
response in cancer into the spectrum of non-coding transcripts. These results
will provide new insights on functional and clinical potential of hypoxia
regulated lncRNAs which may act as novel therapeutic targets in future.
55 Nuclear RNAi and cell fate
Monday 7 July 2014
10:45−12:30
Symposium
New Strategies in Early Detection
56 The NCI early detection research network: Charting the course
of biomarker research
S. Srivastava1 . 1 National Cancer Institute, Bethesda MD, USA
Early detection of cancer can dramatically improve outcomes. In 2000,
NCI’s Division of Cancer Prevention created Early Detection Research
Network (EDRN), an investigator-driven network designed to conduct
translational research that identified markers both for the early detection of
cancer and for cancer risk. EDRN focuses on the goal of creating validated
biomarkers ready for large-scale clinical testing and eventual application.
Without a doubt, real progress has been made by this consortium of more
than 300 investigators and 40 private sector and academic institutions.
These scientists represent diverse disciplines including genomics, proteomics,
metabolomics, bioinformatics and public health. Research collaborations take
place within an environment of teamwork across different disciplines and
laboratories focused on achieving common goals, such as:
• Developing and testing promising biomarkers and technologies to obtain
preliminary information to guide further testing;
• Evaluating promising, analytically proven biomarkers and technologies, such
as measures of accuracy, sensitivity, specificity and, when possible, potential
predictors of outcomes or surrogate endpoints for clinical trials;
• Analyzing biomarkers and their expression patterns to serve as background
for large, definitive validation studies;
• Collaborating with academic and industrial leaders to develop highthroughput, sensitive assay methods;
• Conducting early phases of clinical and epidemiological biomarker studies;
and
• Encouraging collaboration and dissemination of information to ensure
progress and avoid fragmentation of effort.
EDRN is a leader in defining and using criteria for the validation of biomarkers −
an essential condition for scientific progress. Today, EDRN is recognized
nationally and internationally for its interdisciplinary approach to biomarker
validation which has led to approval of five biomarkers by FDA. It has
developed collaborations with China, Cancer Research UK and European
Advanced Translational Research Infrastructure (www.eatris.eu), and Turkey
on biomarker research. EDRN is in discussion with Cancer Institute/Hospital
of the Chinese Academy of Medical Sciences to establish an EDRN-like
structure in China. The speaker will describe the collaborative structure of
EDRN and opportunities for collaboration. Examples of recently approved
biomarker tests will be presented to illustrate the guidelines for biomarker
discovery, development and validation.
57 Early detection biomarkers for oesophageal cancer
R. Fitzgerald1 , R.C. Fitzgerald2 . 1 Hutchinson/MRC Research Centre,
Cambridge, United Kingdom, 2 Hutchinson/MRC Research Centre, MRC
Cancer Unit, Cambridge, United Kingdom
Survival rates in cancer are directly related to the degree of disease spread
at the time of diagnosis and cancer can become advanced before any
symptoms are manifest. Oesophageal cancer is a prime example of this
problem. However, oesophageal adenocarcinoma has a premalignant state
called Barrett’s oesophagus which can gradually progress through stages of
low and high grade dysplasia towards invasive cancer at a rate of 0.3% per
year. The holy-grail for this disease would be to have objective biomarkers
which could identify the small subgroup of patients at highest risk who require
endoscopic therapy prior to the development of symptomatic disease. At the
same time patients deemed to be very low risk could have their surveillance
intervals prolonged or even be discharged.
The state of the biomarker field for this disease will be reviewed including
previously published data to identify progression biomarker panels including
LOH and aneuploidy as well as methylation biomarkers. Recent work using
genome wide technologies has begun to catalogue the mutations and copy
number changes in Barrett’s oesophagus with dramatic changes suggesting
chromosome instability and diversity as progressors approach the diagnosis
of cancer.
In the Fitzgerald laboratory we have investigated several approaches to
developing clinical applicable biomarkers including: (1) development of a
biomarker panel to predict cancer progression comprising histochemistry
for a lectin (AOL), image cytometry for abnormal ploidy and a consensus
diagnosis of LGD. The risk increases by 2.99 for each additional factor
present (95% CI 1.75–5.20), (Gastroenterology 2012); (2) development of a
90 gene panel suitable for a customised fluidics RNA chip to detect which
cases with low grade dysplasia are likely to behave like HGD, (unpublished);
(3) a molecular imaging approach using a fluorescently labelled lectin to
delineate inconspicuous areas at endoscopy (Nature Medicine 2012) and
(4) a non-endoscopic cell collection device called the Cytosponge™, which
when coupled to molecular markers such as TFF3 and marker of risk, is a
much less invasive and cost-effective alternative to endoscopy (BMJ 2010
and new data). These data will be discussed.
We hope that these novel approaches could enable us to identify the patients
at greatest risk for oesophageal cancer and ultimately improve outcomes for
patients.
Conflict of interest: Ownership: None. Advisory board: None. Board of
directors: None. Corporate-sponsored research: GSK research collaboration.
Other substantive relationships: The MRC has licensed Cytosponge
technology and associated biomarkers to Covidien. RCF could stand to benefit
in the future under the terms of the license agreement.
58 Proffered Paper: Noninvasive monitoring of tumour mutations
and dynamics in circulating DNA of non-small cell lung cancer
patients treated with EGFR inhibitors
D. Tsui1 , A.S.C. Wong2 , M. Murtaza1 , T. Forshew1 , R. Soo2 , H.L. Lim3 ,
B.C. Goh2 , D. Gale1 , T.M. Chin2 , N. Rosenfeld1 . 1 Cancer Research UK
Cambridge Institute, University of Cambridge, Cambridge, United Kingdom,
2
National University Cancer Institute, Department of Haematology-Oncology,
Singapore, Singapore, 3 Parkway Cancer Center, Singapore, Singapore
Targeted cancer therapies, (e.g. tyrosine kinase inhibitors, TKI), benefit a
subset of patients whose tumours harbour mutations in EGFR. Acquired
resistance develops, associated with mutations that emerge in response to
selective pressures. Identifying mutational changes during treatment is crucial
for timely decisions, but repeat tumour biopsies are invasive and rarely
available. Analysis of circulating tumour DNA (ctDNA) in plasma offers an
opportunity to continuously screen for mutations and measure their relative
levels, identifying predominant clones.
We collected serial plasma samples from non-small cell lung cancer patients
receiving EGFR-targeted therapy (gefitinib), and quantified mutations in EGFR,
TP53, PTEN and PIK3CA in plasma by digital PCR and tagged-amplicon
deep sequencing of ctDNA. EGFR mutations in plasma and tumour biopsies
showed excellent concordance. Dynamics of EGFR activating mutation in
plasma generally agree with radiological progression, whereas in some
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cases discordance was accompanied by increasing plasma fractions of other
potential drivers, for example mutations in TP53, suggesting selection of
predominant tumour clones as a result of the targeting agents. The EGFR
resistance-conferring T790M mutation was detected in plasma in over half of
the patients with previous EGFR-TKI resistance, as well as a subset of TKInaı̈ve patients months before radiological disease progression. One patient
who progressed on gefitinib had a ctDNA resistance-conferring/activating
mutation ratio of over 50% during first-line treatment. After switching to
chemotherapy, upon completion of treatment the ratio fell below 2%. On rechallenge with gefitinib the disease showed renewed sensitivity.
Serial quantitative mutational analysis in plasma can be used for monitoring
of the changes in relative abundance of activating and resistance-conferring
mutations, as well as to detect the emergence of alternative actionable targets.
It provides a rational framework for adaptive sequential therapy to improve the
management of lung cancer.
59 CTCs and cfDNA, will they be useful for early detection of cancer?
C. Dive1 , P. Crosbie2 , R. Shah3 , R. Metcalf4 , F. Blackhall5 . 1 CRUK
Manchester Institute, Clinical and Experimental Pharmacology, Manchester,
United Kingdom, 2 University of Manchester and University Hospital of South
Manchester, Respiratory Medicine, Manchester, United Kingdom, 3 University
Hospital of South Manchester, Thoracic Surgery, Manchester, United
Kingdom, 4 CRUK Manchester Institute University of Manchester, Clinical and
Experimental Pharmacology, Manchester, United Kingdom, 5 University of
Manchester, Institute of Cancer Sciences, Manchester, United Kingdom
Background: ‘Liquid biopsies’ for the detection of early cancers present
signiicant challenges regarding assay sensitivity. Certainly the prevalence of
circulating tumour cells (CTCs) in the peripheral blood of early non small lung
cancer (NSCLC) patients is low. We are evaluating sereval CTC technologies
to increase sensitivity and testing the hypothesis that number, phenotype or
genotype of CTCs in the pulmonary vein of patients with early stage, resectable
NSCLC may predict disease recurrence.
Surgical resection of NSCLC can produce long-term survival (5 year survival:
Stage I 58−73%, Stage II 36−46%, Stage IIIA 24%). However, recurrence
occurs in 50% of cases, most commonly at distant sites when it is almost
universally fatal. Standard staging methods do not reliably detect micrometastatic disease therefore CTC analysis may identify patients most at risk
of recurrence. We initiated a study to explore CTC prevalence in pulmonary
venous blood at the time of resection of early stage NSCLC. As the pulmonary
veins directly drain blood from the lungs we postulated that CTC number would
be higher than in matched peripheral blood.
Aim: To compare CTC number in peripheral and pulmonary vein blood at the
time of surgical resection of NSCLC by cellSearch and marker independent
technology platforms.
Methods: Ethically approved surgical case series of patients undergoing
resection of NSCLC at the Thoracic Surgical Department, University Hospital
of South Manchester. Pre-operative peripheral blood (10ml) was compared to
intra-operative pulmonary vein blood (10ml), taken prior to vessel ligation or
tumour manipulation. Sample (7.5ml) processing and analysis was undertaken
using CellSearch and by filtration.
Results: 34 patients were recruited (20M/14F). CTC count ranged from
0−4 in peripheral and 0–3093 in pulmonary vein blood, with CTC count
significantly higher in pulmonary vein blood (p = 0.002). CTCs were detected
more frequently in pulmonary compared to peripheral vein samples (1 CTC:
16/34, 47% vs. 7/32, 22%; p = 0.041) (2 CTCs: 14/34, 41% vs. 2/32, 6%;
p = 0.001) and circulating microemboli (CTM) only in pulmonary vein blood
(6/34, 18%). Pulmonary vein CTC count was positive (2 CTCs) in 4/12
(33%) patients with stage I and 10/22 (46%) patients with stage II−IV disease
(p = 0.49). Mesenchymal and epithelial CTCs were observed by filtration.
Pulmonary vein sampling was safe with no significant adverse events and
was possible irrespective of surgical approach.
Conclusion: CTC prevalence is significantly higher in pulmonary vein blood
compared to matched peripheral vain samples in patients undergoing surgical
resection of early stage NSCLC.
Ongoing studies and future Goals: A workflow from CellSearch enrichment
of CTCs to their isolation by DEPArray has been optimised. Single cell
molecular analysis of early CTCs is underway and matched plasma is being
collected for ctDNA analysis. The ability of pulmonary CTCs to generate
explant tumour models in immune-compromised mice will also be evaluated.
Monday 7 July 2014
10:45−12:30
Symposium
AEK Supported Symposium: Invasion and
Metastasis
60 Selection and adaptation during metastatic cancer progression
C. Klein1 . 1 Abteilung für Onkogenomik, Regensburg, Germany
Cancer is often regarded as a process of asexual evolution driven by genomic
and genetic instability. Mutation, selection and adaptation are by convention
thought to occur primarily within, and to a lesser degree, outside the primary
tumour. However, we previously noted in several cancers, including breast and
prostate cancer, that metastatic dissemination occurs often early, suggesting
that metastatic founder cells may lodge and evolve considerable periods of
time outside the primary tumour. The evidence for this was derived from
comparative genetic studies (primary tumour versus disseminated cancer cells,
DCC), analysis of DCIS patients and from transgenic animal models. We now
investigated a large cohort of 1027 melanoma patients and determined the
thickness of the primary melanoma at dissemination. We then investigated
melanoma DCCs and corroborated the breast cancer-derived concept of early
dissemination of immature cancer cells. Strikingly, upon earliest evidence of
metastatic colonization, early DCCs acquire critical somatic mutations, which
appear to drive the lethal metastatic process. Acquisition of these properties
enhanced the ability to engraft in immunodeficient NSG mice, providing strong
evidence that colonization in addition to dissemination is a fundamentally
critical step of metastasis. Abrogation of ectopic progression may provide
new opportunities for therapeutic intervention, which may change diagnostic
procedures and adjuvant therapies.
61 Role of the Hippo transducers YAP and TAZ in
mechanotransduction and cancer stem cells
S. Piccolo1 . 1 University of Padova, Department of Medical Biotechnologies
Section of Histology and Embryology, Padova, Italy
Cells perceive their microenvironment not only through soluble signals but
also in term of physical and mechanical cues, such as extracellular matrix
(ECM) stiffness or cell shape. By mechanotransduction systems, cells translate
these stimuli into biochemical signals controlling multiple aspects of cell
behavior, including growth, differentiation and malignant progression; but how
rigidity mechanosensing is ultimately linked to activity of nuclear transcription
factors remains poorly understood. Here we report evidence that the Yorkiehomologues YAP and TAZ as nuclear relays of mechanical signals exerted by
ECM rigidity and cell-shape. Crucially, YAP/TAZ emerge as universal mediators
of the biological effects of these structural and unsoluble cues.
YAP and TAZ are also regulated by EMT and disturbed cell polarity, a
hallmark of cancer. We found that TAZ regulates biological traits operationally
associated with breast Cancer Stem Cells (CSCs): TAZ is required to sustain
self-renewal and tumor-initiation capacities in cellular models of breast cancer
progression. Gain-of-TAZ in non-CSCs induces them to adopt CSCs-like
behaviors and promotes the transition from low- to high-grade tumors. TAZ
is downstream of EMT. Central to this regulation is the formation of an
endogenous complex between TAZ and the cell polarity determinant Scribble:
induction of EMT, or loss-of-Scribble, disrupts the association of TAZ with the
core Hippo kinases MST and LATS, leading to TAZ activation. This links breast
CSCs to the Hippo pathway, and reveals a mechanistic basis of the control of
Hippo kinases by cell polarity.
62 Proffered Paper: Cell-specific proteomic analysis of
contact-initiated tumour-endothelial signalling identifies novel
regulators of transendothelial migration
M. Locard-Paulet1 , L. Lim1 , J. Sinclair1 , C. Jorgensen1,2 . 1 The Institute
of Cancer Research, London, United Kingdom, 2 Cancer Research UK
Manchester Institute, The University of Manchester, United Kingdom
Background: Most cancer related deaths are caused by metastatic disease,
presenting formidable challenges to improved treatment. Haematological
spread rely on the ability of cancer cells to both intra- and extravasate. Although
these two events are essential for the spread of tumour cells, little is known
of the molecular networks underpinning these processes. Here we present
a novel approach to interrogate key processes underlying metastatic spread.
Critically, using a novel mass spectrometry technique, we describe the cellspecific analysis of contact-initiated signalling between tumour and endothelial
cells, and identify EPHA2 as a novel regulator in extravasation.
Methods: To identify regulators of tumour-endothelial cell adhesion (TEA) and
transendothelial cell migration (TEM) we conducted a phospho-proteomics
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analysis of contact-initiated signalling between tumour and endothelial cells.
To this end we utilized an in vitro model where highly metastatic human
breast cancer cells (MDA-MB-231s) were seeded onto a confluent monolayer
of human umbilical vein endothelial cells (HUVECs). Contact-initiated signals
were interrogated in a cell-specific manner through stable isotopic labelling in
cell culture (SILAC). Co-cultured cells were lysed and subjected to phosphoenrichment and were analysed by LC-MS/MS. Two different labelling strategies
allowed the establishment of a bidirectional map of the major phosphorylation
dependent signalling networks in tumour and endothelial cells respectively.
Results and Conclusion: We have identified 3266 unique phosphosites,
of which 43 and 21 were significantly regulated in tumour and endothelial
cells, respectively. Interestingly, we observed that the EPHA2 activation
loop phosphorylation is decreased specifically in cancer cells following their
interaction with endothelial cells. Silencing or overexpression of EPHA2
validated its involvement in both TEA and TEM in vitro. Moreover, the effect
was dependent on the identified phosphorylation site, where a site-specific
mutant abrogates the effects. Importantly, we confirmed this observation
in vivo, where decreased EPHA2 levels increase lung colonisation of MDAMB-231s. These studies propose a novel function of EPHA2, and its sitespecific phosphorylation in the regulation of adhesion and transendothelial
migration.
63 Tumour–stroma interactions in breast cancer progression
C. Isacke1 . 1 The Institute Of Cancer Research, London, United Kingdom
The interaction of tumour cells with stroma cells and stromal components not
only promotes tumour progression and metastasis, but also influences tumour
cell responses to chemotherapy, endocrine therapy and targeted agents. As a
consequence, there is an urgent need to identify strategies to efficiently target
these interaction pathways for the prevention or suppression of metastatic
disease and to overcome treatment-resistant tumour progression in advanced
breast cancer.
We are taking two main approaches to address this issue. First, we are
interrogating the tumour–stroma crosstalk pathways underpinning the striking
heterogeneity between different breast cancers in their ability to recruit and
activate a pro-tumourigenic stroma. These studies are focussed on the mechanisms involved in the activation of cancer-associated fibroblasts (CAFs) and
the different functional roles played by activated and non-activated CAFs. A key
finding from these studies has been the identification of Wnt7a as a key factor
secreted exclusively by aggressive tumour cells that promotes the acquisition
of an activated CAF phenotype and the remodelling of the extracellular matrix
as a permissive environment for tumour cell invasion. Second, we are probing
the later stages of the metastatic pathway and the response of metastatic
tumour cells to chemotherapy by performing functional in vivo RNAi screens.
A key finding from these studies has been the identification of ST6GalNAc2 as
metastasis suppressor that functions by sialylation of O-linked glycans on the
tumour cell surface to inhibit binding of galectin-3 and decreasing tumour cell
clustering and binding to the vasculature at metastatic sites. New data from
these projects will be presented at the meeting.
Monday 7 July 2014
10:45−12:30
Symposium
Mechanisms of Tumour Suppression
64 Role of TP53 and other tumour suppressors in breast cancer
development and progression
A. Børresen-Dale1 . 1 Oslo University Hospital Radiumhospitalet, Institute for
Cancer Research Department of Genetics, Oslo, Norway
In breast cancer, the TP53 gene harbors somatic mutations in around 25% of
the patients, and TP53 mutations have been associated with poor prognosis.
The prognostic impact of the different types of TP53 mutations across the
different molecular subtypes of breast cancer is poorly understood. We have
characterized the spectrum and prognostic significance of TP53 mutations
with respect to the PAM50 subtypes and Integrative Clusters (IC) in 1420
tumor samples from the METABRIC cohort. TP53 mutations were found in
28.3% and conferring a worse overall and breast cancer specific survival
(p < 0.001), and were also found to be an independent prognostic marker
in estrogen receptor (ER) positive cases. The mutation spectrum of TP53
varied between the breast cancer subtypes, and individual alterations showed
subtype specific association. TP53 mutation status was prognostic in patients
with Luminal B, HER2-enriched and Normal-like tumors, but not in patients
with Luminal A and Basal-like tumors. Similar observations were made in ICs,
where a prognostic effect of mutation status was found in IC1, IC4 and IC5.
TP53 mutated tumors also had a significant overall higher rate of genomic
instability, with most distinct difference in Basal-like cases. Generally higher
rates of these complex alterations (‘firestorms’) measured by CAAI were
also observed in mutated tumors predominantly in the Basal-like and HER2enriched subgroups. No difference in genomic instability could be observed
between different types of TP53 mutations. A synergistic effect of TP53
mutation, TP53 LOH and MDM2 amplification on survival was observed.
TP53 has been implicated in facilitating immune response, and in this study
the prognostic impact of mutations in combination with the measure of
lymphocytic infiltration was investigated. We identified an association in Basallike breast cancer between lymphocytic infiltration and the number of copies
of wild-type TP5, and demonstrated that ER negative patients with severe
infiltration and wild type TP53 had better outcome. Using the Nanodissect
algorithm we showed that elevated expression of a cytotoxic T lymphocytes
CTL gene signature is associated with longer survival outcome in Basal-like
tumors. These findings identify a new link between the TP53 pathway and
beneficial adaptive immune response in breast tumors not expressing the
estrogen receptor, supporting a connection between a lack of TP53 function
and the failure of tumor immunosurveillance.
To search for other tumor suppressors operating in breast and other
cancers, a large number of tumors (>2000) was systematically screened
for homozygous deletions using data from SNP arrays. We found several
established tumor suppressors, evidence of several others emerging (including
FAT1, BIRC2/BIRC3, TET1, MGMT and USP44), and also several novel
tumor suppressors (including CASP3, CASP9, RAD17, BAZ1A, CPEB3 and
SETD1B). Their importance in breast cancer and possible connections with
TP53 mutation status needs to be established.
65 Functional interplay between the DNA damage response and ARF
pathways in human cancer
V.G. Gorgoulis1,2,3 . 1 Molecular Carcinogenesis Group, Department of
Histology and Embryology, School of Medicine, Greece, 2 Biomedical
Research Foundation, Academy of Athens, Greece, 3 Faculty Institute of
Cancer Sciences, University of Manchester, Manchester Academic Health
Science Centre, United Kingdom
The mammalian cell has evolved over time defense responses to deal
with stressogenic signals and avoid malignant transformation. The DNA
damage response (DDR) checkpoint is vital in this process sensing
genotoxic/oncogenic insults and imposing the antitumor barriers of apoptosis
and senescence. Another pathway dealing with oncogenic stress is that
involving the Alternative Reading Frame (ARF) protein produced by the tumor
suppressor locus INK4/ARF. For a long time the DDR and ARF signaling
routes were thought to act independently. Recent evidence emanating from
our lab and others challenged this view demonstrating that the ATM and ATR
branches of DDR cross-talk with ARF forming a new landscape in anti-tumor
orchestrated response. ATM was shown to suppress ARF in a transcriptionindependent manner rendering the latter as a secondary defense line in case
of ATM inactivation. The functional nature of the ATR-ARF interplay is less clear
and warrants more investigation. Potential therapeutic interventions based on
the DDR-ARF interrelationship are proposed.
66 Proffered Paper: Regulation of NF-kB, inflammation and cancer
by the E3 ligase RNF20
O. Tarcic1 , M. Oren1 . 1 Weizmann Institute of Science, Molecular Cell Biology,
Rehovot, Israel
Chronic inflammation is known to mediate and affect cancer progression and
is evoked by many different extracellular stimuli, such as cytokines. One of
the main inducers of inflammation is tumor necrosis factor a (TNF-a) and
its downstream signaling cascade, the NF-úB pathway. When activated, the
NF-úB pathway leads to transcription of its target genes, a process mediated
through many coactivators and histone modifications. RNF20 is an E3 ubiquitin
ligase that monoubiquitylates histone H2B (H2Bub1) and plays a pivotal role
in transcription regulation of a subset of genes. Specifically, RNF20 represses
the transcription of EGF-inducible genes. Interestingly, one of the RNF20repressed genes identified in HeLa cells was IL-8, which is also a major NF-úB
target gene. This observation led to the hypothesis that RNF20 may engage
in a molecular interplay with NF-úB.
Materials and Methods: MCF10A mammary gland cells were subjected to
transient RNF20 knockdown and then stimulated with TNF-a. Activation of
NF-úB was measured by chromatin immunoprecipitation (ChIP) of different
NF-úB subunits, as well as qRT-PCR analysis of major NF-úB target gene
transcripts.
Additionally, RNF20+/− heterozygous mice were subjected to DSS treatment.
DSS-induced inflammation and cancer were scored by colonoscopy, as well
as mouse weight measurement. Additionally, mice colons were analyzed
histologically, and histochemically.
Results and Discussion: The aim of this study was to identify histone
modifications that may favor DNA binding of specific NF-úB dimers, and more
generally to investigate the connection between RNF20 and inflammationdriven cancer. Our studies revealed a role for RNF20 in regulating an
inflammatory response mediated through NF-úB. Specifically, RNF20 and
H2Bub1 were found to reduce NF-úB-dependent transcription in noncancerous cells. We identified a possible mechanism by which RNF20
promoted the binding of homodimers of the p50 NF-úB subunit to target gene
promoters, thus repressing target gene expression. Conversely, depletion of
RNF20 and H2Bub1 led to recruitment of p65 and induction of target gene
expression. Consistent with these in vitro findings, RNF20+/− mice were prone
to more severe acute and chronic inflammation after dextran sodium sulfate
(DSS) treatment, and succumbed more rapidly to inflammation-associated
colorectal tumors. Importantly, human colorectal tumors often exhibit reduced
levels of H2Bub1. Taken together, these results demonstrate that RNF20 may
be an important regulator of inflammation, and an inhibitor of inflammationinduced cancer.
Conclusion: Our findings suggest that RNF20 and H2Bub1 are negative
regulators of inflammation and inflammation-induced cancer. Furthermore,
constitutively low H2Bub1 levels may predispose humans to inflammatory
disease and cancer.
67 Unexpected cancer-related properties of BRCA1
Y. Hu1 , S. Dimitrov1 , H. Wang1 , D. Livingston1 . 1 Dana Farber Cancer
Institute, Emil Frei Professor of Genetics and Medicine Harvard Medical
School, Boston Massachusetts, USA
The BRCA1 tumor suppressor gene encodes multiple, alternatively spliced
products, one of which, p220, is its prime cancer-suppressing protein.
p220 is known to suppress breast and ovarian cancer, and germ line
BRCA1 mutation-carrying males are largely protected against developing
BRCA1-driven disease.
p220 is a large, multifunctional, chromatin-binding E3 ubiquitin ligase that binds
numerous partner proteins and participates in a multitude of processes that
support the maintenance of genome integrity. Some of these activities arise in
response to active genome damage and support proper DNA damage repair
and checkpoint activation. Others engage in regulating centrosome duplication
and normal mitotic events like spindle pole development and outcomes
like proper chromosome segregation. Yet others are directed at proper
heterochromatin development and the repression of satellite RNA synthesis.
A long-standing paradigm is that BRCA1 cancer results from a loss of BRCA1
function in the cells of the breast and ovarian/fallopian tube epithelium, a notion
that is underscored by the fact that virtually all inherited BRCA1 cancers are
known to have undergone LOH at BRCA1 with maintenance of the mutant
allele.
That said, we have recently accumulated evidence that, much like what
happens when p220 abundance is greatly reduced, too much p220, which
can arise either in G1 or in S/G2 from a defect in its mRNA turnover, is
also associated with chronic genome damage. Similarly, we have identified
another biochemical process that normally results in the poly ADP ribosylation
of a specific domain of p220 in cells and that, when defective, leads to an
exaggerated p220 response to the development of double strand genomic
breaks. This p220-dependent hyperrecombinational DNA damage repair
response is, unexpectedly, associated with the development of gross genome
disorder, much like what happens when p220 expression is lost in cells.
Since the development of chronic genome disorder is a major contributor to the
development of BRCA1 cancer, these results trigger speculation that BRCA1
can elicit a tumorigenic response in two ways − (a) by its loss of function and
(b) by the retention, deregulation, and hyperactivity of one or more of its key
DNA damage response functions. If shown to be true, such a bimodal model
would significantly alter the paradigm that explains the genesis of BRCA1
cancer.
Monday 7 July 2014
14:00−15:45
Symposium
Novel Cancer Therapies
68 Novel therapeutic approaches to lung cancer
69 The TGF-beta pathway as a therapeutic target
J. Seoane1 . 1 Vall D’Hebrón University Hospital, Barcelona, Spain
During the past decades, the TGF-beta pathway has recently emerged as a
putative therapeutic target against cancer. However, TGF-beta has a complex
role in cancer. During tumour progression TGF-beta becomes an oncogenic
S17
factor inducing proliferation, angiogenesis, invasion, and metastasis, as well
as suppressing the anti-tumoral immune response. The precise understanding
of the role of TGF-beta in oncogenesis is required in order to design optimal
therapeutic approaches and select the patient population that may benefit from
an anti-TGF-beta therapy. We will review the rationale for evaluating TGF-beta
signalling inhibitors as cancer therapeutics, and the progress made in the
preclinical and clinical testing of anti-TGF-beta compounds in the context of
glioblastoma.
70 Proffered Paper: Inhibition of RNA Polymerase I transcription
by CX-5461 as a completely new approach to treat highly refractory
haematological malignancies
R. Hannan1 , N. Hein1 , S.E. O’Brien2 , D. Drygin3 , C. Cullinane1 , G. Matthews1 ,
R.W. Johnstone4 , R.B. Pearson4 , G. McArthur4 , S. Harrison4 . 1 Peter
MacCallum Cancer Centre, Research, Melbourne Victoria, Australia,
2
Senhwa Biosciences Inc, Research and Development, San Diego CA,
Australia, 3 Pimera Inc, Research and Development, San Diego CA, USA,
4
Peter MacCallum Cancer Centre, Resarch, Melbourne Victoria, Australia
Background: Recent findings by our group have been instrumental in the
development of the novel selective inhibitor of RNA Polymerase I (Pol I),
CX-5461 (Drygin et al., Cancer Research, 2011; Bywater et al. Cancer Cell,
2012). This work has led to the fundamental discovery that ribosomal gene
transcription by Pol I is not simply a ‘house keeping’ process in cancer cells but
is highly regulated to maintain their viability (Bywater et al., Nature Reviews
Cancer 2013). Strikingly, inhibition of Pol I transcription shows a profound
selectivity for malignant over normal cells in preclinical studies.
Material and Methods: To explore the therapeutic potential of Pol I
transcription inhibition via CX-5461 in hematological malignancies refractory
to standard chemotherapy, we employed mouse models of highly aggressive
MLL-driven Acute Myeloid Leukemia (AML) (MLL/AF9 + Nras and MLL/ENL +
Nras) and V*ú-Myc-driven Multiple Myeloma (MM).
Results: CX-5461 administration significantly increased the overall survival
time of AML-bearing mice (MLL/ENL Nras median17 days for vehicle vs 36
days for drug, P < 0.0001; MLL/AF9 Nras median 15 days for vehicle vs
23 days for drug, P 0.0009) without severe adverse effects. The extended
survival of MLL-driven AML bearing mice was associated with, induction of
cell death and an aberrant G2/M cell cycle progression. In contrast, treatment
of Nras AML-bearing mice with Cytarabine/Doxorubicin (5:3) resulted in
a significantly lower survival advantage as compared to animals treated
with CX-5461 therapy. Similarly, in MM-bearing mice, chronic CX-5461
administration robustly reduced the secretion of serum monoclonal Ig, and
significantly prolonged the overall survival time (V*ú-Myc median 102 days for
vehicle vs 210 days for drug, ongoing) demonstrating that CX-5461 exhibits
potent anti-tumour activity in MM model.
Conclusions: These data demonstrate that hyperactivated Pol I transcription
can be successfully targeted through small molecule inhibitors to treat models
of human AML and MM that are refractory to standard therapy. Based on
these and previously published results (Bywater et al., Cancer Cell, 2012) and
a favorable toxicology profile, we have recently initiated a first-in-human phase
I clinical trial of this first-in-class drug, CX-5461 in patients with advanced
hematological malignancies.
71 Targeted cancer genetics
C. Von Kalle1 , S. Fröhling2 , H. Glimm1 , S.M. Pfister3 , T. Zenz2 . 1 National
Center for Tumor Diseases (NCT) and German Cancer Research Center,
Translational Oncology DKFZ-Heidelberg, Heidelberg, Germany, 2 National
Center for Tumor Diseases (NCT) German Cancer Research Center and
Heidelberg University Hospital, Translational Oncology DKFZ-Heidelberg,
Heidelberg, Germany, 3 German Cancer Research Center and Heidelberg
University Hospital, Pediatric Neurooncology DKFZ-Heidelberg, Heidelberg,
Germany
Many recurrent genetic aberrations have been detected through cancer
genome sequencing, however, their associated functional dependencies
remain elusive in many cases. Moreover, tumors that are currently classified
according to pathology often display heterogeneous biologic characteristics
and clinical course. We have implemented a multi-pronged approach
to systematically investigate genotype-phenotype correlations in a broad
spectrum of human cancers through comprehensive genomic profiling,
functional annotation of genetic variants, and ex vivo drug screening. This
multidimensional strategy will enable stratification of patients according to
specific, context-dependent vulnerabilities that can be exploited for therapeutic
benefit.
We have determined the functional consequences of multiple genetic
abnormalities occurring in patients with acute myeloid leukemia and B-cell
chronic lymphocytic leukemia using in vitro and in vivo experimental systems.
In addition to these directed investigations, we have performed unbiased
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functional studies, employing tools such as high-throughput RNA interference,
to discover “secondary” pathway dependencies that arise in consequence of
particular genetic alterations, such as oncogenic KRAS mutations, and we
have devised strategies to target such context-specific liabilities for therapeutic
benefit. We have extensively characterized the dynamic composition of the
tumor-initiating cell compartment in xenograft models of human colorectal
and pancreatic cancer, providing a unique resource for investigations into
the functional requirements of “cancer stem cells” as well as the genetic
basis of the functional heterogeneity observed in human cancers. Finally, we
have established a platform for ex vivo treatment of genetically annotated
primary human lymphoma specimens with a wide range of pathway inhibitors
to systematically correlate mutational profiles with patterns of drug response.
“Undruggable” genetic alterations were addressed in the context of gene
fusions involving the H3K4 methyltransferase MLL, which define an aggressive
subtype of acute myeloid leukemia (AML). Using a systematic functional
genomic approach, we discovered that MLL fusion-driven AML cells are
exceptionally reliant on the cell cycle regulator CDK6, but not its functional
homolog CDK4, providing the rationale for a clinical trial of the smallmolecule CDK6 inhibitor palbociclib in patients with relapsed or refractory MLLrearranged leukemia. Moreover, we systematically investigate heterogeneity of
drug response in primary chronic lymphocytic leukemia (CLL) and lymphoma
using a cell-based high-throughput drug-screening platform and associate the
response phenotype with genetics and other omics technology. The work offers
a novel functional classification of CLL based on drug sensitivity and identifies
drugs with preferential activity for TP53 mutant CLL.
While cancer specific aberrations in adults tend to incur a multitude of genetic
aberrations, the pediatric tumor genome is less complex and thus predestined
for treatment by targeted agents. We identified mutations impacting MAPK
signaling as the genetic hallmark in low-grade gliomas in children classifying
this indication as a MAPK disease. In pediatric medullablastoma we identified
the pivotal role of aberrant SHH signaling − which in turn serves as genetic
predictor of response to SMO inhibition.
The clinical implementation of a center-wide NCT MASTER (Molecularly Aided
Stratification for Tumor Eradication) umbrella protocol provides all components
of a clinical workflow for high-throughput molecular diagnostics. A pilot protocol
for adult cancer patients with a particularly challenging disease course and
surprise responders has enrolled more than 100 patients and identified
actionable genetic lesions in a substantial proportion of cases. The nationwide
INFORM study aims to enroll all children and adolescents with relapsed or
refractory cancers in Germany to systematically enable personalized treatment
based on comprehensive molecular profiling of individual tumors.
Monday 7 July 2014
14:00−15:45
Symposium
Inflammation and Cancer
72 Inflammation in colorectal and liver tumorigenesis
M. Karin1 . 1 University of California San Diego, Laboratory of Gene Regulation
and Signal Transduction Department of Pharmacology, La Jolla CA, USA
Inflammation plays important roles in the pathogenesis of colorectal and liver
cancers. Chronic inflammation of both tissues increases cancer risk in part
through elevated expression of IL-6 and other pro-tumorigenic cytokines such
as IL-1 and TNF. IL-6 exerts its tumorigenic activity by signaling via the receptor
subunit gp130 to activate transcription factor STAT3. Curiously, activating
gp130 mutations were found in human inflammatory hepatic adenoma, that
when combined with activating mutations in the Wnt-b catenine pathway
can lead to hepatocellular carcinoma (HCC). We expressed such a form of
gp130 throughout the intestinal tract to determine its impact on development
and tumorigenesis in the organ. Surprisingly, we found that activated gp130
has a strong impact on tissue homeostasis and leads to activation of a
regenerative pathway responsible for the repair of mucosal injury, which if
left unrepaired can lead to chronic intestinal inflammation. Interestingly, this
pathway is independent of STAT3 and instead it relies on activation of YAP,
a transcriptional co-activator that controls tissue growth and is frequently
activated in HCC. These results suggest that IL-6 family members promote
colon and liver tumorigenesis through multiple mechanisms, all of which need
to be targeted to achieve an effective therapeutic effect.
73 Parainflammation in cancer
A. Lasry1 , H. Hamza1 , E. Kadosh1 , E. Elyada1 , A. Pribluda1 , K. Alitalo2 ,
T. Stiewe3 , M. Oren4 , E. Pikarsky5 , Y. Ben-Neriah1 . 1 The Hebrew
University of Jerusalem, School of Medicine-IMRIC-Immunology and
Cancer Research, Jerusalem, Israel, 2 Biomedicum Helsinki and the Finnish
Institute for Molecular Medicine, University of Helsinki, Helsinki, Finland,
3
Philipps-University, Marburg, Germany, 4 Weizmann Institute, Molecular Cell
Biology, Rehovot, Israel, 5 Hebrew University, Immunology and Pathology,
Jerusalem, Israel
Inflammation has many faces, most commonly observed as an acute
reaction in response to pathogen or another insult, or a chronic phase,
accompanying chronic infection and chronic remittent inflammatory disease,
such as inflammatory bowel disease [1]. Yet, there is another type of
smoldering inflammation, harder to notice or monitor, which appears to
underlie some of the major human diseases, cancer, diabetes type 2
and certain neurodegenerative diseases [2]. We have developed mouse
models of cancer based on inducible CKIa knockout [3], which exhibit
smoldering inflammation, and demonstrate how a low grade, infiltrate-free
inflammatory reaction to persistent DNA damage response translates to an
aberrant growth. We determined this unusual inflammatory repertoire denoted
parainflammation [4] in the knockout mice and gut explants, demonstrated
its association with cellular senescence and showed how in the absence
of p53, parainflammation is converted from a growth inhibitory to growth
promoting mechanism, both in vitro and in vivo. Anti-inflammatory reagents
capable of blocking parainflammation reverse a tumor-related crypt proliferative
phenotype of mutant intestinal organoids in vitro and prevent carcinogenesis
in mutant mice. Our studies may explain the anti-carcinogenic effects of nonsteroidal anti-inflammatory drugs in human cancer patients [5]. We will discuss
mechanisms and evolutionary aspects connecting inflammation and growth.
Reference(s)
[1] Medzhitov, R. Origin and physiological roles of inflammation. Nature 454,
428−35 (2008).
[2] Ben-Neriah, Y. & Karin, M. Inflammation meets cancer, with NF-kappa B
as the matchmaker. Nature Immunology 12, 715–723 (2011).
[3] Elyada, E., Pribluda, A., et al. CKI alpha ablation highlights a critical role
for p53 in invasiveness control. Nature 470, 409-U208 (2011).
[4] Pribluda, P., Elyada, E., et al. A senescence-inflammatory switch from
cancer-inhibitory to cancer-promoting mechanism. Cancer Cell 24,
242–256 (2013).
[5] Chan, A.T. & Lippman, S.M. Aspirin and colorectal cancer prevention in
Lynch syndrome. Lancet 378, 2051–2052 (2011).
74 Proffered Paper: Investigation of the spatial heterogeneity of
specific immune cell phenotypes in the tumor microenvironment
of follicular lymphoma
J. Mansfield1 , L. Nelson2 , B. Wendik3 , C. Van der Loos4 , C. Rose2 ,
R. Byers2 . 1 CRi, Woburn, USA, 2 University of Manchester, Manchester,
United Kingdom, 3 PerkinElmer, Hopkinton, USA, 4 Academic Medical Center,
Amsterdam, Netherlands
Background: Tumor-infiltrating lymphocytes (TILs) are present in the tumor
microenvironment of many cancers, with consequent tumor immunogenicity
and association with survival. In particular, increased levels of regulatory
T cells (Tregs) are associated with poorer prognosis in some cancers. However,
given the complexity of TIL phenotype their visualization in situ is difficult,
and impossible without multiplex immunophenotyping. An understanding
of both the phenotype and spatial distribution of TILs in situ within the
tumor microenvironment would be advantageous to understanding their
role in tumour immunobiology, especially given growing interest in tumour
immunotherapy. Here we present a multi-marker, computer-aided method
for analysing the distributions of CD3/FOXP3 (Treg) and CD3/CD69 (Tact)
T cells in follicular lymphoma sections using a multispectral imaging (MSI)
and automated analysis approach. An hypothesized interaction distance (HID)
analysis was used to determine whether the spatial patterns of Tregs and Tacts
were prognostically significant.
Design: A single section of a tissue microarray containing triplex follicular
lymphoma cores from 40 subjects [24 male, 16 female, age 35 to 75 years
at diagnosis, median 55 years, 2–171 months follow-up] was stained for CD3,
FOXP3, CD69 and hematoxylin. Each core was imaged using MSI and the
individual staining of each marker separated from each other using spectral
unmixing. CD3+ TILs were located using automated image analysis. The
FOXP3 and CD69 status of each CD3+ TIL was then determined and the
spatial distributions of the CD3/FOXP3 and CD3/CD69 cells were used as
input into the HID analysis.
Results: Multiplexed IHC staining, MSI and automated per-cell quantitative
analysis was successful. Kaplan–Meier analysis demonstrated favourable
outcome with higher numbers of CD3+, CD3+/FOXP3+ and CD3+/CD69+
cells. HID analysis demonstrated the association of favourable outcome with a
high entropy, representative of a diffuse spatial pattern, of FOXP3+ and CD69+
positive T cells.
Conclusion: In this study we report that higher Treg cell counts in a diffuse
pattern was associated with favorable prognosis. This supports the importance
of Tregs in the tumour microenvironment. It is pertinent to mention that
contradictory findings are routinely reported from studies investigating the
role of Tregs in solid and haematological malignancies. This is due to the
complex interactions between pro-/anti-tumour immune factors present in the
tumour microenvironment. The resultant effects are due to the summation of
the activities of these factors. It is therefore even more relevant that a method
such as exhibited here, capable of defining and measuring the effect on patient
outcome of the spatial patterns of multiple cellular phenotypes in the tumor
microenvironment is available.
Conflict of interest: Other substantive relationships: PerkinElmer employees.
75 Role of inflammation in epidermal carcinogenesis
F.M. Watt1 . 1 King’s College London, Centre for Stem Cells and Regenerative
Medicine, London, United Kingdom
Multilayered epithelia such as the epidermis and oral mucosa are maintained
throughout adult life by self-renewal of stem cells and differentiation of their
progeny. I will present evidence that both stem cells and differentiating cells
can contribute to tumour development. I will describe how aberrant gene
expression by differentiated cells can recruit stem cells, fibroblasts and cells
of the bone marrow to collaborate to form tumours.
Monday 7 July 2014
14:00−15:45
Symposium
Imaging
76 Multiparametric imaging in cancer research
A.K. Buck1 . 1 Nuklearmedizinische Klinik und Poliklinik, Universitätsklinikum
Würzburg, Germany
Besides detection of cancer, staging, restaging and surveillance, modern
imaging technologies play a pivotal role for the assessment of cancer
therapies. Given the recent developments in magnetic resonance imaging
and the multitude of molecular imaging probes becoming available, a wide
range of image-derived parameters can be used for guiding the process of
treatment individualization. It remains to be determined which parameter or
which combination of parameters represents the most effective predictors
of response and outcome in varying clinical scenarios. This presentation
will highlight the most recent developments in anatomic, functional and
molecular imaging (i.e., dual-source CT, multi-parametric MRI, PET, SPECT
and fluorescence imaging devices). A particular focus is laid on established
and more recent hybrid devices (PET/CT, SPECT/CT, and MR/PET). Based
on the non-invasive identification of therapeutic targets, imaging contributes to
the decision making process in individual cancer patients. When integrated
early in the course of anticancer drug development, these technologies
can also aid in selecting the most efficient compounds for evaluation
in early clinical trials. Furthermore, with recent technologies, imaging of
tumor heterogeneity becomes feasible, including hallmarks of cancer such
as deregulated metabolism, hypoxia, receptor expression, proliferation and
others. In the near future, multimodal or multiparametric imaging will become
reality not only in early preclinical and clinical research but also in the clinical
arena.
77 Molecular imaging for personalised treatment of cancer
W.J.G. Oyen1 . 1 Radboud University Medical Center, Dept. of Radiology and
Nuclear Medicine, Nijmegen, Netherlands
Molecular imaging using radiolabeled agents for SPECT and especially PET
has been evolving rapidly in the last decade. Especially the introduction of
hybrid cameras, combining SPECT or PET with CT, has significantly increased
the acceptance of these techniques in clinical practice and the use in research
protocols.
PET using the glucose-analogue FDG, reflecting tumor metabolism and
its changes during therapy, has gained wide acceptance for staging,
radiation treatment planning and therapy response monitoring. Beyond
metabolic imaging, a large number of agents has been developed to depict
features of tumors, such as tumor cell proliferation (FLT), hypoxia (e.g.
FMISO, FAZA, ATSM), protein synthesis (e.g. FET) and angiogenesis (e.g.
labeled bevacuzimab, RGD). These radiopharmaceuticals allow more specific
assessment of tumor characteristics and of their changes early during local
or systemic therapeutic interventions. This allows tailoring of therapy to the
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individual patient before or early after the start of treatment, before a reduction
of tumor size becomes apparent on conventional anatomical imaging with CT
or MRI.
More recently, PET and SPECT imaging of receptor expression and modulation
with radiolabeled antibodies and peptides has been introduced as a tool
for noninvasive in-vivo assessment of receptor presence, accessibility and
heterogeneity between lesions. A typical example is the development of Zr-89
labeled trastuzumab to assess expression of HER2-receptors in metastatic
breast cancer.
The principle of imaging radiolabeled anti-cancer drugs can also be applied for
small molecules such as chemotherapeutics and targeted therapies with TKIs.
When these drugs are amenable to labeling with e.g. C-11 it can be visualized
whether these drugs actually reach the tumor and accumulate there. Examples
are C-11 labeled docetaxel and C-11-lapatinib.
In conclusion, the use of molecular imaging in clinical oncology, both for patient
care as well as experimental applications opens new perspectives for more
detailed assessment of tumor characteristics, paving the way for individualized
treatment of cancer patients. New hybrid technology combining PET with MRI
will further boost research to develop novel indications.
78 Proffered Paper: Dual wavelength near-infrared fluorescence
imaging of VEGF and IGF-1R in ovarian cancer patient-derived
xenografts
T. Tomar1 , N.G. Alkema1 , A.G. Terwisscha van Scheltinga2 , J.A.L. Visser3 ,
G.J. Meersma1 , E.W. Duiker4 , E.G.E. De Vries3 , A.G.J. Van der Zee1 ,
G.B.A. Wisman1 , S. De Jong3 . 1 University Medical Center Groningen,
Gynecologic Oncology, Groningen, Netherlands, 2 University Medical Center
Groningen, Hospital and Clinical Pharmacy, Groningen, Netherlands,
3
University Medical Center Groningen, Medical Oncology, Groningen,
Netherlands, 4 University Medical Center Groningen, Pathology and Medical
Biology, Groningen, Netherlands
Background: Treatment of ovarian cancer patients with personalized targeted
therapies would benefit of upfront selection of patients based on the expression
of targets within the tumor, like growth factors or growth factors receptor;
and early monitoring of tumor responses. Multiple wavelength fluorescent
molecular imaging allows assessment of the targeted proteins and therapeutic
response at the same time. The aim of our study was to test feasibility and
application of dual wavelength near-infrared fluorescence (NIRF) molecular
imaging in patient-derived xenograft (PDX) model of mice. We focused on
imaging of the secreted vascular endothelial growth factor (VEGF) and the
membrane-bound insulin-like growth factor 1 receptor (IGF-1R), both known
targets of therapy, in multiple ovarian cancer PDX model.
Methods: Monoclonal antibody bevacizumab (anti-VEGF-A) and MAB391
(anti-IGF-1R) were coupled to NIRF dyes IRDye-800CW and IRDye-680RD,
respectively. After in-vitro evaluation of fluorescence tracers, specific tumor
uptake was determined in a panel of ovarian cancer PDX models in NOD
SCID gamma (NSG) mice (n = 10 patients) during 1 week after tracer injection,
followed by ex-vivo fluorescence microscopy and pathologic examination.
Imaging results were compared with histology and immunostaining of VEGF
and IGF-1R on PDX and matched patient tissue.
Results: We detected the different fluorescence signals separately of
both VEGF (bevacizumab-800CW) and IGF-1R (MAB391–680RD) tracers
within the same PDX model simultaneously, which were co-localized
with immunostaining. We found background corrected maximum average
radiance of 2.53–23.3×106 p/s/cm2 /sr for bevacizumab-800CW and 1.5–
15.5×107 p/s/cm2 /sr for MAB391–680RD. Bevacizumab-800CW NIRF signal
intensity was maximal after 24 h and rapidly decreased. MAB391–680RD
showed longer residence lifetime in tumors with a constant NIRF signal
intensity for 6 days. These results were consistent over all PDXs,
except for one. This last PDX showed NIRF signal for bevacizumab800CW but not for MAB391–680RD, which was related to the negative
immunohistochemical IGF-1R staining. All results were supported by standard
histology, immunohistochemistry, and fluorescence microscopy analysis of
tumors derived from PDXs as well as from corresponding patients.
Conclusion: These findings encourage future preclinical applications of PDX
as reliable cancer models for development of novel cancer therapeutic targets
and their validation using molecular optical imaging.
This study is supported by the Dutch Cancer Society, KWF Kankerbestrijding
(grants RUG 2010-4833 & RUG 2011-5231).
79 Imaging as tool for translational research in oncology
M. Schwaiger1 . 1 Klinikum rechts der Isar, Nuklearmedizinische Klinik und
Poliklinik, Munich, Germany
With the advent of multimodality imaging biological information can be
visualized in-vivo with high spatial as well as temporal resolution. The excellent
structural information obtained by modern CT and MRI instrumentation
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is increasingly complimented by molecular imaging signals, using either
radioactive probes or optical agents. Combining structural and molecular
information allows the definition of biological processes in the tumor cells, but
also provides means for characterizing the micromilieu of tumor and normal
tissue. Specific markers for cell migration may help to identify angiogenesis as
well as tumor metastasis. It is hoped that the combination of various imaging
biomarkers will help us to not only detect tumor at an earlier stage, but also
provide important prognostic information. Furthermore, imaging of specific
therapy targets may support a better selection and monitoring of therapy in
the context of personalized medicine.
Monday 7 July 2014
17:15−18:45
Plenary Symposium: Cancer Genomics
80 Recent advances in cancer genomics
E. Mardis1 . 1 Washington University School of Medicine, Genetics/Molecular
Microbiology, St Louis, USA
The implementation of next generation sequencing in the study of cancer
genomes has dramatically changed our understanding of the mutational
landscape of the disease and how incredibly variable it is from patient to
patient. By further integrating sequencing data from RNA expression into the
mutational spectrum, we have further enhanced understanding of the genes
that are expressed at significantly higher levels even if the reason for RNA
overexpression isn’t evident in the DNA itself (e.g. amplification). In addition to
characterizing cancer genomes and transcriptomes as static entities, we have
used these approaches to compare primary to metastatic disease from the
same patient for several tumor types, furthering our understanding of tumor
evolution. My lecture will present a current view of cancer genomics and how
the attendant approaches are now informing clinical care aspects.
81 Oncogenomics of brain tumors: Integrated approaches reveal
novel pathomechanisms
P. Lichter1 . 1 Deutsches Krebsforschungszentrum, Division of Molecular
Genetics, Heidelberg, Germany
Recent molecular profiling studies of brain tumors uncovered a wealth of
genetic changes resulting in the identification of novel markers, signatures
and molecular pathways relevant for tumor pathophysiology. We have carried
out comprehensive genome, methylome and transcriptome analyses in
large cohorts of medulloblastoma and glioma applying next-generation DNA
sequencing. Integration of these data revealed a wealth of new findings
including (i) refinement of tumor classification schemes, (ii) elucidation
of novel pathomechanistic aspects of genome and epigenome biology,
and (iii) identification of novel pathways and actionable targets. Recent
findings related to current challenges in translational neuro-oncology will be
presented.
ScienceDirect
Tuesday 8 July 2014
Tuesday 8 July 2014
08:45−09:30
The EMBO Lecture: Hallmarks of Cancer:
Applications to Cancer Medicine?
82 Hallmarks of cancer: applications to cancer medicine?
D. Hanahan1 . 1 Ecole Polytechnique Fédérale de Lausanne (EPFL), ISREC
School of Life Sciences, Lausanne, Switzerland
The hallmarks of cancer provide a framework from which to consider the
complexity (and underlying commonality) of human cancers. The proposition
is that most forms of human cancer acquire, by different ways and
means, a similar set of complementary hallmark capabilities that collectively
cause the disease. While current and future refinements of the hallmarks
conceptualization are arguably useful as a heuristic tool for research designed
to elucidate mechanistic underpinnings of the diverse manifestations of the
human disease, one can also ask whether there will be beneficial applications
to cancer medicine? Is there rationale for therapeutic co-targeting hallmarks
of cancer, reasoning that resistance might be harder to acquire if several
hallmarks are concurrently impaired? This hypothesis is being tested, both
in preclinical models and in clinical trials. Moreover, there are clues that
resistance to therapies targeting single hallmarks can be afforded by ‘hallmark
switching’, whereby dependence on one hallmark is partially shifted to another
so as to evade the functional inhibition, further motivating multi-targeting
strategies aimed to circumvent such adaptive resistance. Examples of these
emerging applications to cancer medicine will be discussed.
Reference(s)
Hanahan, D. & Weinberg, R. (2000). The hallmarks of cancer. Cell 100:
57−70.
Hanahan, D., & Weinberg, R.A. (2011). Hallmarks of cancer: the next
generation. Cell 144: 346–674.
Hanahan, D., & Coussens, L.M. (2012). Accessories to the crime: functions of
cells recruited to the tumor microenvironment. Cancer Cell. 21: 309–322.
Conflict of interest: Advisory board: SAB − Oncology Pfizer, Inc.
Tuesday 8 July 2014
09:30−10:15
The Pezcoller Foundation − EACR
Cancer Researcher Award Lecture
83 Mechanisms of metastasis by colorectal cancer stem cells
E. Batlle1 . 1 Institute for Research in Biomedicine IRB Barcelona, Oncology
Program & ICREA, Barcelona, Spain
The inner layer of the intestinal tube, the intestinal epithelium, is in a constant
process of renewal. Hundreds of millions of terminally differentiated intestinal
cells are replaced by new cells every day during the life of an adult organism.
This tremendous regenerative power is ultimately sustained by a small
population of intestinal stem cells (ISCs). We have recently discovered that
most human colorectal cancers (CRCs) are constituted by cell populations with
phenotypes similar to either ISCs or intestinal differentiated cells organized
into well-defined compartments. ISC-like cells purified from primary CRC
samples generate tumors in immunodeficient mice with high efficiency and
display both self-renewal and differentiation capacity whereas differentiated
CRC cells are not capable of propagating the disease. Our observations imply
that CRC shares a common hierarchy with the intestinal mucosa and that the
acquisition of an ISC-like gene program is a central process in the development
of metastatic and recurrent CRC. Here I will present our latest data on the
mechanisms employed by CRC stem cells to regenerate a new tumor at the
metastatic site. We have demonstrated that metastasis relies on a tumor cell
non-autonomous program driven by TGF-beta in the microenvironment. This
stromal program confers a survival advantage to the disseminated CRC stem
cells during the initial phase of metastasis. We have now investigated the
dichotomy of TGF-beta signaling in epithelial versus stromal cells during CRC
progression and interrogated mouse models about the efficacy of anti-TGFbeta therapies for CRC treatment.
Tuesday 8 July 2014
10:45−12:15
Plenary Symposium: Stem Cells
84 Mesenchymal and MDS stem cells shape an interactive disease
unit in the bone marrow
A. Trumpp1 , H. Medyouf2 , W.K. Hofmann3 , D. Nowak3 . 1 DKFZ − German
Cancer Research Center, and HI-STEM GmbH, Heidelberg, Germany,
2
DKFZ − German Cancer Research Center, Heidelberg, Germany, 3 University
Hospital, Mannheim, Germany
Myelodysplastic syndromes (MDS) are hematologic disorders characterized
by ineffective hematopoiesis, dysplastic bone marrow and increased risk of
progression to acute leukemias. Here we show that co-transplantation of
lin− CD34+ CD38− cells in-conjunction with mesenchymal-stromal cells both
derived from lower risk MDS patients re-establishes long-term disease
engraftment in NSG mice. Molecular tracking of xenografted MDS cells
allows interrogation of variegated MDS clones. Disease pathogenesis is
reliant on specific interactions between MDS hematopoietic cells and MDS
MSCs as the latter display greater capability for disease initiation compared
to MSCs of healthy subjects. MDS MSCs display deregulated expression
of niche regulators including VEGFA, IGFBP2, LIF and N-Cadherin. The
“re-programing” of MDS MSCs can be mediated by exposure to MDS
hematopoietic cells indicative of microenvironment shaping. This suggests the
presence of a MDS stem cell-niche unit in lower risk MDS patients, which if
re-transplanted allows the establishment of a xenograft platform opening novel
avenues for both MDS analysis and treatment.
85 Stem cells in homeostasis and cancer
E. Fuchs1 . 1 The Rockefeller University, Howard Hughes Medical Institute,
New York, USA
Stem cells (SCs) have the ability to self-renew long term and differentiate
into one or more tissues. Typically, SCs are used sparingly to replenish cells
during normal homeostasis. However, even SCs that are quiescent must be
able to respond quickly to injury in order to fuel rapid tissue regeneration. How
SCs balance self-renewal and differentiation is of fundamental importance to
our understanding of normal tissue maintenance and wound repair. Increasing
evidence suggests that the regulatory circuitry governing this balancing act is
at the root of some types of tumors both in mice and in humans.
The skin is an excellent model system to understand how SCs function
in normal tissue generation and how this process goes awry in cancer.
We’ve identified SC niches with in the epidermis, hair follicle, sebaceous
glands and sweat glands. We’ve learned that different niches become
activated in response to different types of injury, and that during normal
homeostasis, SC behavior is controlled not only through cues received
from their microenvironment but also through signals emanating from their
differentiating lineages. We’ve been dissecting how extrinsic signaling to SCs
trigger a cascade of transcriptional changes that govern SC activation during
tissue development, homeostasis and hair regeneration. Our findings provide
new insights into our understanding of the process of SC activation, and in so
doing have revealed mechanisms which are also deregulated in a variety of
different human cancers. Our goal is to understand how SCs start and stop
making tissue, and how this changes in cancer. Our recent discoveries on
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EACR-23 Oral Presentations, Tuesday 8 July 2014 / European Journal of Cancer 50, Suppl. 5 (2014) S21–S22
this topic have led us to the realm of identifying and characterizing cancer
SCs (tumor-initiating cells) of squamous cell carcinomas (SCCs) of the skin.
We developed a new method to knockdown genes specifically in skin and oral
progenitors, enabling us to screen not only the differences between cancer and
normal SCs, but also the myriad of gene alterations surfacing from the Human
Cancer Sequencing project. Our screens have illuminated new oncogenes and
tumor suppressors for SCCs, among the most prevalent and life-threatening
cancers world-wide that include cancers of lung, esophagus, breast, cervix,
prostate, throat and oral tissues. Our findings are unearthing new targets for
cancer therapeutics.
Tuesday 8 July 2014
12:50−13:30
Mike Price Gold Medal Award Lecture
86 Infections causing human cancers − mechanisms and perspectives
H. zur Hausen1 , E.M. De Villiers1 . 1 German Cancer Research Center,
Heidelberg, Germany
At present slightly more than 20% of the global cancer incidence can be linked
to infections. Although approximately two thirds of infection-linked cancers are
linked to virus infections, Helicobacter pylori, as a bacterium, also plays a
significant role. In addition, in Africa and South East Asia parasitic infections
are of local importance for carcinogenesis. The mechanism by which infectious
factors contribute to carcinogenesis is increasingly understood. Some agents
act as direct carcinogens, where persistence and expression of specific genes
is a necessary precondition for the resulting malignant phenotype. Others
seem preferentially to act as indirect carcinogens, commonly by inducing
inflammatory reactions with the production of mutagenic byproducts. Immune
suppression induced by human immunodeficiency viruses frequently activates
other latently persisting potential tumorviruses (e.g. Epstein–Barr virus, human
Herpesvirus type 8 and others) and could result in cancer induction after about
3 to 15 years.
None of the known infections induces cancer as a direct consequence
of infection. The long latency periods between initial infection and cancer
development, ranging in part up to 60 years, result from the requirement for
additional modifications in host cell genes or within the persisting viral genome.
Without modifications, the affected genes apparently play a protective role
in preventing cancer and developed during a long co-evolution between the
human host and potentially carcinogenic agents.
Epidemiological data suggest the involvement of additional infections in
widespread human cancers. This accounts in particular for cancers of
the colon, possibly also for breast cancer and childhood leukemias and
lymphomas. For colon and breast cancer nutritional factors have been
incriminated, in particular the consumption of red meat. The available data
suggest a pivotal role of beef (Bos taurus) meat and possibly dairy product
consumption. The isolation of novel viral DNAs from cattle serum and
successful transfection of this DNA into human cells will be reported.
ScienceDirect
Poster Sessions (Sunday 6 July & Monday 7 July 2014)
Sunday 6 July 2014
Poster Session
Cell and Tumour Biology I
100 IKKb dependent NF-kB activation suppresses progression of
lung metastases during breast carcinogenesis
H.Y. Fang1 , O.C. Olson2 , J.A. Joyce2 , M. Quante1 , F.R. Greten1,3 .
1
Klinikum rechts der Isar Technische Universitat Munchen, Molekulare
Gastroenterologie II, Martinsried/Munich, Germany, 2 Memorial SloanKettering Cancer Center, Cancer biology & genetic program, New York, USA,
3
Georg-Speyer-Haus, Tumor biology and experimental therapy, Frankfurt,
Germany
Introduction: Inflammatory cells, which are abundant in tumor microenvironment, influence tumorigenesis and metastatic progression in cancers but
the mechanisms underlying remain obscure. The inflammation-responsive
IKKb and its target NF-kB have important tumour-promoting functions within
inflammatory cells and we recently demonstrated that loss of IKKb in myeloid
cells prevents the development of lymph node metastases in colon cancer. In
this study, we aim to investigate the effect of NF-úB in myeloid cells during
breast tumor development.
Material and Method: Mice bearing a myeloid restricted deletion of IKKb
(IKKbDmye ), which will lead to inhibition of NF-KB p65/p50 activation, were
crossed with breast cancer mouse model (MMTV-PyMT).
Results and Discussion: PyMT mice with functional IKKb in myeloid cells
and PyMT mice bearing a targeted deletion of IKKb in myeloid cells (PyMTIKKbDmye ) did not show any difference in primary breast tumor mass.
Surprisingly, however, PyMT-IKKbDmye mice developed significantly more
pulmonary metastases. The enhancement of pulmonary metastases in PyMTI IKKbDmye mice was not associated with changes in the recruitment of
inflammatory cells or differences in the number of cancer stem cells in
tumors. Interestingly, we found that PyMT-IKKbDmye tumors expressed higher
level of cathepsins and IKKb deficient TAMs secreted elevated amounts
of cathepsins into the tumor microenvironment. This suggests that NF-kB
negatively regulates release of cathepsins in PyMT mice and suppresses lung
metastasis development.
Conclusion: Our data underscore the context dependent role of NF-kB
in tumorigenesis and highlight the importance of autochthonous preclinical
models to evaluate potential IKKb/NF-kB inhibitors for therapy of different
cancer types.
This work was supported by Alexander von Humboldt-Foundation fellowship
and grants from the European Research Council (ROSCAN-281967).
101 SWAP-70 is required for spontaneous transformation of mouse
embryo fibroblasts
Y.T. Chang1 , C.L. Shu1 , J.Y. Lai1 , C.Y. Lin1 , C.P. Chuu1 , T. Ichikawa2 ,
K. Morishita2 , R. Jessberger3 , Y. Fukui1 . 1 National Health Research
Institutes, Institute of Cellular and System Medicine, Miaoli County, Taiwan,
2
University of Miyazaki, Faculty of Medicine, Miyazaki, Japan, 3 Dresden
University of Technology, Faculty of Medicine, Institute of Physiological
Chemistry, Germany
Introduction: SWAP-70 is a protein that regulates F-actin rearrangement in
a phosphatidylinositol 3-kinase dependent manner. It plays various roles in
many cell responses. We found that some Swap-70 mutants could transform
mouse fibroblasts.
Mouse embryo fibroblasts (MEFs) undergo transformation after certain periods
of cultivation. This may be due to mutations occurring in some of the cells. If
these mutations hit sequences relevant for transformation of the cells, the cells
become transformed, grow faster than other cells, and become dominant in
the culture. Here we describe that SWAP-70 is required for this spontaneous
transformation of MEFs.
Materials and Methods: MEF1F2, a Swap-70−/− MEF line, which was
cloned from primary MEFs, was used. SWAP-70 genes were introduced and
expressed in these cells, and the characteristics of these cells such as cell
growth, contact inhibition, and tumor formation in animals, were tested.
Results and Discussion: Swap-70−/− MEFs failed to spontaneously transform
for more than 5 years, suggesting that these cells were deficient in
transformation. After expression of human SWAP-70, these cells underwent
transformation. The phenotypes of two clones were compared. Both of them
grew very rapidly and lost contact inhibition, suggesting that they were
transformed. Moreover, sanguinarine, a putative SWAP-70 inhibitor reversed
transforming phenotypes to normal, suggesting that SWAP-70 plays an
important role for transformation of the cells. However, one clone formed
tumor in nude mice but the other did not. Microarray analysis revealed that the
gene expression patterns were not exactly same. These results suggest that
the transformation was caused by different mutations perhaps together with
transforming activity of human SWAP-70. We expressed mouse SWAP-70
in MEFs to further test whether SWAP-70 is required for transformation
of MEFs. The mouse SWAP-70 gene also induced transformation of
the cells.
Conclusion: SWAP-70 contributes to transformation of primary cells and thus
acts like an oncogene.
102 Growth-inhibitory effect of conjugated linolenic acid on human
eosinophilic leukemia EoL-1 cells
W.N. Liu1 , K.N. Leung1 . 1 The Chinese University of Hong Kong, School of
Life Sciences (Biochemistry Programme), Shatin, Hong Kong
Introduction: Conjugated fatty acids (CFA) refer to the positional and
geometric isomers of polyunsaturated fatty acids with conjugated double
bonds. Examples of naturally-occurring CFA include conjugated linoleic acid
(CLA) from ruminant meats and dairy products and conjugated linolenic acid
(CLN) from plant seed oils. Previous researches have demonstrated that
CFA could inhibit the growth of various cancer cell lines in vitro and in vivo.
Nevertheless, the anti-tumor effects and action mechanisms of CLN on human
myeloid leukemia cells are poorly understood.
Materials and Methods: Anti-proliferative effect of jacaric acid, one of the
CLN isomers, on human eosinophilic leukemia EoL-1 cells was determined
by MTT assay. The cell cycle profile and expression levels of various cell
cycle regulatory proteins in jacaric acid-treated EoL-1 cells were analyzed by
flow cytometry and Western blotting, respectively. Occurrence of apoptosis
was investigated by ELISA assay, Annexin V assay, JC-1 dye staining
and Western blotting. Morphological and functional differentiation of EoL-1
cells were examined by hematoxylin-eosin staining and flow cytometry,
respectively.
Results and Discussion: The anti-tumor effect and molecular action
mechanisms of jacaric acid were examined using the EoL-1 cells. Our results
showed that jacaric acid exhibited anti-proliferative effect on EoL-1 cells in a
time- and concentration-dependent manner. Flow cytometric analysis indicated
that jacaric acid could trigger cell cycle arrest at G0 /G1 phase, accompanied
by a decrease in the protein expression levels of cyclin E and CDK2. Moreover,
jacaric acid was shown to induce apoptosis in EoL-1 cells as revealed
by induction of DNA fragmentation, phosphatidylserine externalization and
mitochondrial membrane depolarization. Up-regulation of Bax and caspase-3
proteins and down-regulation of Bcl-2 protein might account for the induction
of apoptosis. Interestingly, jacaric acid also led to morphological differentiation
in EoL-1 cells and increased the expression levels of two eosinophil-specific
proteins, EPO and MBP.
Conclusion: Our results demonstrated the capability of jacaric acid to inhibit
the growth of human eosinophilic leukemia EoL-1 cells through triggering cell
cycle arrest, and inducing apoptosis and differentiation of the cells. Due to its
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EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
relatively abundance and minimal toxicity, this naturally-occurring compound
is a potential candidate for the treatment of some forms of leukemia.
103 The tumour–stroma niche of ovarian cancer in 3D
D. Loessner1 , B.M. Holzapfel1 , J. Baldwin1 , A. Rockstroh2 , V. Magdolen3 ,
D.W. Hutmacher4 , J.A. Clements1 . 1 Institute of Health and Biomedical
Innovation, Queensland University of Technology, Brisbane Qld, Australia,
2
Australian Prostate Cancer Research Centre − Queensland, Brisbane Qld,
Australia, 3 Clinical Research Unit Department of Obstetrics and Gynecology,
Technical University of Munich, Munich, Germany, 4 Institute for Advanced
Study, Technical University of Munich, Munich, Germany
Introduction: Ovarian cancer is the leading cause of death from gynecologic
malignancies worldwide. The pathological features of this disease provide
special opportunities for experimental modelling, since most tumours are
confined within the abdominal cavity and have a unique path of metastasis −
the formation of multicellular spheroids. The tumour-associated stroma is
known to promote disease progression, but the precise interactions between
cancer cells and their stromal microenvironment are still poorly understood.
We aimed to decipher mediators of metastasis by employing a bioengineered
3-dimensional (3D) approach that mimics the tumour–stroma niche of ovarian
cancer.
Materials and Methods: We have developed an integrated 3D co-culture
model of ovarian cancer spheroids and stromal cells, which we have analysed
by microscopic techniques and proliferation assays in vitro and its contribution
to tumour growth in vivo. In patients, ovarian cancer cells form multicellular
spheroids that accumulate in the tumour fluid and adhere to the stromal
layer. To replicate this interaction, spheroids were grown within polyethylene
glycol-based hydrogels that comprise extracellular matrix features due to
incorporation of protease cleavage sites and integrin-binding motifs. These
were layered onto electrospun-fabricated polycarprolactone meshes that
allowed adhesion of stromal cells, representing the abdominal lining. A whole
human genome microarray was conducted to identify genes differentially
expressed upon 3D co-culture. Genes were grouped by biological processes
according to Gene Ontology and pathways mapped using Ingenuity. Tumour
growth and metastasis were monitored over 8 weeks via bioluminescent
imaging and characterised by immunohistochemistry.
Results and Discussion: Imaging and proliferation analyses revealed
enhanced spheroid growth in the presence of stromal cells compared to
mono-cultures. The fully humanised tumour–stroma niche increased tumour
growth and abdominal spread in mice. More genes were differentially
expressed in co-cultured spheroids than in stromal cells. Regulation of gene
transcription was mostly altered in co-cultured spheroids, while inflammatory,
chemotactic and migratory processes were changed in co-cultured stromal
cells. The prostaglandin-endoperoxide synthase 2 (PTGS2; also known as
cyclooxygenase-2, COX2), network, including fibroblast growth factor 2 (FGF2)
and vascular endothelial growth factor C (VEGFC), was upregulated in
spheroids in 3D co-cultures compared to mono-cultures, which was confirmed
via immunohistochemical analyses of respective tumour sections.
Conclusion: Using this integrated 3D approach, we have unravelled pathways
that are critical for ovarian cancer metastasis, thereby providing new insights
into the tumour–stroma crosstalk responsible for disease progression.
104 ZEB1 modulates expression of CDX1 and CDX2 caudal homeobox
genes in colorectal carcinoma cell lines
O. De Barrios1 , C.A. Orozco1 , E. Sánchez-Tilló1,2 , L. Fanlo1 , A. Castells3,4 ,
A. Postigo1,4,5 . 1 Group of Transcriptional Regulation of Gene Expression,
IDIBAPS, Barcelona, Spain, 2 Miguel Servet Program, Instituto de Salud
Carlos III, Madrid, Spain, 3 Institute of Digestive and Metabolic Diseases,
Hospital Clinic, Barcelona, Spain, 4 CIBERehd, Hospital Clinic, Barcelona,
Spain, 5 ICREA, Barcelona, Spain, 6 James G. Brown Cancer Center,
Louisville, KY, USA
Introduction: In order to leave primary tumor and invade other tissues,
cancer cells have to lose their adhesions and epithelial properties and
acquire a dedifferentiated phenotype through a process called epithelialto-mesenchymal transition (EMT). EMT endows cancer cells increased
invasiveness thus promoting tumor progression and metastasis. Members
of the ZEB family of transcription factors (ZEB1 and ZEB2) trigger an EMT
process by repressing the expression of epithelial genes and upregulation
of mesenchymal ones. ZEB1 is induced in invading cancer cells at the
tumor-host interface of colorectal carcinomas (CRC). CDX1 and CDX2 caudal
homeobox factors play an important role during normal intestinal development
and differentiation. The role of CDX factors during colon cancer progression
remains unclear as they inhibit tumor invasiveness and proliferation while
CDX2 gene has been found amplified in some CRC cell lines.
Materials and Methods: Expression of ZEB1, CDX1 and CDX2 were
assessed in a panel of CRC cell lines by quantitative real-time PCR. ZEB1
expression was knocked down both transiently (with siRNA oligos) and stably
(with shRNA lentiviral particles) in order to investigate its capacity to modulate
the protein and mRNA expression of CDX factors. The promoter regions of
CDX factors were also analyzed for ZEB1 regulation at transcriptional level
bioinformatically and using luciferase based systems.
Results: Using a panel of CRC cell lines, we found that ZEB1 displays
an inverse pattern of expression in comparison to CDX1 and CDX2 mRNA
levels. Both, transient and stable knockdown of ZEB1 in SW480 results in the
upregulation of CDX protein and mRNA levels, thus indicating that CDX1 and
CDX2 factors are repressed by ZEB1. Promoter analysis (in silico) reveales
the existence of consensus binding sites for ZEB factors in the proximal
promoter regions of both CDX factors. Overexpression of ZEB1 inhibits the
transcriptional activity of human CDX1 and CDX2 promoters in vitro.
Conclusions: Our data show that ZEB1 represses the expression of CDX
factors in CRC cells. These results identify a new mechanism by which ZEB1
may regulate cancer cell invasiveness during tumor progression.
OdB and LF are recipients of PhD scholarships from the Ministry of Education,
Culture and Sports (FPU Program). EST is financed by Instituto de Salud
Carlos III (Miguel Servet Program; code CP13/00200; contract MS13/00200).
105 Anoikis of colon carcinoma cells triggered by beta-catenin loss can
be enhanced by Tumor Necrosis Factor Receptor 1 antagonists
K. Rosen1 , O. Masson1 , Y. Li1 , B. Yoo1 . 1 Dalhousie University, Pediatrics,
Halifax, Canada
Background: Detachment of non-malignant epithelial cells from the
extracellular matrix causes their apoptosis, a phenomenon called anoikis. By
contrast, carcinoma cells are anoikis-resistant, and this resistance is thought
to be critical for tumor progression. Many oncogenes trigger not only anti- but
also pro-apoptotic signals. The pro-apoptotic events represent an aspect of a
phenomenon called oncogenic stress, which acts as a safeguard mechanism
blocking tumor initiation. In cancer cells oncogene-induced anti-apoptotic
signals outbalance the pro-apoptotic ones. It is now thought that treatments
blocking the anti-apoptotic events but preserving the pro-apoptotic signals
can be particularly effective in killing tumor cells. Whether or not oncogenes
induce any pro-anoikis signals that can be used for enhancing the efficiency of
approaches aimed at causing anoikis of cancer cells is not known. b-catenin is
a major oncoprotein that is often activated in colorectal cancer and promotes
tumor growth via mechanisms that are understood only in part.
Materials and Methods: To understand how b-catenin controls anoikis of
tumor cells we examined the expression of various regulators of apoptosis
in human colon cancer cells before and after ablation of b-catenin by RNA
interference and the role of these regulators in anoikis resistance of such
cells.
Results: We found that b-catenin triggers both anti- and pro-anoikis signals in
colon cancer cells. We observed that the anti-anoikis signals prevail and the
cells become anoikis-resistant. We established that one pro-anoikis signal in
these cells is triggered by b-catenin-induced downregulation of an apoptosis
inhibitor Tumor Necrosis Factor Receptor 1 (TNFR1) and inactivation of a
transcription factor NF-úB, a mediator of TNFR1 signalling. We also found
that the effect of b-catenin on TNFR1 requires the presence of transcription
factor TCF1, a b-catenin effector. We observed that b-catenin ablation in colon
cancer cells triggers their anoikis and that this anoikis is enhanced even further
if low TNFR1 or NF-úB activity is artificially preserved in the b-catenin-deprived
cells.
Conclusions: b-catenin triggers both anti- and pro-anoikis signals in colon
cancer cells. The latter ones are driven by b-catenin-induced TNFR1
downregulation and NF-úB inactivation. Preservation of low TNFR1 or NF-úB
activity in colon cancer cells enhances anoikis of these cells triggered by the
blockade of b-catenin signalling.
107 The positive feedback between interleukin-8 (IL-8) and matrix
metalloproteinase-9 (MMP-9) in hormone dependent breast cancer
J. Milovanovic1 , N. Todorovic-Rakovic1 , Z. Abu Rabi1 . 1 Insitute of Oncology
and Radiology, Experimental Oncology, Belgrade, Serbia
Background: IL-8 is potent human cytokine and it has prognostic significance
in breast cancer. The mechanisms by which IL-8 contributes to breast cancer
progression are multiple and not completely investigated. It is known that
IL-8 is responsible for the fast chemoattraction of neutrophils, promotion
of angiogenesis and mitogenic effects on various cell types. Gelatinase B
(MMP-9) is secreted upon stimulation of neutrophils and besides its effector
role, MMP-9 might have an important regulatory role in breast cancer as it can
regulate cytokine activity by proteolytic processing. It is known that MMP-9
is able to process IL-8 leading to its potentiation, which could result in an
important positive feedback loop between IL-8 and MMP-9. The aim of the
study was to investigate relation between IL-8 and MMP-9 as well as their
relation to steroid receptor status in hormone dependent breast cancer.
Material and Methods: The study included 150 postmenopausal breast
cancer patients with detectable levels of steroid receptors (ER+PR+) that
should indicate hormone dependent disease. IL-8 and MMP-9 levels were
determined by ELISA in primary tumor tissue lysates.
Results: There was a strong positive correlation between IL-8 and MMP-9
expression (Spearman rank order test, p < 0.001). Furthermore, MMP-9
expression was significantly higher in patients with higher levels of IL-8
according to median IL-8 level (M = 28.42 pg/mg) and IL-8 expression was
significantly higher in patients with higher levels of MMP-9 (M = 1.87 ng/mg,
Mann–Whitney rank sum test, p = 0.05 and p = 0.008, respectively). There was
a significant negative correlation between ER and IL-8, as well as between
PR and IL-8 expression (Spearman rank order test, p = 0.02 and 0.006,
respectively). There was no statistically significant correlation between ER and
MMP-9 expression, neither between PR and MMP-9.
Conclusions: Positive feedback loop between IL-8 and MMP-9 might be
mechanism of promotion of angiogenesis and progression of hormone
dependent breast cancer.
108 Potential effects of telomerase activity and Bcl-2 expression
on the apoptosis of the human brain tumors
C. Kim1 , J.H. Cheong1 , J.M. Kim1 . 1 Hanyang University Hospital, Department
of Neurosurgery, Seoul, Korea
Background: Apoptosis is regulated by several gene products including Bcl-2,
which has been known to be anti-apoptotic property. Additionally, telomerase,
a ribonucleoprotein that synthesizes telomeres, has been identified in various
human neoplasms and its potential roles should be clarified. In the present
study, we investigated the apoptotic effect of Bcl-2 and telomerase activity in
the surgical specimens of human brain tumors.
Material and Methods: A total of 76 cases of surgically resected brain
tumors were included in this study. Telomerase activity was examined by the
telomeric repeat amplification protocol assay, and Bcl-2 was characterized by
the Western blot analysis. Apoptosis of the specimens was detected by DNA
fragmentation analysis.
Results: Telomerase activity was detected in 65.8% (50/76) of the brain
tumors, which induced apoptosis in 20.0% (10/50). Bcl-2 was also expressed
in 23.7% (18/76) of the brain tumors, which induced apoptosis in 11.1%
(2/18). In 14 cases with Bcl-2 expression and negative telomerase activity,
apoptosis was detected in 21.4% (3/14). However, apoptosis was not induced
in all 4 cases with Bcl-2 expression and positive telomerase activity. These
results suggested that apoptosis was enhanced in the brain tumors with Bcl-2
expression and negative telomerase activity (p < 0.05). In the 24 cases without
Bcl-2 expression and telomerase activity, apoptosis was found in 25% (6/24).
Apoptosis was induced in 23.4% (8/34) of brain tumors, which Bcl-2 was
not expressed and telomerase activity was positive. Their difference was not
significant statistically.
Conclusions: Our results suggest that apoptosis of the human brain tumors
with Bcl-2 expression may be influenced by telomerase activity, however,
telomerase activity may not affect on apoptosis of the human brain tumors
without Bcl-2 expression. These indicate that telomerase activity may have a
dependent effect on apoptosis of the human brain tumors.
110 Biological activity of novel platinum(II)–iodido complexes
L. Filipovic1 , A. Savic2 , S. Arandjelovic1 , T. Sabo2 , S. Grguric-Sipka2 ,
S. Radulovic1 . 1 Institute of Oncology & Radiology of Serbia, Department of
Experimental Oncology, Belgrade, Serbia, 2 University of Belgrade, Faculty of
Chemistry, Belgrade, Serbia
Introduction: Novel platinum(II)–iodido complexes of general formula
[PtI2 (L1−3 )], (complexes C1−C3, ligands L1−L3): where L1−3 are isobutyl,
n-pentyl and isopentyl esters of (S,S)-propylenediamine-N,N -di-2-(3-cyclohexyl)propanoic acid have been synthesized and characterized by elemental
analysis, UV/Vis, IR, NMR spectroscopy and mass spectrometry, in order to
investigate their biological activity and to elucidate the mechanism of action.
All studies were performed in comparison to cisplatin.
Material and Method: The cytotoxic activity of the complexes C1−C3 and
ligands L1−L3 was investigated by MTT assay for 48 h of continual action on
four tumor cell lines HeLa, LS-174, MDA-MB-231; one transformed endothelial
cell line EA.hy 926; and one normal MRC-5 cell line. Quantitative analysis of
cell cycle phase distribution was performed by flow-cytometric analysis of the
DNA content in fixed HeLa cells, after staining with propidium iodide. Analyses
of the mode of cell death were performed using flow cytometry by Annexin-VFITC assay, and fluorescence microscopy.
Results and Discussion: Complexes C1−C3 exhibited activity comparable
to cisplatin, with the highest potential in HeLa, LS-174 and EA.hy 926
cells. Precursor ligands (L1−L3) showed approximately two- to fourtimes less activity comparing to the corresponding complexes irrelevant
to the target cell line, with the highest activity observed in EA.hy 926.
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MRC-5 and A549 cells were the least sensitive to the action of
complexes and ligands. C1−C3 induced apoptotic changes characterized
by externalization of phosphatidylserine, generation of considerable subG1 peak and some apoptotic morphological alteration. Only C1 showed
higher potential for apoptosis induction in comparison to the precursor L1
and cisplatin.
Conclusion: Structure-activity comparison in this study revealed that coordination of (S,S)-propylenediamine-N,N -di-2-(3-cyclohexyl)propanoic acid
esters to platinum(II) metal center resulted in increased cytotoxicity of
complexes, comparing to precursor ligands. Cytotoxic activity of C1−C3, was
comparable or higher to those observed for cisplatin. Analyses of the mode
of cell death by flow cytometry and fluorescence microscopy, suggested
different mechanism of action of novel iodido-platinum complexes compared
to cisplatin. Altogether novel iodide-platinum complexes showed promising
biological activity and their potential in cisplatin resistant cell lines should be
further investigated.
111 RANK pathway as a new therapeutic target in primary breast
cancer
P. Pellegrini1 , A. Cordero1 , E. González-Suarez1 . 1 Biomedical Research
Institute IDIBELL, Cancer Epigenetics and Biology Program, L’Hospitalet de
LLobregat, Spain
Background: RANK is a key pathway of mammary gland differentiation and
mediates progesterone induced mammary tumorigenesis in mice. However,
the therapeutic impact of targeting RANK signaling in established tumors is
unknown.
Materials and Methods: Expression profile of RANK and RANKL was
characterized by quantitative PCR and immunohistochemistry in MMTV-neu
and MMTV-PyMT normal tissues and tumors. For primary cultures, tumors
were plated in 3D matrigel cultures and RANK signaling was stimulated with
RANKL. For in-vivo assays 3D colonies where dispersed into single cells and
injected in mammary glands (tumor growth assays) or tail vein (metastasis
assays) of immunodeficient mice.
Results: RANK is highly expressed in tumors of two widely used models
of spontaneous mammary tumorigenesis: MMTV-neu and MMTV-PyMT.
Stimulation of RANK signaling by RANKL in cells derived from MMTV-neu and
MMTV-PyMT carcinomas promotes cell proliferation, survival and increased
tumor growth rate. In addition, RANKL treatment results in increased invasion
of tumor cells and increased metastasis formation ability.
Conclusions: RANK signaling plays an important role in mammary tumor
progression and targeting RANK may be beneficial for breast cancer therapy.
113 Expression of voltage-gated sodium channel beta1 subunits
in breast cancer: Promotion of tumor growth and metastasis
M. Nelson1 , R. Millican-Slater2 , L.C. Forrest1 , W. Brackenbury1 . 1 University
of York, Department of Biology, York, United Kingdom, 2 St James’s University
Hospital, Department of Histopathology, Leeds, United Kingdom
Introduction: Voltage-gated Na+ channels (VGSCs) are heteromeric proteins
composed of pore-forming alpha subunits and smaller beta subunits. The beta
subunits are channel modulators and are also cell adhesion molecules (CAMs).
Beta1, encoded by SCN1B, is best characterized in the central nervous system
(CNS), where it regulates electrical excitability, neurite outgrowth and migration
during development. Beta1 is also expressed in breast cancer cell lines, where
it regulates adhesion and migration in vitro. Here, we show that beta1 plays a
functional role as a CAM in regulating tumour growth and metastasis.
Materials and Methods: We studied beta1 expression using Oncomine and
immunohistochemical analysis of patient tissue specimens. We investigated
the effects of beta1 on tumour growth and metastasis by orthotopic
injection of MDA-MB-231 cells into mice, followed bioluminescent imaging,
immunohistochemistry and confocal microscopy. The effect of beta1 on
process outgrowth was assessed by immunocytochemical analysis of breast
cancer cells grown on fibroblast monolayers.
Results: SCN1B mRNA and beta1 protein were up-regulated in breast
tumours, compared with normal tissue. Over-expression of beta1 in MDAMB-231 cells increased tumor growth and metastasis in a xenograft model
of breast cancer. Beta1 overexpression also increased VEGF secretion and
vascularisation, but reduced apoptosis. Beta1 potentiated outgrowth of neuritelike processes from breast cancer cells cultured with fibroblasts. Process
outgrowth in breast cancer cells specifically required the extracellular adhesion
domain of beta1. Beta1-mediated process outgrowth also required Na+ current
and fyn kinase activity.
Conclusions: Beta1-mediated process outgrowth in breast cancer cells
recapitulates the mechanism by which beta1 regulates neurite outgrowth
in CNS neurons. We conclude that when present in breast tumors, beta1
enhances pathological growth and cellular dissemination by recapitulating
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its well-defined role in CNS ontogeny. Targeting beta1-mediated adhesion
interactions may have potential as a novel anti-cancer therapy.
114 An aqueous extract of Limoniastrum guyonianum gall induces
anti-tumor effects in melanoma-injected mice via modulation
of the immune response
M. Krifa1 , I. Skandrani1 , A. Pizzi2 , N. Nasr1 , K. Ghedira1 , L. Chekir3 . 1 Faculty
of Pharmacy, Monastir, Tunisia, 2 ENSTIB/LERMAB Epinal, ENSTIB, Monastir,
Tunisia, 3 Faculty of Dental Medicine, Monastir, Tunisia
Background: Several studies have reported that plant-derived natural
products have cancer chemopreventive and chemotherapeutic properties. The
objectives of this study were to evaluate the in vitro and in vivo anti-tumor
potential of the aqueous gall extract (G extract) from Limoniastrum guyonianum
and to elucidate its immunological mechanisms, in part, by assessing its effects
on the growth of transplanted tumors and the immune response in these tumorbearing mice. Effects of G extract on cellular anti-oxidant activity in the mice
were also studied.
Materials and Methods: Mice were inoculated with B16F10 mouse tumor
cells and then treated intraperitoneally with G extract at 25 or 50 mg extract/kg
BW for 7, 14, or 21 days. At each timepoint, effects of the extract on the growth
of the tumor, splenocyte proliferation, natural killer (NK) cell activity, and CTL
activity among splenocytes isolated from the mice were measured.
Results: The G extract-induced tumor growth inhibition was associated with
characteristic apoptotic changes in the tumor cells, like nuclear condensation.
In addition, the extract inhibited melanin synthesis and tyrosinase activity
among the melanoma cells in a concentration-related manner. In situ, G extract
did not only significantly inhibit the growth of the transplantable tumor, but also
remarkably increased splenocyte proliferation and both NK and CTL activities
in tumor-bearing mice. The extract was also seen to have promoted lysosomal
activity of host macrophages and gave rise to enhanced cellular anti-oxidant
activity in several cell types (blood, splenocytes and macrophages) in the
mice.
Conclusions: Results indicated that use of the G extract could improve
cellular and humoral immune responses in situ and that the extract could
potentially be employed as an anti-tumor agent that acts, in part, by causing
immunostimulatory and anti-oxidant-status enhancing effects. These effects
could be ascribed to the presence of condensed tannins and polyphenols
such as epicatechin and epigallocatechin gallate.
Abbreviations: G extract: aqueous gall extract; NK: natural killer; CTL: cytotoxic
T-lymphocyte.
115 The role of sumoylation in the regulation of p53 response
D. Marouco1 , N.A. Barlev1 . 1 University of Leicester, Biochemistry, Leicester,
United Kingdom
Background: p53 is a major tumour suppressor protein implicated in many
cellular processes, regulating cell cycle arrest, apoptosis and DNA repair. The
regulation of p53 can be achieved by post-translational modifications (PTMs)
which alter the function of the protein in the cell. Among such PTMs, p53 can
be modified via sumoylation − the covalent binding of SUMO (Small Ubiquitin
MOdifier) protein − on lysine 386.
Materials and Methods: The aim of this project is to define the role of
sumoylation in regulation of the activity of p53. For that, we combined realtime qPCR and luciferase reporter assays to analyse the function of p53
sumoylation in cellulo. In vitro binding and modification studies were used
to investigate the interplay between p53 sumoylation and other PTMs.
Results: Here we show that sumoylation of p53 with both SUMO-1 and
SUMO-2 significantly decreases the transcriptional ability of p53 for its target
genes p21 and PUMA, in both U2OS and HCT116 cell lines. Furthermore,
the overexpression of a wild type p53 protein fused with the sumoylation
conjugating enzyme ubc9 (p53-ubc9) in p53 null cells H1299 shows a reduced
ability to activate p53-target genes, when compared to the sumoylation
deficient mutant (p53K386R-ubc9). In addition, in vitro assays show that p53
acetylation by CBP/p300, as well as the methylation of lysine 372 by Set9, is
affected by the presence of sumoylation of lysine 386.
Conclusion: These results highlight the importance of Sumoylation as
a modulator of p53 activity, and confirm the idea of a post-translational
modifications network, where covalent modifications interplay with other
modifications for the regulation of p53.
116 The mRNA-binding protein LARP1 is a pro-survival factor that
promotes tumourigenicity and chemotherapy resistance in ovarian
cancer
T.G. Hopkins1 , J. Weir2 , M. Mura1 , N. Abd-Latip1 , K. Sweeney1 ,
S. Ghaem-Maghami1 , G. Gabra1 , S.P. Blagden1 . 1 Imperial College, Surgery
and Cancer, LONDON, United Kingdom, 2 Imperial College Healthcare NHS
Trust, Cellular Pathology, LONDON, United Kingdom
Background: There is growing evidence that mRNA-binding proteins can be
key post-transcriptional drivers of cancer progression. We have recently shown
that LARP1 is highly expressed in cervical and lung malignancies. In cervical
cancer cells, LARP1 is in complex with an mRNA interactome enriched for
key cancer-related transcripts, including mTOR. We investigated the extent to
which LARP1 regulates cancer progression in ovarian malignancies and used
high-throughput strategies to identify novel LARP1-regulated genes.
Material and Methods: Expression array data was obtained from the TCGA,
Oncomine and kmplot repositories. For in vivo xenograft studies, SKOV3
cells with stable expression of a control or LARP1-targeting short hairpin
sequence were injected subcutaneously into NOD-SCID mice. Apoptosis was
assessed with Annexin V and Caspase 3/7 assays. Stem marker expression
was assessed by flow cytometry. For transcriptome analysis, total RNA was
extracted from OVCAR8 cells following treatment with control or LARP1targeting siRNA. Three independent repeats underwent deep sequencing
using the Illumina HiSeq200 platform. Gene expression was assessed by RTPCR and western blotting.
Results: In silico analysis of publicly available mRNA expression data
demonstrated that LARP1 was highly expressed in ovarian cancers (n = 735)
compared to the normal ovary, and high levels were predictive of poor outcome
(n = 557).
Xenograft experiments, in which ovarian cancer cell lines were implanted
subcutaneously into immunocompromised mice, demonstrated that stable
LARP1 knockdown dramatically reduced tumour growth. In vitro, we found that
reduced LARP1 expression was associated with a reduction in clonogenicity
and anchorage-independent growth. Knockdown of LARP1 led to reductions
in populations positive for putative cancer stem cell markers, including CD133,
and with high aldehydyde dehydrogenase activity. Transient knockdown of
LARP1 was sufficient to restore platinum sensitivity in resistant lines.
To identify LARP1-regulated targets we performed transcriptome deep
sequencing. There was a significant enrichment (p < 0.001) for LARP1
interactome partners in the genes that displayed altered expression following
knockdown. Functional annotation clustering revealed multiple genes linked
to survival and evasion of apoptosis, notably the anti-apoptotic protein BCL2.
We confirmed that LARP1 knockdown led to reduction in BCL2 mRNA and
protein expression. BCL2 transcripts are in complex with LARP1 protein in
ovarian cancer cells and LARP1 is required for transcript stability.
Conclusions: LARP1 appears to be an important driver of malignant
progression in ovarian cancer. LARP1 promotion of BCL2 transcript stability
identifies it as a key pro-survival factor, and the protein may have potential as
a therapeutic target.
118 Interaction of C-FABP and PPARS in prostate cancer
F. Seyed Forootan1 , S. Seyed Forootan Shiva1 , Y. Ke1 . 1 University of
Liverpool, Molecular and Clinical Cancer Medicine (Pathology), Liverpool,
United Kingdom
Background: How C-FABP promotes tumourigenicity of prostate cancer is not
fully understood. We hypothesize that C-FABP may transport an excessive
amount of intracellular fatty acids into cancer cells to activate their nuclear
receptor PPARs to trigger a chain of molecular events which may lead to an
facilitated malignant progression of the cancer cells.
Method: Expression of C-FABP, PPARb/d and PPARg in cell lines were
detected by Western blot at protein level and by RT-PCR at mRNA level. The
expression of C-FABP, PPARb/d and PPARg in BPH and in prostate carcinoma
tissues were detected by immunohistochemical staining. Then the relevant
PPAR gene was suppressed transiently (siRNA technique) and stably (shRNA
technique) to assess its effect on malignant progression in P Ca, in vitro
(Proliferation assay, Invasion assay, Soft agar assay) and in vivo.
Results: Although the expression of PPARb/d in carcinoma tissues was
significantly higher than that in BPH, no significant difference in its expression
between benign and malignant cell lines was observed. For PPARg and
C-FABP, not only that their expression levels in malignant cell lines and tissues
(cytoplasm and nucleus) were significantly higher than those expressed in
benign cells and in BPH respectively, it appeared that their expression levels
were increased as the increasing malignancies of the cell lines and tissues.
While the increased expression of PPARb/d in carcinomas was not correlated
with the prostate cancer patient survival time, the increased expression of
both C-FABP and PPARg were significantly correlated to a reduced survival
period of time. Multi variant analysis further showed that C-FABP and PPARg
were not independent when used as markers to predict the patient survival.
Suppression of PPARg showed to be correlated with significant reduction of
proliferation rate, invasiveness and the ability of colony formation of P Ca cells
in vitro. Furthermore, suppression of PPARg expression in the highly malignant
prostate cancer cells significantly inhibited the tumourigenicity in nude mice.
Conclusion: This study showed that the prognostic value of PPARg is
dependent to C-FABP in prostate carcinomas so they are two independent
factors which can be used to predict the patient outcomes. Our previous
work on C-FABP and this study suggested that suppression of both CFABP and PPARg genes can cause similar degree of reductions on
the tumourigenicity of P Ca cells. These results strongly support our
hypothesis that C-FABP promotes tumourigenicity by transporting an excessive
amount of intracellular fatty acids into cancer cells to activate their nuclear
receptor PPARg which may then activate the down-stream cancer-promoting
genes.
120 Autocrine human growth hormone promotes the oncogenic
behaviour of hepatocellular carcinoma cells
Y.J. Chen1 , X.J. Kong2 , Z.S. Wu2 , Y.C. Lau1 , J.J. Wang1 , T. Zhu2 ,
P.E. Lobie1,3 . 1 National University of Singapore, Pharmacology, Singapore,
Singapore, 2 University of Science and Technology of China, Hefei National
Laboratory for Physical Sciences at Microscale, Hefei, China, 3 National
University of Singapore, Cancer Science Institute, Singapore
Introduction: The liver is an essential target tissue of growth hormone (GH),
which is involved in somatic growth promotion and metabolic processes.
Aberrant GH signalling has been implicated in the development of metabolic
liver diseases, as well as liver cancer. In addition to its endocrine actions,
GH is widely recognized as a local growth factor in a number of tissues.
The oncogenic potential of autocrine human GH (hGH) has been intensively
investigated in human mammary and endometrial cancer cells. However, the
oncogenic potential of autocrine hGH in hepatocellular carcinoma (HCC) is still
unclear. We have recently observed that tumour expression of hGH associated
with poor clinical outcomes in HCC patients. Hence, we have proceeded to
determine whether autocrine hGH promoted oncogenic behaviour in HCC
cells, and deciphered the potential mechanisms.
Material and Method: HCC cells were stably transfected with the pcDNA3hGH plasmid or vector control. Would healing and Transwell assays were
performed to investigate the invasive potential of autocrine hGH in HCC cells.
The cancer stem cell (CSC) like properties promoted by autocrine hGH were
investigated by spheroid and Aldefluor assays. Western blot, luciferase reporter
assay and chromatin immunoprecipitation (ChIP) assays were performed to
determine the potential signalling mechanism utilized by autocrine hGH in
HCC cells.
Results and Discussion: Using wound healing and Transwell assays, we
observed that autocrine hGH significantly increased the invasive potential
of HCC cells. Furthermore, autocrine hGH increased spheroid formation
in suspension culture, indicative of CSC-like activity. Aldefluor assay
demonstrated that autocrine hGH increased the ALDH1 positive population
of HCC cells. Western blot analysis showed that autocrine hGH activated
STAT3 in HCC cells. Autocrine activation of STAT3 in HCC cells reduced the
expression of Claudin-1, a tight junction protein. Luciferase reporter assays
demonstrated that autocrine hGH activated STAT3 inhibited the transcriptional
activity of the Claudin-1 promoter. ChIP assay revealed that STAT3 directly
bound to the promoter region of Claudin-1 gene. And this binding was
enhanced by autocrine hGH. We identified three putative STAT3 binding sites
in the Claudin-1 promoter. Furthermore, AG490, an inhibitor of JAK2, restored
Claudin-1 expression in HCC cells with forced expression of hGH. Forced
expression of Claudin-1 abrogated autocrine hGH stimulation of invasive and
CSC-like behaviour.
Conclusion: These results indicate that autocrine hGH induces invasive and
CSC-like properties in HCC cells. hGH-induced oncogenicity in HCC cells is
mediated by STAT3 dependent inhibition of Claudin-1 expression.
122 Poly(I:C) and chemotherapeutics synergistically induce cell death
in head and neck cancer cell lines
T. Matijevic Glavan1 , B. Verillaud2 , P. Busson2 , J. Pavelic1 . 1 Rudjer Boskovic
Institute, Department of Molecular Medicine, Zagreb, Croatia, 2 Gustave
Roussy Institute, UMR 8126, Paris-Villejuif, France
Introduction: Toll-like receptors (TLRs) recognize pathogen-derived molecules and also products of inflamed tissue, resulting in the activation of the
immune response. TLR ligands are already being used in clinical studies
because of their ability to induce apoptosis and trigger the immune system.
In a previous study we showed that TLR3 activation by synthetic dsRNA
[poly(I:C)] in cancer cell line Detroit 562 (pharynx carcinoma) induced caspasedependent apoptosis. Additionally, when combined with chemotherapeutics
this treatment acts synergistically by enhancing cancer cell death. In this study
we tried to clarify the mechanism of the observed synergy.
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Material and Method: Cell lines used were Detroit 562 and SQ20B. Cell
survival was determined by using cytotoxicity assay and colony formation
assay while protein expression was obtained by western blot.
Results and Discussion: We have shown here that the combination of
poly(I:C) and cisplatin cytotoxicity is TLR3-dependent while methotrexate
and hydroxyurea act through other dsRNA receptors (RIG-I and MDA5).
Additionally, poly(I:C) has a dual effect in Detroit 562 cells: it induces both
pro-apoptotic and anti-apoptotic (an increase in the expression of c-IAP2)
events. Thus, combined treatment by poly(I:C) and c-IAP inhibitor significantly
decreased cell survival even at low concentrations. However, our results
showed that cisplatin has the ability to down regulate the c-IAP2 expression
induced by poly(I:C) in the Detroit 262 cells thus increasing the pro-apoptotic
response. In this study we also used laryngeal cell line SQ20B stably
transfected with inducible shRNA for TLR3 which is a good model for
poly(I:C) combinational therapy research. We confirmed that synergistic effect
of poly(A:U) and cisplatin on cell death is TLR3-dependent. Additionally, as
SQ20B is radiation resistant cell line, we explored here whether it can be
sensitized to radiotherapy by poly(I:C)/cisplatin and which molecular pathways
are involved in this process.
Conclusion: Synthetic dsRNAs, like poly(I:C), have the potential to help
overcoming the resistance of some human malignant cells to classical
modalities of radiotherapy and chemotherapy.
123 M30 assay may not be an accurate method for apoptosis in the
cancer cells expressing low level of cytokeratin
B. Cevatemre1 , F. Ari1 , M. Sarimahmut1 , A. Yilmaztepe Oral2 , E. Ulukaya2 .
1
Faculty of Arts and Sciences, Biology, Bursa, Turkey, 2 Faculty of Medicine,
Medical Biochemistry, Bursa, Turkey
Background: Caspase-cleaved fragment of cytokeratin 18 (M30), which is
present only in epithelial cells, has been regarded as a biomarker of apoptotic
cell death because it is released from the cells during apoptosis. It is
therefore believed that it reflects cell death of epithelial tumors. Based on
this, studies suggest that M30 may have important clinical biomarker utility, as
increased levels of M30 may be prognostic and/or predict tumour response
to chemotherapy. However, it does not increase in the sera of some patients
although those patients respond to the treatment well. In the current study
A549, H1299 and PC3 lung cancer cell lines were used to determine the
correlation between apoptosis and M30 levels.
Material and Methods: The MTT and ATP viability assays were used to
determine the cytotoxic activity of some anti cancer agents that are also
used in the clinics. We used fluorescence imaging of nuclei (Hoechst 33342
staining) as well as Annexin V-FITC staining to detect apoptosis. M30 levels
were measured by ELISA.
Results: M30 levels were clearly increased in A549 cells, but remained
unchanged in H1299 and PC3 cells although these cells underwent apoptosis.
These two cell lines were subsequently found to express low level of cytokeratin
18.
Conclusions: Our results suggest that M30 may not be a universal marker
for apoptosis in all cancer types. Therefore the data should be interpreted with
caution.
124 Vincristine induces autophagy-mediated HMGB1 release via
transcriptional regulation of Mcl-1 antagonizes apoptosis in
human oral cancer cells
S. Yang1 , C. Lin2 , M. Hsieh3 . 1 Chung Shan Medical University, Institute of
Medicine, Taichung, Taiwan, 2 Chung Shan Medical University, Institute of
Oral Sciences, Taichung, Taiwan, 3 Changhua Christian Hospital, Cancer
Research Center, Changhua, Taiwan
Background: The autophagy-associated release of HMGB1 (high-mobility
group box 1) is known to protect cancer cells from numerous chemotherapeutics. However, the related molecular mechanism, involved in the protection
of oral cancer cells remains not clear.
Material and Methods: Cell viability was examined by MTT assay, whereas
cell cycle analysis and quantification of acidic vesicular organelle (AVO)
formation were measured by flow cytometry. Real-time PCR confirmed the
effects of vincristine on HMGB1 mRNA level in SCC-9 cells. Western blotting
was performed for detecting changes of autophagy-associated proteins.
Results: In this study, we determined that HMGB1 released by oral
cancer cells protected the cells against apoptosis caused by vincristine by
upregulating the transcription of Mcl-1. Extracellular HMGB1 seem to be
required for the autophagy-mediated inhibition of apoptosis because HMGB1
knockdown by siRNA abolished the effect of autophagy protective. Vincristine
treatment increased the expression of Mcl-1 mRNA, but did not alter the
protein expression levels of Mcl-1. Inhibiting HMGB1 expression blocked
the increase in the Mcl-1 transcription and reduced Mcl-1 protein levels,
demonstrate that HMGB1-mediated signaling is required for the upregulation
S28
of Mcl-1 transcriptional, thereby maintaining Mcl-1 protein expression at levels
necessary for the survival of oral cancer cells exposed to vincristine.
Conclusions: These results identify the HMGB1-mediated upregulation of
Mcl-1 transcription as a key mechanism by which autophagy protects oral
cancer cells against apoptosis induced by vincristine.
125 Autophagy effects of pterostilbene on human oral cancer cells
through modulation of Akt and mitogen-activated protein kinase
pathway
C. Lin1 , S. Yang2 , M. Hsieh3 . 1 Chung Shan Medical University, Institute of
Oral Sciences, Taichung, Taiwan, 2 Chung Shan Medical University, Institute
of Medicine, Taichung, Taiwan, 3 Changhua Christian Hospital, Cancer
Research Center, Changhua, Taiwan
Background: Extensive research supports the administration of herbal
medicines or natural foods during cancer therapy. Pterostilbene, a naturally
occurring phytoalexin, has various pharmacological activities, including
antioxidant activity, cancer prevention activity, and cytotoxicity to many cancers.
However, the effect of pterostilbene on the autophagy of tumor cells has
not been clarified. In this study, the unique effects of pterostilbene on the
autophagy of human oral cancer cells were investigated.
Material and Methods: The effects of pterostilbene-induced autophagy on
SCC-9 and OECM-1 oral cancer cells were detected by using MTT test,
immunofluorescence, electronic microscope, and flow cytometry. Western
blotting was performed for detecting changes of autophagy-associated proteins
and state of activation of Akt, JNK1/2, Erk1/2 and p38-MAPK.
Results: The results of this study showed that pterostilbene effectively
inhibited the growth of human oral cancer cells by inducing cell cycle arrest
and apoptosis. In addition, the formation of acidic vesicular organelles and
LC3-II production also demonstrated that pterostilbene induced autophagy.
Administering 3-methylamphetamine (3-MA) and bafilomycin A1 (BafA1)
exerted differing effects on the pterostilbene-induced death of human oral
cancer cells. Pterostilbene-induced autophagy was triggered by activation of
JNK1/2 and inhibition of Akt, ERK1/2, and p38.
Conclusions: In conclusion, this study demonstrated that pterostilbene
caused autophagy and apoptosis in human oral cancer cells, and
pterostilbene-induced autophagy played different roles in these cells,
suggesting that pterostilbene could serve as a new and promising agent for
treating human oral cancer.
126 Metabolic and motile reprogramming of ER positive breast cancer
cells following long-term estrogen deprivation
A. Morandi1 , M. Bacci1 , L.A. Martin2 , M.L. Taddei1 , E. Giannoni1 , P. Chiarugi1 .
1
University of Florence, Department of Experimental and Clinical Biomedical
Sciences, Florence, Italy, 2 The Institute of Cancer Research, Breakthrough
Breast Cancer Research Centre, London, United Kingdom
Introduction: Approximately 2/3 of the breast tumors are positive for estrogen
receptor (ER)-a (called hereafter ER) expression and aromatase inhibitors (AI)
have become the first-line endocrine treatment choice for postmenopausal
women with ER+ breast cancers. However, de novo or acquired AI resistance
still limits their benefit for many patients. Metabolic reprogramming is now
considered a hallmark of cancer and a primary and fundamental aspect of cell
transformation. To elucidate whether metabolic reprogramming is linked to AI
resistance we analyzed the metabolic profile of parental and AI resistant cells
and their behavior when glycolysis or oxidative phosphorylation (OXPHOS)
are impaired. Ultimately, motility and aggressiveness were evaluated in both
cell types to correlate metabolic adaptation to cancer cell plasticity.
Material and Method: Long-term estrogen deprived MCF7 (MCF7-LTED) and
MCF7 overexpressing aromatase (MCF7−2A) were used as in vitro models of
AI resistance and sensitivity, respectively. Radioactive assays were used to
determine the levels of Glucose and Lactate uptake and to monitor OXPHOS
activity. Western blot analysis was used to investigate the levels of key
metabolic players in the different cell lines. The changes in phenotype and
metabolism of the cancer cells were investigate in response to 2-Deoxyglucose (2-DG) and Metformin. 2-DG is an analog of glucose that cannot
be metabolized, hence causing a glycolytic block. Conversely, Metformin is an
inhibitor of mitochondrial respiratory chain complex 1 that induce a block of the
OXPHOS. Finally, data mining of publicly available gene expression database
on tumor samples was used to confirm the in vitro experiments.
Results and Discussion: Androgen treated MCF7−2A cells were subject to
different concentration (0.1 mM and 0.1 nM) of the AI, letrozole. The glycolysis
inhibition and the cell viability inhibition induced by letrozole treatment were
shown to be positively correlated, suggesting a role for letrozole in impairing
glycolysis. Furthermore, metabolic comparison of LTED-MCF7 cells versus
parental MCF7 cells showed an important increase in glycolysis dependence
of the AI-resistant cells. When both cell lines were exposed to either 2-DG
or to Metformin, only the parental MCF7 cells showed a decrease in cell
viability. MCF7-LTED cells were able to switch to either OXPHOS or glycolysis
when 2-DG or metformin were used, respectively: only the combination of
2-DG and metformin was able to induce MCF7-LTED cell death suggesting
a high metabolic plasticity of MCF7-LTED. Importantly, MCF7-LTED cells are
more motile than parental MCF7 and once MCF7-LTED cells were subjected
to 2-DG treatment, they further increase their motile ability. Finally, datasets
analysis of patients that were treated with AI in a neo-adjuvant setting revealed
an enhanced glycolytic phenotype in patients that did not respond to AI
treatment.
Conclusion: Glycolytic metabolism is associated with enhanced breast cancer
aggressiveness and tumor cell growth in the absence of estrogen. The data
presented here suggest that the metabolic plasticity is intimately linked to
cancer aggressiveness. Thus, metabolic reprogramming could be a potential
therapeutic target if used in combination with current therapeutic regimes.
128 Loss of far upstream element (FUSE) binding protein (FBP)interacting repressor (FIR) function supports HCC growth
M. Malz1 , M. Bovet1 , J. Samarin1 , D.F. Calvisi2 , S. Rössler1 , S. Singer1 ,
M. Weber3 , M. Zörnig4 , P. Schirmacher1 , K. Breuhahn1 . 1 Institute of
Pathology, Heidelberg, Germany, 2 Institute of Pathology, Greifswald,
Germany, 3 Institute of Surgical Pathology, Zürich, Switzerland,
4
Chemotherapeutisches Forschungsinstitut Georg-Speyer Haus, Frankfurt,
Germany
Introduction: Overexpression of far upstream element (FUSE) binding
protein-1 (FBP-1) stimulates HCC cell proliferation and significantly correlates
with poor overall survival of HCC patients (Malz et al., 2009, Hepatology).
Under physiological conditions transcriptional activity of FBP-1 is tightly
regulated by the FBP-interacting repressor (FIR). We therefore ask if the
loss of FIR expression allows FBP-1 to unfold its full oncogenic properties
in hepatocarcinogenesis.
Materials and Methods: FIR expression, subcellular localisation, and
splice variant abundance were analysed at the mRNA and protein levels
in HCC tissues and HCC cells (real-time PCR, western immunoblot,
immunhistochemistry). Functional impact of FIR on HCC cell proliferation
(MTT- and BrdU-assay), apoptosis (FACS, PARP cleavage) and lateral
movement (time-lapse microscopy) was defined after siRNA-mediated
inhibition. Downstream effectors of FIR were identified using microarray
analysis. Results were confirmed using functional epistasis experiments and
luciferase reporter assays. FBP-1 expression was defined on transgenic mice
with liver-specific E2F1 expression. Subcutaneous tumor growth after lentiviralbased inhibition of FIR splice variants in HCC cells was analysed (xenograft).
Results and Discussion: FIR overexpression correlates with chromosomal
gains of the fir gene locus (chr. 8q24.3) in human HCC tissues. Nuclear FIR
accumulation in premalignant lesions (15%) and HCCs (60%) significantly
correlates with tumor dedifferentiation and cell proliferation. In vitro, FIR
silencing drastically reduced HCC cell viability, proliferation, and migration but
did not affect tumor cell apoptosis. Thus, nuclear FIR enrichment in HCC cells
supports tumor growth and HCC cell dissemination.
FIR induced FBP-1 expression in HCC cells was detected together with a
positive correlation between nuclear FIR and FBP-1 in primary human HCCs.
FIR supports HCC cell proliferation partly via FBP-1 induction. We confirmed
that FIR stimulates FBP-1 expression in an E2F1/DP1-dependent manner
in vitro and in livers from E2F1 transgenic mice. Thus, FIR loses its repressor
function in HCC cells and stimulates rather than inhibits FBP-1 expression and
activity.
Four FIR splice variants with and without exon 2 and/or 5 were detected in
HCC cells/tissues (42%) but not in normal hepatocytes at the transcript and
protein levels. All analysed splice variants accelerate tumor growth in vitro and
in vivo. Thus, different FIR isoforms support HCC cell tumorigenicity.
Conclusion: High-level nuclear enrichment of FIR splice variants does not
facilitate repressor properties but supports HCC growth. Pharmacological
inhibition of the onocgenic FIR/FBP-1 pathway may represent a promising
therapeutic strategy for a subgroup of HCC patients.
129 Semaphorin7A increases growth and metastasis of mammary
tumours by promoting tumour cell survival and motility
R. Garcia-Areas1 , S. Libreros1 , S. Amat2 , C. Castro-Silva1 , P. Robinson1 ,
E. Wojcikiewicz1 , K. Schilling3 , V. Iragavarapu-Charyulu1 . 1 Florida Atlantic
University, Schmidt College of Medicine, Boca Raton, USA, 2 Max Planck
Florida Institute for Neuroscience, Jupiter, USA, 3 Boca Raton Regional
Hospital, Christine E. Lynn Women’s Health & Wellness Institute, Boca
Raton, USA
Introduction: The study of breast cancer relies heavily upon the identification
of tumour-associated proteins involved in tumour growth and metastasis. Our
laboratory discovered that mammary tumour cells express high levels of the
axonal guidance molecule Semaphorin7A (SEMA7A). SEMA7A expression is
induced by activation of the PI3K/AKT pathway, an important cell survival
pathway. SEMA7A may promote tumour cell survival as an effector of the
PI3K/AKT pathway.
Material and Method: Non-metastatic 67NR cells were transfected to overexpress SEMA7A and the SEMA7A gene was silenced in 4T1 mammary
tumour cells using shRNA. Cytoskeletal changes and motility were assayed
by F-actin staining and atomic force microscopy. In vitro proliferation and
apoptosis were quantified using flow cytometry. BALB/c mice were inoculated
with mammary cells with altered SEMA7A expression. Immunohistochemistry
and qPCR were used to correlate SEMA7A and ki67 expression in human
breast tumours.
Results and Discussion: Overexpression of exogenous SEMA7A induced
a mesenchymal morphology in 67NR cells, while gene silencing of SEMA7A
in 4T1 cells decreased mesenchymal qualities. SEMA7A silenced 4T1 cells
showed decreased ki67 proliferation, decreased activation of the PI3/AKT
pathway and increased apoptosis. In vivo, inhibition of SEMA7A resulted in
reduced tumour growth, reduced metastasis and enhanced survival. Increased
SEMA7A expression positively correlated with high ki67 (>25%) in human
breast tumours biopsies.
Conclusion: Our findings suggest a novel role for SEMA7A in promoting
tumour cell survival and proliferation. Further characterization of SEMA7A
could expose new mechanisms and pathways involved in breast cancer
progression.
130 Eight microRNAs as biomarkers for metastatic spread in triple
negative breast cancer
A. Mathe1 , K.A. Avery-Kiejda1 , M. Wong-Brown1 , J.F. Forbes2 , S.G. Braye3 ,
R.J. Scott1 . 1 University of Newcastle & Hunter Medical Research Institute,
Medical Genetics, Newcastle NSW, Australia, 2 Calvery Mater Newcastle
Hospital, Surgical Oncology, Newcastle NSW, Australia, 3 John Hunter
Hospital, Hunter Area Pathology Service, Newcastle NSW, Australia
Background: Triple negative breast cancer (TNBC) is known to be more
aggressive than other breast cancer subtypes. More frequent and rapid
metastatic spread occurs after initial diagnosis, which is associated with a
significant decrease in survival. For TNBC patients, there is currently no
targeted therapy available and new approaches for the treatment of this breast
cancer subtype are required.
microRNAs (miRNAs) are small non-coding RNAs, that are able to alter
gene expression post-transcriptionally. Multiple studies provide evidence that
miRNAs are involved in breast tumour initiation as well as metastases. The
aim of this study is to identify miRNAs as biomarkers for metastatic spread and
study their functional role in the process of cell proliferation and migration.
Material and Methods: We used a sample cohort of 31 TNBC samples to
run miRNA microarrays (Sanger release 14.0 − Agilent), which were analysed
with Genespring GX v 12.1 (Agilent Technologies) and Genomic Suite v 6.6
(Partek).
We used a TNBC cell line (MDA-MB-231) to transiently over-express the
miRNAs of interest, which were validated by real-time PCR.
Proliferation and migration play a major role in the process of metastatic spread
therefore we examined the behaviour of a TNBC cell line (MDA-MB-231) after
over-expressing miRNAs using CellTiter-Glo Luminescent Cell Viability Assay
(Promega) and a scratch assay.
Results and Discussion: Eight microRNAs were found to be differentially
expressed in lymph node positive cases and lymph node metastases
compared to lymph node negative tumours, confluent in both microarray
analyses.
Real-time PCR demonstrated that the transfected miRNAs were significantly
overexpressed compared to a non-targeting miRNA transfection control.
Nonetheless, none of our miRNAs of interest showed a change in cell
proliferation following overexpression either alone or in combination with other
miRNAs. This might be explained by the sensitivity of the performed assay. The
first run of the migration assay revealed that overexpression of our miRNAs can
have variable effects on migration when transfected alone or in combination.
Three miRNAs promoted, and 4 potentially inhibited migration.
Conclusion: We demonstrated that miRNAs can impact migration in TNBC.
Further experiments will be necessary to validate these results. This study has
the capacity to identify important pathways that are regulated by microRNAs
and that are involved in cancer progression in TNBC.
131 Diacylglycerol kinase alpha regulates Src and promotes 3D growth
in breast cancer
P. Torres-Ayuso1 , M. Daza-Martin1 , A. Ávila-Flores1 , I. Mérida1 . 1 Centro
Nacional de Biotecnologia-CSIC, Immunology and Oncology, Madrid, Spain
Introduction: Diacylglycerol kinases (DGK) are a family of lipid enzymes
that phosphorylate diacylglycerol to produce phosphatidic acid, two key
intermediates of signaling and metabolic pathways. To date, the DGK family
S29
consists of ten isoforms. The DGKa isoform has been widely implicated in
cancer promotion. This isoform is required for the survival, migration and
invasive properties of tumors. In addition, DGKa is an important effector of
receptor tyrosine kinases and Src functions. However, the exact mechanisms
by which DGKa-contributes to these processes have not been thoroughly
determined.
Methods: Using different types of breast cancer cell lines (luminal, Her2+ and
triple negative), we have investigated here the contribution of DGKa to the
regulation of Src tyrosine kinase and evaluated the therapeutical opportunities
of targeting this isoform. We used three-dimensional (3D) matrigel cell
cultures. These culture conditions mimic more closely the in vivo environment
and permit to explore more in depth mechanisms of drug response and
resistance.
Results: We found that DGKa was required the in vivo growth of grafted breast
tumors, and that siRNA-mediated downregulation of its levels compromised
tumor cell proliferation in a 3D context. DGKa expression increased when
cells were culture on matrigel. DGKa silencing or pharmacological inhibition
resulted in incomplete Src activation. Moreover, DGKa was found to be under
the control of the PI3K-Akt-FoxO axis, and its levels increased when PI3K was
inhibited, thus suggesting that DGKa upregulation can contribute to resistance
to PI3K-Akt inhibitors. Pharmacological targeting of DGKa was effective in
reducing tumor growth, both in vitro and in vivo. This effect was further
increased when combined with PI3K or Src inhibitors. Inhibition of DGKa did
not affect the survival of non-transformed breast cells.
Conclusions: Our results suggest important roles for DGKa in promoting
tumor progression and provide additional mechanisms that link malignant
transformation with lipid metabolism. Our data indicate that DGKa could
represent an effective target for anti-cancer therapies.
132 Autocrine human growth hormone suppression of E-cadherin
via p44/42 MAPK promotes epithelial-to-mesenchymal transition
of colorectal carcinoma cells
J. Wang1 , Z. Wu2 , V. Pandey3 , Y. Chen1 , T. Zhu2 , P. Lobie1,3 . 1 National
University of Singapore, Pharmacology, Singapore, Singapore, 2 University of
Science and Technology of China, Hefei National Laboratory for Physical
Sciences at Microscale, Hefei, China, 3 Cancer Science Institute of Singapore,
Singapore, Singapore
Introduction: Human growth hormone (hGH) has been reported to
be associated with lymph node metastasis and poor survival outcome
in patients with mammary and endometrial carcinomas. Functionally,
autocrine expression of hGH has been demonstrated to promote oncogenic
transformation, and phenotypic conversion of human mammary carcinoma
cells associated with increased cell migration and invasion. hGH and the hGH
receptor have been reported to be expressed in colorectal carcinoma (CRC).
However, the clinicopathologic association of hGH with CRC and its potential
functional role in CRC remains largely to be determined.
Materials and Methods: We generated two human colorectal adenocarcinoma cell lines DLD-1 and Caco2 stably transfected with the pcDNA3.1
expression vector containing hGH cDNA or empty pcDNA3.1 as control.
Cell motility was determined by several cell function assays, such as wound
healing, transwell migration and invasion assays. Quantitative PCR (qPCR)
and Western blot analysis were performed to determine the expression of
markers of epithelial-to-mesenchymal transition (EMT).
Results and Discussion: In situ hybridization and immunohistochemical
analysis demonstrated that hGH was frequently (50%) expressed in CRC
compared to normal tissue. The expression of hGH was positively associated
with tumor size and lymph node metastasis. Functionally, we observed
that autocrine hGH altered CRC cellular morphology from an epithelial
phenotype with copious intercellular contact to a more scattered and
mesenchymal morphology. Wound healing and transwell assays demonstrated
that autocrine hGH significantly increased cell motility. Furthermore, autocrine
hGH stimulated CRC cell invasion through matrigel. qPCR and western
blot analysis demonstrated that autocrine hGH up-regulated the expression
of mesenchymal markers (vimentin, fibronectin 1) and down-regulated the
expression of epithelial markers (occludin, E-cadherin). Autocrine hGH
activated p44/42 MAP kinase to decrease E-cadherin promoter activity and
expression in CRC cells. Treatment with the MEK1/2 inhibitor PD98059
prevented autocrine hGH suppression of E-cadherin and inhibited autocrine
hGH stimulated cell invasion. Concordantly, forced expression of E-cadherin in
CRC cells significantly inhibited autocrine hGH stimulated CRC cell migration
and invasion.
Conclusion: hGH is expressed in CRC and associated with clinicopathologic
features of the tumor. Autocrine hGH altered cell morphology, stimulated EMT
and promoted cell motility in CRC cells. hGH promoted EMT in CRC cells is
mediated by the decreased expression of E-CADHERIN in a p44/42 MAPKdependent manner.
S30
133 Oral cavity changes in lymphoma patients
I. Besu Zizak1 , S. Jelic2 , S. Matkovic2 , B. Mihaljevic3 , L.J. Jankovic4 .
1
Institute of Oncology and Radiology of Serbia, Belgrade, Serbia, 2 Institute
of Oncology and Radiology of Serbia, Medical Oncology, Belgrade, Serbia,
3
Clinical Center of Serbia, Institute of Hematology, Belgrade, Serbia, 4 Faculty
of Dental Medicine, Clinic of Periodontology and Oral Medicine, Belgrade,
Serbia
Introduction: Non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL)
represents a heterogeneous disease groups. The aim of this study was to
investigate the status of the oral cavity in these patients, before therapy
(because chemotherapy can result in changes of the oral cavity) or after first
relapse before new therapeutic cycle.
Materials and Methods: The study included 66 patients, of which 59 with
NHL and 7 with HL. Among NHL patients, 40 patients (40/59) were diagnosed
before therapy, while 19 patients (19/59) were diagnosed in the relapse stage
before undergoing new therapeutic cycle. Four out of seven (4/7) patients with
HL were diagnosed before therapy while three (3/7) patients were in relapse
stage. Dental examinations were performed using a dental mirror and probe.
Results and Discussion: Changes in the oral cavity were noted in 34 out of
66 patients (51.5%). Among 23 newly diagnosed lymphoma patients and 11
patients in the relapse stage of the disease changes in the oral cavity were
present while for other 32 patients (48.5%) no visible changes were observed.
In the group of patients that presented with changes in the oral mucosa, 24
out of 34 (70.6%) had a single change, while 10 out of 34 (34.4%) presented
with two or more. The most common oral manifestation, 15 cases (44.1%),
was pale oral mucosa. In 9 patients (26.5%) atrophic glossitis was detected.
In 3 cases (9.4%) we’ve observed coated tongue and purpura while in groups
of 2 patients each (5.9%) the following changes were observed respectively:
enantem of the oral mucosa, necrotizing periodontal disease, dry mouth and
white changes. In addition, in single patients (2.9%) hemangiomas, hyperplasia
of the oral mucosa, opalescent oral mucosa, hyperkeratosis of the tongue,
ulcerations and hematoma were noted.
Conclusion: Here we propose that described changes in the oral cavity of NHL
and HL patients are linked to the disease onset. Currently, we are focusing
on analysis of molecular mechanism that might be responsible for oral cavity
alterations observed in NHL and HL patients.
134 The role of Delta-40p53 and p53 in Estrogen Receptor-a signalling
pathways in breast cancer
B. Morten1 , R.J. Scott1 , K.A. Avery-Kiejda1 . 1 University of Newcastle &
Hunter Medical Research Institute, Medical Genetics, Newcastle, Australia
Introduction: The tumour suppressor gene p53 is an important transcription
factor that is essential for maintaining genomic stability. However, only ~25%
of breast cancers harbour p53 mutations, suggesting other mechanisms are
causing the inactivation of p53. In breast cancer, the interaction between p53
and Estrogen Receptor-a (ERa) drives tumourigenesis through the imbalance
of growth promoting and inhibitory pathways. Small transcriptional variants of
p53, known as p53 isoforms, can mediate p53 signalling pathways and have
been shown to be abnormally expressed in a range of human cancers. Our
laboratory has recently published data demonstrating that the p53 isoform,
D40p53, was the most highly expressed isoform in breast cancer and was upregulated in the tumour samples when compared to matched normal adjacent
tissue. In the current study, we aimed to examine the effect of altering the
level of D40p53 relative to full length-p53 (FLp53) on p53 and ERa signalling
pathways, and to further elucidate the role of this isoform in breast cancer.
Material and Method: The expression of the p53 isoforms was compared with
ERa, and target genes in a cohort of 113 ER-positive tumour tissue samples.
Following this, two ER-positive, WTp53 breast cancer cell lines, MCF-7 and
ZR75−1, were transfected with siRNAs to study the effects of knocking down
D40p53, FLp53 or the combination of both isoforms. Gene expression and
protein expression analyses were performed, following transfection, on a
number of genes involved in the p53 and ERa signalling pathways.
Results and Discussion: FLp53 and D40p53 were correlated with ERa
expression and its target genes in a cohort of breast tumour tissues. To
investigate whether D40p53 could modulate ERa expression, we knocked
down D40p53 and FLp53 in breast cancer cells using siRNAs, demonstrating
40−60% and 50−80% knockdown, respectively. Following knockdown of
D40p53 or both isoforms, we found an upregulation of ERa and its target
genes. This suggests that D40p53 is capable of mediating ERa expression,
and may be essential in affecting downstream signalling of ERa in breast
cancer.
Conclusions: These results propose that the transcriptional regulation of
ERa by p53 is more complex than first described. D40p53 may act as an
intermediary in the feedback loop between p53 and ERa in breast cancer,
and may be important in mediating the loss of ERa expression seen in some
breast cancers.
135 A novel monoclonal antibody allowing for the isolation and analysis
of Lgr5 positive cells from cell lines and primary human tissue
D. Agorku1 , N. Chelius1 , A. Bosio1 , O. Hardt1 . 1 Miltenyi Biotec GmbH, R&D,
Bergisch Gladbach, Germany
Introduction: Cancer stem cells (CSCs), also called tumor initiating cells,
have gained substantial interest in the research field over the past few years.
CSCs have been isolated from multiple tumor entities and were shown to play
a crucial role during tumor growth and metastasis. However, there is still a
major debate about specific cell surface markers capable of identifying CSCs
in most tumor entities.
The leucine-rich repeat-containing G-protein-coupled receptor (LGR) Lgr5 and
its close homologues Lgr4 and Lgr6 associate with Wnt-receptors and act
as R-spondin receptors thereby playing a central role in the modulation of
Wnt/b-catenin signaling in normal and neoplastic stem cells (de Lau et al.,
2011, Carmon et al., 2011). Initially described as a highly specific marker
for stem cells in the small intestine, colon, hair follicle, stomach, and during
kidney development (Barker et al., 2007, Jaks et al., 2008, Barker et al.,
2010, Barker et al., 2012), Lgr5 positive cells were also shown to be crucial
during the development and progression of cancer. It was shown that Lgr5
positive crypt stem cells are the cells-of-origin in intestinal cancer and that
CSCs in human colorectal cancer can be identified and isolated based on Lgr5
expression (Barker et al., 2009, Kemper et al., 2012). However, the analysis
of Lgr5 expressing cells is hampered by the lack of highly specific monoclonal
antibodies.
Materials and Methods: We have developed a novel rat monoclonal antibody
specifically binding to the extracellular domain of human Lgr5. Fluorochrome
conjugates were generated allowing for the direct detection, enumeration, and
analysis of Lgr5 expressing cells in cell lines and primary tissues. Furthermore,
the antibody was evaluated for use in immunohistochemistry and western blot
based detection of Lgr5. Subsequently, we generated conjugates of the novel
antibody with superparamagnetic nanoparticles (MicroBeads) and titrated it to
optimize the separation efficiency by using magnetic cell sorting (MACS® ).
Results: Using stable transfectants for Lgr4 and Lgr6, we could prove that
the antibody is not cross reactive with the close homologues of Lgr5. This is
of particular importance for Lgr4, as this protein is also expressed on more
differentiated progenitor cells. It was possible to detect cells expressing Lgr5
even at low levels in cell lines as well as in primary tissues using flow cytometry,
immunohistochemistry, and western blot analysis. In addition, we generated a
new magnetic cell separation (MACS® ) protocol that can be used as an easy
and fast method to isolate Lgr5 positive cells from cell lines and primary human
tissue. Using this method Lgr5 positive colon carcinoma cells as well as human
skin stem cells were isolated at purities >95% and >75%, respectively.
Conclusion: Taken together, we have developed a highly specific monoclonal
antibody allowing for the analysis and isolation of Lgr5 positive cells from cell
lines and primary tissues.
Conflict of interest: Other substantive relationships: All authors are full time
employees of Miltenyi Biotec.
137 Antitumor activities of some macrofungi extracts
Z. Zizak1 , A. Klaus2 , M. Kozarski2 , J. Vunduk2 , M. Niksic2 , Z. Juranic1 .
1
Insitute of Oncology and Radiology, Experimental Oncology, Belgrade,
Serbia, 2 Faculty of Agriculture, Institute for Food Technology and
Biochemistry, Belgrade, Serbia
Introduction: Methanol and hot water extracts as well as hot alkali extracted
polysaccharides of the fruiting bodies of seventeen macrofungi collected at
various localities in Serbia were screened for cytotoxic activity against three
cancer cell lines with a validated MTT assay.
Material and Methods: Fungi were identified by authors from an examination
of macro- and micromorphology in comparison to standard descriptions in
the monographs and taxonomic treatments. The aerial parts of the fungi
were air-dried, powdered, and extracted with methanol, hot water or hot
alkaline water solution. Water extracts were dissolved directly in nutrient
medium and methanol extracts were dissolved in DMSO at concentrations of
150 mg/ml, and afterwards diluted by nutrient medium to final concentrations
in the range from 0 to 3 mg/ml. Target tumor cells were malignant human
breast cancer MDA-MB-453, cervix adenocarcinoma HeLa and myelogenous
leukemia K562 cells. Antiproliferative activity of investigated compounds was
assessed, measuring cell survival in standard, 72 h MTT test.
Results and Discussion: Methanol extracts derived from, Piptoporus
betulinus, Ganoderma lucidum, and Ganoderma applanatum, displayed
significant cytotoxic activity and a dose dependent antiproliferative action
towards all investigated tumor cell lines. Moderate cytotoxic effects showed
methanol extracts derived from a Cantharellus cibarius, Trametes versicolor ,
Hericium erinaceus, Fomes fomentarius and alkali extracted polysaccharides
from Fomes fomentarius. The most cytotoxic effect showed methanol extract
of the Ganoderma applanatum growing on spruce tree (IC50 = 0.023 mg/ml
for K562 cells) and Piptoporus betulinus (IC50 = 0.039 mg/ml for K562 and
HeLa cells). The least cytotoxic species evaluated in this study were Phelinus
S31
linteus, Agaricus bisporus, Hericium erinaceus, Auricularia auricula-judae and
Grifola frondosa. Generally methanol extracts showed significantly stronger
effect than water extracts. In fact only hot alkali extracted polysaccharides from
Fomes fomentarius showed moderate cytotoxicity toward tumor cells (IC50 =
0.564 mg/ml for HeLa, 0.312 mg/ml for K562 and 0.879 mg/ml for MDA-MB-453
cells).
Conclusion: Results obtained showed that some compounds of methanol
extracts from Piptoporus betulinus, Ganoderma lucidum and Ganoderma
applanatum, could be promising agents for the treatment of human tumors and
are candidates for further analyses on experimental animals, in vivo. Our study
provides a first comprehensive evaluation of the cytotoxicity of various Serbian
fungi species and forms an important basis for the isolation and structural
elucidation of cytotoxic compounds from these species in the future.
with data in original bulk tumors, xenospheres harboring a mutated KRAS
gene were resistant, while those harboring wild-type RAS pathway genes
(RASwt ) were sensitive. However, EGFR inhibition in sensitive CC-ICs could be
counteracted by cytokines secreted by cancer-associated fibroblasts. Among
such cytokines, HGF, the ligand of the MET receptor, was particularly active
in supporting in vitro CC-IC proliferation and resistance to EGFR inhibition.
Consistently, in spheropatients genetically engineered to produce human
HGF, so as to achieve endocrine and paracrine activation of human MET in
xenografted CC-ICs, MET inhibition enhanced and sustained tumor regression
induced by anti-EGFR antibodies, leading to a virtually complete and durable
response. These data show that RASwt CC-ICs rely on both EGFR and MET
signaling, and provide a strong proof of concept for concurrent targeting of the
two pathways in the clinical setting.
138 Induction of cellular senescence and hair follicle stem cell
dysfunction upon p14ARF and p16Ink4a expression in the skin
141 RET/PTC1 in vitro models unveil a novel tumor suppressor miRNA
in papillary thyroid carcinoma
N. Azazmeh1 , R. Tokarsky-Amiel1 , I. Ben-Porath1 . 1 Institute for Medical
Research − Israel − Canada Hadassah School of Medicine The Hebrew
University of Jerusalem, Department of Developmental Biology and Cancer
Research, Jerusalem 91120, Israel
E. Minna1 , P. Romeo1 , L. De Cecco2 , M. Dugo2 , G. Cassinelli3 , C. Lanzi3 ,
S. Pilotti4 , M.A. Pierotti5 , A. Greco1 , M.G. Borrello1 . 1 Istituto Nazionale
Tumori, Molecular Mechanisms Unit Department of Experimental Oncology
and Molecular Medicine, Milan, Italy, 2 Istituto Nazionale Tumori, Functional
Genomics and Bioinformatics Unit Department of Experimental Oncology
and Molecular Medicine, Milan, Italy, 3 Istituto Nazionale Tumori, Molecular
Pharmacology Unit Department of Experimental Oncology and Molecular
Medicine, Milan, Italy, 4 Istituto Nazionale Tumori, Department of Pathology
and Laboratory Medicine, Milan, Italy, 5 Istituto Nazionale Tumori, Scientific
Directorate, Milan, Italy
Introduction: Cellular senescence is a physiologic stress response program,
in which cells cease to proliferate and undergo dramatic alterations in
morphology, metabolic activity and protein secretion. Despite its importance
in aging and tumor suppression, many fundamental aspects of cellular
senescence remain poorly elucidated. Senescence is executed most often
by the p19ARF /p53 and p16Ink4a /Rb pathways, which are interconnected and
mediate partially redundant effects, yet their distinct contributions to the
senescence program are not fully understood.
Materials and Methods: We developed mice that allow inducible activation of
the two central mediators of senescence, p14ARF and p16Ink4a , in the epidermis
of the skin. We set out to test the comparative ability of the two genes to induce
senescence, and their effects on tissue structure and stem cell function.
Results and Discussion: We found that both p14ARF and p16Ink4a acutely
reduced proliferation in the epidermis and induced the formation of senescent
cells. However, activation of p14ARF gave rise to substantially more senescent
cells than p16Ink4a activation. The endogenous Cdkn2a products − p19ARF
and p16Ink4a − were induced upon the activation of both transgenes, revealing
a senescence-promoting feed-forward loop. Activation of p14ARF , but not
of p16Ink4a , also caused a rapid, but transient, wave of apoptosis, prior to
the generation of senescent cells. The influence of p14ARF and p16Ink4a on
hair follicle stem cell function was also distinct: while the proliferation of
the stem cells and the entry of the hair follicle to anagen were completely
prevented upon p14ARF activation, leading to a severe hair loss (alopecia),
a partial arrest was detected upon p16Ink4a activation. We found that hair
follicle bulge stem cells were resistant to p53-induced apoptosis, and instead
underwent senescence, causing an irreversible blockage of proliferation
potential. Interestingly, induction of skin hyperplasia prevented the appearance
of senescent cells in the epidermis, yet was not sufficient to overcome bulge
stem cell dysfunction.
Conclusion: Our findings indicate that the p19ARF /p53 pathway is a more
powerful inducer of senescence in the epidermis than the p16Ink4a /Rb pathway,
and can execute complete blockage of stem cell function. This, despite the
interconnectivity of the two pathways. These data shed light on one of the
most important cellular tumor suppression mechanisms.
140 MET signaling in colorectal cancer-initiating cells blunts response
to EGFR inhibition: Implications for targeted therapy
P. Luraghi1 , G. Reato1 , E. Cipriano1 , E. Menietti1 , F. Sassi1 , V. Bigatto1 ,
F. Orzan1 , A. Bertotti1 , L. Trusolino1 , C. Boccaccio1 . 1 Candiolo Cancer
Institute − FPO (IRCCS) University of Turin, Experimental Clinical Molecular
Oncology, Candiolo (TO), Italy
Metastatic colorectal cancer remains largely incurable, although in a subset
of patients survival is prolonged by new targeting agents such as antiEGFR antibodies. Reportedly, colorectal cancer contains a tumorigenic cell
hierarchy sustained by a subpopulation of colorectal cancer-initiating cells
(CC-ICs), which may confer therapeutic resistance. However, how CC-ICs
respond to EGFR inhibition has not been fully characterized. From a
previously set-up platform of molecularly annotated patient-derived xenografts
of metastatic colorectal cancer (xenopatients), we derived sphere cultures
endowed with properties of CC-ICs (xenospheres), which faithfully retain the
same genetic alterations as the tumor of origin, and generate secondary
tumors (spheropatients), highly resembling those of both xenopatient and
patient of origin. Xenospheres and spheropatients offered the unprecedented
opportunity to investigate the molecular and biological response to targeted
therapies at cancer-initiating cell level. Interestingly, we found that xenospheres
retained the genetic determinants of response to anti-EGFR therapy: in line
Background: Thyroid cancer (TC) is the most common endocrine malignancy,
with increasing incidence. The majority of TCs are classified as papillary
thyroid carcinomas (PTCs) and RET/PTC oncogene is the second most
frequently identified driving genetic lesion in this tumor histotype. In
addition, several studies have reported aberrant miRNA expression in PTC.
Nevertheless, the mechanisms leading to miRNA deregulation in this neoplasia
as well as the consequences of their altered expression are still poorly
understood.
Methods: MiRNA/gene microarray expression profiles were defined in two
PTC cell models: thyrocytes infected with RET/PTC1 oncogene and TPC1
cells, harbouring endogenous RET/PTC1, treated with the inhibitor RPI. The
expression of miR-199a-3p was evaluated by meta-analysis of miRNA profiles
of a large cohort of PTCs/non-neoplastic thyroids publicly available on The
Cancer Genome Atlas and by qRT-PCR in PTC surgical specimens and in
thyroid derived cell lines. The functional role of this miRNA was then assessed
by transfecting a miR-199a-3p mimic in thyroid derived cell lines and analysing
proliferation, migration and cell death.
Results: We identified a total of 30 miRNAs and 301 coding genes significantly
and concordantly de-regulated in the two cell models accordingly with the
presence of an active RET/PTC1 oncoprotein. Among de-regulated miRNA
we focused on miR-199a-3p. We showed that this miRNA is under-expressed
in PTC surgical samples and in PTC-derived cell lines, and its restoration
in PTC cells causes MET and mTOR protein levels reduction. Moreover,
we demonstrated that miR-199a-3p can exert oncosuppressor functions in
PTC cell lines by impairing migration and proliferation and, more interesting,
inducing lethality through an unusual form of cell death similar to methuosis,
caused by macropinocytosis dysregulation. However, neither MET nor mTOR
specific silencing recapitulate miR-199a-3p-induced cell death, suggesting the
involvement of additional target genes.
Conclusions: Cancer cell modeling and functional approaches lead to the
identification of miRNAs relevant in thyroid carcinogenesis as suggested by
our results that indicate miR-199a-3p as a novel tumor suppressor miRNA in
PTC.
142 Proteolytic regulation of the EphB4 receptor in prostate cancer
J. Lisle1 , I. Mertens-Walker2 , C. Stephens2 , S. Stansfield2 , A. Herington2 ,
J. Clements2 , S. Stephenson2 . 1 Princess Alexandra Hospital, Translational
Research Institute, Woolloongabba, Australia, 2 Translational Research
Institute, Cancer Program, Woolloongabba, Australia
Introduction: EphB4, a receptor tyrosine kinase, is over-expressed in
66% of prostate cancers (PCa) where it promotes tumour angiogenesis,
increases cancer cell survival and facilitates cell invasion and migration;
however, the mechanism of action is still unclear. Recently, we developed
an overexpression model to study EphB4 in PCa using the cell line 22Rv1
(22Rv1-B4) and identified the presence of novel EphB4 fragments. Using
EphB4-specific antibodies directed to either the C- or the N-terminus of the
protein we predicted these were the result of sequential specific proteolytic
cleavage events releasing both extracellular (ECF − 70kDa) and intracellular
(ICF − 47kDa) fragments. This study focused on the possible mediators of
fragment generation and the function of the ICF in PCa.
S32
Material and Methods: Western immunoblotting was used to identify protein
fragments in various PCa cell lines which both endogenously and exogenously
over-express EphB4. Based on a proteomic modelling approach the PCaassociated Kallikrein-like peptidase 4 (KLK4) was tested as a candidate
protease responsible for the first cleavage event by treating cells with
recombinant KLK4 and KLK4 inhibitors. The involvement of a second protease,
g-secretase, often linked to further processing of cleaved surface proteins, was
tested using the specific inhibitor, compound E. Subcellular fractionation of
the KLK4-positive PCa cell line LNCaP was used to determine the cellular
localisation of the ICF. The ICF was overexpressed in PC3 and DU145 cells
to examine the potential function of this fragment in PCa.
Results: Addition of recombinant KLK4 to 22Rv1-B4 cells increased the
amount of the EphB4 fragments, confirming KLK4 as a candidate protease.
Treatment of these cells with a KLK4 inhibitor or compound E prevented
release of the 47kDa ICF confirming that g-secretase mediates the second
cleavage event. The ICF was localised primarily in the nuclear fraction of
LNCaP cells, suggesting it may have nuclear actions. DU145 and PC3
cells engineered to over-express the ICF had a more mesenchymal-like
morphology compared to the vector only control cells, suggesting a role in
the transformation of these cells to a more aggressive cancer phenotype.
Conclusion: This study provides the first evidence of proteolytic regulation
of EphB4 in PCa whereby the PCa-associated protease KLK4 cleaves the
ectodomain of EphB4 leading to a subsequent second intracellular cleavage by
g-secretase, releasing a potentially bioactive nuclear fragment. We predict this
may be a novel mechanism by which EphB4 contributes to the development
of PCa. Understanding the functions of the released EphB4 fragments will
determine whether the development of strategies for inhibition of proteolysis
and/or nuclear translocation may prove to be a potential novel avenue for anticancer therapies.
143 Zoledronate suppresses human osteosarcoma (U2OS) cells
invasion and migration by affecting cell morphology and
cytoskeletal organization via FAK, RhoA, Ras, vimentin and
E-cadherin
K. Lu1 , S. Yang2 . 1 Chung Shan Medical University Hospital, Department of
Orthopedics, Taichung, Taiwan, 2 Chung Shan Medical University Hospital,
Department of Medical Research, Taichung, Taiwan
Background: In addition to skeletal fractures in patients with cancers,
zoledronate can be used to treat hypercalcemia of malignancy and pain from
bone metastases. Here, we tested the hypothesis that zoledronate suppresses
human osteosarcoma U2OS cellular migration and invasiveness and also
investigated its signaling pathways.
Material and Methods: The effects of zoledronate on cell viability, migration
and invasion in U2OS cells were investigated. The levels of vimentin,
E-cadherin, focal adhesion kinase (FAK), Ras homolog gene family, member
A (RhoA) and Ras were tested by western blotting. Reverse transcription
polymerase chain reaction was used to detect the mRNA level of E-cadherin.
Matrix metalloproteinase (MMP)-2 and MMP-9 levels were examined by gelatin
zymography and western blotting.
Results: In the absence of cytotoxicity, the inhibitory effects of zoledronate
on the cell–matrix and cell–cell interactions, migration potential, and
invasive activity in U2OS cells were reversed by GGOH (geranylgeraniol),
not by FOH (farnesol). Effects of zoledronate on cell morphology and
cytoskeletal organization in U2OS cells by decreasing vimentin expressions
and phosphorylation of FAK 397 and FAK 925, and increasing E-cadherin,
RhoA and Ras expressions were also reversed by GGOH, not by FOH. No
significant effects of zoledronate on MMP-2 and MMP-9 levels were noted.
Conclusions: Our findings suggested that zoledronate suppresses the cell–
matrix and cell–cell interactions, the cell migration potential, and the cell
invasive activity of U2OS cells by interfering with cell morphology and
cytoskeletal organization via inducing E-cadherin, decreasing the vimentin
expression, and inhibiting FAK-mediated RhoA activation. These effects are
reversed by GGOH, not by FOH.
144 Breast cancer tumor secreted factors induce both endothelial
activation and increment of vascular permeability in vitro
M.A. Gallardo Vera1 , J.L. Ventura Gallegos1 , A. Zentella Dehesa2 . 1 Institute
for Biomedical Research UNAM, Genomic Medicine and Environmental
Toxicology, Distrito Federal, Mexico, 2 National Institute of Nutrition and
Medical Sciences, Unit of Biochemistry, Distrito Federal, Mexico
Background: In worldwide women, breast cancer is the main cancer-related
death cause. Furthermore, more than 90% of cancer patients die because
of metastatic dissemination of primary tumor cells to distant organs. Despite
clinically represents one of the most relevant steps in tumor progression,
metastasis it remains a poorly understood event in cancer biology. Tumor cell
dissemination is a multistep process that involves: local invasion, intravasation,
survival in the circulation, adhesion to the vascular wall, extravasation and
colonization. Changes in vascular permeability have been associated as
a prerequisite to metastatic spreading of primary tumor cells. Additionally,
endothelial overexpression of several adhesion molecules has been related
with an in vivo increment of metastasis. Here we describe the effect of natural
mixture of the soluble factors secreted by tumor cells over endothelial cells
activation and changes in vascular permeability.
Material and Methods: Cell culture: Human umbilical vein endothelial cells
(HUVEC) were isolated from umbilical cords as previously described by Jaffe
et al. 1973. Cells were pooled and maintained in M199 supplemented medium.
Breast cancer cell lines MDA-MB-231, ZR75.30, MCF-7 and monocyte cell
line U937 were obtained from ATCC. U937 were radioactively labeled before
adhesion assay.
Preparation of tumor-secreted factors (TSFs): Breast cancer cell lines were
cultured in T175 tissue culture plates to confluence. Then complete media
was removed and serum-free media were added. After 48 h incubation, the
supernatant was collected, centrifuged, filtered and stored (−20ºC) up to use.
Adhesion assay: HUVEC were seeded in 48-well plate and incubated to
confluence. Stimulated or not (TNF-a [10 ng/ml] or TSFs [10 mg/ml]) HUVEC
monolayers were co-cultured with a suspension of U937 cells. After 3hr
incubation all wells were rinse. Attached U937 were lysed and radioactivity
was measured.
Vascular permeability assay: Sealed HUVEC monolayers were cultured in
Boyden chambers (96-well plate). Cells were stimulated or not with TNF-a
[10 ng/ml] or TSFs [10 mg/ml] and incubated per 12hr. Then cells were washed,
a dextran-FITC solution was added (20 min) and the bottom well fluorescence
was quantified.
Results and Discussion: Overexpression of adhesion molecules is an
endothelial activation marker that results in an increasing of the adhesive ability
of endothelial cells. Adhesion assay revealed that HUVEC stimulated with
MDA-MB-231 and ZR75.30 TSFs attached U937 cells over 2.5 fold respect to
the control, similar like-TNF-a did. The same two factors were able to increase
the HUVEC monolayer permeability and the ZR 75.30 TSFs even exceeded
about 50% of the TNF-a induced permeability.
Conclusion: Tumor secreted factors derived from high metastatic potential
cell lines such as MDA-MB-231 and ZR 75.30 are capable to inducing an
endothelial activation state as well as vascular permeability enhancement. This
could be relevant in tumor metastasis.
145 Tumor microenvironment influencing 18 F-FDG uptake − an in vitro
study in three different colorectal carcinoma cell lines
A. Abrantes1 , A.S. Pires2 , M. Laranjo3 , A.C. Mamede4 , A.F. Brito2 ,
J. Casalta-Lopes5 , A.C. Gonçalves6 , A.B. Sarmento-Ribeiro6 , M.F. Botelho3 .
1
Biophysics Unit − IBILI, IBILI-Faculdade de Medicina, Coimbra, Portugal,
2
Biophysics Unit CIMAGO FCTUC, Faculty of Medicine University of Coimbra,
Coimbra, Portugal, 3 Biophysics Unit CIMAGO IBILI, Faculty of Medicine
University of Coimbra, Coimbra, Portugal, 4 Biophysics Unit CIMAGO IBILI
CICS, Faculty of Medicine University of Coimbra, Coimbra, Portugal,
5
Biophysics UnitRadiotherapy department, Faculty of Medicine University
of Coimbra, Coimbra, Portugal, 6 Applied Molecular Biology Biochemistry
CIMAGO, Faculty of Medicine University of Coimbra, Coimbra, Portugal
Background: Recent clinical data indicate that tumor hypoxia negatively
affects the treatment outcome of radiotherapy and chemotherapy in various
cancers, emphasizing the need for noninvasive detection of tumor hypoxia. The
application of 18 F-FDG PET imaging in oncology is based on the upregulation
of glucose transporters and glycolytic enzymes, and tumor hyperglycolysis.
In this context, the main objective of this study is to determine the pattern
of uptake of 18 F-FDG in three colorectal carcinoma cell lines in normoxia
and hypoxia conditions and correlate these results with the expression of
GLUT-1, -3 and p53.
Material and Methods: Studies were performed in colorectal carcinoma
cell lines (WiDr, C2BBe1 and LS1034). Uptake studies with 18 F-FDG
were carried out. After incubation in a cell suspension of 2×106 cells/ml
(25 mCi/ml), samples were collected, radioactivity of pellets and supernatants
was measured and percentage of uptake calculated. Experiments were
conducted in normoxia and hypoxia environment. GLUT-1, -3, as well as p53
expression was assessed by flow cytometry and western blot, respectively.
Results: p53 protein quantification revealed that C2BBe1 cell line does not
express p53 while WiDr and LS1034 do. Related to 18 F-FDG uptake, LS1034
and WiDr cell lines increased the uptake in hypoxic conditions in contrast with
C2BBe1. Concerning GLUT-1 and -3 expression we observed that hypoxia (2
and 48 hours) induced an increase of these glucose transporters.
Conclusions: With these results, we can conclude that in solid tumors, as
colorectal cancer, the uptake of current radiotracers is influenced by tumor
microenvironment. Besides that, characteristics like GLUTs, the main glucose
transporters, can be responsible for these results. However, the genetic
background also reveals be a key role in cells uptake.
147 Exploring the metabolic profile of colorectal cancer cells from
different origin
A. Abrantes1 , L. Tavares2 , A.S. Pires3 , M. Laranjo4 , J. Casalta-Lopes5 ,
R.A. Carvalho2 , M.F. Botelho4 . 1 Biophysics Unit − IBILI, IBILI-Faculdade de
Medicina, Coimbra, Portugal, 2 Life Sciences Department, Faculty of Science
and Technology University of Coimbra, Coimbra, Portugal, 3 Biophysics Unit
CIMAGO IBILI FCTUC, Faculty of Medicine University of Coimbra, Coimbra,
Portugal, 4 Biophysics Unit CIMAGO IBILI, Faculty of Medicine University of
Coimbra, Coimbra, Portugal, 5 Biophysics Unit CIMAGO IBILI Radiotherapy
Department, Faculty of Medicine University of Coimbra, Coimbra, Portugal
Background: Due to temporal and spatial heterogeneity of oxygenation that
occurs in solid tumors the adaptation to the variability of its microenvironment
is critical in cancer cells. Therefore, tumor hypoxia negatively affects the
treatment outcome of radiotherapy and chemotherapy. In this work we aimed
to characterize and compare the metabolic profile of three colon cancer cell
lines of different localization.
Material and Methods: Colorectal cancer cell lines (LS1034, WiDr and
C2BBe1-ATCC) were characterized concerning glycolytic and oxidative flows
using NMR isotopomer analysis upon incubation with [U-13 C]glucose. Citrate
synthase and complex IV activities were also evaluated. All experiments were
performed in normoxia and hypoxia with 5 mM (low) or 25 mM (high) glucose.
Results and Discussion: 1 H NMR analysis showed that hypoxia resulted
in an increase in the rate of lactate production with high glucose in all cell
lines, being LS1034 the most glycolytic, but for low glucose cells behaved
differently − WiDr reduced lactate production while LS1034 and C2BBe1 had
an increase. Under normoxia, WiDr is the most glycolytic cell line followed
by C2BBe1 and finally LS1034. 13 C NMR isotopomer analysis revealed very
distinct 13 C3-Lac/13 C3-Ala ratios, indicative of very different cytosolic redox
states, and very distinct 13 C3-Lac/13 C4-Glu ratios, denoting major differences
in metabolic coupling between glycolytic and Krebs cycle fluxes. Concerning
the C4Q/C4D45 ratio, there is a decrease on hypoxia condition in WiDr and
LS1034 cell lines. In C2BBe1 such ratio remains approximately constant, in
accordance with the reduced oxidative character of this cell line. These results
are in accordance with those obtained for the complex IV and CS, in which
LS1034 revealed the higher activity.
Conclusions: Results of lactate production, even in aerobic conditions, reveal
the occurrence of the Warburg effect. Hypoxia proved to have a distinct
effect on the three colorectal cell lines. This microenvironment alteration
demonstrates that LS1034, the cell line resistant to chemotherapeutic drugs,
has dramatically increased anaerobic metabolism to account for its energy
requirements. For the remaining cell lines, WiDr and C2BBe1, as they are
less dependent on oxidative metabolism they are less likely to change their
metabolic phenotype due to the hypoxic insult.
149 Is 18 F-FDG uptake in three different colon cancer cell lines
affected by incubation with sodium butyrate?
T.J. Gonçalves1 , A.S. Pires2 , J.C. Encarnação1 , R. Teixo3 , A.F. Brito4 ,
J.E. Casalta-Lopes3 , A.M. Abrantes4 , M.F. Botelho4 . 1 Biophysics Unit/FFUC,
Faculty of Medicine University of Coimbra, Coimbra, Portugal, 2 Biophysics
Unit/FCTUC/IBILI/CIMAGO, Faculty of Medicine University of Coimbra,
Coimbra, Portugal, 3 Biophysics Unit, Faculty of Medicine University of
Coimbra, Coimbra, Portugal, 4 Biophysics Unit/IBILI/CIMAGO, Faculty of
Medicine University of Coimbra, Coimbra, Portugal
Background: Butyrate is a short chain fatty acid (SCFA) and it’s produced by
decomposition of dietary fiber by intestine’s bacteria, being the main energy
source of colonocytes. It is related with colon cancer mostly because of its
capacity to inhibit histone deacetylases (HDAC) and induce apoptosis and
differentiation in contrast to normal cells. Some studies suggest that the
Warburg effect may explain why the cancer cells use preferentially glucose
rather than other energy sources, inducing butyrate accumulation in the tumor
cells. Other studies also suggest that butyrate can be used by tumor cells as
energy source when glucose levels are reduced. The aim of this study is to
evaluate if butyrate interferes with uptake of the radiolabeled glucose analogue
(18 F-FDG) and the increased glycolysis in colorectal cancer cells.
Methods: WiDr and C2BBe1 cell lines were cultured in DMEM with high
glucose (HG − 25 mM) and low glucose (LG − 5 mM) content and LS1034
cell line was cultured in RPMI with the same levels of glucose. To perform
the uptake studies, cells were incubated with or without butyrate, 3 mM for
WiDr cells, 6 mM for Ls1034 and 15 mM for C2BBe1 cells during 1 and 4
hours, before the incubation with 18 F-FDG (25 mCi/ml). At 5, 30, 60, 90, and
120 minutes, duplicate samples of 200 ml of cell suspension were collected for
eppendorfs with iced phosphate buffer solution. The samples were centrifuged
at 10,000 rpm for 60 seconds, separating the pellet from the supernatant.
In order to calculate the 18 F-FDG uptake percentage, radioactivity of both
fractions was measured in a well-type gamma counter, in counts per minute.
Results: When cells were cultured in LG content, 18 F-FDG uptake is greater
in WiDr than in the other two cell lines. In general, we observed that incubation
S33
with butyrate decreases 18 F-FDG uptake in C2BBe1 and WiDr cell lines and
there seems to be a trend to a decrease in 18 F-FDG uptake with the increase
of butyrate exposure time. When cells were cultured in HG conditions, butyrate
seems to induce a decrease in 18 F-FDG uptake by C2BBe1 cells, although less
pronounced than with LG content. In WiDr cell line there were no differences
in the uptake maybe because of the large difference between glucose (25 mM)
and butyrate (3 mM) concentrations. No changes were observed in 18 F-FDG
uptake in LS1034 cells (at LG and HG levels), probably related with the
upregulation of P-glycoprotein (an efflux transporter protein).
Conclusions: Our study suggests that butyrate can reduce the 18 F-FDG
uptake and may interfere with the Warburg effect, which influences the
agressiveness of the tumor. The largest differences are seen with glucose
low levels, which is the condition that better mimics the clinical situation. The
results also suggest that butyrate can act in cancer cells in an advanced phase
of development and could contribute to the understanding of the importance
of our diet in advanced tumor stages.
150 High selective cytotoxic activity of sesamol induced mitochondrialmediated apoptosis pathway in lung adenocarcinoma cells
B. Siriwarin1 , N. Weerapreeyakul2 , S. Barusrux3 . 1 Khon Kaen University,
Khonkaen, Thailand, 2 Khon Kaen University, Pharmaceutical Chemistry,
Khonkaen, Thailand, 3 Khon Kaen University, Clinical Immunology, Khonkaen,
Thailand
Sesame seed (Sesamum indicum L.) has long been used in the traditional
medicine of China, India, German and Southeast Asian countries for cancer
treatment. Sesame lignan sesamol has been reported to be one among the
high potent antioxidant existed in sesame seeds. However, antioxidants are
recently reported to promote lung cancer progression by reducing reactive
oxygen species (ROS) and DNA damage. Lung cancer is the leading cause
of death in both genders and was reported to have low success treatment
rate. In order to achieve successful treatment, one preference mechanism
of drug action is apoptosis induction effect. So far, the cytotoxic effect and
apoptosis mechanism of sesamol in lung cancer cells has not yet been
clarified. Due to the ROS involved with mitochondrial-mediated apoptosis
pathway, thus the mitochondrial-mediated apoptosis mechanism of sesamol
was investigated in the human lung adenocarcinoma cell line (SK-LU1).
The cytotoxicity was performed by neutral red assay. Activation of caspases
was determined using a luminogenic substrate specific to either caspase-9
or caspase-3. The alteration of mitochondrial transmembrane potential was
determined by flow cytometry using 3,3 -dihexyloxacarbocyanine dye. The
protein expression of Bcl-2 and Bax were detected by western blot. Sesamol
was shown to be high selectively cytotoxic to the SK-LU1 with an IC50
value of 2.7 mM with complementary selectivity (selective index = 2.9)
compared to the Vero cell line at 48 hours. Sesamol at 1IC50 concentration
induced apoptosis in the SK-LU1 cell line. Activation of caspase-9 and
the subsequent activation of caspase-3 were detected at 24 hours. The
mitochondrial transmembrane potential was significantly decreased (41.5%)
at 48 hours. The high expression of Bax/Bcl-2 proteins was clearly evidence
in the cell treated with sesamol at 2IC50 concentration. Our finding indicated
that sesamol at this millimolar concentrations evidently induced apoptosis cell
death by triggering mitochondrial-mediated apoptosis in the SK-LU1 cell line
through the activation of caspases. Additional anticancer activity of sesamol
especially via apoptosis signaling pathway in the lung cancer cells is worth for
further investigation.
152 Impact of the microenvironment on therapeutics in breast cancer
C.J. Lovitt1 , V.M. Avery1 . 1 Eskitis Institute for Drug Discovery, Griffith
University, Brisbane, Australia
Background: The presence of the tumour microenvironment can have
implications for the efficacy of anti-cancer agents. An integral component
of the tumour microenvironment is the extracellular matrix (ECM). Signalling
resulting from ECM-to-cell interactions have been implicated in the resistance
of various cancer cell types exposed to selected chemotherapeutics. Cells
attach to ECM through integrins, with b1-integrin the most common subunit
in integrin heterodimers. The objective of this research was to investigate the
factor/s contributing to the resistance observed in breast cancer cells cultured
in a three-dimensional (3D) ECM-containing microenvironment in response to
doxorubicin (Adriamycin® ) exposure.
Materials and Methods: Generating 3D cell culture conditions involved a
single cell suspension seeded on top of Matrigel™ or PuraMatrix™ in 384well microtitre plates. Doxorubicin was applied to cell cultures for a period of
6 days, followed by measurement of the metabolic activity via the EnVision™
Multilabel Plate Reader and imaging of the 3D cell culture morphology utilising
the Operetta™ High Content Imaging System. To measure the proliferation rate
and the penetration of doxorubicin, cells were imaged with the Opera™ High
Content Imaging System and analysed with Volocity™ and ImageJ. Protein
S34
levels following exposure to a b1-integrin function blocking antibody and/or
doxorubicin were measured using Western blotting.
Results: To determine the underlying mechanisms of resistance in breast
cancer cells cultured in a 3D ECM-containing microenvironment upon
exposure to doxorubicin, numerous elements potentially involved in causing
this reduction in sensitivity were investigated. These factors included the
cellular proliferation rate, drug diffusion and the involvement of cell-to-ECM
signalling. Results revealed that the decreased doxorubicin activity may have
been affected by a reduction in the proliferation rate of cells grown in 3D
(in comparison to cells cultured in a 2D monolayer) and altered cellular
signalling downstream from the ECM-to-cell interactions. Specifically, when
these breast cancer cells were cultured in 3D and then exposed to doxorubicin,
the pro-survival proteins of Bcl-2 and Bcl-XL were up-regulated, however, when
b1-integrin signalling was inhibited in the presence of doxorubicin, Bcl-2 and
Bcl-XL were down-regulated. In addition, inhibition of b1-integrin in 3D cell
culture conditions in combination with doxorubicin exhibited dose-dependent
enhanced anti-breast cancer activity.
Conclusion: Specific mechanisms underlying doxorubicin resistance in breast
cancer cells have been elucidated utilising 3D cell culture methodology. The
reduced proliferation rate and adhesion of cells to ECM through of b1-integrin
may function in mediating doxorubicin resistance. Further evaluation of novel
combination therapeutics involving inhibition of b1-integrin signalling together
with doxorubicin is warranted.
154 Antithetic effect of PDGFRbeta-induced miR-9 and ZEB-1-repressed
miR-200cin vasculogenic mimicry of triple negative breast cancer
E. D’Ippolito1 , I. Plantamura1 , P. Casalini2 , S. Baroni1 , C. Piovan1 ,
R. Orlandi2 , F. De Braud3 , E. Tagliabue2 , M.V. Iorio1 . 1 Fondazione IRCCS
Istituto Nazionale Tumori, Start Up Unit Experimental Oncology and
Molecular Medicine, Milan, Italy, 2 Fondazione IRCCS Istituto Nazionale
Tumori, Molecular Targeting Unit Experimental Oncology and Molecular
Medicine, Milan, Italy, 3 Fondazione IRCCS Istituto Nazionale Tumori, Medical
Oncology, Milan, Italy
Background: Vasculogenic mimicry (VM) is defined as the ability of
aggressive tumor cells to form de novo vasculogenic-like networks in threedimensional matrices in vitro. Our preliminary data showed that triple negative
breast cancers (TNBCs) acquire the ability to perform VM, contributing to the
establishment of the tumor vasculature, and that PDGFR-b plays a crucial role
in mediating this phenomenon.
With the aim to identify microRNAs involved in the PDGFR-b-mediated VM,
we investigated miR-9 and miR-200c. The existence of a negative regulatory
loop between PDGFR-b and miR-9 has been previously reported, where
the up-modulation of miR-9 mediated by PDGFR-b activation resulted in
turn in repression of the receptor itself; moreover, PDGF-D ligand is able
to mediate the ephitelial-to-mesenchymal transition through up-modulation
of ZEB1, transcriptional factor associated to VM, and subsequent miR-200c
down-modulation.
Material and Methods: VM ability was assessed in vitro in TNBC cell lines
by tube formation assay on Matrigel, and the loops were quantified with
Image Pro Plus software. Transient transfections with pre-miRs (Ambion),
LNAs (Exiqon) and siRNAs (Dharmacon) were performed with 10–100nM
oligonucleotides using RNAiMax or Lipofectamine 2000 (Invitrogen), according
to manufacturer’s instructions. Modulation of molecules of interest was
evaluated by western blot and/or qRT-PCR. Expression levels of miR-9 and
miR-200c were evaluated in 90 breast cancer samples (43 luminal, 24 HER2
and 21 TNBC) and 75 TNBC specimens by qRT-PCR.
Results: Ectopic expression or inhibition of miR-9 accelerated or impaired
VM ability, respectively; the inhibitory effect obtained with miR-9 repression
was partially rescued by stimulation of PDGFR-b. Furthermore, silencing of
CREB but not MYC, both positive transcriptional regulators of miR-9 acting
downstream PDGFR-b, reduced loop formation, suggesting that PDGFR-bmediated VM at least partially relies on CREB-miR-9 axis.
Ectopic expression of miR-200c, as well as silencing of ZEB1, strongly
impaired VM; interestingly, silencing of ZEB1 slightly induced miR-200c upmodulation but consistently repressed PDGFR-b.
Finally, analysis of miR-9 and miR-200c levels in a series of breast cancer
specimens indicated a subtype-dependent expression of miR-9, up-modulated
in TNBC in comparison to luminal and HER2+ patients, and a negative
association, even though not statistically significant, of both microRNAs with
disease-free-survival.
Conclusions: PDGFR-b-miR-9 axis and miR-200c play an opposite role on
vasculogenic mimicry of TNBC. ZEB1 could represent the link between these
two microRNAs, acting through the negative regulation of miR-200c and
maintenance of PDGFR-b expression.
155 Novel CSF-1 receptor ligand IL-34 modulates macrophage-breast
cancer cell crosstalk
S. Gunawardhana1 , K. Zins1 , T. Lukas1 , D. Abraham1 . 1 Medical University of
Vienna, Center for Anatomy and Cell Biology, Wien, Austria
Background: Interaction between stromal cells and malignant cells within
the tumor microenvironment is important for tumorigenesis. According to
current clinical and experimental evidence, tumor associated macrophages
(TAMs) play a major role in tumor progression to metastasis in different
cancers. In breast cancer, increased number of TAMs is associated with poor
prognosis. Previous work in our laboratory has shown that depletion of TAMs
by blocking colony-stimulating factor-1 (CSF-1) suppresses tumor growth,
matrix metalloprotease production and further recruitment of macrophages in
a MCF-7 breast cancer xenograft model. In this study, we aim to investigate
Interleukin-34 (IL-34), a novel ligand for the colony stimulating factor-1 receptor
(CSF-1R). IL-34 functions as a specific and independent ligand and as an
agonist to the CSF-1R. Furthermore it is known to induce proliferation through
mitogen activated protein kinase (MAPK) activation in macrophages and
cancer cells. To date, nothing is known about the role of IL-34 in breast cancer
and its effects on downstream signalling pathway modulation in cancer cellmacrophage crosstalk.
Material and Methods: Methods used are realtime PCR, migration assays
using Boyden chambers, Western blotting and cell proliferation assays using
WST-1 reagent.
Results and Discussion: Our real-time PCR data shows that in cocultures with murine macrophages, human MCF-7 and MDA-MB-231 breast
cancer cells express increased levels of IL-34. In addition, in vitro migration
assays demonstrated that recombinant human and murine IL-34 significantly
increases the migration of murine macrophages, MCF-7 and MDA-MB-231
breast cancer cells. IL-34 also increased the proliferation of murine
macrophages and breast cancer cells. Experiments on the protein level
demonstrated the activation of MAPK/ERK in murine macrophages and breast
cancer cells by recombinant IL-34. These preliminary data indicate that
TAM–breast cancer cell interactions induce IL-34 production associated with
induction of proliferation and the migratory capacity of tumor and stromal cells,
which together may influence the invasiveness of the developing tumor.
Conclusion: Here we propose IL-34 as a target for the treatment of breast
cancer which may affect the accumulation of TAMs and improve breast cancer
prognosis.
156 Reduced expression of GRHL genes in human non-melanoma
skin cancers
A. Kikulska1 , B. Wilczynski2 , T. Rausch3 , V. Benes3 , P. Rutkowski4 ,
T. Wilanowski1 . 1 Nencki Institute of Experimental Biology, Department of
Cell Biology, Warsaw, Poland, 2 Institute of Informatics University of Warsaw,
Computational Biology Group, Warsaw, Poland, 3 European Molecular
Biology Laboratory (EMBL), Genomics Core Facility, Heidelberg, Germany,
4
Maria Sklodowska Curie Memorial Cancer Centre and Institute of Oncology,
Department of Soft Tissue/Bone Sarcoma and Melanoma, Warsaw, Poland
Background: The balance between proliferation and differentiation of
keratinocytes in the epidermis is regulated by a complex interplay of
transcription factors, including the evolutionarily conserved Grainyhead-like
proteins (GRHL1, GRHL2, GRHL3). Many genes regulated by GRHL TFs
(like CDH1, DSG1, PTEN, hTERT, PCNA, miR-21) were previously implicated
in carcinogenesis. Based on literature and our preliminary results we
hypothesized that skin cancers are accompanied by disrupted expression of
GRHL genes or impaired GRHL protein function.
Material and Methods: Surgical specimens, including tumour and adjacent
unaffected skin, were resected from 32 patients with histologically confirmed
non-melanoma skin cancers. Nucleic acids were purified using appropriate
methods. Target enrichment was performed with HaloPlex Kit (Agilent) and
targeted fragments of GRHL genes were sequenced with MiSeq Illumina
System, 100-fold coverage depth. TaqMan Real-Time PCR of GRHLs was
carried out with Applied Biosystems chemistry.
Results: To find potential mutations in skin tumour samples, targeted deep
sequencing was performed but no de novo mutations were found in GRHL
genes. To determine the relationship between skin cancer occurrence and
SNP distribution in the GRHL genes, SNP frequencies in the patient group
were compared to frequencies in 1000 Genomes Database and outlier
SNPs were pointed. Next, indicated SNPs were assigned to functional DNA
fragments: coding regions (non-synonymous SNPs), promoter regions (SNPs
in sequences recognised by transcription factors; CpG rich regions), DNAseI
hypersensitive sites and to 3 UTRs where miRNAs may potentially bind.
Quantitative real-time PCR has shown statistically significant downregulation
of GRHL1 and GRHL3 genes in human skin cancers. It was also shown that
there is a significant correlation between the levels of expression of both genes
which may indicate a common regulation of their expression.
Conclusions: In human non-melanoma skin cancers both GRHL1 and GRHL3
genes are downregulated. There were no de novo mutations in these genes
in tumour samples. SNPs in promoter regions which may affect GRHL gene
expression were pointed. Continued efforts are aimed at finding molecular
regulators of GRHL gene expression.
157 Unravelling the role of Caveolin-1 in metabolic reprogramming
during hepatocellular carcinoma
Z. Nwosu1 , S. Dooley2 , C. Meyer2 . 1 Manheim University Hospital, Department
of Medicine II Molecular Hepatology Section, Mannheim, Germany, 2 Manheim
University Hospital University of Heidelberg, Department of Medicine II
Molecular Hepatology Section, Mannheim, Germany
Introduction: Hepatocellular carcinoma (HCC) remains a major killer cancer
worldwide. In order to rationalise an effective anti-cancer therapy, detailed
knowledge of cancer survival and metastatic mechanisms is pivotal. A
traditional view is that tumour cells generate ATP via an inefficient
aerobic glycolysis (‘Warburg effect’) and circumvent mitochondrial oxidative
phosphorylation. In the absence of glucose, cancer cells can also uniquely
generate ATP by deploying alternative energy-rich metabolites. This metabolic
reprogramming enables evasion from cell death, even under stressful
conditions like hypoxia, nutritional insufficiency or drug treatment. Clinical
benefits of targeting this phenomenon in HCC are hugely understudied. The
oncogene Caveolin-1 (Cav-1), expressed in plasma membrane caveolae and
regulated during cancer progression, modulates a variety of cellular pathways.
We aimed at unravelling the role of Cav-1 in alterations of cancer metabolism
that are potential therapeutic targets in HCC.
Material and Methods: We have strategically knocked down Cav-1 in
primary hepatocyte using siRNA, stimulated with transforming growth factor
beta (TGF-b), and carried out gene expression profiling studies in order to
understand Cav-1 effects on metabolism in physiologic context. For HCC,
we used lentiviral gene transduction to modulate Cav-1 expression in a
panel of cell lines. Furthermore, we stimulated HCC cell lines cultured in
modified nutritional conditions with TGF-b, and examined time-course and
dose-dependent effects of blocking glycolysis with 2-deoxy-D-glucose (2-DG,
a glucose analogue). As readout, fluorescent microscopy, FACS, systems
biology tools (e.g. Ingenuity Pathway Analysis, KEGG, DAVID), metabolic
assays, proliferation/viability assays, migration assays, immunoblotting, and
qPCR were applied.
Results: Downregulation of Cav-1 in hepatocytes led to changes in genes
involved in autophagy, oxidative phosphorylation, cell cycle, MAPK and notably
in cancer pathways, including IRS1, mTOR and STAT5A. In addition, TGF-b
stimulation altered genes related to cancer cell death as well as glycolysis,
glutathione metabolism, oxidative phosphorylation and cell cycle. Furthermore,
TGF-b regulates Cav-1 expression in HCC. In HLE, a HCC cell line, we
observed a significant dose-dependent reduction in cell proliferation following
inhibition of glycolysis with 2-DG. Cell proliferation was also reduced in
glucose-containing media that lack glutamine, implying that HLE cells display
sustained survival by exploring other metabolic pathways beyond glycolysis.
Conclusion: We show that Cav-1 expression affects glycolysis in HCC cells.
Our findings provide a rational base for further investigations of Cav-1 and
associated mediators of metabolic rewiring that could be novel druggable
targets in hepatocellular carcinoma patients.
159 11beta-hydroxysteroid dehydrogenase in human colon and
colonic tumors
J. Pácha1 , P. Ergang1 , M. Moravec2 , J. Bryndová1 , S. Zbánková2 , M. Kment3 ,
V. Mandys4 . 1 Institute of Physiology Academy of Sciences, Epithelial
Physiology, Prague 4, Czech Republic, 2 Institute of Physiology Academy of
Sciences and 3rd Faculty of Medicine Charles University, Internal Medicine,
Prague 4, Czech Republic, 3 3rd Faculty of Medicine Charles University,
Internal Medicine, Prague 4, Czech Republic, 4 3rd Faculty of Medicine
Charles University, Pathology, Prague 4, Czech Republic
Introduction: Colorectal cancer is an important cause of cancer death
worldwide. It is the result of colonic epithelium transformation into neoplastic
tissue accompanied by a delay or inhibition of cellular differentiation
and apoptosis and hyperproliferation of colonic crypts. This transformation
progresses through a series of stages, including adenomas that can be
transformed into malignant tumors. Glucocorticoids (GCs) have been used
for decades in the treatment of various malignancies and in experimental
studies GCs have been shown to prevent the development of various tumors.
Effect of GCs on the target tissue depends not only on the number of
GC receptors and the plasma concentration of GCs, but also on the local
metabolism of GCs, which is determined by the enzyme 11beta-hydroxysteroid
dehydrogenase (11HSD), which exists in two types. Type 1 (11HSD1) converts
biologically inactive cortisone into active cortisol whereas type 2 (11HSD2)
converts cortisol into inactive cortisone. We have studied therefore, whether
there are differences of local metabolism of GCs in neoplastic and autologous
S35
non-neoplastic tissue in various stages of malignant transformation of colonic
epithelium.
Materials and Methods: Samples were retrieved from patients who underwent
surgical resection (colorectal adenocarcinomas, CRC) or patients who
underwent polypectomy (adenomas with low grade (LGA) or high grade (HGA)
dysplasia). As a control tissue was used the macroscopically normal area
of colonic epithelium at the margin of the resection and the biopsies from
the normal rectosigmoid mucosa of patients undergoing polypectomy. The
expression of 11HSD1 and 11HSD2 was measured by real-time PCR and
the enzyme activity by radiometric assay.
Results: In CRC, 11HSD2 mRNA expression and enzyme activity were
downregulated whereas 11HSD1 mRNA and activity were mostly increased.
11HSD2 mRNA was decreased already in the early stages of neoplastic
transformation such as LGA and HGA but the levels of 11HSD1 mRNA were
not changed.
Conclusions: The results demonstrate that (1) downregulation of 11HSD2 is
a typical feature of the development of colorectal polypous lesions and their
transformation into CRC and that (2) colorectal tumor cells seem to have a
decreased capability of autocrine inactivation of glucocorticoids already in premalignant stages.
160 Pterostilbene inhibit hepatocellular carcinoma cells proliferation
by induce autophagy and apoptosis in vitro
H. Chiou1 , S. Yang2 , M. Hsieh3 . 1 Chung Shan Medical University, School
of Medical Laboratory and Biotechnology, Taichung, Taiwan, 2 Chung Shan
Medical University, Institute od Medicine, Taichung, Taiwan, 3 Changhua
Christian Hospital, Cancer Research Center, Changhua, Taiwan
Background: Hepatocellular carcinoma (HCC) is the sixth most common
cancer worldwide, however, it is the third leading cause of cancer-related
death. Pterostilbene is a naturally-derived antioxidant mainly found in
blueberries, grapes, and tree wood. Pterostilbene is an analogue of the
resveratrol, but is significantly more bioavailable when ingested. The aim
of this study is to investigate the anti-proliferation effect of pterostilbene on
hepatocellular carcinoma cells.
Materials and Methods: We investigated the effect of pterostilbene on
proliferation of the human hepatocellular carcinoma cell lines, Huh 7 and SKHep-1. We used DAPI and acridine orange staining to observe the morphology
changes caused by pterostilbene treatment. Western blotting was used to
examine the protein expression level. We also used flow cytometer to verify
the effect of pterostilbene on cell cycle and apoptosis.
Results: Our result demonstrated that pterostilbene can inhibit cell proliferation
in dose-dependent and time-dependent manner in Huh 7 and SK-Hep-1
cells (p < 0.001). Nuclear condensation and the formation of acidic vesicular
organelles were also observed under fluorescence microscopy in the treated
cells. Results from the western blotting showed that pterostilbene can increase
the conversion of LC3-I into LC3-II, indicating that autophagy was activated.
And the cleavage of caspase 9 indicating that apoptosis was also induced by
pterostilbene.
Conclusion: Our data indicated that pterostilbene can inhibit cellular
proliferation of human HCC cell lines by the induction of apoptosis and
autophagy.
161 Role of Id1–IGF2–VEGF–VEGFR relay in esophageal
tumorigenesis
A.L.M. Cheung1 , W.W. Xu1 , B. Li1 , S.W. Tsao1 , K.W. Chan2 . 1 University of
Hong Kong, Anatomy, Hong Kong, Hong Kong, 2 University of Hong Kong,
Pathology, Hong Kong, Hong Kong
Introduction: It is increasingly evident that the close interactions between
cancer cells and other cells in the tumor microenvironment help drive tumor
progression. Esophageal squamous cell carcinoma (ESCC) is a highly lethal
disease, and there is an association between poor clinical outcome and
Id1 overexpression in ESCC. We previously reported that Id1 induces the
expression and secretion of insulin-like growth factor-2 (IGF2) which promotes
esophageal cancer progression and metastasis in an autocrine manner. In
this study, we aim to investigate the paracrine effect of Id1-induced IGF2 on
esophageal fibroblasts, and the stimulatory effects and roles of fibroblastsecreted vascular endothelial growth factor (VEGF) on endothelial cells
and VEGF receptor (VEGFR)-positive bone marrow cells during esophageal
tumorigenesis.
Materials and Methods: Human ESCC cell lines with ectopic expression of
Id1, co-expression of Id1 and shRNA against IGF2 (shIGF2), or empty vector
were established. Bone marrow was harvested from femurs and tibias of
nude mice for isolation of VEGFR1+ bone marrow cells by flow cytometry.
Cell proliferation and migration were determined using MTT and chamber
migration assays respectively. Western blot and ELISA were used to determine
the protein expression and concentration in cell lysates and conditioned
S36
medium (CM). Tube formation assay was used to analyze the in vitro
angiogenic ability of endothelial cells. Human ESCC cells were subcutaneously
injected into the flanks of nude mice to establish the tumor xenografts.
Results and Discussion: Our data from Western blot and ELISA showed
that the addition of CM from the ESCC cells with overexpression of Id1
or recombinant human IGF2 alone could promote esophageal fibroblasts to
produce VEGF in vitro, and co-expression of shIGF2 or neutralizing antibody
against IGF2 greatly diminished this effect. We also found that the CM collected
from IGF2-activated fibroblasts could enhance the proliferation, tube-formation
and migration abilities of human umbilical vein endothelial cells (HUVECs). The
addition of VEGF-neutralizing antibody effectively attenuated these stimulatory
effects. In addition, our results showed that the CM collected from IGF2activated fibroblasts increased the mobility of VEGFR1+ bone marrow cells.
Furthermore, data from in vivo experiment showed that bone marrow cells
harvested from nude mice bearing Id1-expressing tumor xenografts had tumorpromoting activity when co-injected subcutaneously with untransfected ESCC
cells into new groups of nude mice.
Conclusions: Id1-overexpressing ESCC cells secrete IGF2 and educate
stromal fibroblasts to secrete VEGF, which may in turn activate and mobilize
endothelial cells as well as bone marrow-derived hematopoietic-progenitor
cells to promote tumor angiogenesis.
This study was supported by the Research GrantsCouncil of the Hong Kong
SAR, China, GRF Projects No. HKU762610M and HKU763111M.
162 Specific EMT-inducers signature associates with oncogenic
events in breast tumour progression
C. Moyret-Lalle1 , E. Ruiz1 , S. Courtois-Cox1 , C. Bardel2 , A. Veron3 . 1 Cancer
Research Center of Lyon, Centre Léon Bérard, Lyon, France, 2 LBBE UMR
CNRS 5558, Public Health, Lyon, France, 3 Cancer Research Center of Lyon,
Molecular Epidemiology, Lyon, France
Background: Mammary epithelial cancer cells are able to reactivate
an embryonic program, the Epithelial-to-Mesenchymal Transition (EMT),
to acquire motility, renewal and de-differentiation capacities. EMT drives
a profound genetic reprograming including reactivation of embryonic
transcription factors (ETFs) that may cooperate with specific mitogenic
stresses during tumorigenesis. Our goal is to determine whether specific
expression networks between ETFs and mitogenic stresses exist in breast
tumours, representing a driving force towards transformation in breast
tumorigenesis.
Material and Methods: We performed oncogenic cooperation assays in
immortalized human mammary epithelial cells (HMEC-hTert) by using an ETF
expression library in combination with various mitogenic stresses (activated
b-catenin, overexpression of EGFR, deletion of PTEN and p53). In softagar colony assays, these mitogenic stresses are not sufficient to transform
cells and form colonies. In contrast, the combination of ETFs and mitogenic
stresses led to transformation of mammary human cells. In order to determine
whether the ETFs signature was specific to the mitogenic insult, EMT-inducer
expression was analysed in a large number of colonies. We have used a Chitwo and arborescences analyses to determine how expression of ETFs was
interconnected in different mitogenic insult conditions.
Results: We found that expression of Goosecoid and Zeb1 was specifically
enriched in colonies generated when PTEN or p53 is silenced. Twist1 and
Twist2 expression was selected following b-catenin activation whereas random
ETF combinations were found to be expressed in colonies expressing a high
level of EGFR. The impact of PTEN/Goosecoid signature was evaluated in vivo
in a large cohort of TNBC (Triple Negative Breast Cancer).
Conclusions: We have identified specific ETF signatures, depending on
mitogenic insult during tumour progression. Therefore, the impact of EMT
in mammary transformation might depend on mitogenic alterations already
present in cells. A better understanding of how ETFs cooperate with mitogenic
insults might allow us to propose new combined therapeutics.
163 Metabolic activity of tumor cells in a variable microenvironment:
combinations of glucose, glutamine, lactate and pH
A.M. Otto1 , C. Janzon2 , J. Hutterer1 . 1 Munich Technical University, Institute of
Medical Engineering (IMETUM), Garching, Germany, 2 Klinikum Leverkusen,
Leverkusen, Germany
Introduction: The variability of the tumor microenvironment requires high
metabolic plasticity to maintain cell growth and survival. With the wealth of
knowledge on the molecular components in energy metabolism, the question
often remains as how do these act and interact in quantitative terms. Such
information is paramount for evaluating metabolic imaging. Therefore, a series
of investigations have been started in-vitro using different tumor cell lines
level to study the effects of (1) limiting nutrient conditions and (2) variable
extracellular pH on selected parameters of tumor cell growth and metabolism.
Material and Methods: The human breast cancer cell lines MCF-7 and MDAMB231 were cultured 3−4 days in ‘low growth’ medium (DMEM with reduced
serum) containing tumor-relevant concentrations of glucose, glutamine and
lactate, and set at different pH (6.6−7.4). Metabolic activity was measured with
the WST-1 assay, a tetrazolium-based dehydrogenase assay, which reflects
the activity of cell surface membrane NADH-oxidases (Berridge and Tan,
1998) and is an indicator of cytosolic NADH-levels. Rotenone was used to
inhibit mitochondrial complex I. The activities of pyruvate kinase and lactate
dehydrogenase (LDH) were measured in cell homogenates and compared with
a count of viable cells in culture. Glucose consumption and lactate production
were determined in cell culture supernatants.
Results and Discussion: (1) Tumor cell growth and metabolic activity do
not correlate and are highly dependent on the ratio of low glucose/glutamine
concentrations. Foremost, there is an increase in NADH-oxidase activity at
2.5 mM glucose with low (0.1 mM) glutamine, which is transiently enhanced
by rotenone, indicating an increase in cytosolic NADH level. Similarly, pyruvate
kinase and LDH activities increased, with LDH activity being 2−3-fold lower.
In MCF-7 cells, glucose consumption was higher with 1.0 mM than with low
glutamine, and <10% was released as lactate. Adding lactate enhanced the
activity of cell surface NADH-oxidases, suggesting that lactate can contribute
to the cytosolic NADH levels. Therefore, defined glucose/glutamine/lactate
combinations can induce a switch in glycolytic metabolism leading to pyruvate
being channelled into anabolic and/or oxidative pathways.
(2) Metabolic activities are highly sensitive to extracellular pH. The metabolic
variability described above occurs at pH 7.4. With increasing acidity, tumor
cells become increasingly less dependent on the variable levels of nutrients,
with no glucose/glutamine dependency at pH 6.6. Thus, the extracellular pH
changes the metabolic phenotype of tumor cells.
Conclusions: Tumor cells can rapidly adapt their metabolic activity through
NADH-dependent processes depending on the pH and levels of glucose,
glutamine, and lactate found in the tumor microenvironment.
164 Interplay between EMT-inducers and miRNAs during breast
tumorigenesis
C. Moyret-Lalle1 , B. Vitton-Méa1 , E. Ruiz1 , C. Hermann2 , E.W. Lam3 ,
A. Puisieux4 . 1 Cancer Research Center of lyon, Centre Léon Bérard,
Lyon, France, 2 DKFZ German Cancer Research Center, Bioinformatics,
Heidelberg, Germany, 3 Imperial College of London, Hammersmith, London,
United Kingdom, 4 Cancer Research Center of Lyon, Centre Léon Bérard,
Lyon, France
Background: Growing evidence suggests that breast cancer cell plasticity
arises due to a partial reactivation of the embryonic epithelial–mesenchymal
transition (EMT) program in order to give cells pluripotency leading to
a stemness-like phenotype. EMT drives a profound genetic reprograming,
including reactivation of embryonic transcription factors (ETFs) and severe
changes in miRNA expression. Cross-regulation between ETFs and miRNAs
has been previously described, such the Zeb1-miR-200 feedback loop. Our
goal is to determine whether specific expression networks between ETFs and
miRNAs exist in tumours, representing a driving force towards transformation
in breast tumorigenesis.
Material and Methods: We developed an in silico approach to identify miRNAs
targeting ETFs and identified one novel miRNA, miR-A, able to regulate the
four major families of ETFs (Zeb, Twist, Snail, FoxC). Using bioinformatic tools,
we identified different sequences of ETFs binding in the miR-A promoter. We
have realized dual luciferase assays using promoter constructs and using ETF3 UTR constructs, in different breast cancer cell lines. To study the impact
of miR-A on EMT we have established breast cancer cell lines expressing
miR-A.
Results: We have shown that miR-A is down regulated in basal B
breast cancer cell lines in contrast to EMT markers and ETFs expression.
Overexpression of miR-A in a mesenchymal cell line induces an inhibition of
ETFs’ expression (FoxC1, Snail, Slug, Twist1 and Zeb1) and a shift of EMT
markers expression. We have shown that miR-A down-regulates expression
of Slug, FoxC1, Zeb1 and Zeb2. We have shown that ETFs belonging to the
Fox, Snail, Twist and Zeb families inhibit the transcription of miR-A, especially
Twist1 and Zeb1. Furthermore, miR-A is down regulated after a treatment with
TGF-b, an EMT inducer.
Conclusions: We have identified a novel miRNA involved in EMT regulation
by targeting ETFs. We have found a new feedback loop between miR-A and
Zeb1. Deciphering the ‘interactome’ existing between ETFs and miRNAs is
crucial to elucidate the regulation of EMT during transformation.
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165 The role of the transcription factor ecotropic viral integration
site 1 in prostate cancer
167 Role of trefoil factor-3 peptide (TFF3) in prostate cancer
progression
A. Queisser1 , S. Hagedorn1 , W. Vogel1 , D. Böhm1 , A. Von Mässenhaussen1 ,
C. Lengerke2 , S. Perner1 . 1 Institute of Pathology, Department of Prostate
Cancer Research, Bonn, Germany, 2 University Hospital of Basel, Department
of Biomedicine, Basel, Switzerland
M. Nowak1 , C. Merz1 , A. Von Maessenhausen1 , W. Vogel1 , D. Boehm1 ,
N. Wernert2 , G. Kristiansen2 , S. Perner1 . 1 Institute of Pathology, Department
of Prostate Cancer Research, Bonn, Germany, 2 Institute of Pathology, Bonn,
Germany
Background: Prostate cancer is the most common cancer in men in the
western world. It is known that the accumulation of molecular changes
is essential for prostate carcinogenesis. For example, mutations in tumor
suppressorgenes like PTEN and TP53 as well as in the oncogenes MYC
and EGFR are important for prostate cancer progression. However, the
role of transcription factors with stem cell marker properties during prostate
carcinogenesis is insufficiently examined. Due to gene rearrangements and
inversion the stem cell marker ecotropic viral integration site 1 (EVI1) is often
overexpressed in several types of leukaemia but also in solid tumours such
as ovarian cancer and breast cancer. The aim of this study was to investigate
the role of EVI1 in prostate cancer (PCa).
Material and Methods: The expression level of EVI1 was investigated on
paraffin-embedded tissues of a prostate cancer progression cohort (primary
PCa n = 121, localized lymph node metastases n = 11, distant metastases
n = 31). Staining against EVI1 was done with standard immunohistochemistry
(IHC). The staining intensity as well as the quantity of positively stained
cells were determined using the semi quantitative image analysis system
(Definiens). PCa cell lines were screened for EVI1 mRNA expression by qRTPCR and for protein expression by western blot. To stably down regulate EVI1,
EVI1-expressing PC3 cells were transduced with EVI1-shRNA or scrambled
shRNA by lentiviral mediated gene transfer. Anchorage-independent growth
was determined using a soft agar assay. Proliferation was assed with the
xCELLigence system and migration using a scratch assay.
Results: In primary prostate cancer samples we could detect a heterogeneous
expression level of EVI1. The number as well as the intensity of the stained
cells increased with the progression to lymph node and distant metastasis.
qRT-PCR and western blot analysis revealed that the PCa cell line PC3
expressed EVI1 on RNA and protein level. Down regulation of EVI1 in PC3
cells using EVI1-shRNA led to smaller and lesser colonies in soft agar assays
compared to scrambled controls. Furthermore, EVI1 inhibited cells showed an
impaired proliferation capacity as well as a reduced migratory potential.
Conclusion: Our data indicate that EVI1 plays an important role during
prostate cancer progression.
Introduction: Prostate cancer (PCa) is the second most abundant cancer
diagnosis in men and accounts for about 10% of cancer-related deaths.
Whilst established therapeutic options for primary tumors exist, long-term
remissions inevitably lead to the development of castration-resistant prostate
cancer (CRPC). One of the most frequent mutations in CRPC is the gene
fusion of the Ets-family transcription factor ERG with the promoter region of
the serine protease TMPRSS2. This aberrant expression of ERG in prostate
cells perturbs normal gene expression.
The ability of tumors to metastasize depends on the coordinated expression
of extracellular matrix degrading enzymes (MMPs) and adhesion molecules.
TFF3 (ITF, intestinal trefoil factor) is a 18 kDa protein with a characteristic
trefoil structure containing three conserved cysteine residues. To date, neither
the exact function nor its receptors are well defined. Aim of this study was to
identify the receptor for TFF3 and its role in prostate cancer progression.
Material and Method: Expression of TFF3 was assessed by immunohistochemistry. Functional assays were performed using prostate (tumor) cell lines
BPH-1, DU145, PC-3, and LnCaP.
Results: Based on the unresolved regulation and receptor for TFF3 we tested
the basal expression in cells from benign prostate hyperplasia (BPH-1) and
three cell lines from prostate cancer metastasis (DU145, PC3, LnCaP). Among
the tested cell lines BPH-1 cells showed the highest expression. LnCaP cells
were TFF3 negative, PC3 and DU145 showed intermediate concentrations
within this spectrum. Which soluble factors regulate the expression of TFF3
in prostate cancer cells is not known. Using antibody-mediated blockage of
the interleukin-6 receptor (IL-6R), we show that TFF3 expression is driven
by autocrine IL-6. Incubation with TFF3 mobilized calcium from intracellular
stores and led to phosphorylation of STAT3, independent of the EGFR, which
has been discussed as a potential TFF3 receptor. Furthermore, using boydenchamber migration assays we show that TFF3 is a chemokine, which is
mediated by the chemokine receptor CXCR4.
Conclusion: Our results point to a complex autocrine loop between IL-6,
TFF3, and CXCR4 as a receptor driving the expression of TFF3.
166 FoxF1 is a potential oncogene in prostate cancer
M. Nowak1 , R. Menon1 , F.A. Kunze1 , M.A. Svensson2 , J. Carlsson2 ,
N. Wernert3 , G. Kristiansen3 , O. Andrén2 , S. Perner1 . 1 Institute of Pathology,
Department of Prostate Cancer Research, Bonn, Germany, 2 University
Hospital of Örebro, Department of Urology, Örebro, Sweden, 3 Institute of
Pathology, Bonn, Germany
Introduction: Castration resistant prostate cancer (CRPC) is the most
aggressive form of prostate cancer (PCa) with a poor prognosis, and remains a
significant therapeutic challenge. Key to the development of novel therapeutic
strategies is to identify molecular targets of this lethal disease. FoxF1 belongs
to the family of forkhead transcription factors. However, the exact function of
FoxF1 remains unclear. The aim of the study was to assess the role of FOXF1
within prostate cancer.
Material and Method: Using tissue microarrays of a prostate cancer
progression cohort we analysed the protein expression of FoxF1. mRNA and
protein expression of FoxF1 were analysed by qRT-PCR and Western Blot,
respectively. For assessment of FoxF1 function in prostate tumor cell lines,
cell lines stably expressing FoxF1 or respective knockdown cell lines were
established.
Results: Primary tumors and distant metastases exhibited a significantly
higher FoxF1 expression compared to benign tissue. mRNA and protein
expression analysis revealed a considerable expression range amongst
CRPC-derived cell lines, with PC3 cells showing the lowest FoxF1
expression.
Forced expression of FoxF1 in prostate tumor cells stably transfected with
FoxF1 resulted in loss of E-Cadherin and gain of Vimentin expression,
indicative of epithelial-to-mesenchymal transition (EMT). Furthermore, overexpression of FoxF1 led to an increased migration rate in vitro without affecting
the proliferation rate.
Conclusion: In summary, our results point to role of FoxF1 as a potential
oncogene in prostate cancer.
168 Regulation of S-adenosylmethionine synthesis in a sequential
model of cirrhosis-hepatocellular cancer by adenosine derivative
L.R. Marı́a Guadalupe1 , V.L. Nora Gabriela1 , D.L. Mariana1 , C.S. Victoria1 .
1
Instituto de Fisiologı́a Celular, Biologı́a Celular y Desarrollo, Mexico City,
Mexico
Hepatocellular cancer (HCC) is a very common disease, with a high index
of mortality, which is originated in 80−90% from cirrhosis. We showed
that an adenosine derivative (IFC305) (UNAM Patent US8,507,459 B2)
has anticarcinogenic effect and we studied the effect of this compound in
S-adenosylmethionine (SAM) synthesis. SAM is the main biological methyl
donor and its synthesis and degradation occurred mainly in the liver. The
synthesis of SAM is catalyzed by methionine adenosyltransferase (MAT),
MAT1A is expressed only in the liver, and it is the product of gene expression
of the Mat1a, while isoform MAT2A is widely distributed and it is result of
gene expression Mat2a. MAT isozymes differ in kinetic parameters and in
their regulatory properties. In addition to this, AMPK is the essential regulator
which is activated by cancer and it is an important regulator of Mat2a gene
expression.
We investigated the effects of IFC305 in a sequential model of cirrhosisHCC described by Schiffer (2005). Male wistar rats were treated with DEN
for cirrhosis induction for 12 weeks or for cancer induction for 16 weeks, and
two different groups were simultaneously treated with IFC305. All animals were
treated according with the rules of Institutional Vivarium. The liver expression of
MAT1A, MAT2A, and AMPK were measured by Western blot; gene expression
of these isoforms of MATs by qPCR and cellular SAM levels were measured
by HPLC at the cirrhosis and cancer stage.
The results of this assays showed significant decrease in the SAM levels in
cirrhosis and cancer in tumoral and no-tumoral sites; while the administration
of IFC305 showed an increment of SAM levels. Therefore, in a protein level, it
showed significant increase of MAT2A and decrease of MAT1A in cirrhosis and
cancer stage, while the administration with this compound increased levels of
liver-specific MAT and decreased levels of non-liver-specific MAT. Furthermore,
our compound prevents activation of AMPK induced by DEN treatment.
The assays of gene expression levels showed significant changes decreasing
Mat1a and increasing Mat2a in HCC; these effects were reversed by treatment
with IFC305.
The result suggests that the modification in SAM levels observed in cirrhosis
and cancer in the absence and presence of the compound respond to two
S38
possible mechanisms, gene and post-transcriptional regulation of the enzymes
involved in SAM synthesis. Further studies are needed to clarify it.
Thanks to support PAPIIT of DGAPA-UNAM.
169 Autoschizis: a cell death induced by the anticancer, pro-oxidant
stress of ascorbate:menadione combination
J. Gilloteaux1 , J.M. Jamison2 , J.L. Summers2 . 1 St George’s University,
Anatomical Sciences, Newcastle upon Tyne, United Kingdom, 2 Summa
Research Foundation, Apatone Research Centre, Akron Ohio, USA
Background: Carcinoma incidence and carcinogenesis are in direct
relationship with the repressed activities of nucleases. Hence, reactivating
these enzymes would render more easily susceptible cancers to radiation
and chemotherapy. Taper and collaborators (1981–2008) found that a coadministration of ascorbate (VC) with menadione (VK3) to a variety of tumour
cell lines resulted in specific antitumour activity associated with DNAse and
RNase reactivations at doses that were 10 to 50 times lower than if those
vitamins were administered alone.
Material and Methods: Light and ultrastructure, cytochemistry, flow cytometry
and molecular techniques characterised the cytotoxic effects of VC, VK3, and
VC:VK3 combination on bladder (T24, RT4), ovary (MDAH), and prostate
(DU145) human carcinoma cell lines following a 1−4 h treatment by 24 h
incubation in culture medium. Xenotransplanted DU145 solid tumours in nu/nu
mice allowed through Animal Review Committee were treated similarly and left
to survive at least 2 weeks before studying tumour morphology.
Results: Oxidative stress-induced injuries of tumour cells encompassed
decrease protective enzymes against ROS, decreased ATP, and other
repair mechanisms. Organelles such as nucleus were shown to display
segregation of nucleolus components along with progressive karyorrhexis
to chromatolysis and karyolysis; these injuries were verified by DNA gel
electrophoresis producing smears of DNA, not internucleosomal laddering as
in apoptosis. Altered mitochondria became associated with endomembranes
and lysosomes to form complex autophagosomal deposits. Cytoskeleton
damage was responsible for superficial and peripheral, cytoplasmic autoexcisions resulting in significant diminution of cell size. However the cell pieces
never contain organelles, contrarily to the apoptotic bodies. Tumour cells were
reduced to their altered nucleus and a rim of cytoplasm. Tumour cell death
ensues through oncotic ruptures of plasma membrane and of the nuclear
envelope. This VC:VK3 treatment killed tumour cells efficiently with a main
cell death classified historically by us as autoschizis (Gilloteaux 1998–2014),
certainly not by apoptosis or other type of cell death. It contributed to tumour
shrinking without any other organ damage.
Conclusion: These observations support that the combined vitamins or
Apatone® could be used as a safe adjuvant or treatment against not only
uro-gynecologic cancers but others as early Clinical Trials I/II with the drug
have been successful.
Studies sponsored by St George’s University School of Medicine UK-NY,
Summa Research Foundation, Akron OH and IC Med Tech, San Diego CA,
USA.
References: Taper HS et al., 1981, Cancer 47: 523; 1987, Int J Cancer 40:
575; Anticancer Res 12: 1651; 2001, J Histochem Cytochem 49: 109; 2008,
Anticancer Res 28: 2727. Gilloteaux et al. 1998, Scanning 20: 564; 2001,
Ultrastruct Pathol 25: 183; 2006, Anat Rec Mol Cell Biol 296: 40; 2004: Tissue
Cell, 6: 197; 2010: Ultrastruct Pathol 34: 140; 2014, PMID 24460713.
170 A novel Zn(II)-compound reactivates mutant p53 protein in cancer
cells: molecular mechanisms and therapeutical implications
A. Garufi1 , D. Pucci2 , M.L. Avantaggiati3 , G. D’Orazi4 . 1 Regina Elena National
Cancer Institute, Dept Experimental Oncology, Roma, Italy, 2 University of
Calabria, Dept of Chemistry, Cosenza, Italy, 3 Georgetown University, Dept of
Oncology, Washington DC, USA, 4 University “G. d’Annunzio”, Dept Medical
Oral and Biotechnologic Sciences, Chieti Scalo (Chieti), Italy
Background: TP53 oncosuppressor is frequently mutated in cancer
contributing to tumor progression and resistance to therapies. Impairment
of the DNA binding and transcription ability of wild-type (wt) p53 is largely
responsible for tumor outgrowth and resistance to chemotherapy in cancers
carrying p53 mutations. Mutant p53 (mutp53) protein is often highly expressed
in cancers due to its increased half-life. Mutp53 proteins are prone to loss
of the Zn(II) ion bound to the core DNA binding domain, and this in turn
favours protein unfolding, aggregation and impairment of DNA binding and
transcription ability. Therefore, reactivation of wild-type function(s) from mutp53
may have therapeutic significance.
Materials and Methods: Here we used a novel fluorescent curcuminbased Zn(II) complex [Zn(II)-curc] and assessed its ability to induce mutp53
reactivation in cancer cells. Biochemical and molecular biology studies were
carried out.
Results: Zn(II)-curc restored the folded conformation of mutp53 proteins
(R175H), inducing wtp53 DNA binding and transactivation, as assessed by
chromatin immunoprecipitation and reverse-transcription (RT)-PCR assays.
Consequently, Zn(II)-curc triggered apoptosis in mutp53-carrying cell lines. In
addition, Zn(II)-curc promoted mutp53H175 degradation through autophagy.
Suppression of autophagy, by pharmacologic or genetic menas, prevented
Zn(II)-curc-induced mutp53H175 degradation and restoration of wild-type p53
oncosuppressor activities. On the contrary, the proteasome inhibitor MG132
failed to do so, suggesting that autophagy was the main route for p53H175
degradation by Zn(II)-curc. Mechanistically, Zn(II)-curc restored the wtp53
ability to induce the expression of the p53 target gene DRAM (damageregulated autophagy modulator), a key regulator of autophagy, leading
to autophagic induction. Accordingly, inhibition of wtp53 transactivation by
pifithrin-a (PFT-a) impaired both autophagy and mutp53H175 degradation
induced by Zn(II)-curc.
Conclusions: These results uncover a novel mechanism employed by Zn(II)curc to reactivate mutp53H175 which involves, at least in part, mutp53
degradation via wtp53-mediated autophagy. We thus propose that due to its
effect in reducing the levels of accumulated mutp53H175, together with the
ability of ameliorating mutp53H175 misfolding, Zn(II)-curc may serve as a key
lead compound for the development of anticancer drugs to effectively treat
mutp53H175-carrying tumors.
171 Cycling hypoxia amplifies tumor microenvironment inflammation
C. Michiels1 , C. Tellier1 , C. Graux2 , L. Finet2 , M. Raes1 , O. Feron3 . 1 University
of Namur, URBC-NARILIS, Namur, Belgium, 2 CHU UCL Dinant-Godinne,
Biobank, Yvoir, Belgium, 3 UCL, FATH − IREC, Bruxelles-Woluwé, Belgium
Background: Cycling hypoxia, that is periods of hypoxia followed by
reoxygenation, occurs in solid tumors due to transient blood flow in the
dysfunctional tumor blood network. These pO2 fluctuations may influence
tumor cell as well as stromal cell (e.g. endothelial cell) physiology. Another
feature of tumor microenvironment is inflammation. Indeed, tumor-promoting
inflammation is a new enabling characteristic of tumor progression.
Material and Methods: EAhy926 endothelial cells and HUVEC were
incubated under cycling hypoxia for 6 hours with or without TNFa at 0.1 ng/ml in
order to mimic the tumor pro-inflammatory microenvironment. Biopsies from
human colon carcinomas were also analyzed. LLc tumor-bearing C57BL/6
mice were exposed to cycling hypoxia. Gene expression level was assessed
by RT-qPCR while protein levels were measured by western blot, ELISA or
immunohistochemistry.
Results and Discussion: Cycling hypoxia increased IL-6 and IL-8 secretion
induced by TNFa as well as stabilized a high ICAM-1 protein expression
induced by TNFa. NF-úB seems to be the transcription factor involved in these
effects as its nuclear abundance and transcriptional activity were increased by
cycling hypoxia and p65 silencing led to an abrogation of ICAM-1 expression
and IL-6/IL-8 secretion. Finally, we observed an increase in THP-1 monocyte
adhesion induced by TNFa to HUVEC exposed to cycling hypoxia supporting
the active state of endothelial cells. These results indicate that endothelial cells
exposed to cycling hypoxia display an amplified pro-inflammatory phenotype.
The role of cycling hypoxia was also assessed on global tumor inflammation
in vivo in mice. Results showed that cycling hypoxia enhanced inflammation in
tumors as COX2, IL6, KC and MIP-2 (murine IL8 homologs) mRNA expression
was increased, validating the specific inflammatory phenotype associated
to cycling hypoxia. Furthermore, we evidenced a more important leukocyte
infiltration in tumors, reflecting the amplification of tumor-related inflammation.
The specific gene signature was also observed in human colon carcinoma
biopsies and correlated with poor prognosis.
Conclusion: Cycling hypoxia seems to be an important connection between
inflammation and cancer. Our findings evidenced an innovative mechanism
initiated by cycling hypoxia that accounts for the amplification of tumor-related
inflammation, and that could thus favor tumor aggressiveness.
172 Serum endostatin levels are elevated in colorectal cancer and
correlate with invasion and systemic inflammatory markers
A. Tuomisto1 , T. Kantola1 , J.P. Väyrynen1 , K. Klintrup2 , J. Mäkelä2 ,
S.M. Karppinen3 , T. Pihlajaniemi3 , H. Autio-Harmainen4 , T. Karttunen1 ,
M. Mäkinen1 . 1 University of Oulu, Pathology, Oulu, Finland, 2 University of
Oulu, Surgery, Oulu, Finland, 3 University of Oulu, Institute of Biomedicine
and Biocenter Oulu, Oulu, Finland, 4 Oulu University Hospital, Pathology,
Oulu, Finland
Background: Endostatin − proteolytically cleaved fragment of collagen XVIII −
is an endogenous angiogenesis inhibitor. Despite of the well-known antitumor functions of endostatin, elevated circulating endostatin concentrations
have been found in several human cancers. Here we wanted to evaluate the
significance of serum endostatin levels incolorectal cancer (CRC).
Materials and Methods: Serum endostatin levels were measured by enzymelinked immunoassay from a prospective series of 113 patients with CRC and
84 age- and sex-matched controls. Clinicopathological parameters of CRC
including the densities of specific types of tumor infiltrating inflammatory cells,
serum leukocyte differential count and C-reactive protein (CRP) levels were
determined. Expression of collagen XVIII in tumor tissue was assessed with
immunohistochemistry.
Results: CRC patients had higher serum endostatin levels than the controls
(p = 0.005), and high levels associated with advanced age, tumor invasion
through the muscularis propria and poor differentiation, but not to local or
distant metastases. Endostatin levels showed a positive correlation with the
markers of systemic inflammatory response (mGPS, CRP and neutrophillymphocyte ratio) and negative correlation with the densities of tumor infiltrating
mast cells and dendritic cells. Collagen XVIII was expressed in tumor stroma
most strikingly in blood vessels and capillaries, and in the muscle layer of the
bowel wall between muscle cells, but not in tumor cells.
Conclusion: Elevated endostatin levels are present in CRC and correlate
with systemic inflammation and invasion through the muscularis propria.
We suggest that in CRC patient serum endostatin levels increase when
cancer cells invade through collagen XVIII expressing bowel wall and release
endostatin into the circulation. The negative correlations between serum
endostatin and intratumoral mast cells and immature dendritic cells may reflect
angiogenesis inhibition by endostatin.
173 Role of NANOS family members in tumor progression
E. De Keuckelaere1 , V. Andries1 , K. Staes1 , S. Grelet2 , B. Nawrocki-Raby2 ,
P. Birembaut2 , F. Van Roy1 . 1 VIB & Ghent University, Biomedical Molecular
Biology, Ghent, Belgium, 2 INSERM UMRS URCA, Plasticité de l’épithélium
respiratoire dans les conditions normales et pathologiques, Reims, France
Introduction: Nanos genes comprise a small family of homologous genes
coding for (CCHC)2 zinc finger proteins and showing conserved evolutionary
functions in germ cell development. In Drosophila melanogaster, the single
Nanos homolog is an RNA-binding protein that forms multi-subunit translationinhibitory complexes with Pumilio. In human cancer, several lines of evidence
indicate that hNanos members have the potential to confer malignancy by
increasing the invasive and migratory abilities.
Material and Method: Pull-down experiments were used to identify new
Nanos/Pumilio-interacting proteins. RNA-immunoprecipitations are ongoing in
order to identify potential mRNA targets associated with both Nanos and its
interacting protein(s). Target transcripts identified in vitro will be validated
by expression analysis of mRNAs and encoded proteins in our unique and
conditional transgenic mouse models for oncogenic activities of each of the
three hNanos members.
Results and Discussion: Pull-down experiments identified an ATP-dependent
RNA helicase as a new interacting protein for hNanos-1 and hNanos-3.
Interestingly, RNA helicases constitute a large group of enzymes involved
in all aspects of RNA metabolism. Several of them have been shown to
be dysregulated in cancer, including over-expression in various types of
tumors. Systematic identification of the RNAs associated with this RNA-binding
protein complex is therefore needed to identify the potential mRNA targets
under influence of a hNanos/RNA-helicase complex. To this end, we are
currently performing RNA-immunoprecipitations, in which we intend to capture
in different cancer cell lines the hNanos- and RNA-helicase-bound transcripts,
followed by Illumina sequencing of the purified mRNAs (VIB Nucleomics
core).
Conclusion: The search for additional Nanos/Pumilio-interacting proteins (and
mRNAs) might bring more insight into the pathways in which these important
regulatory proteins are involved, and it might help us to unravel the functions
of these proteins in cancer.
174 Vasoactive intestinal peptide decreases MYCN expression and
AKT activity via a PKA-dependent pathway in neuroblastoma cells
M. De Boisvilliers1 , A. Garnier1 , A.C. Meunier2 , S. Bensalma1 , J.M. Muller1 ,
C. Chadeneau1 . 1 University of Poitiers, Equipe émergente “Récepteurs
régulation et cellules tumorales” (2RCT), Poitiers Cedex 9, France, 2 University
of Poitiers, ERL CNRS/University of Poitiers nº7368, Poitiers Cedex 9,
France
Introduction: Neuroblastoma (NB) is an embryonic tumor derived from
the neural crest. This cancer accounts for about 15% of deaths due to
pediatric tumors. Several critical genetic aberrations are associated with a
poor prognosis in NB. A major one is the amplification of the MYCN gene
encoding a transcription factor and present in about 20−30% of NB cases.
Other genetic alterations are found in the anaplastic lymphoma kinase (ALK )
gene. The most common ALK mutation, ALK F1174L , is preferentially associated
with MYCN amplification in NB and induces constitutive activation of the
PI3K/AKT pathway, among others. These two alterations in MYCN and ALK
S39
genes associated with low stage of neuronal differentiation define patients
with a very poor outcome. The Kelly NB cells have MYCN amplification
and the ALK F1174L mutation. Here we assessed the effects of the vasoactive
intestinal peptide (VIP) on these cells. VIP is a neuropeptide known to induce
differentiation of human NB cell lines and to reduce MYCN expression. In NB
tumors, VIP level increases with the degree of differentiation.
Materials and Methods: Kelly NB cells treated with VIP were photographed
and neurite length was quantified using the ImageJ software. The MYCN
expression in VIP-treated cells was analyzed by RTq-PCR and western
immunoblotting. To determine the signaling pathways involved in the effects
of VIP, inhibitors of PKA, PKC and PI3K were tested and AKT phosphorylation
was analyzed using western immunoblotting.
Results and discussion: In Kelly cells, VIP increased neurite length starting
from 1 hour of treatment. This peptide reduced the expression of MYCN mRNA
for 1- to 6-h treatments, with a maximal inhibition of about 50% at 3 hours. At
the protein level, VIP decreased MYCN expression by about 45% after 3 hours
of treatment, an effect persisting for 48 h. The VIP-induced neuritogenesis and
decreased MYCN expression were both PKA-dependent. VIP also reduced
the phosphorylation of the AKT kinase, whose constitutive activation by the
ALK F1174L mutation leads to MYCN stability. VIP effect on AKT phosphorylation
was PKA-dependent.
Conclusion: The neuropeptide VIP induces neuritogenesis, decreases MYCN
expression sustainably and inhibits AKT activity in Kelly NB cells via the PKA
signaling pathway. VIP is thus able to act on main potential therapeutic targets
of high-risk NB.
175 miR-27a is a functional biomarker for tamoxifen treatment of
luminal A/B breast tumors
B. Ljepoja1 , J. Garcia-Roman1 , F. Kopp1 , E. Wagner1 , A. Roidl1 . 1 LMU
Munich, Department of Pharmacy, Muenchen, Germany
Introduction: Despite the benefits of estrogen receptor alpha (ERa)-targeted
endocrine therapies in breast cancer, resistance development is still a major
obstacle to successful therapy. Thus, functional biomarkers to predict the
efficacy of endocrine therapies are urgently needed. MicroRNAs (miRNAs)
have been suggested as promising biomarkers and here we analyzed whether
miR-27a is suited as such.
Material and Method: We overexpressed a miR-27a mimic in several breast
cancer cell lines and analyzed the resulting molecular and physiological effects
by Western blotting, qPCR, cytotoxicity- and profileration assays. Additionally,
we performed an in silico analysis of the expression of miR-27a and the 5-year
survival rate in the study of Lyng et al. on ER-positive breast cancers.
Results: Our study demonstrates an increasing tamoxifen-sensitivity of tumor
cells dependent on the presence of ERa and the expression level of miR27a. Overexpression of miR-27a leads to an upregulation of the ERa and
thus increased tamoxifen susceptibility. Accordingly, cell proliferation is also
increased by miR-27a overexpression. An in-vitro-resistance assay consisting
of 6 cycles tamoxifen treatment shows a loss of miR-27a and subsequent
downregulation of the ERa leading to tamoxifen resistance. Additionally,
in silico analysis of ERa positive tumors displayed an increased 5-year survival
of breast cancer patients when miR-27a expression is elevated.
Conclusion: We suggest miR-27a as novel functional biomarker for ERapositive breast cancer patients indicating which patients benefit most from
a endocrine cancer therapy. High expression of miR-27a suggests a good
prognosis for tamoxifen treated patients, whereas low expression or loss of
miR-27a during therapy implicates downregulation of ERa finally leading to
tamoxifen resistance.
176 Characterization and analysis of deregulation of signaling pathways
of cancer stem cells derived from oral squamous cell carcinoma
N. Andrade1 , M.F.S.D. Rodrigues1 , C.O. Rodini1 , F.D. Nunes1 . 1 College of
Dentistry − University of São Paulo, Pathology, São Paulo, Brazil
Oral squamous cell carcinoma (OSCC) is the oral cavity most common
cancer. No important improvements on treatment and survival rates have been
achieved for the last decade. Recently, it was reported that OSCC presents
a subpopulation of tumor-initiating cells that correspond to cancer stem cells
(CSC), also responsible for tumor growth and recurrence. This knowledge
raised the perspective of new therapeutic approaches targeting this subset of
cells and a better understanding of OSCC development mechanisms. The main
purpose of the present study was to sort CSC from an OSCC cell line based
on CD44 expression, characterize these cells, and investigate the expression
of other genes related to the CSC subpopulation. After FACS-sorting SCC4
CD44high (CSC) and CD44low (non-CSC) subpopulations cells were plated for
colony formation assays, morphology analysis, immunofluorescence for CD44
and total RNA extraction for analysis of CD133 and OCT-4 expression by
qRT-PCR. In order to assess the differential expression level of several genes
related to stem cell signaling in both subpopulations, a commercially available
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RT2 Profiler™ PCR Array was used. Results revealed that CD133 and OCT-4
transcripts were indeed highly expressed in CD44high subpopulation. The
colony formation assay showed greater proliferation of CD44high cells that also
present the formation of holoclones, whereas colonies derived from CD44low
cell showed more fusiform and scattered cells. Also, the gene expression
screening revealed that NOTCH, Hedgehog, TGFB and WNT signaling pathways were at least two fold overexpressed in SCC4 CD44high subpopulation
when compared to CD44low cells. In conclusion, the CD44high subpopulation
present morphologic characteristics of CSC and at the molecular level the
expression of markers related to stem cell signaling pathways.
177 ADAM9 coordinates genes in anoikis resistance for lung cancer
metastases
Y. Sher1 , C. Lin2 , C. Huang3 , L. Lai4 , T. Kuo3 , G. Tseng5 , M. Hung6 . 1 China
Medical University, Taichung, Taiwan, 2 China Medical University, Graduate
Institute of Clinical Medical Science, Taichung, Taiwan, 3 China Medical
University Hospital, Center for Molecular Medicine, Taichung, Taiwan,
4
National Taiwan University, Graduate Institute of Physiology, Taipei, Taiwan,
5
China Medical University Hospital, Department of Pathology, Taichung,
Taiwan, 6 The University of Texas MD Anderson Cancer Center, Department
of Molecular and Cellular Oncology, Houston, USA
Background: Brain metastasis is a major cause of morbidity and mortality
in lung cancer. A disintegrin and metalloprotease 9 (ADAM9) is a member of
the ADAM family of type I transmembrane proteins and plays an important
role in cell adhesion and migration. Overexpression of ADAM9 is observed in
many cancers and correlates with lung cancer brain metastasis. However, the
molecular mechanism is not clearly understood.
Material and Methods: By comparing our established brain-metastatic lung
cancer sublines and their parental cancer cells, we found ADAM9 (a disintegrin
and metalloprotease 9) was overexpressed in metastatic sublines. To further
understand the mechanisms by which ADAM9 promotes lung cancer brain
metastasis in addition to its role in brain endothelial cell adhesion, we analyzed
the differential gene expression between control and ADAM9 knockdown brain
metastatic lung cancer cells and investigated the ADAM9-related pathways
required for lung cancer brain metastasis. Lung adenocarcinoma patient
samples were also used to investigate the clinical relevance of ADAM9.
Results: A transcriptome microarray analysis reveals a set of genes that are
associated with ADAM9 such as CDCP1, a regulator of anoikis resistance.
We demonstrate ADAM9 enhances active form of CDCP1 via tPA activation
for cell metastasis. Blocking ADAM9-mediated downstream signaling offers a
synergistic cytotoxic effect in lung cancer cells. Analysis of clinical samples
shows that patients with high level of these genes and ADAM9 correlate with
poor prognosis. Therefore, ADAM9 regulates a complicated network in lung
cancer brain metastasis through tPA-mediated CDCP1 cleavage.
Conclusions: The primary cause of death for most cancer patients’
metastases, and the most common primary malignancy that gives rise to brain
metastases is lung cancer. The current study provides critical insights into the
mechanism of lung cancer brain metastasis through ADAM9-regulated CDCP1
activation via tPA-mediated CDCP1 cleavage and may have therapeutic value
for lung cancer patients with metastasis.
178 COX-2 independent induction of apoptosis by two synthetic COX-2
inhibitors in breast cancer cell line
M. Salimi1 , S. Norouzi2 , M. Norouzi2 , M. Amini3 , A. Amanzadeh4 . 1 Pasteur
Institute of Iran, Physiology and Pharmacology, Tehran, Iran, 2 Faculty
of Science Kharazmi University, Biochemistry, Tehran, Iran, 3 Faculty of
Pharmacy Tehran University of Medical Sciences, Medicinal Chemistry,
Tehran, Iran, 4 Pasteur Institute of Iran, National Cell Bank of Iran, Tehran,
Iran
Background: Breast cancer is considered to be the most familiar malignant
tumor in females in western countries; and is becoming more and more
widespread in Asia. Apoptosis is an intricate molecular program with favorable
application in many tumor treatments. Cyclooxygenase (COX)-2 inhibitors
may induce apoptosis through the COX-2-independent mechanism via a
mitochondrial pathway. In view of the reported antiproliferative activities of compounds 1 (3-(4-chlorophenyl)-5-(4-fluorophenyl)-4-phenyl-4,5-dihydro-1,2,4oxadiazole) and 2 (3,5-bis(4-chlorophenyl)-4-phenyl-4,5-dihydro-1,2,4-oxadiazole) as two COX-2 inhibitor derivatives in breast cancer cells (MCF-7), the
present study was undertaken to evaluate the potential of these compounds
to induce apoptosis and unravel the associated mechanisms.
Material and Methods: The apoptotic activities of compounds 1 and 2 were
assessed using flowcytometry. Protein expression was determined by western
blot analysis and immunoassay kit.
Results: Compounds 1 and 2 induced MCF-7 cells to undergo apoptosis,
which was demonstrated by annexin-V and propidium iodide staining. Further
investigation showed that compounds 1 and 2 inhibited nuclear factor kappalight-chain-enhancer of activated B cells (NF-úB), ferritin heavy chain (FHC)
and extra cellular signal-regulated kinase (ERK) activation, whereas it could
not change COX-2, c-Myc and early growth response protein 1 (EGR-1)
expression dramatically.
Conclusion: Altogether, these results revealed that compounds 1 and 2
may be potential and promising chemotherapeutic agents to treat breast
cancer through COX-2-independent and NF-úB-dependent pathways, which
sequentially inhibit P-ERK and FHC expressions.
179 Antiproliferative effects of different fractions obtained from
Anthemis nobilis leaves on human oral cancer cell
M. Salimi1 , N. Gheisarzadeh2 , K. Azadmnaesh3 , N. Rastkari4 , M. Salimi5 .
1
Islamic Azad University of Pharmaceutical Sciences branch, Biotechnology,
Tehran, Iran, 2 Islamic Azad University of Pharmaceutical Sciences branch,
Pharmacognosy, Tehran, Iran, 3 Pasteur Institute of Iran, Virology, Tehran,
Iran, 4 University of Medical Sciences, Center for Air Pollution Research
(CAPR) Institute for Environmental Research (IER), Tehran, Iran, 5 Pasteur
Institute of Iran, Physiology and Pharmacology, Tehran, Iran
Background: The global burden of cancer continues to increase largely
because of the aging and growth of the world population alongside an
increasing adoption of cancer-causing behaviors. Major issues relating to
the conventional anticancer chemotherapy are the occurrence of side effects
induced by the non-specific targeting of both normal and cancer cells, and
the emergence of drug-resistant cancer cells. In this regard, plants are an
invaluable source of potential new anti-cancer drugs. Here, we investigated
the cytotoxic activity of n-hexane, chloroform and ethyl acetate leaf fractions
of Anthemis nobilis on human oral squamous cancer cell line (BHY).
Material and Methods: Antiproliferative activity was evaluated by MTT assay
against human oral cancer cells at 24, 48 and 72 h and the 50% inhibitory
concentration (IC50 ) value was measured from dose–response curves. HGF
(human gingival fibroblast) cells were used as a non-tumoral cell line in this
experiment. The apoptotic activity of the most effective fraction was assessed
using flowcytometry.
Results: Our present study indicated that chloroformic fraction had the
lowest IC50 value (0.05±1.1 mg/ml) and induced BHY cells to undergo
apoptosis, which was demonstrated by annexin-V and propidium iodide
staining. Additionally, this fraction increased the cell population at subG1phase.
Conclusion: Our findings reveal that A. nobilis chloroformic extract exhibits
promising anti-cancer activity by triggering both cell cycle arrest and apoptosis,
suggesting that this plant may contain potential anti-cancer agents for single
or combinatory cancer therapy against oral cancer.
180 Cellular and molecular regulations of head and neck
carcinogenesis under diabetic environment
W.J. Chang1 , C.Y. Chen2 , T.Y. Chen1 , P.L. Lee1 , W.C. Li2 . 1 Institute of Oral
Biology, School of Dentistry National Yang-Ming University, Taipei, Taiwan,
2
Department of Dentistry, School of Dentistry National Yang-Ming University,
Taipei, Taiwan
Background: Head and Neck (H&N) cancer is one of the most prevalent
neoplasias worldwide. Aging associated physiology in company with potent
dietary stimulations and environmental challenges were considered as
oncogenic cues for developmentof H&N cancer. In contrast to other factors,
the impact of hyperglycaemia/diabetes mellitus (DM) induced imbalanced
metabolism during carcinogenesis was less emphasized. Recent study
described an anti-cancer effect of glucose-lowing agent metformin during
H&N carcinogenesis suggesting a potential link between hyperglycaemia
and H&N cancer formation. Although several preclinical and epidemiological
investigations have shown that hyperglycaemic/DM condition possibly
correlated with greater incidence and poorer prognosis in subjects with H&N
cancer, the outcomes from different groups are contradictive and underlying
mechanisms require to be explored.
Materials and Methods: To better define progressive hyperglycaemia/DMmediated regulation for H&N cancer development, current experimental
scheme was two-folds. In the aspect of in vitro analysis, dynamic changes
for cell malignancy using H&N cancer cells from different origins (tongue,
oropharyngeal or oral squamous cells) in response to prolonged glucose
challenge were examined. Further histological analysis for tumour lesions and
molecular expression using animals bearing 4-Nitroquinoline 1-oxide (4-NQO)
induced oral cancer with/without administration of DM inducer streptozotocin
(STZ) was also carried out.
Results: Current results demonstrated that hyperglycaemia promoted H&N
cancer malignancy in through (1) modulation of cell growth; (2) suppression
of cell differentiation; (3) enhancement of epithelial mesenchymal transition
(EMT) mediated cell motility and (4) up-regulation of nutrient mediated
oncogenic molecules. In vivo study detected tongue epithelial thickness
after 12-week exposure of 4-NQO as histological analysis indicated
progressive hyperplasiadysplasia correlated with longer 4-NQO treatments.
Under combinational experimental scheme using 4-NQO and multiple lowdose STZ injection, greater tumour mass was observed on tongues of
DM mice confirming DM-mediated tumour promotion. Interestingly, the
detection of accelerated establishment of hyperglycaemia in mice treated
with 4-NQO suggested reciprocal interplay between hyperglycaemia and oral
carcinogenesis.
Conclusions: Present study confirmed that elevated glycaemic challenge
enhanced tumour malignancy in H&N cancer both in vitro and in vivo.
The outcomes are of great benefit in understanding how hyperglycaemic
insult regulates H&N cancer development and in providing better anti-cancer
treatment strategy for DM patients.
181 IGF family genes expression in clear cell renal cell cancer and
their corresponding adjacent non-cancerous kidney tissues
R.S. Braczkowski1 , M. Bialozyt2 , B. Braczkowska3 . 1 Silesian Medical
University Katowice, Public Health, Bytom, Poland, 2 E. Michalowski Memory
Hospital, Urology, Katowice, Poland, 3 Silesian Medical University Katowice,
Epidemiology, Katowice, Poland
Background: In Poland, as well as in Czech Republic renal cancer is burdened
with a high mortality level due to metastasis, and therefore it is considered
one of the most important urological cancers in Central and Eastern Europe.
Renal cell carcinoma (RCC) is the most common type of kidney cancer. Clear
cell renal cell carcinoma (ccRCC) is a distinct and the most frequent subtype
of RCC. Renal cancer develops as a result of disturbances occurring in the
genetic background, whose base has not yet been elucidated. Tumor stage
and grade are still considered as the only widely accepted prognostic markers
of RCC. Avalanche increasing discoveries in the field of molecular biology give
hope of better understanding the molecular problems contributing to disease
development and progression. Insulin like growth factors are peptides with
strong promitotic and antiapoptotic effect. The action of both is mediated
through the activation of receptor IGF type I. IGF system is implicated in growth
regulation of the kidney and it is also involved in kidney pathological process.
Against this background we may suspect that IGFs promote the development
and growth of renal cell cancer.
Objective: To find the differences in the expression of mRNA for IGFs,
IGFBP-3 and IGF receptors between clear cell renal cell carcinoma and and
their corresponding adjacent non-cancerous kidney tissues.
Material and Methods: 62 patients suffered from kidney cancer qualified to
radical nephrectomy were included to the study. Only materials obtained from
patients with tumors qualified as ccRCC were included to further examination.
Finally 52 were qualified. Expression of genes for IGFs, IGF receptors and
IGFBP-3 were evaluated by RT-PCR. Non-parametric Mann–Whitney U test
was used for statistical analyses because the Kolmogorov–Smirnov test
indicated that the data were not normally distributed.
Results: There are no significant differences in the incidence of expression of
genes for IGF-1, and IGF-2 between ccRCC and corresponding non-cancerous
kidney tissues. A small but not statistically significant difference has been
noted in the case of IGFBP-3. Differences have been observed in the case of
receptors. Incidence of expression of IGFR-1 gene is higher in ccRCC than in
free of cancer tissue of kidney with ccRCC.
The IGFR-2 gene expression is rarely observed in both tissues. In ccRCC it
is observed only in 3 cases, The expression in free of cancer tissue of kidney
with ccRCC is observed in 11 cases. The difference is statistically significant.
Interesting that only in one case, the latter expression has occurred in both
tissues.
Conclusions:
1. Incidence of expression of IGFR-1 gene is higher in ccRCC than in their
corresponding adjacent non-cancerous kidney tissues
2. The IGFR-2 gene expression is rarely observed in both tissues, but the
incidence is higher in non cancerous tissue of kidney with ccRCC.
182 Class III beta-tubulin is a target gene of HIF-2alpha in glioblastoma
cells exposed to hypoxia
K. Bordji1 , A. Grandval1 , L. Cunha-Alves1 , E. Lechapt-Zalcman2 ,
M. Bernaudin1 . 1 GIP CYCERON, UMR CNRS 6301-ISTCT, Caen, France,
2
CHU de Caen, Service d’Anatomie et Cytologie Pathologique, Caen,
France
Background: Glioblastoma multiforme (GBM) is the most common,
chemorefractory and deadliest form of primary brain cancer in adults. Several
reports indicated aberrant levels of class III b-tubulin (bIII-t), a neuron-specific
protein also known as Tuj-1, in human GBM. bIII-t overexpression has been
linked to increasing malignancy in glial tumors and was described to determine
the apparition of resistance to chemotherapy with tubulin-binding agents.
Moreover, a linkage was suggested between the induction of bIII-t expression
and hypoxia, a hallmark of GBM. In the present study, we investigated the
S41
respective role of HIF-1a and HIF-2a in the regulation of TUBB3 (gene coding
for bIII-t) and the production of bIII-t in human GBM cell lines (GL15 and U87)
cultured in normoxia or in hypoxia.
Material and Methods: GBM cell lines (GL15 and U87) were cultured in
normoxic or hypoxic conditions (1% O2 ) for different times (up to 24 h). siRNAs directed against HIF-1a or HIF-2a were used in cell cultures to assess
the role of both isoforms in the expression of bIII-t. HIF-1a, HIF-2a and bIII-t
mRNA and protein expression was measured by real-time PCR and Westernblot. Cells were transfected with reporter constructs containing the promoter
or the 3 UTR region of TUBB3, before to be exposed to hypoxia and cell
extracts were prepared for gene-reporter analysis. The involvement of hypoxia
response elements (HRE) located in both regulation sequences was assessed
by site-directed mutagenesis. The binding of HIF proteins on HRE sequences
was visualized by EMSA and super-shift experiments.
Results: We observed that hypoxia-induced bIII-t expression is well-correlated
to the kinetic of HIF-2a protein stabilization in GBM cells cultured for a
prolonged time in hypoxia. This suggests a role of this HIF isoform in bIII-t
induction. Indeed, with siRNA experiments, we found that HIF-2a, but not
HIF-1a, is involved in hypoxia-induced bIII-t expression. By gene-reporter
experiments and site-directed mutagenesis, we identified two overlapping
hypoxia response element (HRE) located in the 3 untranslated region of
the gene as being involved in the transcriptional activation of TUBB3. This
occurred through an enhanced binding of HIF-2a to these HRE, as revealed by
EMSA and supershift assays. In contrast, the HRE present in the promoter of
TUBB3 were shown to be inactive. Furthermore, RNA interference experiments
showed that HIF-1a exhibits a repressive effect on bIII-t expression in cells
cultured in normoxia. These results strongly suggest that both HIF-a isoforms
have opposing effects on bIII-t expression in GBM cells by downregulating
(HIF-1a) or upregulating (HIF-2a) the TUBB3 gene.
Conclusions: We reveal for the first time the involvement of HIF-2a in the
overexpression of bIII-t induced by hypoxia in cultured GBM cells. The evidence
of a direct linkage between HIF-2a and elevated expression of bIII-t by hypoxia
in glial tumors suggests that an anti-HIF-2a strategy, by downregulating bIII-t,
may be of potential interest to improve GBM treatments.
183 From ERa66 to ERa36: a new predictive marker for cancer
progression and therapeutic response in breast tumors?
C. Chamard1 , A. Jung2 , A. Chesnel1 , J. Abecassis2 , S. Flament1 ,
C. Macabre2 , S. Ledrappier2 , T. Boukhobza1 , H. Dumond1 . 1 Université de
Lorraine, CRAN UMR CNRS 7039, Vandoeuvre-lès Nancy, France, 2 Centre
Paul Strauss, EA 3430, Strasbourg, France
Background: Breast cancer is the main cause of cancer-induced morbidity
and mortality in women. Breast tumors are usually classified according to
the canonical estradiol receptor alpha (ERa66) expression status, noticed as
[ER+] standing for ERa66 expressing tumors or [ER-] for ERa66 negative.
Such a classification led to the use of endocrine therapeutic agents against
[ER+] tumors. Nevertheless, numerous therapeutic failures are observed due
to unclear resistance mechanism.
ERa66 was considered as the unique functional estrogen receptor in hormone
sensitive breast tumor until the recent identification of membrane bound
new estrogen receptors: the G protein coupled estrogen receptor (GPER)
and the 36kDa ERa splice variant (ERa36). Surprisingly, ERa36 stimulates
cell proliferation in response to tamoxifen treatment and could therefore be
involved in the acquired resistance to this compound. Moreover, a high
ERa36 expression level correlates with a short term survival for ERa66negative patients as well as enhanced tumorigenesis and metastatic potential
of triple-negative cells in vitro. Therefore, we addressed (i) the involvement
of ERa36 in stimulating cancer progression mechanisms in non cured [ER+]
and [ER-] tumor biopsies and (ii) the ability of ERa36 to trigger cancerous
phenotype in normal epithelial breast cell in response to realistic doses of
4-hydroxytamoxifen.
Methods: In a retrospective study of 118 biopsies, we tried to decipher
underlying mechanisms by using an original nonlinear correlation analysis
and mutual information computations in order to identify the relationship
between ERa36, other estrogen receptors and metastatic marker expression.
In vitro, specific pharmacological inhibitors, gene-silencing strategies and gene
network analyses were used in order to characterize the molecular phenotype
of the MCF-10A cell line under exposition to OHT.
Results: Patient biospsies analyses led to (i) better define the field of
acceptance of [ER+] versus [ER-] classification and (ii) characterize a complex
gene network connecting non-genomic ERa36-dependent to metastatic
process. In vitro analysis revealed that ERa36 is involved in OHT-dependent
proliferation and survival.
Conclusion: This study indicates that a high expression level of ERa36 could
be a key node for both metaplastic transformation of mammary epithelial cells
and metastatic progression of breast cancer cells exposed to OHT.
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184 Roles of digoxin and digitoxin in hepatocellular carcinoma
W. Huang1 , Y. Jeng2 , I. Fong3 , H. Hsu4 . 1 Hsin Sheng College of Medical Care
and Management, Nursing, Taoyuan, Taiwan, 2 National Taiwan University
Hospital and College of Medicine, Pathology, Taipei, Taiwan, 3 College of
Medicine National Taiwan University, Pathology, Taipei, Taiwan, 4 Department
of Internal Medicine Taipei City Hospital, Cardiology, Taipei, Taiwan
Background: Digoxin (DG) and digitoxin (DT), composed of digitalis, were the
main drugs for heart disease. DG is widely used in the treatment of various
heart conditions, namely atrial fibrillation, atrial flutter and sometimes heart
failure. DG and DT were mainly metabolized by kidney and liver. It has been
known that DG may be a possible therapy for prostate cancer. DT inhibits
the growth of cancer cell lines at concentrations commonly found in cardiac
patients. hypoxia-induced factor (HIF) activity is involved in angiogenesis
required for cancer tumor growth. It has been known that DG down-regulates
VEGF through the inhibition of HIF-1alpha under hypoxic conditions in some
human cancer cells lines. The aim of this study is to find the role of DG and
DT in hepatocellular carcinoma (HCC) cells by observing the proliferation,
apoptosis, and morphology in DG and DT treatment.
Material and Methods: Several HCC cancer cell lines were treated with DG
or DT in dose- and time-responses. Cells apoptosis was observed by MTT
assay, cells containing transforming oncogenes grown in focus-forming assay,
and cell migration was proved by Wound healing assay. Western blotting also
showed the HIF-1 alpha expression in DG and DT treatment.
Results: Our preliminary data in vitro study with MTT and focus-forming assay
showed that treatment with DG (0.1−1 uM) in PLC5, HA22T and Hep3B liver
cancer cell lines, as well as treatment with DT (0.1−1 uM) in PLC5 and HA22T
liver cancer cell lines, significantly inhibited the survival and growth in liver
cancer cells in 24 hrs and 48 hrs. The morphology of cells was transformed
normal to round shape in DG or DT (0.1−1 uM) compared to normal cells.
Cell migration decreased significantly under DG at higher doses (0.05−0.2
uM). Western blotting data showed that HIF-1 alpha over-expressed under the
treatment of DG at dose of 1 uM after 24 hrs.
Conclusions: Our study investigate that DG and DT might promote the cell
apoptosis, inhibit migration, and change the cell morphology by the HIF-related
conditions in HCC. This finding may be a basis for clinical HCC therapy.
185 Regulation of Panax notoginseng extract on metastasis in human
colon cancer cells
C.C. Wu1 , Y.H. Kuo2 , C.Y. Lee3 , C.C. Tsai4 , S.L. Hsieh2 . 1 Chang Jung
Christian University, Nutrition and Health Sciences, Tainan City, Taiwan,
2
National Kaohsiung Marine University, Department of Seafood Science,
Kaohsiung City, Taiwan, 3 E-DA Hospital, Department of Chinese Medicine,
Kaohsiung City, Taiwan, 4 I-Shou University, College of Medicine The School
of Chinese Medicine for Post Baccalaureate, Kaohsiung City, Taiwan
Background: Panax notoginseng is a traditional Chinese medicine for
cardiovascular disease, but it is limited on anti-metastasis study.
Materials and Methods: To investigate the regulation of Panax notoginseng
alcohol extract (PNAE) on metastasismigration and adhesion of colon cancer.
The human colon cancer cells (HCT-116) cultured was as an experimental
model, and wound healing assay, adhesion reaction and regulate molecular
expression were analyzed in this study.
Results: According to the results, the inhibition percentage of migration on
HCT-116 cells were significantly increased after 0.05, 0.1 or 0.5 mg/mL PNAE
treatment for 24 and 48 hrs (P < 0.05). From gelatin zymography analysis result
show that 0.1 and 0.5 mg/mL PNAE groups were significantly decreased the
activity of MMP-2 after 24 hrs incubation (P < 0.05). In adhesion reaction assay
results shown 0.1 and 0.5 mg/mL PNAE groups were significantly decreased
HCT-116 cells adhesion to endothelial cells (EA. hy926 cells). E-selectin
protein levels of EA. hy926 cells were significantly decreased after 0.1 or
0.5 mg/mL PNAE treatment.
Conclusion: These results demonstrate the anti-metastatic properties of
PNAE. Furthermore, the mechanism is through the inhibition of cell migration
and adhesion.
186 The anti-cancer effects of clioquinol on oral cancer
M.W. Lee1 , P.C. Lin2 , W.C. Tsai2 . 1 Chung Shan Medical University,
School of Medical Laboratory and Biotechnology, Taichung City, Taiwan,
2
Kaohsiung Medical University, Department of Medical Laboratory Science
and Biotechnology, Kaohsiung City, Taiwan
Background: Clioquinol (iodochlorhydroxyquin) is an antifungal drug and
antiprotozoal drug. It is neurotoxic in large doses. It is a member of a family of
drugs called hydroxyquinolines which inhibit certain enzymes related to DNA
replication. The drugs have been found to have activity against both viral and
protozoal infections. In a recent study, researchers found that the zinc-binding
compound, clioquinol, was able to kill cancer cells. The addition of zinc, which
would be expected to reverse the activity/property of a sequestering agent,
was found to potentiate the cytotoxic effects of clioquinol, and direct evidence
that clioquinol was acting as a zinc ionophore was obtained. More recently,
CQ is a newly discovered anticancer agent. In this study, we focus on the
anti-cancer effects of CQ on human oral squamous cell carcinoma (OSCC).
Material and Methods: OC-2 oral cell lines established from primary tumors
from adult male OSCC patients from Taiwan. HSC-3 derived from human
tongue carcinoma with lymph node metastasis was from the Taiwan Collection
of Research Bioresources. Cell viability was determined via MTT assay. Cell
apoptosis, caspase-3 activation, mitochondria membrane potential change and
ROS production were analyzed by flow cytometer and analyzed by WinMDI
analysis software. Protein expression was detected by western blot.
Results: We found that CQ with copper could enhance the cytotoxicity of CQ in
two kinds of oral cancer cells, OC-2 and HSC-3 cells. Further, CQ with copper
would induce cell apoptosis, decrease mitochondria membrane potential and
increase ROS production in OSCC cells. Moreover, we also found the effect
of CQ with copper caused aberrant expression of apoptosis-related protein in
intrinsic cell apoptosis pathway.
Conclusions: CQ with copper could enhance the cytotoxicity of CQ in oral
cancer cells compared to CQ alone. CQ with copper could also induce ROS
production, mitochondria disruption, and trigger cell apoptosis via intrinsic
(mitochondrial) cell apoptosis pathway in OSCC cells. Our results supported
that CQ would act as a potential selective anti-cancer drug due to the
accumulation of copper in tumor part but not normal tissue, and induce OSCC
cells apoptosis by intrinsic apoptosis pathway.
187 The expression of the human Sprouty protein-1 (hSpry1) inversely
correlates with proliferation, migration and invasion of the SKOV-3
and 1A9 human ovarian cancer cells
S. Masoumi-Moghaddam1 , A. Amini1 , D.L. Morris1 . 1 St George Clinical
School, The University of New South Wales, Surgery, Sydney NSW,
Australia
Background: Sprouty proteins are regulators of MAPK/ERK pathway. We have
already shown that the human ovarian cancer cell lines SKOV-3 and 1A9
express low and high levels of the Sprouty protein 1 (Spry1), respectively.
In the present study, the effect of the Spry1 overexpression or silencing on
behaviour of these malignant cells was investigated.
Materials and Methods: Spry1 was silenced in 1A9 cells using the specific
siRNA. In parallel, SKOV-3 cells were transiently transfected with either the
Spry1 plasmid or the pcDNA3.1 vector. The effect of the altered expression
was then functionally investigated using proliferation, MTT, scratch-wound,
migration and invasion assays. To evaluate the effect of the Spry1 transfection
on the SKOV-3 cell survival, the stably-transfected clones were also selected.
Results and discussion: The Spry1 silencing led to a significant increase
in growth and proliferation of 1A9 cells at 48 h and 72 h time points as
shown by growth (48 h, p: 0.0365; 72 h, p: 0.0228) and MTT assays (48 h,
p: 0.0011; 72 h, p: 0.0024) as well as a higher percentage closure of the
scratch at the 40 h endpoint (p: 0.0259). In the migration assay, the number of
the migrated cells in the silenced group was significantly higher than control
examined at hours 14 (p: 0.0125) and 20 (p: 0.0090). Similarly, our invasion
assay showed an increased number of the invaded cells in test group at
hours 14 (p-value: 0.0298) and 20 (p-value: 0.0373). In SKOV-3 cells, the
proliferation of the transfected cells was significantly lower than control on day
3 post-transfection as evaluated by growth (p-value: 0.0003) and MTT (p-value:
0.0042) assays. In the migration assay, the number of the migrated cells in
the transfection group was significantly lower examined at hours 6 (p-value:
0.0090) and 12 (p-value: 0.0002). Invasion assay similarly showed a decreased
number of the invading cells in the Spry1 group assayed at hours 6 (p-value:
0.0159) and 12 (p-value: 0.0005). Also, a significantly-decreased percentage
of the scratch closure was observed at hours 20 and 24 (p-values of 0.0232
and 0.0046, respectively). Finally, the Spry1 stably-transfected clones were
almost undetectable after two weeks post selection. Given its pivotal role in
modulation of MAPK/ERK, Sprouty regulates key cellular events, including
cell proliferation, differentiation and apoptosis. In agreement with our earlier
study indicating differential expression of Spry1 in a range of ovarian cancer
cells with different invasiveness, our results argue that the Spry1 deregulation
confers functional changes in human ovarian cancer cells.
Conclusion: Here, we report for the first time that the expression of Spry1
inversely correlates with proliferation, migration, invasion and survival of the
human ovarian cancer cells. The clinicopathological relevance of the Sprouty
deregulation in ovarian cancer is currently under investigation.
188 Bromelain and N-acetylcysteine induce cytotoxic effects and
reduce the expression of mucin in mucin-producing carcinoma
cell lines of gastrointestinal origin
A. Amini1 , S. Masoumi-Moghaddam1 , D.L. Morris1 . 1 St George Hospital
University of New South Wales, Surgery, Sydney NSW, Australia
Background: To enhance the efficacy of the current standard of care in
patients with peritoneal mucinous tumors, in particular those with mucinous
ascites as the dominant feature, our research group at St George Hospital
with an established Peritoneal Surface Malignancy Program intends to develop
a novel formulation for locoregional targeting of the mucin-producing tumor
cells and mucin, itself. In the present study, the potential utility of bromelain
and N-acetylcysteine (NAC) in such a therapeutic approach was evaluated in
in vitro models.
Materials and Methods: The KATO-III, MKN45, HT29−5F12 and HT29-FM21
mucin-expressing gastrointestinal carcinoma cells were seeded into 96 well
plates. At desired confluence, cells were treated with a range of concentrations
of bromelain and/or NAC. After an incubation period of 72 hours, the effect of
treatment on the growth and proliferation of the cancer cells was determined
using sulforhodamine B (SRB) assay. Moreover, the expression patterns of
the MUC1 and MUC5AC mucins in KATO-III and MKN45 cells before and
after 24 hours of treatment were visualized with immunocytochemistry. Finally,
treatment-induced apoptosis after 24 hours was examined in MKN45 cells by
TUNEL test.
Results and Discussion: Data from SRB assay indicated that bromelain at
the concentrations of 40 (p < 0.0001), 40 (p < 0.0001), 100 (p = 0.0018) and
100 mg/ml (p < 0.0001) and NAC at the concentrations of 10 (p = 0.0011),
5 (p = 0.0091), 5 (p = 0.0006) and 25mM (p < 0.0095) significantly inhibited
proliferation of HT29−5F12, HT29−5M21, MKN45, and KATO-III cells,
respectively. When combined together, synergistic effects were evident.
Mechanistically, apoptosis was shown in TUNEL test to mediate cell death.
Confocal microscopy also revealed the decreased expression the MUC1 and
MUC5AC mucins after treatment. Bromelain and NAC are two mucolytic agents
of plant origin with a good safety profile. At the concentrations within the safe
range reported in the literature, they were proved here to significantly inhibit
growth and proliferation of and to induce apoptosis in the cancer cells studied.
The treatment was also found to target MUC1 and MUC5AC. Since both
membrane-associated and secreted mucins play critical roles in the biology
of the mucin-expressing carcinoma cells, this formulation with dual effects on
the malignant cells and their mucin product could be of potential value in
targeted therapies.
Conclusion: Here we report that bromelain and NAC, on their own and in
combination, induce cytotoxicity and reduce the expression of MUC1 and
MUC5AC in the mucin-producing gastrointestinal carcinoma cells. Further
investigations are underway to preclinically confirm efficacy and safety of this
novel formulation before proceeding to clinical evaluation.
189 Characterization of ovarian carcinoma-associated fibroblasts:
The capability in predicting tumor aggressiveness
C. Liu1 , T. Mao2 . 1 Taipei Medical University, School of Medical Laboratory
Science and Biotechnology College of Medical Science and Technology,
Taipei, Taiwan, 2 National Taiwan University Hospital, Department of
Pathology, Taipei, Taiwan
Introduction: Ovarian carcinoma is the most lethal female malignancy.
The grave prognosis is due to presentation of most ovarian carcinomas at
advanced stage and the lack of effective therapy for recurrent tumors. Various
approaches had been attempted in developing biomarkers for detection and
treatment of ovarian carcinomas, but with limited progress. In this study,
we searched for factors related to the aggressiveness of ovarian carcinoma
through ovarian carcinoma-associated fibroblasts (CAFs). Based on in vitro
system and animal models in various tumors, CAFs are thought to be related
to tumor initiation, growth and metastasis. However, how CAFs react in the
microenviroment in ovarian carcinomas is largely unknown. We hypothesize
that some ovarian CAFs have the ability in promoting tumor growth and
increasing the aggressiveness of the tumor.
Material and Method: We applied transwell invasion assay and classified
CAFs into low aggressiveness, intermediate aggressiveness, and high
aggressiveness based on the capability in promoting the transwell activity of
ovarian cancer cell line TOV21G. CAFs with high and low-invasion promoting
activity were subjected to cDNA microarray. Genes with at least 2 fold changes
were selected and verified by quantitative real-time RT-PCR.
Results: CAFs from 51 tumors (25 from high-grade serous carcinoma, 12
from clear cell carcinoma, 4 from endometrioid carcinoma, 8 from mucinous
tumor and 2 from neuroendocrine tumor) were successfully studied and
classified into CAFs with high, intermediate and low invasion promoting
activity. CAFs from high-grade serous carcinoma and clear cell carcinoma
have significantly higher invasion promoting activity than CAFs from other
types of ovarian tumor, consistent with the high-grade behavior of these two
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types of tumors. By cDNA microarray, we identified 92 upregulated genes
and 52 downregulated genes in CAFs from high-grade serous carcinoma and
69 upregulated genes and 37 downregulated genes in CAFs from clear cell
carcinoma. Four differentially expressed genes including SRPX, hemicentin
1, fibronectin 1 and SFRP2 were selected for verification. Quantitative realtime RT-PCR revealed significant overexpression of SRPX and hemicentin 1
in CAFs with high invasion promoting activity than in CAFs from low invasion
promoting activity.
Conclusion: Our result revealed that CAFs in ovarian tumor microenvironment
interact with tumor cells and may produce factors related to the aggressiveness
of the tumor. Further in vitro and in vivo studies are needed to investigate the
mechanism in regulating the behavior of tumor cells.
190 Involvement of CHK2 kinase in pre-mRNA splicing
H.H. Choi1 , H.K. Choi1 , Y. Bae2 , S.T. Kim1 , T. Kim3 . 1 Sungkyunkwan
University School of Medicine, Molecular Cell Biology, Seoul, South Korea,
2
Seoul National University College of Medicine, Parasitology, Seoul, South
Korea, 3 Sungkyunkwan University School of Medicine, Immunobiology,
Suwon, South Korea
Checkpoint kinase 2 (CHK2) kinase, which is frequently mutated in a rare
familial cancer syndrome, Li–Fraumeni syndrome, is a key mediator in many
cellular responses to genotoxic stresses, including ionizing radiation (IR) and
topoisomerase inhibitors. Upon IR, CHK2 is activated by ataxia telangiectasia
mutated kinase and regulates the S-phase and G1-S checkpoints, apoptosis
and DNA repair by phosphorylating downstream target proteins, such as
p53 and Brca1. In addition, CHK2 is thought to be a multi-organ cancer
susceptibility gene.
In this study, we used a tandem affinity purification strategy to identify proteins
that interact with CHK2 kinase. Cyclin-dependent kinase 11 [CDK11 (p110)]
kinase, implicated in pre-mRNA splicing and transcription, was identified as
a CHK2-interacting protein. CHK2 kinase phosphorylated CDK11 (p110) on
serine 737 in vitro. Unexpectedly, CHK2 kinase constitutively phosphorylated
CDK11 (p110) in a DNA damage-independent manner. At a molecular level,
CDK11 (p110) phosphorylation was required for homodimerization without
affecting its kinase activity. Overexpression of CHK2 promoted pre-mRNA
splicing. Conversely, CHK2 depletion decreased endogenous splicing activity.
Mutation of the phosphorylation site in CDK11 (p110) to alanine abrogated its
splicing-activating activity.
These results provide the first evidence that CHK2 kinase promotes pre-mRNA
splicing via phosphorylating CDK11 (p110).
191 Heterogeneity among early-stage K-Ras driven lung
adenocarcinoma predicts tumor aggressiveness and identifies
Ddr1 as a therapeutic target
C. Ambrogio1 , G. Gomez2 , D. Santamaria1 , M. Barbacid1 . 1 CNIO, Molecular
Oncology, Madrid, Spain, 2 CNIO, Structural Biology & Biocomputing
Programme, Madrid, Spain
Introduction: K-Ras driven lung adenocarcinoma is a devastating disease
with unmet medical needs. Analysis of early tumor stages could facilitate
the characterization of essential oncogenic mediators thereby potentially
identifying valuable therapeutic targets. Since this approach is not feasible in
humans we have studied K-RasG12V -driven early cancer lesions in a genetically
engineered mouse (GEM) model that faithfully recapitulates human disease.
Materials and Methods: The K-RasLSLG12Vgeo strain used in this study allows
controlled expression of a resident K-RasG12V oncogene coupled to a color
marker that allows identification of K-RasG12V expressing cells at the single
cell level. This property facilitates the isolation and characterization of very
early hyperplastic lesions by laser capture microdissection techniques.
Results and Discussion: K-RasG12V -driven early hyperplastic lesions
clustered into two distinct subtypes (T1 and T2) defined by their gene
expression profiles. Importantly, the T2 signature overlaps with the gene
expression profile of human aggressive lung adenocarcinomas that carry
K-Ras activating mutations, thus suggesting that tumor malignancy is
determined at a very early stage of tumor development. Among the top
ranking genes within the T2 signature we identified Ddr1, an epithelial-specific
membrane receptor tyrosine kinase previously implicated in the activation
of the PI3K/Akt and Ras/ERK cascades. To validate Ddr1 as a therapeutic
target, we inserted Ddr1 null alleles in our GEM model. Genetic ablation
of Ddr1 resulted in substantial attenuation of tumor burden and increased
life span. Interfering with Ddr1 activity by means of a selective chemical
inhibitor triggered cell death and decreased overall tumor burden in mice
bearing K-RasG12V lung tumors. Importantly, tumor response correlated with
high expression levels of the Ddr1 receptor. Taken together, these data indicate
that Ddr1 is an early marker of malignant potential of K-Ras-driven lung
adenocarcinomas as well as a valuable therapeutic target.
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Conclusions:
1. Morphologically indistinguishable K-RasG12V early hyperplastic lesions
display two distinct gene expression profiles (T1 and T2 signatures).
2. T2 signature overlaps with that of aggressive human lung adenocarcinomas, suggesting that tumor malignancy is determined at an early stage.
3. A prominent hit in the aggressive T2 signature is the tyrosine protein kinase
receptor Ddr1.
4. Ddr1 is required for K-RasG12V tumor progression and maintenance.
5. Pharmacologic inhibition of Ddr1 triggers an apoptotic response and
decreases tumor burden, indicating that lung adenocarcinoma patients
displaying DDR1 overexpression (>70% by IHC studies using TMAs) can
benefit from DDR1 inhibitors.
192 Pinus morrisonicola Hayata extracts inhibit cell proliferation and
promote apoptosis of human promyelocytic HL-60 leukemia cells
G.Y. Liu1 , Z.W. Wang1 . 1 Institute of Microbiology & Immunology, Chung Shan
Medical University, Taichung, Taiwan
Background: The ancient Chinese books Shen Nong Ben Cao Jing and
Bencao Gangmu mention that plants such as pines, woods and grasses are the
best source of drugs to lengthen human life and to be health foods. People
have all along been seeking to prolong life. Pinus morrisonicola Hayata is
one of original coniferous species in Taiwan. Many previous studies indicate
pine’s physiological activities and therapeutic effects, including as a remedy for
carcinoma. But there are few reports regarding the biological effects of Pinus
morrisonicola Hayata that have been found so far.
Material and Method: The objective of this study is to investigate the effects
on inhibiting cell proliferation and promoting apoptosis of Pinus morrisonicola
Hayata extracts and its active compounds.
Results and Discussion: Results from this study indicate that Pinus
morrisonicola Hayata extract, the partition of water phase and its compounds
inhibited cancer cell growth and promoted apoptosis. In addition, our data
show that Pinus morrisonicola Hayata extract at 500 mg/ml did not have
significant cytotoxicity on normal peripheral blood mononuclear cells and
zebrafish embryos.
Conclusion: In conclusion, we have shown that Pinus morrisonicola Hayata
extract exerts a significant effect on inhibition of cancer cell growth and
induction of apoptosis in leukemia cancer cells mediated by cell cycle and
apoptosis regulatory proteins. These data suggest that Pinus morrisonicola
Hayata extract, as natural substances with powerful growth inhibition and
inducing apoptosis effects on leukemia cells, will be a good candidate for
chemoprevention or chemotherapeutic adjuvant in the future.
193 Tetraindole suppresses breast cancer growth and metastasis
by reversing epithelial–mesenchymal transition state associated
with up-regulation of the miR-200 family
W. Li1 , C. Wang1 , C. Chen1 , S. Jao2 . 1 Academia Sinica, Institute of
Chemistry, Taipei City, Taiwan, 2 Academia Sinica, Institute of Institute of
Biological Chemistry, Taipei City, Taiwan
Background: The results of recent studies have shown that metastasis, the
most common malignancy and primary cause of mortality promoted by breast
cancer in women, is associated with the epithelial-to-mesenchymal transition
(EMT). The results of the current study show that SK228, a novel tetraindole
substance, exhibits anti-cancer activity. In addition, the effects of SK228 on the
regulation of EMT in breast cancer cells as well as the underlying mechanism
have been explored.
Material and Method: The inhibition of in vitro migration and invasion activities
of SK228 were examined employing three breast cancer cell lines by using a
transwell assay. The effects of this tetraindole derivative on cell viabilities were
determined by using the MTT assay. The expressions of EMT inducers, along
with epithelial and mesenchymal markers were examined by using western
blotting and reverse transcription-PCR. In addition, the expression of members
of the miRNA-200 family was examined by utilizing real-time qPCR and HDAC
activities were determined by using a commercial kit. Therapeutic efficacy of
SK228 was evaluated using a breast cancer xenograft animal model by using
tail vein injection (12.5 mg/kg/mouse/week).
Results: SK228 was observed to induce a fibroblastoid to epithelial-like
change in the appearance of various breast cancer cell lines and to suppress
the migration and invasion of these cancer cells in vitro. Moreover, expression
of E-cadherin was found to increase following SK228 treatment whereas ZEB1
expression was repressed. Expression of other major EMT inducers, including
ZEB2, Slug and Twist1, is also repressed by SK228 as a consequence of
up-regulation of members of the miR-200 family, especially miR-200c. The
results of animal studies demonstrate that SK228 treatment leads to effective
suppression of breast cancer growth and metastasis in vivo.
Conclusions: The observations made in this investigation show that SK228
reverses the EMT process in breast cancer cells via an effect on the miR-
200c/ZEB1/E-cadherin signalling pathway. In addition, the results of a detailed
analysis of the in vivo anti-cancer activities of SK228, carried out using a
breast cancer xenograft animal model, show that this substance is a potential
chemotherapeutic agent for the treatment of breast cancer.
195 Platycodi Radix facilitates autophagy through increasing ROS
formation in NCI-H460 human non-small lung carcinoma cells
S.H. Hong1 , C. Park2 , M.H. Han1 , Y. Choi1 . 1 Dongeui University College
of Oriental Medicine, Department of Biochemistry, Busan, South Korea,
2
College of Natural Sciences Dongeui University, Department of Molecular
Biology, Busan, South Korea
Introduction: The root of Platycodon grandiflorum, Platycodi Radix (PR), is
widely used in traditional Oriental medicine for the treatment of many chronic
inflammatory diseases. However, little is known about the molecular events
linking apoptosis to the regulation of PR inducing autophagy in cancer cells. In
this study, we examined the effects of PR on the production of reactive oxygen
species (ROS) and evaluated the association of these effects with apoptotic
and autophagic tumor cell death using a human non-small lung carcinoma
NCI-H460 cell line.
Material and Method: MTT assay and flow cytometry analysis were carried
out to determine the cell proliferation and apoptotic cell death, respectively.
Protein expression was measured by immunoblotting. Autophagic vesicular
organells (AVOs) were performed by Cyto-ID detection. Production of
intracellular ROS was checked using FACS analysis.
Results and Discussion: PR decreased cell proliferation and increased
apoptotic cell death in NCI-H460 human non-small lung carcinoma cells in
a dose- and a time-dependent manner. PR treatment led to clear formation of
AVOs, the conversion of microtubule-associated protein 1 light chain 3 a, upregulation of Beclin-1 and several autophagy-related genes. Pretreatment of
autophagy inhibitors, 3-methyladenin and bafilomycin A1, increased the PRmediated apoptotic effect. In addition, PR treatment significantly enhanced
intracellular ROS and nuclear factor erythroid-2-related factor 2 (Nrf2)
translocation to nuclei, which was associated with the increase the expression
of heme oxygenase-1 (HO-1) and NAD (P) H: quinone oxidoreductase 1.
However, scavenging of ROS by anti-oxidants, N-acetylcysteine or Vitamin
C, reduced PR-induced autophagy and recovered Nrf2 translocation.
Conclusion: These results suggest that PR induces cell protective autophagy
in NCI-H460 cells, which mediate by increasing intracellular ROS formation
and Nrf2 translocation.
NRF grant funded by the Korea government (No. 2013R1A1A2065537 &
2012046358).
196 System-wide characterization of dynamics of cytosolic
macromolecular protein complexes during oncogene-induced
cell transformation
B. Diedrich1 , K.G. Rigbolt2 , S. Bestmann2 , T. Brummer3 , J. Dengjel1 .
1
Freiburg University Medical Center, Dept. of Dermatology, Freiburg,
Germany, 2 Zentrum für Biosystemanalyse (ZBSA) University of Freiburg
Germany, Freiburg, Germany, 3 University of Freiburg Germany, Institute of
Molecular Medicine and Cell Research, Freiburg, Germany
Introduction: The majority of human cancers are caused by the malignant
transformation of epithelial cells, one of the most frequently occurring
malignancies being colorectal carcinoma (CRC). Activating mutations of the
oncogene BRAF contribute decisively to the development of carcinomas.
Recent studies suggest that the use of B-Raf inhibitors in BRAF mutant
CRC cells yield less uniform responses than in melanoma reflecting the
heterogeneous character of CRC. These studies highlight the need for a better
understanding of normal and mutant B-Raf signalling at the molecular level,
in particular a solid understanding of B-Raf-dependent interaction networks,
their structure and regulation.
We hypothesized that B-Raf-dependent oncogenic cell transformation leads to
global alterations in protein–protein interactions driving tumor development.
Material and Methods: To address this we used human epithelial colorectal
adenocarcinoma cells (CaCo-2) inducibly expressing oncogenic or wild type
BRAF as CRC model system. We studied direct B-Raf-dependent as well
as global alterations in protein interactions upon oncogene expression.
(1) Combining affinity purification with quantitative proteomics differences
between interaction partners of B-RafWT and B-RafV600E were characterized.
(2) To further expand our search we applied a combination of sizeexclusion chromatography and high-accuracy, high-resolution quantitative
mass spectrometry for unbiased high-throughput screening of changes in
protein interactions providing a platform for identification of altered complex
formation of proteins downstream of B-Raf.
Result: Using these different approaches we were able to identify several
thousand protein–protein interactions including several that demonstrate
differences between the wild type and oncogenic form of B-Raf.
Conclusion: This global, unbiased investigation of the BRAF-dependent
interactome elucidates new players in malignant cell transformation that
represent potential new therapeutic drug targets.
197 Tetraarsenic hexaoxide induces apoptosis in human bladder
cancer 5637 cells via reactive oxygen species generation and
DNA damage
M.H. Han1 , Y. Choi1 , S.H. Hong1 , W.J. Kim2 , C. Park3 . 1 Dongeui University
College of Oriental Medicine, Department of Biochemistry, Busan, South
Korea, 2 Chungbuk National University College of Medicine, Department of
Urology, Cheongju, South Korea, 3 Dongeui University College of Natural
Sciences, Department of Molecular Biology, Busan, South Korea
Introduction: Tetraarsenic hexaoxide (As4 O6 ) has been used in traditional
medicine for the treatment of cancer, and arsenic trioxide (As2 O3 ) is currently
used as a chemotherapeutic agent. However, the evidences suggest that
As4 O6 -induced cell death pathway was different from that of As2 O3 , and the
anticancer effects and mechanisms of As4 O6 are not fully understood. In this
study, we investigated the pro-apoptotic actions of As4 O6 in human bladder
cancer 5637 cells.
Material and Method: Cytotoxicity was evaluated MTT assay. Apoptosis was
detected using DAPI staining, DNA fragmentation assay and flow cytometry.
Reactive oxygen species (ROS) level was measured using DCFHDA staining.
DNA damage was measured by comet assay. The protein levels were
determined using Western blotting.
Results and Discussion: Exposure of 5637 cells to As4 O6 resulted in
apoptosis induction. As4 O6 treatment caused DNA damage and increased the
phosphorylated form of histone variant H2AX (gH2AX). As4 O6 treatment also
led to increased intracellular ROS formation, which was inhibited by N-acetyl-lcysteine (NAC) pretreatment. Furthermore, NAC pretreatment inhibited As4 O6 induced apoptosis and DNA damage.
Conclusion: These observations provide novel mechanisms and potential
targets for better understanding of the anti-cancer mechanisms of As4 O6 .
NRF grant funded by the Korea government (No. 2008–0062611).
198 Effects of pro-apoptotic Bcl-2 on morin-induced apoptosis in
human leukemia U937 cells
C. Park1 , Y. Choi2 , M.H. Han2 , W.J. Kim3 , S.H. Hong2 . 1 College of Natural
Sciences Dongeui University, Department of Molecular Biology, Busan, South
Korea, 2 Dongeui University College of Oriental Medicine, Department of
Biochemistry, Busan, South Korea, 3 Chungbuk National University College of
Medicine, Department of Urology, Cheongju, South Korea
Introduction: Flavonoids represent a group of polyphenolic compounds
isolated from plants and have long been recognized for their general healthpromoting properties. Morin (3,5,7,2 ,4 -pentahydroxyflavone), a flavonoid
isolated from Oriental medicine of the Moraceae family, exhibits many
bioactivities especially anti-hyperlipedimia, anti-hyperinsulinemia and antiinflammation, however the molecular mechanisms responsible for its anticancer activity are largely unknown. In the present study, we investigated the
pro-apoptotic effects of morin in human leukemia cell lines.
Material and Method: The inhibition of cell growth was evaluated by MTT
assays, and the apoptotic cells were determined by the DAPI staining, DNA
fragmentation and DNA flow cytometry. JC-1 fluorescence probe was used
to examine the mitochondria membrane potential (MMP, Dym). The protein
levels and caspases activity were measured using Western blot analyses and
colorimetric assay, respectively.
Results and Discussion: It was found that the anti-proliferative effects of
morin were more sensitive in U937 cells than HL-60, K562 and THP-1
cells, which was associated with the induction of apoptotic cell death. The
apoptotic cell death by morin was connected with an up-regulation of Bad,
down-regulation of Bcl-XL and cIAP-2, and increased the percentage of
cells with a loss of MMP. Morin treatment also induced the proteolytic
activation of caspases (-3 and -9), and degradation of caspase-3 substrate
proteins. Both the cytotoxic effects and apoptotic characteristics induced
by morin were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor,
which demonstrates the important role that caspase-3 played in the
observed cytotoxic effects. Although, the levels of pro-apoptotic Bcl-2 proteins
were remained unchanged, Bcl-2 overexpression significantly reversed the
morin-induced apoptosis via inhibition of the MMP collapse and caspases
activation.
Conclusion: Taken together, these findings demonstrate that the pro-apoptotic
effect of morin is mediated through mitochondria dysfunction and activation of
caspases in leukemia cells.
NRF grant funded by the Korea government (No. 2013R1A1A2065537 &
2012046358).
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199 ATM kinase expression regulates breast cancer stem-like
phenotype
M. Antonelli1 , M. Sambucci2 , R. Brandi3 , I. Arisi3 , M. D’Onofrio3 , D. Barilà1 ,
V. Stagni1 . 1 Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS)
Fondazione Santa Lucia, Laboratory of Cell Signaling, Roma, Italy, 2 Istituto
di Ricovero e Cura a Carattere Scientifico (IRCCS) Fondazione Santa Lucia,
Neuroimmunology Unit, Roma, Italy, 3 European Brain Research Institute
(EBRI) ‘Rita Levi-Montalcini’, Genomics Facility, Roma, Italy
Introduction: Recent studies have provided strong support for the cancer
stem cell (CSC) hypothesis, which suggests that many cancers, including
Breast Cancer (BC), are driven by a subpopulation of cells that display stem
cell properties and by virtue of their relative resistance to anti-cancer drugs,
contribute to treatment relapse. HER2 is a RTK overexpressed in 20−25% of
BC and this overexpression seems to be associated with an increase of the
fraction of CSCs pool within BC.
The role of serine threonine kinase ATM in cancer development and therapy
is still largely debated. ATM may act as tumor suppressor as well as tumor
promoter gene and the modulation of its activity may exert positive or negative
effects in cancer therapy. Recently in our lab we identified a new tumorigenic
role of ATM in HER2 positive BC. The role of ATM in regulation of CSCs
has not been investigated yet, although recent studies suggest that ATM may
contribute to the maintenance of the pool of normal stem cell. Based on
literature and our data, the aim of this study is to identify a new possible
function of ATM in the maintenance of the ‘stem-like’ phenotype in BC.
Material and Method: Lentivirus infection to genetically interfere ATM
expression; mammosphere assay; immunoblotting analysis; real time-PCR,
microarray, ALDH assay.
Results and Discussion: As model system for BCSCs we have isolated
and characterized cell population with stem-like features from BC cell
lines as MCF-7 and MCF-7HER2, by using mammosphere assay. Our
preliminary data show an increase in ATM protein and mRNA levels in cells
grown as mammospheres, compare to the same cells grown in adherent
conditions, suggesting a role of ATM in regulation of mammospheres formation.
Interestingly, downregulation of ATM expression significantly reduces spheres
formation and the activity of ALDH-1, a well-known marker of BCSCs features.
Moreover Real-Time experiments could show that downregulation of ATM
correlates very well with a modulation of the expression of genes involved
in the stemness pathway. For these reasons, we will analyse different gene
expression profiles obtained by microarray analysis comparing sphere versus
differentiating MCF-7/MCF-7HER2 cells in presence or absence of ATM.
Conclusions: Our findings suggest a crucial role of ATM in the maintenance
and in the regulation of the ‘Cancer stem-like’ phenotype in BC and its inhibition
may represent a novel strategy to selective target BCSCs.
200 Potential therapeutic targets for the hypoxic niche in glioblastoma
A. Dirkse1 , M. Sanzey1 , J. Bohler1 , R. Bjerkvig1,2 , A. Golebiewska1 ,
S. Niclou1 . 1 Centre de Recherche Public de la Sante (CRP-Sante),
Department of Oncology Norlux Neuro-Oncology Laboratory, Luxembourg,
Luxembourg, 2 University of Bergen, Department of Biomedicin Norlux
Neuro-Oncology, Luxembourg, Luxembourg
Like most solid tumors glioblastomas are heterogeneous tumors exposed to
fluctuating oxygen levels that require a dynamic adaptation of cancer cells
to varying environmental conditions. Hypoxia in glioblastoma is associated
with shorter patient survival, and perinecrotic hypoxic niches were reported
to contain cancer stem-like cells with increased resistance to radio- and
chemotherapy. Moreover, we have recently shown that anti-angiogenic
treatment increases tumor hypoxia. Using transcriptomic analysis and RNA
interference screen, we set out to identify putative therapeutic targets to tackle
hypoxic cancer cells. We show that glioma stem-like cells and primary cultures
adapt to severe oxygen deprivation by upregulating glycolysis and autophagy.
shRNA depletion of candidate genes in the tumor enhanced sensitivity to
hypoxia in vitro and led to a survival benefit of xenografted mice. Thus
we provide evidence that glycolysis and autophagy may represent important
therapeutic targets to sensitize hypoxic tumors to treatment.
201 The aging suppressor hormone klotho: A novel tumor suppressor
and regulator of estrogen signaling in ovarian cancer
I. Lojkin1 , O. Schwarzman2 , T. Rubinek1 , B.Y. Karlan3 , I. Wolf4 . 1 Ichilov
Medical Center, Oncology, Tel-Aviv, Israel, 2 Sackler Faculty of Medicine Tel
Aviv University, Oncology, Tel-Aviv, Israel, 3 Cedars-Sinai Medical Center,
Gynecologic Oncology, Los Angeles, USA, 4 Tel Aviv Sourasky Medical
Center, Oncology, Tel Aviv, Israel
Introduction: Klotho is a transmembranal protein, composed of two domains,
KL1 and KL2 that can be cleaved, shed and act as a hormone. It is expressed
in kidneys, brain and hormone-responsive tissues, including the ovaries.
Our group identified klotho as a tumor suppressor in several malignancies,
S46
including breast and pancreatic cancer. The precise mechanisms responsible
for klotho anti-tumor effects are not clear. However, we and others showed that
it is a potent inhibitor of the insulin-like growth factor-1 (IGF-1). As klotho is
expressed in the ovaries, and as the IGF-1is implicated in the development of
ovarian cancer (OC) we hypothesized that klotho may serve as a novel tumor
suppressor in OC.
Materials and Methods: Klotho expression in OC cell lines was analyzed by
RT-PCR and by immunohistochemistry (IHC) in OC tissue assay (277 OC and
30 normal ovary tissues). The effect of klotho on OC cell growth and viability
was assessed by MTT and colony formation assays. Signaling proteins were
detected by Western blot analysis.
Transcriptional activity of estrogen response elements (EREs) were measured
by a luciferase reporter assay and by RT-PCR of down-stream genes.
Results and Discussion: High klotho expression was observed in normal
ovary, while reduced expression was noted in 30% of the tumors. Reduced
expression was not associated with histology, tumor location or stage.
Klotho mRNA levels were reduced in most ovarian cancer cell lines.
Overexpression of klotho or KL1, as well as treatment with soluble klotho,
reduced clonal growth and viability of OVCA-432, SKOV3 and ES2 cell lines.
Moreover, klotho treatment enhanced the cisplatin activity.
E-cadherin is a marker of differentiation, and we revealed that klotho mediates
upregulation of E-cadherin expression in SKOV-3, OVCA-432 and ES-2 cells.
We aimed to elucidate the mechanism that mediates these effects, and studied
the effect on the IGF-1 pathway. Klotho inhibited IGF-1 pathway activation in
ES2, OVCA-432 and SKOV3 cells.
ERa signaling plays a role in OC development. Our studies revealed that klotho
inhibits ERa expression and transcriptional activity and inhibits expression of
downstream genes such as progesterone receptor.
Conclusion: These data indicate klotho as a potential tumor suppressor
in OC. Treatment with klotho, either alone or in combination with standard
chemotherapy, may serve as a novel therapy for this subset of tumors.
202 Simultaneous measurements of multiple injected contrast agents
using multispectral optoacoustic tomography (MSOT) in phantoms
and in vivo
T. Sardella1 , N.C. Burton1 , W.H.P. Driessen1 , J. Claussen1 , S. Morscher1 ,
D. Razansky2 , V. Ntziachristos2 . 1 iThera Medical, R&D, München, Germany,
2
Helmholtz Zentrum, IBMI, München, Germany
Background: MSOT is an emerging imaging modality which provides high
spatial resolution in deep tissue. A key feature of MSOT in vivo imaging
is its ability to illuminate at multiple wavelengths and spectrally unmix the
contributions of a probe together with the endogenous absorbers present in
tissue including oxyhemoglobin, deoxyhemoglobin and melanin. This makes it
possible to determine the spatial distribution and intensity of each individual
absorber in the cross-sectional plane through the entire length of the
mouse.
A thrilling MSOT imaging potential for the field of tumor biology research is to
contemporarily multiplex two or more exogenous probes with differing spectra
in the near infrared (NIR) range together with the endogenous absorbers.
In this study we aimed to quantitatively define the spectral unmixing accuracy
of MSOT with known concentrations of dyes with distinct spectra.
Materials and Methods: The NIR probes used in this study included DY-704,
DY-750, DY-831 and IRDye800.
These were first imaged in the MSOT inVision 256-TF tomographic system
(iThera Medical GmbH) using agar scattering phantoms. In vivo work was
carried out using CD1 nude mice injected though the tail vein with known
amounts of an individual dye or with multiple dyes.
Results: In the first part of the study, we performed highly repeatable and
controlled imaging in phantoms on serial dilutions of NIR dyes placed alone or
in combination with other NIR dyes having close or distant absorbance peaks.
This allowed for the determination in optimally defined conditions of the lowest
limit of detection of each dye alone and in combination with other probes.
In the second part of the study we used a selection of the above probes
to define the accuracy of the unmixing process for multiple exogenous dyes
in vivo. For this purpose mice were injected with one probe only through the
tail vein while acquisition was taking place and the pharmacokinetic properties
of each probe was measured with high temporal resolution over time in defined
vessels, in the kidneys, the liver and the spleen. Finally, combinations of
probes with similar or different excretion routes where injected together and
the accuracy of the MSP signal in these defined conditions was determined
quantitatively in the relevant anatomical regions.
Conclusions: Multiplexing contrast agents has long been an advantage of
optical imaging. In the current study, the ability of MSOT to simultaneously
detect up to four chromophores in vivo with distinct absorption spectra is
also demonstrated. Sensitivity studies further demonstrate that multispectral
unmixing allows an investigator to maintain high sensitivity despite a
background of other absorbers. Based on this study in vivo deep live tissue
multiplexing can significantly contribute to pave the way to new research
opportunities in the field of cancer research involving anatomical, functional
and molecular imaging.
Conflict of interest: Ownership: iThera Medical.
203 The ethanolic extract of Phellinus linteus inhibits breast cancer
cell growth through autophagy-related cell death
W. Lee1 , T. Chiang2 . 1 Chi Mei Medical Center, Department of Pathology,
Tainan, Taiwan, 2 Chung Hwa University of Medical Technology, Department
of Medical Laboratory Science and Biotechnology, Tainan, Taiwan
Background: Phellinus linteus (PL), a medicinal mushroom in traditional Oriental medicine, has the most potent anti-cancer effect among basidiomycetes.
PL has been shown to have anti-cancer effects in vitro and in vivo, but the
underlying mechanism remained to be elucidated. The aim of the present
study was to evaluate the inhibitory effects of the ethanolic extract from the
PL fruiting bodies on the highly invasive andmetastatic human breast cancer
cell line MDA-MB-231 and to determine the mechanism of cell death.
Material and Method: Cell viability was determined by measuring
modifiedtetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTS) dye absorbance of living cells. Potential cell death mechanisms
in cells treated with PL extract (30 mg/mL) for 24 h were investigated in
apoptosis and autophagy.
Results and Discussion: The ethanolic extract of PL significantly inhibited
the proliferation of MDA-MB-231 cells in a dose-dependent manner, with an
IC50 value of 40 mg/mL. PL did not induce apoptosis, as demonstrated by the
DNA fragmentation assay, the sub-G1 population, and staining with annexin
V-FITCand PI. The exposure of the breast cancer cells to PL extracts resulted
in several confirmed characteristics of autophagy, including the appearance of
autophagic vacuoles revealed by monodansylcadaverine (MDC) staining, the
formation of acidic vesicular organelles (AVOs), autophagosome membrane
association of microtubule-associated protein lightchain 3 (LC3) characterized
by cleavage of LC3 and its punctuatere distribution, and ultrastructural
observation of autophagic vacuoles by transmission electron microscopy.
Traditional cancer therapies primarily enhance apoptosis. However, itis wellestablished that many cancers are chemo-resistant and defective inapoptosis
induction. Recent studies have reported that various anti-cancer therapies,
including natural products, can inhibit tumor growth through autophagy
induction. Taken together, these results provide a new perspective on the
exploitation of additional natural compounds as potential novel anti-cancer
drugs by targeting the autophagy pathways.
Conclusion: We report for the first time that PL ethanolic extracts inhibited
MDA-MB-231 metastatic and triple-negative breast cancer cells proliferation
by inducing autophagy-related cell death. Our results suggest a new potential
therapeutic strategy involving the use of PL to induce autophagy-related cell
death in apoptosis-defective cancer cells and triple-negative breast cancer
cells.
204 Pre-mRNA alternative splicing controls VEGF-A autocrine functions
and response to bevacizumab in non small cell lung carcinoma
A. Boudria1 , S. Gout1 , M. Keramidas2 , J.L. Coll2 , S. Lantuejoul1 ,
E. Brambilla1 , S. Gazzeri1 , B. Eymin1 . 1 INSERM U823, Team 2 Molecular
Basis of Lung Cancer Progression, Grenoble Cedex 09, France, 2 INSERM
U823, Team 5 Targeted Therapies and Vectorisation, Grenoble Cedex 09,
France
Background: VEGF-A is highly subjected to pre-mRNA alternative splicing,
that generates several splice variants with both pro- (VEGFxxx ) and
anti- (VEGFxxx b) angiogenic properties. Owing to its crucial role during tumor
progression, therapies targeting VEGF-A have been developped, among them
the humanized anti-VEGF-A monoclonal antibody Bevacizumab (AvastinR ).
However, clinical results on overall survival are disappointing as some patients
do not respond or acquire resistance. Recent studies have highlighted a
potential role of VEGF-A splice isoforms in tumor response to Bevacizumab
treatment. As we previously demonstrated that overexpression of the splice
variant VEGF165 b is associated with reduced angiogenesis in nude mice
xenografts derived from lung adenocarcinoma cell lines (Merdzhanova et al.,
Oncogene, 2010), we further investigated the status and the biological
functions of VEGF165 b and VEGF165 splice variants in primary and cellular
models of Non Small Cell lung Carcinoma (NSCLC).
Material and Methods: VEGF-Atotal and VEGF165 b expression was analyzed
in situ by immunohistochemistry in a series of 16 normal lung parenchyma
and 74 NSCLCs including 38 adenocarcinomas and 36 squamous cell
lung carcinomas. Various cellular models derived from lung adenocarcinoma
and expressing detectable amounts of VEGFR1, VEGFR2, neuropilin-1
and neuropilin-2 proteins were used to analyze the effects of rhVEGF165
or rhVEGF165 b recombinant ligands on cellular proliferation, apoptosis
and/or migration. Activation of VEGFR1/2-dependent signaling pathways was
analyzed by western blotting. In vivo experiments in nude mice were performed
according to ethical procedures.
Results: We provide the first evidence that NSCLC patients exhibit various
pattern of VEGF165 b tumor expression and that high levels of VEGF165 b
correlate with lymph node metastasis. At the molecular level, we identify
a VEGFR1/VEGFR2-activated autocrine loop by which VEGF165 b triggers
a more invasive phenotype in NSCLC cells. Consistently, VEGF165 b and
P-VEGFR1 (Tyr1213) expression levels are significantly associated in NSCLC
(p = 0.0061). Furthermore, we show that VEGF165 inhibits this VEGFRdependent autocrine loop and triggers apoptosis of NSCLC cells, unraveling
an unexpected anti-invasive function of this specific isoform in NSCLC.
Finally, we demonstrate that bevacizumab treatment combined or not with
chemotherapy increases VEGF165 b expression, leading to activation of the
VEGFR-dependent autocrine pathway.
Conclusions: It is known since one decade that VEGF-A pre-mRNA
alternative splicing generates splice isoforms with antagonist activities on
endothelial cells. Here, we demonstrate that VEGF-A pre-mRNA alternative
splicing also controls VEGF-A autocrine functions on tumor cells in an opposite
manner and more importantly is regulated by anti-angiogenic therapies.
206 GDF15 knockdown induces resistance to temozolomide treatment
in glioblastoma cell line
M. Baroni1 , P.F. Fedatto2 , A.F. Andrade3 , V.K. Suazo2 , R.G.P. Queiroz2 ,
L.G. Tone2 , C.A. Scrideli2 . 1 Medical School of Ribeirão Preto-USP, Clinical
Oncology Stem Cells and Cell Therapy, Ribeirão Preto − SP, Brazil, 2 Medical
School of Ribeirão Preto-USP, Pediatrics, Ribeirão Preto − SP, Brazil,
3
Medical School of Ribeirão Preto-USP, Genetics, Ribeirão Preto − SP,
Brazil
Background: Glioblastoma (GBM) is one of the most frequent and malignant
human tumors. GBM displays high heterogeneity with complex genetic
alterations, characterized by resistance to traditional treatments such as
radiotherapy and treatment with the drug Temozolomide (TMZ). Among the
genetic changes, our group observed a high GDF15 expression in GBM
primary tumor samples and cell lines. The GDF15 is a growth factor that
in normal physiological conditions is poorly expressed, but in cases of
inflammation and malignancies your expression is increased. This study aims
to correlate GDF15 knockdown with sensitivity to TMZ treatment in child and
adult cell lines of GBM.
Material and Methods: To examine these correlations, first was analyzed
the GDF15 expression of nine GBM cells lines: U87, T98G, U251, U138,
LN319, MO59K, U343, KNS42 and SF188, by qRT-PCR. For this analysis, it
was used Taqman gene probes for GDF15 and endogenous controls, TBP
and HPRT . For silencing assays, lentiviral shRNA construct for GDF15 and
lentiviral control shRNA in pLKO vector were used. The expression reduction of
GDF15 gene and protein was confirmed respectively by qRT-PCR and Western
Blotting. For cellular proliferation analysis, we realized Resazurin assay in
96 wells plate, in quadruplicate samples in three independent experiments.
Cells were treated with different doses of TMZ (250, 500, 1000 and 1500mM)
and times (24−96 h). One-ANOVA and Bonferoni post-hoc was performed for
statistical analysis with the Package SPSS Statistics 20.0.
Results: Eight from nine GBM cell lines showed GDF15 expression 2.5 times
higher than white matter human controls. The cell lines with highest
expression, when compared to control, was the adult cell line U343 and
the pediatric cell line KNS42, showing 22 and 194 times more expression
of GDF15, respectively. In these two cell lines, the GDF15 was silenced by
shRNA. The efficiency of gene silencing was confirmed by protein levels,
decreasing 56% for KNS42 and 73% for U343. Cell proliferation assay was
performed with GDF15 knockdown cells treated with TMZ. The KNS42 GDF15
silenced cells did not show difference when compared to control cells at all
doses of TMZ and times. It was observed that U343 GDF15 knockdown cells
were more resistant to TMZ treatment. The strongest effect was after 72 hours
at the doses of 250 and 500mM of TMZ (p < 0.05).
Conclusion: This study showed that GDF15 knockdown does not sensitize
pediatric GBM cell line to TMZ while for adult cell line the gene silencing
induces resistance to treatment. To better understand GDF15 correlation with
cell sensitization, further studies will be conducted.
207 A tea polyphenol epigallocatechin gallate (EGCG) displays
a superior effect on enzyme inhibition of human ornithine
decarboxylase
H.C. Hung1 , L. Pan1 . 1 National Chung Hsing University, Department of Life
Sciences, Taichung city, Taiwan
Introduction: Ornithine decarboxylase (ODC) catalyzes the decarboxylation
of ornithine to putrescine and is the rate-limiting enzyme inthe polyamine
biosynthesis pathway. Because ODC activity and the cellular levels of
polyamine are crucial for cell proliferation and are critical for the beginning and
progression of neoplastic diseases, ODChas been recognized as an oncogenic
S47
enzyme. Therefore, ODC inhibitors and negativeregulators of the polyamine
pathway could be beneficial in the treatment of many cancers.
Material and Methods: In the present study, the effects of green tea
polyphenols (catechin, gallic acid EGCG, GCG, ECG and EGC), on ODC
enzyme inhibition were examined. In addition, the size-distribution analysis
measured by analytical ultracentrifugation was performed to examine the
possible change in quaternary structure of ODC in the presence of these
tea catechins.
Results and Discussion: The inhibition studies indicated that EGCG is most
effective to inhibit ODC enzyme activity, GCG and EGC showed secondly
and thirdly, respectively. The IC50 values for these three catechins were 10.2,
30.1 and 66.3 mM, respectively. The other catechins, catechin, gallic acid and
ECG, show little or no enzyme inhibition, indicating this inhibitory effect is
structure-specific. The size distribution analysis showed that EGCG induced
ODC polymerization, especially to form a double dimer. Because ODC is a
dimeric enzyme, and the dimeric structure is essential for ODC enzyme activity.
The inhibitory effect of EGCG may due to its binding toward the dimerinterface
of ODC and then to induce the formation of the double-dimers.
Conclusion: This study provides information to better understand the
molecular mechanisms underlying the biological activities of catechins and
to design new polyphenol-based ODC-specific inhibitors.
208 GALNT1 knockdown suppresses hepatocellular carcinoma
cell malignant behaviours and decreases EGFR activation and
recycling
M. Huang1 , Y.M. Wu2 , M.C. Huang1 . 1 National Taiwan University College of
Medicine, Graduate Institute of Anatomy and Cell Biology, Taipei City, Taiwan,
2
National Taiwan University Hospital, Department of Surgery, Taipei City,
Taiwan
Introduction: N-acetylgalactosaminyltransferase (GALNT) 1 is one of the 20
GALNT isoenzymes that initiate the biosynthesis of mucin-type O-glycosylation
producing GalNAca1-O-serine/threonine, also known as cancer-associated
Tn-antigens, that is fundamental for further complex O-glycan formation.
GALNT1 is the major GALNT enzyme expressed in liver cancer. Very few
studies have reported the function of GALNT1 in cancer, in particular,
hepatocellular carcinoma (HCC), a highly lethal cancer that is ranked the third
leading cancer-caused death worldwide. This study aims to investigate the
role of GALNT1 mediated O-glycosylation in HCC pathogenesis as the basis
for potential therapeutic drug development.
Material and Method: Data retrieved from public microarray database and
real-time RT-PCR quantification of GALNT1 of 140 HCC tumours collected
from the National Taiwan University Hospital (NTUH) were statistically
analysed. This study was approved by the NTUH Ethics Committee and patient
informed consents were obtained prior to collection. Western blotting was used
for protein expression and signal transduction analysis. siRNA knockdown
and GALNT1/pcDNA3.1A overexpression of GALNT1 in PLC5 and HA22T
cells were subjected to transwell migration and matrigel invasion assays using
growth factors, including, EGF, FGF, HGF, IGF, PDGF, TGFb, and VEGF as
chemoattractants. Immunoprecipitation and Vicia villosa (VVA) lectin pull down
assays were used to determine the Tn-antigen expression on EGFR. EGFR
recycling was analysed by immunofluorescence microscopy.
Results and Discussion: Public microarray data indicate that GALNT1 is
frequently up-regulated in HCC compared with normal liver tissues. Our
data show that GALNT1 mRNA and protein expression is predominantly
up-regulated in paired HCC tumours and higher GALNT1 expression is
associated with poorer overall survival. GALNT1 knockdown suppressed while
overexpression enhanced PLC5 and HA22T cell migration and invasion.
GALNT1 knockdown most significantly suppressed EGF-induced cell migration
and invasion. GALNT1 knockdown reduced EGFR O-glycan expression and
EGF-induced EGFR activation. GALNT1 knockdown facilitated EGF-induced
EGFR degradation and hindered EGFR recycling.
Conclusion: GALNT1 is frequently up-regulated in HCC and in vitro studies
show that knockdown of GALNT1 suppresses HCC cell malignant behaviours
by decreasing EGFR O-glycosylation, which in turn reduces EGF-induced
EGFR activation and recycling. In conclusion, GALNT1 plays an important
role in HCC pathogenesis rendering it a potential as a target for therapeutic
drug development.
209 K06, a novel CD43 epitope on human thymocytes, is involved
in caspase-independent cell death
T.J. Kim1 , Y. Bae2 . 1 Sungkyunkwan University School of Medicine,
Immunology, Suwon, Korea, 2 Seoul National University College of Medicine,
Parasitology and Tropical Medicine, Seoul, Korea
Background: The K06 antigen is a 120 kDa molecule which is highly
expressed on the double positive thymocytes. We present immunological and
biological evidence demonstrating that K06 is a variant of CD43. The functional
S48
roles of cell surface receptors in T-cell signal transduction and apoptosis have
been studied via the cross-linking of monoclonal antibodies. The aim of this
study is to investigate the mechanism of apoptosis in the Molt-4 T-cells by
CD43 cross-linking with K06 monoclonal antibody.
Materials and Methods: Human leukemic cell lines, Jurkat and Molt4, were
cultured and used for understanding the role of CD43 antigen in apoptosis
by using K06 mAb. The previously developed novel anti-CD43 monoclonal
antibody K06 was treated in Molt-4 and Jurkat human leukemic T-cells.
Apoptosis was detected by flow cytometry.
Results: K06-CD43 expression pattern and biochemical characteristics
suggest its participation in carbohydrate-based interactions in the thymus.
We used specific Ab to mimic putative K06 interactions with natural ligand
and examined the effect on isolated human T cell line, Molt4. Treatment with
K06 had effect on apoptosis of Molt4 cells. Thses data strongly suggest
that engagement of K06 delivers a positive signal that favors apoptosis.
The cell death by K06 is caspase-independent, and bcl-2 is involved in the
pathway of K06-signaling cell death signal. The apoptosis in Molt-4 T-cells
showed several characteristic features of apoptosis, including the exposure of
phosphatidylserine residues (Annexin V staining) with or without incorporation
of vital dye (7-AAD staining), a reduction in mitochondrial transmembrane
potential, Dym, (DiOC6 staining), and a lack of internucleosomal fragmentation
of DNA (DNA laddering and immunoblotting). Apoptosis occurred in a caspaseindependent pathway.
Conclusions: This result suggests that apoptotic signals can be induced via
the specific epitope of CD43 in the specific leukemic cell line.
210 Effect of sesamin, sesamolin and sesamol on P-glycoprotein
mediated efflux
C. Junhom1 , B. Siriwarin2 , N. Weerapreeyakul3 , S. Barusrux4 . 1 Khon
Kaen University, Biomedical Science Program Graduate School Faculty
of Pharmaceutical Sciences Khon Kaen University, Khonkaen, Thailand,
2
Khon Kaen University, Research and Development in Pharmaceuticals
Program Graduate School Faculty of Pharmaceutical Sciences Khon Kaen
University, Khonkaen, Thailand, 3 Khon Kaen University, Center for Research
and Development of Herbal Health Products (CRD-HHP) and Faculty of
Pharmaceutical Sciences Khon Kaen University, Khonkaen, Thailand,
4
Khon Kaen University, Centre for Research and Development of Medical
Diagnostic Laboratories and Faculty of Associate Medical Sciences Khon
Kaen University, Khonkaen, Thailand
Introduction: Mechanisms of resistance to chemotherapeutic drugs are
important causes of cancer treatment failure and the P-gp efflux protein is
an important determinant in intrinsic or acquired resistance of cancer cells.
Various studies have been conducted to search for P-gp modulators that
can enhance accumulation of chemotherapeutic drugs in resistant cancer
cells and lessen the severity of their side effects. Our interest is in sesame
seeds (Sesamum indicum) which are consumed worldwide and contain the
bioactive lignans; sesamin, sesamolin and sesamol. Sesamin has previously
been reported to act as a P-gp inhibitor, however, the effects of sesamol and
sesamolin on P-gp have not been investigated. Here we report the findings of
a comparative study into the effect of these bioactive sesame lignans on P-gp
efflux activity.
Materials and Methods: The HepG2 hepatocellular carcinoma cell line was
induced to become a resistant cell subline (R-HepG2) by stepwise exposure
to cisplatin. Evaluation of P-gp activity was then performed using a method
based on accumulation of Rhodamine123 and the cytotoxicity of the sesame
lignans in the R-HepG2 subline was determined by neutral red assay.
Results and Discussion: The resistant index (RI), calculated from the
cytotoxic IC50 values of the resistant cells (10.9±0.2 mg/ml) versus the
parental cells (4.7±0.2 mg/ml), was 2.3. The three sesame lignans increased
accumulation of Rhodamine123 in R-HepG2 cells 1.6−1.8 fold when compared
to untreated cells. Verapamil, a positive control P-gp inhibitor, exerted less P-gp
inhibition than the three sesame lignans (1.4 fold). Sesamolin exhibited higher
inhibition of P-gp than sesamin and sesamol, respectively. All three sesame
lignans showed no cytotoxicity in either the parental HepG2 cell line or the
R-HepG2 cell subline at the concentrations tested.
Conclusion: This study suggests that the three bioactive sesame lignans
may be useful for overcoming P-gp-mediated multidrug resistance. Therefore,
sesame seed consumption may be beneficial for cancer patients undergoing
chemotherapy. However, more detailed studies of these sesame lignans on
efflux modulation mechanisms in other cancer cell lines are still required.
211 Co-evolution of stroma and neoplastic cells in prostate cancer
is sustained by reprogramming energy metabolism
M. Bologna1 , P. Sanita’2 , A. Angelucci2 , P. Muzi1 , C. Vicentini1 . 1 University
of L’Aquila, Department of Medicine Health and Environment, L’Aquila, Italy,
2
University of L’Aquila, Department of Biotechnological and Applied Clinical
Sciences, L’Aquila, Italy
Introduction: Cancer growth requires altered energy metabolism in order to
sustain the continuous proliferation of cancer cells in oxygen- and nutrientdeprived environment. Altered metabolic profiles involve not only cancer cells
but also stromal associated cells which can be conditioned by metabolites
secreted from cancer cells. One of such metabolic cross-talk systems is based
upon the exchange of monocarboxylic acids whose trafficking through cell
membrane is regulated by monocarboxylate transporters (MCTs). MCT1 and
MCT4 are the best characterized MCTs permitting, respectively, the import
and the export of different monocarboxylic acids, including lactic acid.
Materials and Method: Mitogenic role of MCTs was investigated in vitro
and in vivo using transformed prostate epithelial cells, carcinoma cell
lines and diploid fibroblasts. Prostate tissues from carcinoma and benign
hypertrophy clinical cases were analyzed in order to evaluate clinicalpathological significance of MCT1 and MCT4 expression.
Results and Discussion: MCT1 and MCT4 were detected in prostate
carcinoma (PCa) and transformed prostate epithelial cell lines with MCT4
expression that correlated with aerobic glycolytic metabolism. Exogenous
lactate sustained cell growth in low glucose culture environment and blockade
of MCT1 function performed by silencing via siRNA determined an appreciable
antiproliferative effect when lactate was utilized as energetic fuel. In vitro coculture with prostate cancer cells induces glycolytic metabolism in cancer
associated fibroblasts. MCT1 silencing determined the reduction of tumor
growth in a xenograft model with co-injection of PCa cells together with
high glycolytic human fibroblasts. In non-neoplastic prostate tissue, MCTs
demonstrated a specific distribution, with MCT1 expressed in epithelial cells
and MCT4 localized in stromal cells. In PCa tissue we observed a significant
upregulation of MCT4 in tumoral tissue respect to hypertrophic tissue, with a
positive correlation between stromal MCT4 and tumor MCT1 staining.
Conclusion: Our data demonstrated that PCa progression may benefit of
MCT1 expression in presence of high lactate and low glucose concentration.
A collaborative interaction between PCa cells and prostate stromal cells may
exist based upon lactate shuttle, a phenomenon known as reverse Warburg
effect. Therefore, MCTs may represent a promising therapeutic target in early
phases of neoplastic transformation according to a strategy aimed at interfering
with the energy metabolic adaptation of PCa cells.
212 WIP, a novel negative regulator in WBP2-mediated Wnt pathway
activation
S. Lim1 , Y.P. Lim1 . 1 National University of Singapore, Biochemistry,
Singapore, Singapore
Background: Previous phosphoproteomic analysis of a xenograft-derived,
isogenic cell line model identified WBP2 as a novel EGFR tyrosine kinase
substrate that is associated with breast cancer development. Subsequent
in vitro and in vivo functional studies of WBP2 implicated its potential role
as an oncogene in ER+ breast cancer via ER-Wnt pathway crosstalk. To
understand more about its function and regulation, we aim to identify new
interacting partners of WBP2.
Material and Methods: In the present study, we had employed both the
yeast-two-hybrid cDNA library screening and interactomics mass spectrometry
approach to identify potential novel interacting proteins of WBP2 with
regulatory functions.
Results: WIP (WBP2 Interacting Protein) was discovered as the top hit
among the candidates from both approaches and subsequently validated
and characterized as the novel interacting partner of WBP2. Interestingly,
WIP acts as a negative regulator of WBP2 and tyrosine phosphorylation on
WBP2 also regulates the WBP2–WIP interaction. WIP significantly abolished
WBP2-mediated Wnt pathway activation and plays a role in modulating the
sensitivity of cancer cell to Wnt inhibitor. Mechanistic studies on the role of Wnt
stimulation on WBP2–WIP interaction as well as WBP2 and WIP subcellular
localization will be presented.
Conclusions: WIP emerges as a new player in the negative regulation of
WBP2-mediated Wnt pathway regulatory network.
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213 The direct cytotoxic and P-glycoprotein modulating effects of
Thai indigenous plant extracts in drug resistant HepG2 cells
215 Activated B cell receptor signaling pathway contributes to
bortezomib resistance in mantle cell lymphoma
C. Junhom1 , N. Weerapreeyakul2 , S. Barusrux3 , T. Titimatharoj2 . 1 Khon
Kaen University, Ga Biomedical Science Program Graduate School Faculty
of Pharmaceutical Sciences Khon Kaen University, Khonkaen, Thailand,
2
Khon Kaen University, Center for Research and Development of Herbal
Health Products (CRD-HHP) and Faculty of Pharmaceutical Sciences Khon
Kaen University, Khonkaen, Thailand, 3 Khon Kaen University, Centre for
Research and Development of Medical Diagnostic Laboratories and Faculty
of Associate Medical Sciences Khon Kaen University, Khonkaen, Thailand
A. Kim1 , S. Park2 , D. Shin3 , W. Jang4 , S. Lee4 , S. Lee2 . 1 Korea Institute
of Radiological & Medical Science, Seoul, Korea, 2 Korea Institute of
Radiological & Medical Science, Pathology, Seoul, Korea, 3 Korea Institute
of Radiological & Medical Science, Hematooncology, Seoul, Korea, 4 Korea
Institute of Radiological & Medical Science, Experimental Pathology, Seoul,
Korea
Introduction: Many studies have reported medicinal plants as a source of
natural compounds useful for killing drug resistant cancer cells. We screened
fourteen extracts from eleven Thai indigenous plants for direct cytotoxic activity
and indirect cytotoxic effects based on the reversal of P-glycoprotein efflux
pump function in an induced drug resistant hepatocellular carcinoma HepG2
cell subline.
Materials and Methods: The plants and extracts investigated in this study
were Cratoxylum formosum (C. formosum, twig), Polyalthia evecta (P. evecta,
leaf), Bombax anceps (twig), Catunaregam tomentosa (twig), Diospyros
castanea (twig and leaf), Lannea coromandelica (twig), Erythrophleum
succirubrum (twig), Tetracera loureirii (twig), Cladogynos orientalis (twig),
Amomum villosum var. xanthioides (leaf and rhizome), and Acorus tatarinowii
(leaf and rhizome). The resistant HepG2 subline was induced by exposure to
cisplatin via the intermittent method.
Results and discussion: The resistant index was 2.3, calculated from the
cytotoxic IC50 values of the resistant cells (4.7±0.2 mg/ml) versus the parental
cells (10.88±0.2 mg/ml). Direct cytotoxic activity was determined using the
neutral red assay. The inhibition of P-gp function was determined by using flow
cytometry to measure the accumulation of Rhodamine123. Among the plant
extracts studied, C. formosum showed the highest cytotoxicity in the resistant
subline with an IC50 value of 311.3±11 mg/ml and extracts from P. evecta and
C. formosum increased rhodamine123 uptake into the resistant HepG2 subline
(2 and 1.7 fold, respectively, compared to untreated resistant cells).
Conclusion: Both the P. evecta and C. formosum extracts show promise
as enhancers for anticancer drugs by limiting drug efflux, however, as the
C. formosum extract exhibited an additional cytotoxic effect, the C. formosum
extract is the most potential anticancer candidate. These results warrant further
investigation into the mechanism of the anticancer activity of the C. formosum
extract.
214 The deubiquitinating enzyme USP15 deubiquitinates the E3 ligase
SMURF2
P. Iyengar1 , P. Jaynes1 , P. Eichhorn1 . 1 Cancer Science Institute, National
University of Singapore, Singapore, Singapore
Introduction: Ubiquitin modification of TGFb pathway components is emerging
as a key mechanism of TGFb pathway regulation. All together, 5 different
deubiquitinating enzymes have been identified to regulate the TGFb pathway
with 4 different deubiquitinating enzymes targeting the TGFb receptor alone.
We therefore sought to determine the individual roles of these deubiquitinating
enzymes in TGFb receptor kinetics.
Materials and Methods: Using shRNA and CRISPR technology we
investigated the roles of deubiquitinating enzymes targeting the TGFb
receptor.
Results: We find that while USP4, USP11, and UCH37 appear to directly
deubiquitinate the TGFb receptor, USP15 directly targets the E3 ligase
SMURF2 resulting in decreased TGFb receptor ubiquitination. USP15
deubiquitinates SMURF2 in vivo and in vitro resulting in reduced SMURF2
activity and TGFb receptor ubiquitination. Furthermore, we demonstrate
that USP15 deubiquitinates multiple lysine residues on SMURF2 leading to
deregulation of overall SMURF2 ligase activity.
Conclusion: It has recently been shown that USP15 plays a dual role in
the TGFb pathway. First, USP15 opposes R-SMAD monoubiquitylation and
SMURF mediated polyubiquitination, permitting SMAD promoter recognition.
Secondly, USP15 binds to the SMAD7-SMURF2-TGFb receptor complex
regulating TGFb receptor ubiquitination and stability. Our results suggest that
SMURF2 is a critical target of USP15 and may explain how USP15 targets
multiple nodes in the TGFb pathway.
Background: Although proteasome inhibition with bortezomib (BTZ) is a
validated treatment for relapsed or refractory mantle cell lymphoma (MCL)
many patients show initial or acquired resistance to BTZ. However, the
molecular mechanism of BTZ resistance in MCL has not been elucidated.
In the present study, we examined BTZ-resistant MCL cells in vitro and
in vivo to investigate the mechanism of BTZ resistance. Activated B-cell
receptor (BCR) signaling plays a key pathogenetic role in B-cell malignancies
and has recently been investigated in several lymphoma subtypes. Here, we
demonstrate that BTZ-resistant MCL cells showed increased expression of
BCR signaling components with overexpression of CD79A and CD79B.
Material and Methods: We used MCL cell lines Jeko1, Mino, and Rec1 and
stable BTZ-resistant cell lines were designated Jeko1/BTZ and Mino/BTZ. Cell
viability after CD79A, Src or Lyn silencing was examined in MCL cell lines by
MTT assay. Expression of SFKs after Src and Lyn silencing in MCL cells
was examined by Western blotting. Therapeutic effect of in vivo dasatinib was
examined using SCID mouse.
Results: Activation of the BCR signaling pathway enhanced the activity of
Src family kinases (SFKs) and downstream kinases PI3K/AKT/mTOR in BTZresistant MCL cells. Cell proliferation was enhanced in BTZ-resistant MCL cells
whereas suppression of SFKs by a kinase inhibitor PP2 significantly reduced
tyrosine kinase activity leading to inhibition of proliferation. Moreover, BTZresistant MCL cells were sensitive to silencing of BCR components, indicating
that activated BCR signaling is an important pathway in these cells. The SFK
inhibitor dasatinib had a growth inhibitory effect in BTZ-resistant cells in vitro
and in vivo through suppression of SFKs and PI3K/Akt/mTOR.
Conclusions: Collectively, our data show that overexpression of BCR and its
induced kinases confer BTZ resistance in MCL and suggest that BCR signaling
could be a novel therapeutic target for patients with MCL that has relapsed or
is refractory to treatment with bortezomib.
216 Role of extracellular S100A4 in stimulation of melanoma cells
crossing the blood–brain barrier in vitro and in vivo
N. Herwig1 , S. Wolf1 , C. Haase-Kohn1 , J. Steinbach1 , J. Pietzsch1 .
1
Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical
Cancer Research, Dresden, Germany
Background: Brain metastasis predominantly causes death in melanoma
patients, but the underlying mechanisms are poorly understood. In this
regard, the S100 protein family is increasingly gaining interest as a player in
melanoma metastasis. One member of this family, S100A4, is overexpressed
and oversecreted in melanoma. We hypothesized that extracellular S100A4
promotes brain metastasis through interaction with the receptor for advanced
glycation endproducts (RAGE) by affecting endothelial cell integrity.
Material and Methods: In vitro investigations were performed in a blood–
brain barrier model using human endothelial hCMEC/D3 cells on collagencoated transwell inserts. Cell integrity was studied by transendothelial electrical
resistance (TEER) measurement and detection of expression levels of tight
junction proteins occludin and claudin-5, and, additionally, VE-cadherin by
Western blotting and immunofluorescence staining. Transmigration of human
A375 melanoma cells overexpressing S100A4 and RAGE, respectively, and
coexpressing GFP was studied compared to A375 wild type cells. Influence of
S100A4 was investigated by addition of either human recombinant S100A4 or
soluble RAGE (sRAGE) into cell culture supernatants. In an in vivo pilot-study,
A375 cells (wild type and transgenic) were intracardially injected into NMRInu/nu mice and metastases were analyzed by histological analysis and optical
imaging.
Results: Incubation with S100A4 decreased expression of occludin and VEcadherin revealing a loss of endothelial cell integrity. In contrast, expression
of claudin-5 and TEER were unaffected. However, TEER decreased after coculture with A375 cells. Transmigration of S100A4- or RAGE-overexpressing
A375 cells was significantly increased compared to A375 wild type cells, but
was not further affected by addition of S100A4 or sRAGE. Interestingly, mice
injected with S100A4- or RAGE-overexpressing A375 cells showed a higher
incidence of metastases and lower survival rates compared to A375 wild
type cells with primarily exhibiting bone and ovarian metastases, respectively.
However, brain metastases were only rarely observed.
Conclusion: The findings provide first information on the involvement of
interaction of melanoma-derived S100A4 with endothelial RAGE in melanoma
metastasis.
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217 The identification of novel targets in b-catenin-driven acute
myeloid leukemic stem cells
H. Yi1 , J. Wang2 , M. Kavallaris1 , J.Y. Wang3 . 1 Children’s Cancer Institute
Australia for Medical Research, Sydney NSW, Australia, 2 Black Family Stem
Cell Institute, Mount Sinai School of Medicine, New York, USA, 3 School of
Women’s and Children’s Health, Faculty of Medicine University of New South
Wales, Sydney NSW, Australia
Introduction: Leukemic stem cells (LSCs) are a small population of stem-like
cancer cells that are believed to be responsible for the high rate of patient
relapse in acute myeloid leukemia (AML). Although aberrant Wnt/b-catenin
signaling has recently been shown to be required for the development of LSCs
in AML, key components of the pathway remain elusive.
Material and Methods: Hematopoietic stem cells were transduced with MLLAF9 or Hoxa9/Meis1a to generate pre-LSCs. Stable knockdown was achieved
by lentiviral vector system to express short hairpin RNAs. Tumorigenic
potential was assessed in vitro (by methylcellulose colony forming assays) and
in vivo (by transplantations into syngeneic C57BL/6 mice). Differential gene
expressions were assessed by microarray analysis, and target genes were
confirmed by Western blot. Other techniques such as Wright Giemsa staining
and apoptosis assay were used to examine changes in cell phenotypes.
Results and Discussion: Gene expression analysis of MLL-rearranged
AML patient samples revealed a 3-fold increase in expressions of Lgr4 and
Ctnnb1 compared to normal hematopoietic stem cells. Here we demonstrated
that depletion of Lgr4 impaired pre-LSC growth and significantly prolonged
mouse survival through inhibition of b-catenin expression. Furthermore,
overexpression of Lgr4 promoted cell growth and accelerated leukemia onset.
Differential gene expressions by microarray analysis identified groups of Wnt
target genes and genes that regulate G-protein signaling. We showed that
inhibition of Gaq by a Gaq specific inhibitor caused marked reduction of colony
forming capacity as well as increased differentiation and apoptosis of preLSCs.
Conclusion: Our results suggest Lgr4 and Gaq as potential therapeutic targets
for elimination of b-catenin-driven LSCs in AML.
218 The role of WWOX gene in EMT process in endometrial cancer
E. Pluciennik1 , M. Nowakowska1 , K. Pospiech1 , K. Kosla1 , I. Baryla1 ,
K. Wojcik-Krowiranda2 , A. Bienkiewicz2 , M. Galdyszynska3 , M. Popeda3 ,
A.K. Bednarek1 . 1 Medical University of Lodz, Department of Molecular
Cancerogenesis, Lodz, Poland, 2 Medical University of Lodz, Clinical Division
of Gynecological Oncology, Lodz, Poland, 3 Medical University of Lodz,
Faculty of of Biomedical Sciences and Postgraduate Education, Lodz,
Poland
Background: In Poland endometrial cancer is on the first place in morbidity
statistics and the third in mortality within reproductive track-related cancers
among women. WWOX is a newly described tumour suppressor gene which
is affected in many types of tumor including steroid hormones regulated such
as breast, ovary, prostate cancer. The EMT process is also of importance
in this cancer type with unknown role of WWOX in its regulation. The aim
of this study was to precise the role of WWOX in regulation of epithelial to
mezenchymal transition in endometrial cancer.
Material and Methods: This study was performed on series of 164 endometrial adenocarcinoma patients and well-differentiated steroid-responsive
endometrial cell line − ECC1, which was transducted cDNA WWOX by
retroviral system. We evaluated correlation between expression of WWOX
gene and EMT markers (CDH1, VIM, ZEB1, SNAI1) on mRNA (real-time
RT-PCR) and protein level (Western Blot). The EMT process were also
analysed in in vitro model with biological assays such as adhesion of
cells to six of extracellular matrix proteins, migration through basement
membrane, anchorage-independent growth (soft agar assay), activity of
MMPs (zymography test). Using DNA microarrays (HumanOneArray™;) we
determined WWOX dependent pathways in ECC1 cell line.
Results: In patients positive correlation between WWOX and ZEB1 (Rs = 0.26;
p = 0.00037), and negative correlation with CDH1 and VIM (Rs = −0.5118,
p = 0.0001; Rs = −0.2687, p = 0.0005, respectively) was observed. We also
noticed that expression of WWOX inversely correlated with the risk of
recurrence, i.e. tumors with low risk had higher WWOX expression, than those
with medium and high risk, with the statistically significant difference between
low and medium risk of disease recurrence. The negative correlation with VIM
and positive with CDH1 was confirmed on protein/mRNA level in transfected
cancer cell line. ECC1 cell line with overexpression of WWOX protein revealed
increased migratory capacity, with increased expression of metalloproteinases
MMP2/MMP9. On the other hand, those cells were not able to form colonies in
suspension, which indicate that they loose the aggressive potential. Moreover,
this transductants revealed tendency to decrease adhesion to fibronectin and
statistically significant decrease in adhesion to fibrinogen. Microarray analysis
demonstrated that WWOX has impact on variety of cellular pathway including
cadherin and integrin signalling pathways.
Conclusion: Our results suggest the role of WWOX gene in the regulation of
EMT process in endometrial cancer via regulation of expression of cell motility
associated proteins and thus influencing tissue remodeling, with the important
suppression of mezenchymal marker.
Acknowledgements: This study was funded by the National Center of Sciences
N N407 168940.
219 Inactivation of nucleoside-derived anticancer drugs by catabolic
prokaryotic enzymes
J. Vande Voorde1 , S. Sabuncuoglu1 , J. Balzarini1 , S. Liekens1 . 1 Rega
Institute for Medical Research KU Leuven, Microbiology and Immunology,
Leuven, Belgium
Background: Nucleoside analogues (NAs) target tumor cell replication and
represent ~20% of the approved anticancer drugs. NA treatment efficiency
depends on the expression and activity of nucleo(s)(t)ide-metabolizing
enzymes. Since prokaryotic enzymes are often endowed with a different
activity and substrate specificity compared with their mammalian counterparts,
bacterial presence in the tumor microenvironment may affect NA-based cancer
treatment. Mycoplasmas are part of the normal flora of the human body
and several mycoplasma species (e.g. Mycoplasma hyorhinis) are reported to
preferentially colonize tumors in cancer patients. These prokaryotes interfere
with host cell nucleoside metabolism and may influence the therapeutic
efficacy of NAs.
Material and Methods: The cytostatic activity of different NAs was evaluated
in M. hyorhinis-infected and control tumor cell cultures. The stability and/or
metabolism of radiolabeled cladribine and gemcitabine was monitored in
mycoplasma-infected and control human MCF-7 breast carcinoma cells. The
antitumor activity of gemcitabine and floxuridine was studied in mice bearing
M. hyorhinis-infected or uninfected murine mammary FM3A tumors.
Results: The cytostatic activity of several NAs (e.g. gemcitabine, cladribine
and floxuridine) was severely compromised in various tumor cell cultures
upon infection with M. hyorhinis. Accordingly, a significantly decreased
antitumor effect of gemcitabine and floxuridine was observed in mice
bearing M. hyorhinis-infected tumors. Mycoplasma-mediated drug inactivation
of cladribine, floxuridine and gemcitabine was studied in detail. Cladribine
and floxuridine were catabolised to their (inactive) free nucleobases
by mycoplasma-encoded nucleoside phosphorylases. For gemcitabine, a
tandem-mechanism of action in which two catabolic mycoplasma enzymes
act in concert was identified: (i) mycoplasma cytidine deaminase (Cyd-DA)
catalyzes deamination of gemcitabine to a poorly cytostatic metabolite and
(ii) natural inhibitors of Cyd-DA are catabolised by a mycoplasma pyrimidine
nucleoside phosphorylase. The cytostatic activity of these NAs was restored
by co-administration of a mycoplasma-directed antibiotic or relevant inhibitor
of the mycoplasma-encoded catabolic enzymes. These observations suggest
that the presence of mycoplasmas in the tumor microenvironment may limit
the anticancer efficiency of certain NAs.
Conclusions: Recently, commensals were found to be essential for an
optimal response to immunotherapy and cyclophosphamide- or platinumbased chemotherapy [Iida et al., Science (2013) 342: 967−70 & Viaud et al.,
Science (2013) 342: 971−76]. Such findings stress the potential risks of using
antibiotics prior to, or during, cancer therapy. However, our results indicate
that targeting the microbiome using antibiotics may improve the therapeutic
outcome of NA-based cancer treatment.
220 Up-regulation of C1GALT1 promotes breast cancer cell growth
through MUC1-C signaling pathway
C. Chou1 , M.C. Huang1 . 1 National Taiwan University College of Medicine,
Graduate Institute of Anatomy and Cell Biology, Taipei City, Taiwan
Introduction: Breast cancer is the most frequently diagnosed malignancy
in women and the fifth leading cause of cancer-related death worldwide.
Core 1 b1,3-galactosyltransferase (C1GALT1) is the exclusive enzyme that
catalyzes the formation of T antigens, a cancer associated structure, through
the addition of galactose (Gal) to N-acetylgalactosamine (GalNAc)-Ser/Thr.
C1GALT1 is pivotal in many biological functions in the developmental process.
However, little is known of the function of C1GALT1 in association with
cancer development, particularly, breast cancer. This study aims to correlate
C1GALT1 expression with breast cancer clinicopathological characteristics and
to investigate the role of C1GALT1 in breast cancer malignant behaviors and
its underlying mechanisms.
Materials and Methods: C1GALT1 expression in breast cancer tissue
array (Pantomics) was analyzed by immunohistochemistry. C1GALT1 was
knocked down with specific siRNA and C1GALT1/pcDNA3.1A plasmid was
constructed to overexpress C1GALT1 in breast cancer cell lines. C1GALT1,
MUC1 and MUC1-C expression in cells were analyzed by Western blotting.
Vicia villosa (VVA) and peanut agglutinin (PNA) lectins were used to detect
Tn- and T-antigens, respectively, in lectin pull-down, Western blotting, and
flow cytometry. Cell fractionation was performed to investigate subcellular
localization of MUC1-C. Tumor growth was analyzed in NOD/SCID xenograft
model.
Results and Discussion: Data from public microarrays and our data show
that C1GALT1 is frequently up-regulated in breast cancer and increased
C1GALT1 expression is correlated with higher histological grade and advanced
tumor stage. C1GALT1 is differentially expressed in multiple breast cancer
cell lines. C1GALT1 expression modified O-glycan structures on glycoproteins
in breast cancer cells. Overexpression of C1GALT1 enhanced cell growth
in MCF-7 cells, whereas knockdown of C1GALT1 suppressed cell growth in
T47D cells. In vivo results revealed that C1GALT1 enhanced tumor growth
in xenograft mice. Interestingly, our results showed that C1GALT1 modified
Tn- and T-antigen expression on MUC1 oncoprotein and promoted MUC1-C
translocation to nucleus.
Conclusion: C1GALT1 is up-regulated in breast cancer tissues and
overexpression of C1GALT1 enhances breast cancer cell growth probably
through modification of MUC1 O-glycosylation and MUC1-C localization.
221 The T-box transcription factor, TBX3, promotes tumourigenesis
in soft tissue and bone sarcomas: a possible therapeutic target
T. Willmer1 , S. Prince2 . 1 Groote Schuur Hospital and University of Cape
Town, Human Biology, Cape Town, South Africa, 2 University of Cape Town,
Human Biology, Cape Town, South Africa
Background: Sarcomas comprise a subset of malignant tumours derived
from mesenchymal tissue and while they are rare, they represent some
of the most aggressive cancers due to their highly metastatic nature and
their resistance. Furthermore, they are resistant to conventional chemo- and
radiation therapies which limits the options of their treatment. There has
therefore been a great interest in developing single targeted therapies for
sarcomas and indeed inhibitors to tyrosine kinases and c-KIT have proven
promising. The transcription factor, TBX3, is overexpressed in several epithelial
cancers and plays a direct role in their development which suggests that it may
be a novel target for anti-cancer treatments. However whether this is the case
for cancers of mesenchymal origin is not known and hence this study explores
a possible tumour-promoting role for TBX3 in sarcomas.
Material and Methods: TBX3 protein and mRNA expression were analysed
in a panel of sarcoma cell lines by western blot analysis and quantitative
real time PCR respectively. TBX3 protein expression was also determined in
paraffin embedded sarcoma tissue sections by immunohistochemistry. TBX3
was silenced in chondrosarcoma cell lines using a shRNA approach, and the
impact of this on the cancer phenotype was assessed using proliferation and
cell cycle analyses, soft agar and cell motility assays and western blot analysis
with antibodies to key cell cycle regulators.
Results: We show for the first time that TBX3 is overexpressed in a panel
of sarcoma cell lines and tissue. Importantly, we demonstrate that knocking
down TBX3 levels in two chondrosarcoma cell lines is sufficient to reverse
key features of the sarcoma phenotype. We show that TBX3 depletion
triggers an S-phase arrest and inhibits substrate-dependent and -independent
cell proliferation. This is shown to be accompanied by the activation
of the p38-MAPK pathway and increased levels of p53 and p21 which
may suggest a mechanism by which TBX3 promotes cell proliferation in
sarcomas. Interestingly, knocking down TBX3 also inhibited chondrosarcoma
cell migration which was reproducible in liposarcoma and rhabdomyosarcoma
cell lines.
Conclusions: Our data show for the first time that TBX3 is overexpressed in a
diverse subset of sarcomas and that it is a key driver of the oncogenic process
in these cancers. This work extends our current understanding of the role of
TBX3 in cancer and provides additional support for its use in single targeted
therapies to treat this highly aggressive disease.
222 Chronic intermittent hypoxia triggers adaptive changes that
promote protection against cell death
J. Matschke1 , H. Riffkin1 , R. Handrick2 , L. Klein-Hitpass1 , V. Jendrossek1 .
1
University Hospital Essen, Institute of Cell Biology, Essen, Germany,
2
Institute for Pharmaceutical Biotechnology Biberach, University of Applied
Sciences, Biberach, Germany
Introduction: Hypoxia is considered as one main biological factor driving
malignant progression and promoting tumor cell resistance to chemotherapy
and radiotherapy. We showed in previous work that chronic intermittent hypoxia
drives the evolution of hypoxia-tolerant lung cancer cells that display increased
resistance to stimuli of the intrinsic apoptosis pathway. Aim of the present
study was to gain further insight into the molecular changes responsible for
hypoxia tolerance and to understand the molecular mechanisms that promote
apoptosis resistance in hypoxia-tolerant lung cancer cells.
Methods: Human lung adenocarcinoma cells (NCI-H460) were subjected to 25
cycles of severe hypoxia (48 h <0.1% O2 ) and reoxygenation (120 h 21% O2 ).
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We analyzed tumor cell growth and sensitivity to treatments involving the
generation of toxic reactive oxygen species (ROS), e.g. ionizing radiation. Cell
function was determined by measuring apoptosis, cell death, mitochondrial
membrane potential and ROS under control or starvation conditions. Finally,
we compared gene expression profiles of hypoxia-selected and control cells
by microarray analysis and validated genes of interest by qRTPCR.
Results: The hypoxia-selected NCI-H460 cells were characterized by
decreased formation of ROS and increased survival in response to ionizing
radiation. Moreover, the hypoxia-tolerant cells differed from the control cells
in their response to glucose, glutamine or serum starvation in normoxia and
hypoxia. These changes were linked to complex alterations in gene expression
hinting to adaptive changes in cell metabolism. Finally, the hypoxia-tolerant
cells and the control cells showed distinct responses to drugs targeting cell
metabolism.
Conclusions: Our data suggest that improved ROS-defense of the hypoxia
tolerant NCI H460 cells may contribute to their decreased sensitivity to
ionizing radiation. We speculate that the identification of specific metabolic
dependencies of hypoxia-tolerant cancer cells may offer novel opportunities
for the treatment of chronically hypoxic tumors.
223 HIF prolyl hydroxylase PHD3 maintains hypoxic cell cycle through
cyclin-dependent kinase inhibitor P27
H. Högel1 , P. Miikkulainen2 , L. Bino3 , P.M. Jaakkola4 . 1 Turku Centre for
Biotechnology, Turku, Finland, 2 Turku Centre for Biotechnology, University of
Turku, Turku, Finland, 3 Institute of Biophysics, The Academy of Sciences of
the Czech Republic, Brno, Czech Republic, 4 Faculty of Medicine, University
of Turku, Turku, Finland
Introduction: Hypoxia is a common feature of solid tumours and has multiple
effects on cancer progression. It has a major role in many of the events
considered as hallmarks of cancer. Moreover, hypoxia causes a cell cycle
arrest at G1/S interface which can be overcome by carcinoma cells. Growth
control and cellular survival in hypoxic environment require changes in cellular
signaling many of which are mediated by activity of master regulator of hypoxic
response, hypoxia-inducible factor HIF-1. HIF prolyl hydroxylases (PHDs) are
considered as cellular oxygen sensors as they regulate the activity of HIF-1.
However, one of the three PHD isoforms, PHD3, has been shown having other
substrates as well and it is upregulated under hypoxia. PHD3 is overexpressed
in various cancer types including head and neck squamous cell carcinomas,
renal cell carcinoma and pancreatic cancer. Recently we have shown that
PHD3 is essential for the progression of cell cycle from G1 to S phase in
hypoxia, and that the inhibition of PHD3 expression causes non-apoptotic cell
death. Moreover, we found that PHD3 depletion causes induction of cyclindependent kinase inhibitor p27.
Materials and Methods: Quantitative RT-PCR was used to study transcription
and western blotting to monitor protein expression. siRNA and plasmid
transfections were performed using manufacturers’ protocols. Cycloheximide
chase was used to study stability of p27 at different phases of cell cycle. For
cell cycle analysis with flow cytometry, cells were synchronized using serum
starvation or aphidicolin treatment.
Results and Discussion: We have previously shown that the cellular oxygen
sensor PHD3 enhances hypoxic cell cycle entry at G1 to S transition. Here
we show that PHD3 knockdown stabilizes the expression of cyclin-dependent
kinase inhibitor p27. PHD3 inhibition led to increased p27 expression under
hypoxia and p27 was required for hypoxic cell cycle block induced by PHD3
knockdown. PHD3 depletion increased the p27 half-life in G0 to G1 but
did not affect p27 transcription. PHD3 inhibition led to an increase in p27
phosphorylation at Ser10 without affecting other p27 phosphorylation sites.
Intact Ser10 was required for normal hypoxic degradation of p27.
Conclusions: Our data shows that PHD3 enhances hypoxic cell cycle entry
from G1 to S phase by decreasing the half-life of p27 through regulation of
phosphorylation.
224 Effect of HuIFN-a and Royal jelly on the proliferation of human
colon cancer (CaCo-2) cells in vitro
K. Rihar1 , D. Gregoric Exel1 , S. Sladoljev2 , B. Filipic3 . 1 Ljubljana, Slovenia,
2
Institute of Immunology, Zagreb, Croatia, 3 Institute for Microbiology and
Immunology, University of Ljubljana, Laboratory for Interferon Research,
Slovenia
Background: Royal jelly is a milky substance secreted by the pharyngeal
glands of worker bees. In order to determine its unusual nature, different
studies were conducted where biological properties and possible antitumor
activity were examined. Interferon alpha (IFNa) was used in the treatment of
different types of cancer, although its molecular mechanism remained fairly
unknown. The main role in its antitumor activity is the antiproliferative (AP)
effect. Experiments were performed to measure the effect of HuIFN-a and
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Royal jelly on the proliferation of CaCo-2 cells and effect on the intracellular
Glutathione (GSH) and Lipid peroxidation.
Material and Methods: Royal jelly (0.1 g/10 ml PBS) was used; Human
Interferon-aN3 (IFNa) (1000 IU/ml) was used and 10-HDA (10-hydroxy-2decenoic acid) at 100 mM/ml. The AP activity was measured with number of the
well with 50% cell growth inhibition. Cells were treated: (a) Royal jelly, (b) IFNa,
(c) 10-HDA, (d) Royal jelly + IFNa 1:1, 1:2, 2:1, (e) Royal jelly + 10-HDA 1:1,
1:2, 2:1. The AP activity was determined with the well in the row, where 50%
cell growth inhibition was found. GSH was determined with the Glutathione
assay kit (Sigma-Aldrich) and expressed as nM/mg of GSH. TBA-reactive
substances (RS) produced by lipid peroxidation were measured at 350 nm
according to the TBA method. The results were expressed as malondialdehyde
(MDA) nM/mg of protein.
Results: The following results of AP activity were obtained: (1) Royal jelly: 1;
(2) IFNa: 2.0; (3) DHA: 1.5; (4) Royal jelly + IFNa 1:1 0.5, 1:2 0.5, 2:1 3.5;
(5) Royal jelly + DHA: 1:1 2.0, 1:2 2.0, 2:1 3.0; (6) IFNa + DHA: 1:1 1.5, 1:2 0.5,
2:1 2.5. Royal jelly alone has low AP activity (1.8) with ID50 = 0.005 mg/ml;
HuIFN-a has AP activity 1.5, with ID50 = 208.33 IU/ml. The 10-HDA has AP
activity of 0.8 with ID50 = 37.5mM/ml. The combination HuIFN-a:RJ 2:1 shows
the level of GSH 24.9±2.4 nM/mg and level of MDA 72.3±31 nM/mg.
Conclusions: It can be concluded: (1) Royal jelly alone has low AP
activity: 1.0. (2) IFNa alone has AP activity of 2.0. (3) Best combination
between Royal jelly and IFNa was 2:1 where the AP activity was 3.5. (4) The
most active was the combination HuIFN-a:RJ 2:1, where level of GSH was
24.9±2.4 nM/mg of proteins (70.2±3.2 nM/mg in Control) and level of MDA
was 72.3±3.1 nM/mg of proteins (23.6±9.1 in Control). 10-HDA, as the main
component of the Royal jelly is responsible for the influence of Royal jelly on
interferon’s alpha (HuIFN-a) inhibiton of human colon cancer cells (CaCo-2)
proliferation in vitro.
225 Patient-derived tissue culture and xenograft models for studying
prostate tumor responses to therapy
L. Yu1 , T.E. Kähkönen1 , P. Taimen2 , P. Boström3 , J. Tuomela1 , P. Härkönen1 .
1
University of Turku, Department of Cell Biology and Anatomy, Turku,
Finland, 2 University of Turku, Department of Pathology, Turku, Finland,
3
Turku University Hospital, Department of Urology, Turku, Finland
Introduction: Prostate cancer is a very common malignancy among Western
males but rare in other species. Several experimental models have been
developed for prostate tumorigenesis and various stages of tumor progression.
However, the models often lack typical characteristics of human disease. The
aim of our study was to establish patient-derived models for testing individual,
specific responses of tumor tissue for different treatment options.
Materials and Methods: Clinical prostate tumor specimens were collected
from robotic assisted laparoscopic radical prostatectomy operations in Turku
University Hospital (Turku, Finland). The patient-derived tissues were cut
into 300 mm-thick pieces using a tissue slicer and cultured in vitro for 7
days. In PDX (patient-derived xenograft) model, tissue slices were implanted
subcutaneously into immunodeficient mice. First, PDX samples were implanted
in Balb/c nude, NOD Scid or NOG mice to select an appropriate mouse strain
on the basis of tumor-take. Viability and differentiation of cultured tissues
and xenografts were examined histologically using antibodies against Ki-67,
caspase-3, the androgen receptor and fibroblast growth factor 8 (FGF8).
Test compounds (hormonal therapy and/or FGFR inhibitors) and reference
compounds (androgens and cytotoxic drugs) were administered into the tissue
culture medium and subsequently to xenograft-bearing mice.
Results: Tumor tissue maintained its viability in tissue culture for 7 days and
responds to testosterone treatment. PDXs also maintained their viability in all
explored mouse strains, as shown by staining for the proliferation marker Ki-67,
the apoptosis marker caspase-3, androgen receptor and FGF8. Glandular
epithelium flattened, when tissue grafts are grown without hormone pellets,
as expected. Until now, we have tested effects of testosterone using patientderived models. Next, we expand our analysis to other hormonal therapies
and FGFR inhibitors.
Conclusion: According to our preliminary results, patient-derived xenografts
and tissue cultures maintain viability and appropriate responses to androgen.
Furthermore, PDX models may be useful for testing the individual responses
of prostate cancer patients to therapeutics, which would allow developing a
tool for personalized medicine to be used in prostate cancer research.
226 Role of EGFR-regulated microRNAs in malignant processes of
S. Langsch1 , S. Schäfer1 , M.P. Tschan1 , E. Vassella1 . 1 Bern Universtiy
Hospital, Molecular Pathology, Bern, Switzerland
Background: Epidermal growth factor receptor (EGFR) tyrosine kinase (TK)
mutations occur in up to 15% of Caucasian non-small cell lung cancer (NSCLC)
patients. Mutated EGFR TK domain enables constitutive active downstream
signaling of PI3K/AKT, KRAS/MAPK and JAK/STAT pathways and results in
lung cancer development by uncontrolled proliferation, survival and migration.
Interestingly, NSCLC patients harboring mutated EGFR TK domain respond
well to the small molecule TK inhibitors (TKI) gefitinib and erlotinib. However,
the tumors develop resistances rapidly. Therefore, there is great interest to
increase the knowledge of the resistance mechanisms of NSCLC to EGFR
TKIs in addition to develop new therapeutic approaches. MicroRNAs (miRNAs)
are noncoding regulatory short nucleic acids, and are implicated in lung
cancer progression. Published data demonstrate that miRNAs are involved in
regulating the EGFR signaling pathway. We aim to identify such miRNAs that
play a role in regulating EGFR downstream key molecules and to investigate
their role in malignant processes of NSCLC.
Results and Methods: We demonstrate that transduction of bronchial
epithelial cells (BEAS-2B) with mutated KRAS induces increased CyclinD1
expression which indicates an activated downstream signaling pathway. Next,
a miRNA array profiling of KRAS-transduced cells revealed upregulated miR138 and miR-29b expression, compared to control-transduced cells. miR-138
and miR-29b have been described to promote cell cycle arrest and apoptosis
and have an important tumor suppressive role in lung cancer. Analysis by
RT-qPCR of individual clones confirmed the upregulation of miR-138 and
miR-29b expression in KRAS-transduced cells. Similarly, we confirmed the
upregulated expression levels of miR-138 and miR-29b in NSCLC tissue
harboring KRAS mutations, by comparing it to tumors with wild-type KRAS.
Consistently, treatment of NSCLC cell lines with an EGFR (gefitinib) or a MEK
(U0126) inhibitor lead to downregulated miR-29b expression by RT-qPCR.
However, the expression of miR-138 was only slightly reduced upon EGFR
and MEK inhibition.
Outlook: Next, we will assess whether KRAS-induced overexpression of miR29b is regulated on the transcriptional level by transfecting NSCLC cells with
miR-29b promoter luciferase reporter constructs. In addition, we will analyse
whether the upregulation of miR-29b precursor transcripts (pri-miR-29b and
pre-miR-29b) by RT-qPCR are consistent with the increased luciferase levels.
Furthermore, we will transiently transfect the miRNA precursors into NSCLC
cells in order to study the role of these miRNAs in cell cycle control, apoptosis,
migration and chemoresistance. In summary, these results will help to better
understand the role of miRNAs in regulating key components of the EGFR
signaling pathway leading to NSCLC malignancies.
227 The WWOX gene modulates the adhesion of neural cells to ECM
K. Kosla1 , E. Styczen-Binkowska1 , M. Nowakowska1 , K. Pospiech1 ,
E. Pluciennik1 , I. Baryla1 , A.K. Bednarek1 . 1 Medical University of Lodz,
Department of Molecular Cancerogenesis, Lodz, Poland
Background: The WWOX gene is localized in a common fragile site FRA16D.
It is known to behave as a tumor suppressor. Alteration in WWOX expression
affects main cellular processes such as proliferation and cell death. Our
previous experiments on glioblastoma patients and cell lines showed that
WWOX expression is strongly implicated in brain tumor cancerogenesis.
Growing evidence indicates that its action is not limited only to cell cycle control
or genome integrity maintenance. The aim of the current study was to specify
how WWOX may influence neural cell ability for adhesion to extracellular matrix
proteins. Beside of the essential role of the cell–ECM interactions for tumor cell
surveillance and invasion, they are critical also for normal cell differentiation
and central nervous system development. The experiments were conducted
on the T98G glioblastoma cell line, and human neural stem cells (hNSC). In
order to identify what changes are caused by increase or decrease of WWOX
level we upregulated its expression in T98G and silenced it in hNSC.
Material and Method: The WWOX gene cDNA was introduced into T98G
glioblastoma cells by a retroviral transfection with the pLNCX2 vector. The
WWOX gene expression was stably silenced in hNSC cells by a shRNA
delivered by a lentiviral vector. The changes in cells ability to adhere to
extracellular matrix proteins was assessed by 90 min incubation in a vessel
covered with selected proteins and subsequent colorimetric assessment of a
number of adhesive cells. The level of integrins expression was evaluated by
Western Blot.
Results and Discussion: The attachment assays showed that WWOX
overexpression significantly decreases glioblastoma cell adhesion to fibronectin, collagen I, collagen IV and fibrinogen. Consistently, WWOX
silencing significantly enhanced the attachment of neuronal progenitor cells
to fibronectin and a mixture of ECM proteins. One possible mechanism by
which WWOX forces changes in cellular attachment may be the regulation
of integrin levels. The T98G cells overexpressing WWOX showed reduced
expression of ITGb1. Contrary, the level of the same protein was not altered
in hNSC after silencing of WWOX .
Conclusion: The results of the experiment show that WWOX is able
to modulate neural cell binding to fibronectin and other extracellular
matrix proteins. This property is of great importance for cancerogenesis,
developmental processes and maintaining tissue structure.
228 A novel animal model of phaeochromocytoma for preclinical
therapy evaluation
M. Ullrich1 , R. Bergmann1 , J. Pietzsch1 , M. Cartellieri1 , M. Peitzsch2 ,
G. Eisenhofer2 , S.R. Bornstein3 , C.G. Ziegler3 . 1 Helmholtz-Zentrum
Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research,
Dresden, Germany, 2 Technical University Dresden, Institute for Clinical
Chemistry and Laboratory Medicine, Dresden, Germany, 3 University Hospital
Carl Gustav Carus, Department of Medicine III, Dresden, Germany
Phaeochromocytoma (PHAEO) is a rare but potentially lethal neuroendocrine
tumour arising from catecholamine producing chromaffin cells. One reason for
the lack of appropriate treatment strategies, especially for metastatic PHAEO,
is the lack of a convincing human PHAEO cell line and suitable animal
models. Interestingly, peptide hormone receptors are frequently overexpressed
in PHAEO, and can be targeted by highly effective cytotoxic and radiolabelled
peptide analogues. Here we focused on the establishment and characterisation
of a novel fluorescence-based mouse model in order to evaluate combined
therapies of malignant PHAEO.
In this study we employed a multimodal small animal imaging approach for
preclinical evaluations of diagnostic and therapeutic procedures in vivo. We
generated a fluorescence-tagged mouse phaeochromocytoma (MPC) cell line
named MPC-mCherry, which allows for observing tumour development in a
subcutaneous mouse model of PHAEO by optical imaging. The tumours were
functionally evaluated by multimodal imaging using PET, SPECT, MRI and CT.
Tumour tissue samples were also examined ex vivo. The physiological followup of tumour growth was done by non-invasive blood pressure measurement
and a recently established sensitive LC-MS/MS technique enabling targeted
analyses of urinary monoamine-related biomarkers.
We found MPC-mCherry cells forming a solid tumour after subcutaneous
injection in athymic NMRI nu/nu mice. Fluorescence imaging in the nearinfrared range improved monitoring of tumour development in vivo. The tumour
fluorescence intensities correlated positively with tumour size. Urinary samples
showed a distinct pattern of elevated catecholamines and metanephrines
(>35 mmol/mmol creatinin). Concentrations of dopamine, methoxythyramine
and normetanephrine in urine correlated positively with tumour fluorescence
intensities. The results confirm the tumour predictive value of urinary
monoamine-related biomarkers in PHAEO diagnostic follow-up. Functional
small animal imaging in vivo and histological studies revealed that SSTR2
is abundantly expressed in the MPC-mCherry cell-derived tumours. Relating
to our previous studies, which demonstrated strong anti-tumour effects
of cytotoxic somatostatin analogues in MPC cells in vitro, our animal
model is suitable for the evaluation of a combined SSTR2 targeting
cytotoxic/radionuclide therapy in vivo.
229 The LIM domain protein cysteine-rich protein 2 (CRP2) promotes
breast cancer progression
C. Hoffmann1 , X. Mao1 , M. Dieterle1 , F. Moreau1 , A. Steinmetz1 , C. Thomas1 .
Centre de Recherche Public de la Sante (CRP-Sante), Oncology,
Luxembourg, Luxembourg
1
Background: Central steps in cancer progression and metastasis, e.g.
uncontrolled cell division, loss of adhesion and increased migration, largely
depend on actin cytoskeleton remodeling. Accordingly, the primary actin
cytoskeleton regulators, namely actin-binding proteins (ABPs), represent
attractive candidates for targeted therapy and/or diagnosis. Over the last years,
actin-bundling proteins, a subset of ABPs specialized in the crosslinking and
stabilization of actin filaments into parallel arrays, have been shown to be
frequently deregulated in cancer and to represent novel potential targets. In this
context, we recently identified an actin-bundling protein, namely the cysteine
rich protein 2 (CRP2), whose up-regulation correlates with basal-like breast
carcinoma, a breast cancer subtype associated with poor prognosis.
Material and Method: We use both gain-of-function and loss-of-function (short
hairpin RNA) strategies to manipulate CRP2 levels in human breast cancer cell
lines including the low metastatic, epithelial-like, MCF7 (ER+, PR+, HER2-)
and the highly metastatic, mesenchymal-like, MDA-MB-231 (ER-, PR-, HER2-).
Modifications in actin bundling, cell proliferation (including 3D cell growth),
adhesion, motility, and invasion induced by these manipulations are evaluated
using quantitative methods.
Results and Discussion: Our preliminary data confirm that CRP2 promotes
actin bundling in breast cancer cells and that its expression correlates cell
aggressiveness. Down regulation of CRP2 in MDA-MB-231 cells induces
repression of mesenchymal gene makers such as snail1 and vimentin. In turn,
CRP2 overexpression in MCF7 enhances cell proliferation and extracellular
signal-regulated kinase (ERK) phosophorylation. Other analyses are under
way and the corresponding data will be presented during the meeting.
Conclusion: Our data support that CRP2 promotes breast cancer progression
through actin cytoskeleton remodeling. The underlying molecular mechanism
is currently deciphered in our lab.
S53
230 BRAF V600E-mutated cells in a papillary thyroid carcinoma do
not form a compact ball, but a mesh of cells sparsely embedded
within the stroma
A. Antoniou1 , M. Tarabichi1 , S. Le Pennec1 , V. Detours1 , C. Maenhaut1 .
1
Université Libre de Bruxelles (ULB), IRIBHM, Brussels, Belgium
Introduction: The BRAF V600E mutation drives ~40% of papillary thyroid
cancer (PTC). Several reports, however, have documented tumors in which the
corresponding DNA alteration had a low allelic ratio, suggesting that V600E
was not the initiating mutation (Guerra et al. J. Clin Endo. Metab. 97, p517,
2012; Ghossein et al. J. Clin Endo. Metab. 98, p1414, 2013). To address this
point, we investigated exome-wide allelic fraction in multiple blocks from a
PTC patient and reconstructed specifically the 3D volume occupied by BRAFmutated cells.
Material and Methods: Four primary tumor blocks, 1 contralateral tumor, 3
invaded lymph nodes, 3 normal adjacent thyroid tissues, 2 normal lymph nodes
and 1 blood sample were collected from one PTC patient. Each block was split
in two parts, one for sequencing, the other for 3D reconstruction.
Illumina HiSeq exome sequencing generated 1.5e8 2×100pb paired-end
reads per block. A wider view of BRAF V600E allelic fraction was provided
by 500 PTCs from The Cancer Genome Atlas (TCGA).
For 3D reconstruction, 70 serial sections of 10 mm were prepared from
paraffin-embedded blocks of 0.5×0.6×0.2 cm3 . Slices were stained by
antibody specific for BRAF V600E-derived proteins. The 2D images were
segmented to delimit the stained tumor cells from the stroma. They were then
staked with a non-rigid registration algorithm. Finally, a 3D model of the tumor
was build and visualized.
Results and Discussion: Approximately 10% BRAF-mutated PTC from
TCGA had a BRAF allelic fraction <20%. In our patient, BRAF V600E was
detectable in 4/5 primary tumors blocks and all 3/3 invaded lymph nodes. It
was not found in the normal blood, thyroid or nodal tissues from the same
patient. The negative tumor block came from the contralateral thyroid lobe.
When present, the BRAF mutation allelic ratio ranged from 2% to 31%.
Importantly, none of dozens other somatic mutations had a higher allelic
fraction than V600E in any sample. V600E was therefore unlikely to be a
subclonal mutation. This, in turn, implied that the tumor cell fractions were
much lower than the 70% estimated from pathology examination.
We examined the volume occupied by BRAF-mutated cells. Serial tumor
slices were stained with an anti-V600E antibody. 3D reconstruction of the
V600E clone revealed a mesh of cells deeply, but sparsely, infiltrated in the
stroma. Its volume fraction to the total tumor volume was compatible with the
small allelic fraction inferred from sequencing data. Yet, the entire block was
densely infiltrated by V600E cells, explaining the large pathology-estimated
tumor fraction.
Conclusion: The mesh topology departs dramatically from the textbook image
of a compact ball-like tumor. It implies that the tumor/stroma interface surface
may be much larger than previously thought. It also raises a question: what is
the actual contribution of BRAF-mutated cells proliferation to the total increase
in tissue volume observed during tumor progression?
231 Murine tumors microenvironment after irradiation characterized
by diffusion-weighted, dynamic contrast-enhanced MRI and
immunohistology
A. Drzal1 , M. Gonet1 , T. Skorka2 , M. Elas1 . 1 Biochemistry Biophysics and
Biotechnology, Biophysics, Krakow, Poland, 2 Institute of Nuclear Physics
PAS, MRI, Krakow, Poland
Introduction: Tumor microenvironment can be assessed non-invasively by
several MRI techniques. Two of the most common methods used clinically
are Diffusion-Weighted and Dynamic Contrast-Enhanced MRI. The ability of
these techniques to predict and monitor early response to treatment would
provide an opportunity to optimize individual patient management. The aim of
this study was to find a method to assess a tumor vasculature changes after
a radiotherapy in murine model.
Material and Methods: C57BL/6 mice bearing Lewis Lung Carcinoma
(LLC) were irradiated with 15 Gy using Phillips 300 kVp X radiator. MRI
measurements were performed using 9.4T system (Bruker, Germany).
Diffusion images were obtained using FairEPI sequence. T1 -weighted images
for Dynamic Contrast-Enhanced (DCE) were acquired with FLASH sequence
and Magnevist® as a contrast agent was used. Matlab scripts were used
for image analysis. Tumor sections were stained with 9F1 antibody and
microvessels to tumor area ratio was calculated.
Results and Discussion: Apparent Diffusion Coefficient (ADC) and intravoxel
incoherent motion (IVIM) model derived parameters were compared with
those obtained from pharmacokinetics analysis of DCE-MRI measurements.
Feasibility studies have shown that pseudodiffusion fraction calculated from
IVIM model seems to be correlated with perfusion parameters from DCE.
Furthermore, both methods shown differences in perfusion after 24 h between
treated group and non-treated one. Additionally irradiated group demonstrated
reduction in microvessel to tumor area ratio.
S54
Conclusions: DWI and DCE MRI methods allow to asses environment of
tumor and demonstrate early changes caused by radiotherapy. Also application
of IVIM model to DWI measurements has a potential to be an alternative
or complementary method for DCE MRI without the necessity of applying a
contrast agent in tumor microenvironment study.
232 The glycolytic enzyme GPI/AMF is a stimulator of glioma cell
migration and directly contributes to the pro-migratory phenotype
under hypoxic conditions
A. Kathagen1 , M.H. Holz1 , A.S. Schulte1 , M.W. Westphal1 , K.L. Lamszus1 .
1
University Medical Center Hamburg Eppendorf, Neurosurgery, Hamburg,
Germany
Background: The Warburg effect describes the phenomenon of a metabolic
redirection in tumor cells away from oxidative phosphorylation to aerobic
glycolysis for ATP synthesis. In this context, we recently discovered an oxygen
concentration-dependent reciprocal metabolic switch between glycolysis and
the pentose phosphate pathway, associated with a pro-migratory as opposed
to a pro-proliferative phenotype, respectively (Kathagen et al. 2013). However,
the mechanistic link between glycolysis and cell migration is largely unclear.
Since it has been shown that the hypoxia-induced glycolytic enzyme glucose
6-phosphate isomerase (GPI) and the cytokine autocrine motility factor (AMF)
are identical, we aimed to determine the contribution of GPI/AMF to the promigratory phenotype of glioma cells under hypoxia.
Material and Methods: Functional and expression analysis were performed
using chronically and acutely normoxic (21% O2 ) and hypoxic (1% O2 )
glioblastoma-derived stem-like cell lines (GS cells) as well as glioma cell lines
and human glioma tissues.
Results: Functional analyses using recombinant GPI/AMF revealed a
concentration-dependent pro-migratory effect on GS cell lines. Conversely,
GPI/AMF deceased proliferation of GS cells. The inhibitor of GPI/AMF,
erythrose 4-phosphate, abrogated the motogenic effect of GPI/AMF. Gene
expression profiling as well as confirmatory qPCR and Western Blot analyses
in GS cell lines, glioma cell lines and other cancer cell lines (HuH7, MDAMD231) showed a strong induction of GPI/AMF and its receptor AMFR by
acute hypoxia, whereas ligand and receptor were both downregulated by acute
oxygenation of hypoxic GS cells. In addition, the amount of secreted GPI/AMF
in the supernatant of cultured glioma cells increased under hypoxia. Functional
analysis using conditioned media revealed an auto- and paracrine stimulation
of migration by AMF. Immunostaining of a tissue microarray containing 350
samples of gliomas of different grades and normal brain revealed strong
expression of GPI/AMF and AMFR in hypoxic pseudopalisading cells.
Conclusion: Our findings stress the previously described association between
hypoxia-induced glycolysis and increased migration by showing that secreted
GPI/AMF strongly stimulates migration while reducing cell proliferation. This
observation indicates that the upregulation of GPI/AMF in glioma cells may
enable these cells to evade hypoxic stress and may be a promising target to
inhibit invasion.
233 Clinical and prognostic values of the pretreatment PSA doubling
time in patients with prostate cancer
O. Bogomolov1 , G. Zharinov2 , M. Shkolnik3 . 1 Central Research Institute
of Roentgenology and Ra, Saint-Petersburg, Russian Federation, 2 Central
Research Institute of Roentgenology and Ra, Oncology, Saint-Petersburg,
Russian Federation, 3 Central Research Institute of Roentgenology and Ra,
Urology, Saint-Petersburg, Russian Federation
Introduction: Nowadays PSA doubling time (PSADT) is used in various
clinical cases, including for estimates aggressive phenotype of prostate cancer
(PCa).
Materials and Methods: Pretreatment PSADT and follow-up information
was compiled on 912 men who were treated with external beam radiation
therapy. PSADT were compared with the clinical tumor category, Gleason
score, PSA level at diagnosis, as well as the age and the education level
of patients. The pretreatment PSADT was compared with survival rates of
patients. Patients were divided into groups corresponding to the slow and fast
PSADT. Illustrations of the estimates of time to all-cause mortality following RT
were made using Kaplan–Meier cumulative incidences, respectively, for men
with slow and fast PSADT.
Results: The median PSADT for the patients with local PCa was 24.5
months. For the group of advanced PCa the median PSADT was 12.2 months,
for metastatic PCa − 2.4 months. The differences between groups were
significant.
The median PSADT for patients with Gleason score 6 was 20.8 months.
In groups with Gleason score 7 and 8−10 median PSADT were 9.0 and
3.85, respectively. By comparing the PSADT and PSA level at diagnosis also
revealed significant differences. The median PSADT decreased when PSA
level increased.
The median PSADT depending on the education level and patient’s age were
also significantly different.
The prognostic significance of PSADT was confirmed − short DT is a predictor
of negative outcome, and a prolonged DT implies a prostate cancer-specific
survival advantage.
Conclusion: PSADT is a powerful indicator of tumor biology. A short DT is a
surrogate for rapid tumor growth and a longer DT time implies a more indolent
tumor. Moreover, in the study the prognostic value of PSADT was confirmed.
The statistically and clinically significant associations between the PSADT and
all-cause mortality in the setting of PSA failure following have been described.
234 MMP2 as a molecular biomarker of proficient tumor–stroma
cross-talk in lung cancer
E. Baldoli1 , T. Caputo1 , G. Bertolini1 , M. Moro1 , F. Facchinetti1 , R. Caserini1 ,
U. Pastorino2 , G. Sozzi1 , L. Roz1 . 1 Fondazione IRCCS Istituto Nazionale
dei Tumori, Experimental Oncology and Molecular Medicine, Milan, Italy,
2
Fondazione IRCCS Istituto Nazionale dei Tumori, Thoracic Surgery Unit,
Milan, Italy
Background: Evidence has accumulated to suggest that microenvironment
plays a critical role in modulating development and progression of epithelial
cancers. Among cellular components involved in this process a central role
is played by stromal fibroblasts. In previous in vivo experiments we showed
that co-injection of lung cancer cells and fibroblasts resulted in increased
tumorigenicity associated with changes in expression of specific genes
involved in extracellular matrix (ECM) composition and remodeling including
MMP2, COL6A3 and CTSL. Since ECM remodelling proteinases, such as
matrix metalloproteinases (MMPs), are crucial mediators of the alterations
observed in the microenvironment during cancer progression we focused in
particular on the study of the role of MMP2 in tumor–stroma cross-talk in lung
cancer.
Materials and Methods: To study changes in expression of ECM-related
genes, different lung cancer cell lines were exposed to medium conditioned
by primary fibroblasts isolated from resected lung cancers or seeded in closeproximity transwell assays. Co-culture experiments and cell sorting were used
to evaluate the relevance of physical contact. The expression of ECM genes
in cancer cells was assessed by real time PCR and the protein levels by
western blot. FACS analysis and immunofluorescence assay were used to
study MMP2 protein expression and localization. To evaluate its functional
relevance the endogenous expression of MMP2 was suppressed by RNA
interference and proliferation and invasion assays were performed. Silenced
cells were co-cultured with fibroblasts, sorted and injected in nude mice to
evaluate tumor growth. MMP2 expression in xenografts was evaluated by
immunofluorescence.
Results and Discussion: Conditioned media, close proximity transwell assay
and co-culture with fibroblasts induced modification in ECM-related genes
expression in different lung cancer cells. Levels of MMP2 and COL6A3 in
cancer cells markedly increased after co-cultures, indicating that aspects of
tumor–stroma cross-talk may be regulated by direct interactions. Interestingly
the effects of co-culture with fibroblasts were distinctly higher in specific
subpopulations of cancer cells and resulted in increased tumorigenicity. Shortterm silencing of MMP2 on cancer cells was able to prevent the initial increase
in MMP2 levels after co-culturing but not the increase in tumorigenic potential
or higher MMP2 expression in tumors obtained by injection of sorted cells
suggesting long term persistence of the effects of fibroblasts–cancer cells
interaction.
Conclusion: These data demonstrate that cross-talk between stroma and
cancer cells can dictate ECM composition. Fibroblasts induce their protumorigenic effect at least in part by priming cancer cells through MMP2
upregulation. The identification of MMP2 as a biomarker of proficient tumor–
stroma crosstalk could also have prognostic implications.
235 Identification and characterization of novel cancer-associated
molecules
A. Chernenko1 , P.R. Karhemo1 , M. Reinman2 , Y. Chen3 , S. Goodison4 ,
K. Takkinen2 , P. Panula5 , P. Laakkonen1 . 1 Institute of Biomedicine,
Translational Cancer Biology Research Program, Helsinki, Finland, 2 VTT
Technical Research Centre, Biotechnology, Espoo, Finland, 3 Neuroscience
Centre and Institute of Biomedicine, Anatomy, Helsinki, Finland, 4 MD
Anderson Cancer Center, Cancer Research Institute, Florida, USA,
5
Neuroscience Centre and Institute of Biomedicine, Faculty of Medicine,
Helsinki, Finland
Introduction: Tumor metastasis is a major cause of cancer mortality. Despite
significant advances in the treatment of primary tumors, the ability to predict
metastatic behaviour of cancer, as well as detection and eradication of
metastatic lesions, remains the greatest clinical challenge in oncology.
Knowledge of the molecular mechanisms involved in metastatic spread and
growth is needed to facilitate prognostic evaluation of individual patients and
design therapies to inhibit the metastatic process.
In our study we aim at discovering metastasis-promoting molecules, revealing
their functional role and preparing antibodies against these molecules.
Material and Methods: We use two subclones of the MDA-MB-435 human
melanoma cell line as a model for the metastatic spread of cancer. One
cell clone metastasizes consistently to the lungs whereas the other, equally
tumorigenic and capable of dissemination, fails to give growth to metastatic
lesions at a secondary site in athymic mice.
In order to find new metastasis-associated markers we screened these cell
lines with phage-displayed single-chain variable fragment (scFv) antibody
libraries. Specificity of the selected scFv-antibody clones was confirmed
by FACS analysis. Antigens for the selected antibodies were identified by
immunoprecipitation followed by a mass-spectrometry analysis.
Results and Discussion: We found several scFv-phage clones preferentially
binding to the metastatic cells. The selected scFv-antibodies recognize
antigens on the cell surface and some of them are able to internalize.
We were able to identify the antigen for one of the selected antibodies. This
protein is present in higher amounts on the surface of the metastatic cell
clone compared to the non-metastatic clone. Currently, we study the role of
the discovered KA2 membrane protein in tumor progression and metastasis
as well as in normal development of zebrafish.
Conclusion: Using the antibody-phage display approach we identified a novel
membrane protein which is over-expressed by the highly metastatic cell line.
Nothing is known about the function of this protein. We are currently performing
in vitro and in vivo assays to uncover the role of this protein in normal
development (zebrafish model) and cancer.
Monday 7 July 2014
Poster Session
Cell and Tumour Biology II
236 Novel approaches for dynamic biomarker imaging by multispectral
optoacoustic tomography (MSOT)
W. Driessen1 , S. Morscher1 , N.C. Burton1 , T. Sardella1 , D. Razansky2 ,
V. Ntziachristos2 . 1 iThera Medical, München, Germany, 2 Helmholtz Zentrum
München, IBMI, München, Germany
Background: In designing new biomarker imaging approaches for cancer
research, the heterogeneity and dynamics of the tumor microenvironment
cannot be disregarded. The interplay between the different components of
the tumor stroma and parenchyma play a significant role in tumor progression,
invasion and treatment response. In order to study such dynamic processes
by (molecular) imaging, volumetric imaging modalities with a high temporal
and spatial resolution need to be combined with contrast agents that possess
appropriate pharmacokinetic parameters.
Materials and Methods: In this work, we assessed tumor heterogeneity by
combining multispectral optoacoustic tomography (MSOT) with three different
fast-clearing contrast agents. MSOT is an imaging modality based on the
photoacoustic effect. Multiple wavelengths in the NIR range are used and
by detecting the acoustic waves at 5MHz, cross-sectional images can be
obtained with 150 mm resolution in <1 s. Imaging multiple cross-sections
allows for volumetric reconstruction and multispectral data analysis enables
the specific identification of endogenous absorbers and/or multiple injected
contrast agents. The pharmacokinetic parameters of the imaging agents were
optimized to allow for fast imaging regimens (<1hr) that can be employed
daily.
Results: First, we assessed inter- and intra-tumoral heterogeneity in tumor
perfusion and vascular permeability by systemically administering novel
formulations of ICG. Pixel-by-pixel analysis was then performed to create
parametric maps, thereby revealing a high degree of heterogeneity in
dynamic contrast enhancement within tumors. Secondly, apoptotic regions
within orthotopic tumors were visualized using a caspase-targeted probe and
compared to the hypoxia status of each tumor region. Before treatment,
maximal apoptosis-signal was co-localized with more hypoxic regions in
the tumor. However, overall signal intensity was significantly increased after
systemic treatment with doxorubicin. Lastly, tumor-associated macrophage
within the tumor stroma was visualized by a novel NIR marker specific for
macrophage. More intense TAM-specific signal was observed in areas of
relative hypoxia.
Conclusions: In summary, MSOT offers an imaging modality that can provide
anatomical, functional, molecular and kinetic information at high temporal
and spatial resolution. When combined with (molecular) imaging agents
that possess appropriate pharmacokinetic properties, this modality can be
leveraged for new biomarker imaging approaches in cancer research.
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237 Sequential application of targeted therapies guided by biomarkers
overcomes therapy resistance in rapidly evolving highly
aggressive mammary tumors
O. Sahin1 , Q. Wang2 , S.W. Brady2 , H. Wang2 , C. Chang2 , S.T. Wong3 ,
W.J. Muller4 , F.J. Esteva5 , J. Chang6 , D. Yu2 . 1 Bilkent University, Department
of Molecular Biology and Genetics, Ankara, Turkey, 2 The University of
Texas MD Anderson Cancer Center, Department of Molecular and Cellular
Oncology, Houston, USA, 3 The Methodist Cancer Center, Department of
Systems Medicine and Bioengineering, Houston, USA, 4 Goodman Cancer
Center, McGill University, Montreal, Canada, 5 The University of Texas MD
Anderson Cancer Center, Department of Breast Medical Oncology, Houston,
USA, 6 The Methodist Cancer Center, The Methodist Hospital Research
Institute, Houston, USA
Introduction: Combinatorial targeted therapies are more effective in treating
cancer by blocking by-pass mechanisms or inducing synthetic lethality.
However, their clinical application is hampered by resistance and toxicity. To
meet this important challenge, we developed and tested a novel concept
of biomarker-guided sequential applications of various targeted therapies
using ErbB2-overexpressing/PTEN-low, highly aggressive breast cancer as our
model.
Materials and Methods: HER2/neu overexpressing-PTEN homozygous loss
(PTEN−/− /NIC) mice were treated with different drugs in sequential and combinatorial fashion, and survival analysis was done. Drug resistant human cell
lines cells and mouse primary cells were used to dissect the mechanisms of
resistance in 3D culture, protein stability assays and RPPA/p-RTK array experiments. In vitro and in vivo findings were validated in two breast cancer patient
cohorts by: 1. pathway analysis in neo-adjuvant trastuzumab+chemotherapy
treated patients with known pathological response and 2. gene set enrichment
analysis (GSEA) in pre- and post-lapatinib treated patients.
Results and Discussion: We found that sustained activation of ErbB2
and downstream pathways drives trastuzumab resistance in both PTEN
low/trastuzumab resistant breast cancers from patients and mammary
tumors from genetically engineered mice. Although lapatinib initially inhibited
trastuzumab resistant mouse tumors, tumors by-passed the inhibition by
activating the PI3K/mTOR signaling network which was also observed
in neo-adjuvant lapatinib-treated patients manifesting lapatinib-resistance.
Trastuzumab+lapatinib resistance was effectively overcome by sequential
application of a PI3K/mTOR dual kinase inhibitor (BEZ235) with no significant
toxicity: however, BEZ235 treatment led to increased ErbB2 expression and
phosphorylation in mouse tumors and in 3-D culture leading to BEZ235
resistance. Mechanistically, we identified ErbB2 protein stabilization and
activation as a novel mechanism of BEZ235 resistance which was reversed
by subsequent lapatinib+BEZ235 combination. Remarkably, this sequential
application of targeted therapies guided by biomarker changes in the rapidly
evolving resistant tumors doubled the life-span of mice bearing exceedingly
aggressive tumors.
Conclusion: This fundamentally novel approach of using targeted therapies in
a sequential order can effectively target and reprogram the evolving resistant
cancer signaling networks during treatment.
238 Vascular endothelial growth factor-C modulates proliferation
and chemoresistance in acute myeloid leukemic cells through
an endothelin-1-dependent induction of cyclooxygenase-2
Y. Yang1 , K. Hua2 , M. Chien3 , M. Kuo2 . 1 Graduate Institute of Oncology,
College of Medicine National Taiwan University, Taipei City, Taiwan, 2 Graduate
Institute of Toxicology, College of Medicine National Taiwan University, Taipei
City, Taiwan, 3 Graduate Institute of Clinical Medicine, College of Medicine
Taipei Medical University, Taipei City, Taiwan
Background: High-level expression of vascular endothelial growth factor
(VEGF)-C is associated with chemoresistance and adverse prognosis in
acute myeloid leukemia (AML). Our previous study has found that VEGF-C
induces cyclooxygenase-2 (COX-2) expression in AML cell lines and significant
correlation of VEGF-C and COX-2 in bone marrow specimens. COX-2 has
been reported to mediate the proliferation and drug resistance in several solid
tumors. The molecular mechanisms underlying VEGF-C-mediated COX-2 and
the effect of COX-2 induction in AML remain largely unclear.
Material and Methods: The COX-2 expression level were determined by
Western blot and quantitative real-time PCR after treated with VEGF-C
or endothelin-1 (ET-1) and/or BQ 123 (endothelin receptor antagonist) in
leukemia cells. Flowcytometry were used for detecting the changes of cell
cycle populations. Xenografted THP-1 tumors were generated by injecting
NEO, VEGF-C, VEGF-C/ET-1 shRNA or VEGF-C/COX-2 shRNA cell lines
subcutaneously in mice to investigate the role of ET-1 and COX-2 in VEGF-C
induced chemoresistance.
Results: Herein, we demonstrated that the VEGF-C-induced proliferation of
AML cells is effectively abolished by the depletion or inhibition of COX-2.
The expression of ET-1 rapidly increased following treatment with VEGF-C.
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We found that ET-1was also involved in the VEGF-C-mediated proliferation
of AML cells, and that recombinant ET-1 induced COX-2 mRNA and protein
expressions in AML cells. Treatment with the endothelin receptor A (ETRA)
antagonist, BQ 123, or ET-1 shRNAs inhibited VEGF-C-induced COX-2
expression. Flowcytometry and immunoblotting revealed that VEGF-C induces
S phase accumulation through the inhibition of p27 and the upregulation of
cyclin E and cyclin-dependent kinase-2 expressions. The cell-cycle-related
effects of VEGF-C were reversed by the depletion of COX-2 or ET-1. The
depletion of COX-2 or ET-1 also suppressed VEGF-C-induced increases
in the bcl-2/bax ratio and chemoresistance against etoposide and cytosine
arabinoside in AML cells. We also demonstrated VEGF-C/ET-1/COX-2 axismediated chemoresistance in an AML xenograft mouse model.
Conclusions: Our findings suggest that VEGF-C induces COX-2-mediated
resistance to chemotherapy through the induction of ET-1 expression. Acting
as a key regulator in the VEGF-C/COX-2 axis, ET-1 represents a potential
target for ameliorating resistance to chemotherapy in AML patients.
240 SOX14 downregulates SOX1 expression in HeLa cells
I. Petrovic1 , J. Popovic1 , D. Stanisavljevic1 , M. Schwirtlich1 , A. Klajn1 ,
J. Marjanovic1 , N. Kovacevic Grujicic1 , V. Topalovic1 , M. Mojsin1 ,
M. Stevanovic1 . 1 Institute of Molecular Genetics and Genetic Engineering
University of Belgrade, Laboratory for Human Molecular Genetics, Belgrade,
Serbia
Background: It has been reported that deregulation and/or amplification of
many SOX genes are associated with a large number of tumor types. SOX14
is a largely unexplored member of the SOXB2 subgroup of transcription
factors implicated mainly in neural development, while its closest relative,
SOX21, apart from the role in neural development was also proposed as tumor
suppressor. Having in mind redundant properties of SOX proteins, the similar
role of SOX14 in cancer was not reported, especially in context of functional
cross-talk between SOX14 and SOXB1 members. The aim of this study was
to analyze its activator/repressor properties and to test whether SOX14 may
affect SOXB1 members’ expression in HeLa cells.
Material and Methods: In order to analyze functional role of SOX14
in cancer model system we generated SOX14 expression construct. The
activation/repression property of human SOX14 protein was performed by
co-transfection experiments with SOX14 expression construct and SOXresponsive luciferase reporter gene in HeLa cells. In order to analyze the
potential cross-talk between SOXB1 and SOX14, HeLa cells were transiently
transfected with SOX14 expression construct and the effect of its ectopic
expression on SOXB members was analyzed by Western blot.
Results: Our data demonstrated that SOX14 overexpression reduced SOX1
protein level, while no significant effects on SOX2, SOX3 and SOX21
expression was observed. It was shown that SOX1 is highly methylated in high
grade squamous cell cervical carcinomas. On the other hand, a genome-wide
RNA interference (RNAi) screen in K-ras transformed NIH 3T3 cells identified
that SOX14 is one of 28 genes required for Ras-mediated epigenetic silencing
of the pro-apoptotic Fas gene. Based on those findings, SOX14 could be
involved in the positive or negative regulation of expression of genes implicated
in epigenetic silencing, and its elevated expression could increase promoter
hypermethylation of target genes, such as SOX1.
Conclusion: This is the first report showing that SOX14 could affect the
expression of SOX1. Since SOX1 is considered as tumor suppressor, the
molecular mechanisms involved in the regulation of its expression, that relies
on SOX14 gain additional significance. It would be interesting to explore
how elevated expression of SOX14 influences HeLa cells’ proliferation and
invasiveness, and what impact decreased expression of SOX1 has on the
aforementioned processes.
241 Bcl-xL protein overexpression enhances tumor progression of
human melanoma cells in zebrafish xenograft model: involvement
of interleukin 8
C. Gabellini1 , E. Gómez-Abenza1 , S. De Oliveira2 , D. Del Bufalo3 , V. Mulero1 .
1
University of Murcia, Department of Cell Biology and Histology Faculty of
Biology, Murcia, Spain, 2 University of Lisbon, Microvascular Biology and
Inflammation Unit Molecular Medicine Institute Biochemistry Institute Faculty
of Medicine, Lisbon, Portugal, 3 Regina Elena National Cancer Institute,
Experimental Chemotherapy Laboratory, Rome, Italy
Background: The anti-apoptotic protein bcl-xL enhances metastatic potential
in different tumor hystotypes and promotes tumor angiogenesis through
enhancing pro-inflammatory chemokine interleukin 8 (CXCL8) expression.
Using zebrafish as experimental model, we evaluated the impact of bclxL/CXCL8 axis in promoting melanoma angiogenesis and aggressiveness
in vivo.
Materials and Methods: To evaluate invasive and metastatic capability of
human melanoma M14 cell line stably overexpressing bcl-xL protein, cells
were implanted into the yolk sac of 2 days post-fertilization (dpf) zebrafish
embryos. To analyze the pro-angiogenic activity of recombinant human and
zebrafish CXCL8 proteins, they were injected in the yolk sac of 2dpf transgenic
fli1:EGFP embryos and tested for the capability to induce subintestinal vein
(SIV) sprouting. To test the pro-inflammatory activity of human and zebrafish
CXCL8 recombinant proteins, they were injected in the yolk sac of 2dpf
transgenic lyz:DsRed embryos and tested for neutrophil recruitment ability.
Results: Implantation of M14 trasfectants overexpressing bcl-xL protein into
zebrafish embryos resulted in higher significant dissemination from primary site
of injection and metastatization to distal parts of the larvae when compared
to control cell line. Since the enhanced invasiveness showed by bcl-xL
overexpressing cells might be due to their enhanced CXCL8 protein secretion,
we tested the capability of human recombinant CXCL8 protein to induce
angiogenesis in zebrafish, demonstrating that increasing doses of human
CXCL8 protein induced a significant increased percentage of larvae positive for
SIV sprouting. Next we demonstrated that also zebrafish Cxcl8-L2 elicits proangiogenic activity demonstrating that it is able to induce SIV sprouting, but at
a lesser extent than human CXCL8. However, when comparing the capability
of human and zebrafish CXCL8 to induce neutrophil recruitment, we found
that human CXCL8 is not able to induce neutrophil recruitment to zebrafish
yolk sac, in contrast to its zebrafish homologue. Next, we will investigate the
involvement of CXCL8 receptors Cxcr1 and Cxcr2, highly conserved between
humans and zebrafish, in the capability of bcl-xL protein to enhance melanoma
cell invasion in zebrafish larvae and with the aim of elucidating the signaling
pathways involved in the pro-angiogenic activity of human CXCL8 chemokine
in zebrafish.
Conclusions: These data elucidate the involvement of CXCL8 signalling
in bcl-xL-induced melanoma progression, supporting future studies for
developing new therapeutic approaches for tumors characterized by high bclxL expression. Moreover the conservation of pro-angiogenic activity of CXCL8
molecule in zebrafish establishes the possible use of this experimental model
to study the efficacy of novel compounds inhibiting CXCL8 axis to counteract
tumor progression.
242 Characterisation of retinoic acid effect on breast cancer cell
plasticity
G. Paroni1 , A. Zanetti1 , R. Affatato2 , E. Garattini1 . 1 Istituto di Ricerche
Farmacologiche Mario Negri, Biochemistry and Molecular Biology, Milano,
Italy, 2 Istituto di Ricerche Farmacologiche Mario Negri, Cardiovascular
Research, Milano, Italy
Introduction: In breast cancer, retinoids suppress tumor cell growth and
prevent mammary cancer in rodent models. Their efficacy has been explained
by growth inhibition and induction of apoptosis. Retinoids are required to
maintain the differentiated state of adult epithelia. In cultures of breast cancer
cell lines, we and others have shown that retinoids act as inducers of an
epithelial-like phenotype whose implication in the therapeutic setting has been
poorly investigated.
Increasing evidence supports an aberrant regulation of the epithelial to
mesenchymal transition (EMT) developmental process in breast tumour
progression. Indeed, EMT is associated with augmented motility and invasion.
Understanding the nature of retinoid induced modulation of breast cancer
plasticity represents a crucial issue for the use of retinoids as anti-tumor
agents.
Material and Methods: The effect of ATRA (all-trans retinoic acid) on a panel
of breast cancer cell lines was evaluated in 2D and 3D cultures. Western Blot
and Immunofluorescence analysis of EMT associated genes were performed.
As EMT cell model, the ATRA sensitive SKBR3 cell line was used. In this
cell line EGF and Heregulin induce a mesenchymal phenotype increasing the
migratory ability of the cells. The effect of ATRA in this experimental setting
was investigated. Migration was evaluated by Boyden Chamber assay. qRTPCR of EMT associated genes were performed to identify genes modulated by
ATRA treatment. ATRA mediated regulation of the identified genes was further
validated by Immunofluorescence and Western Blot analysis.
Results: ATRA causes a switch to an epithelial-like phenotype by inducing
a rearrangement in both tight and adherens junction complexes and by
modulating the expression of their constituent proteins. In cell lines amplified for
the retinoic acid receptor a, ATRA induces the formation of polarized/organized
structures reminiscent of mammary gland epithelium. Upon EMT induction
by growth factors the mesenchimal phenotype and the increased migratory
ability of the cells are counteracted by retinoids. A screening of EMT
determinants demonstrated that NOTCH1, Snail and miR200c are regulated
by ATRA. In particular, ATRA decreases EGF and Heregulin induced NOTCH1
expression, inhibits NOTCH1 transcriptional activity and phenocopies the
migration impairment triggered by the g-secretase (NOTCH) inhibitor DAPT.
Conclusion: Overall our data support a role for ATRA in breast cancer
plasticity whose further investigation is essential for a rational use of retinoids
in the clinics.
243 p27 is a haploinsufficient tumor suppressor in MENX rats and this
associates with the development of invasive medullary thyroid
carcinoma
N. Pellegata1 , S. Molatore1 , F. Neff1 , M. Irmler2 , E. Pulz1 , F. Roncaroli3 .
1
Helmholtz Zentrum München, Institute of Pathology, Neuherberg,
Germany, 2 Helmholtz Zentrum München, Institute of Experimental Genetics,
Neuherberg, Germany, 3 Imperial College, Neuro-Oncology Lab, London,
United Kingdom
Introduction: MENX is a multiple endocrine neoplasia (MEN) syndrome that
spontaneously arose in a rat strain. Affected animals develop within their first
year of life bilateral pheochromocytoma (incidence 100%), anterior multifocal
pituitary adenomas (100%), thyroid C-cell hyperplasia, and other tumors.
MENX was first reported to be inherited as a recessive trait. Indeed, we could
later confirm that affected rats are homozygous for a germline loss-of-function
frameshift mutation in Cdkn1b encoding p27. Germline mutations in p27 were
subsequently found also in human patients (MEN4 syndrome).
p27 is a negative regulator of the cell cycle and it is considered a tumor
suppressor. It has been shown that p27 is haploinsufficient for tumor
suppression in mice (i.e. one copy of the wild-type allele is not sufficient for
normal cellular function). Thus, we set out to determine whether p27 behaves
as a dose-dependent suppressor also in MENX rats by studying the phenotype
of heterozygous mutant rats.
Materials and Methods: Rats wild-type for Cdkn1b, or bearing one (p27+/mut)
or two copies (p27mut/mut) of the mutated Cdkn1b allele were sacrificed and
tissues collected. Normal and tumor tissues were analyzed histologically by
experienced pathologists. Expression of p27, hormones, tumor markers and
the proliferation marker Ki67 was assessed by immunohistochemistry. Gene
expression profiling of tumors and normal tissues was performed.
Results: p27+/mut develop the same neuroendocrine tumors (NETs) as
p27mut/mut rats but with slower progression: in p27+/mut rats, tumors at the
3 major organs (adrenal, pituitary and thyroid) are macroscopically detectable
around 16 months of age. Since the p27+/mut rats live significantly longer
than p27mut/mut rats (average survival 512 versus 243 days; p = 5.2e−20 ) their
tumors become very large. In thyroid glands of older p27+/mut rats, carcinomas
showing local invasion and occasionally distant metastases were identified.
The tumors in both p27+/mut and p27mut/mut animals are histologically very
similar, but, at the molecular level, while pituitary adenomas developing in both
rat groups share the main genetic signatures, pheochromocytomas do not.
Conclusion: MENX is a prototype disorder caused by a haploinsufficient
tumor suppressor gene, as the presence of a single functional p27 allele
is not sufficient to prevent NETs development in heterozygous rats. Tumors
in p27+/mut rats can be used to model human tumors with either reduced
p27 expression or bearing heterozygous somatic mutations which impair the
protein function (i.e. small intestine NETs). Our studies have unveiled a novel
model of invasive/metastatic MTC, which may facilitate the identification of
genes promoting the invasive and metastatic potential of MTC in both rats
and humans, thereby opening novel therapeutic avenues for this aggressive
cancer.
244 Aromatase expression contributes to the survival and metastasis
of estrogen receptor positive breast cancer cells
L.Z. Sun1 , K. Mukhopadhyay1 , Z. Liu1 , A. Bandyopadhyay1 . 1 UTHSCSA,
Cellular & Structural Biology, San Antonio Texas, USA
Introduction: In postmenopausal women, the local estrogen produced by
the adipose stromal cells in the breast is implicated for fueling the growth of
breast cancer. This raises the question of how estrogen receptor alpha (ERa)
positive metastatic breast cancer cells survive after they enter blood and lymph
circulation, where estrogen level is very low in postmenopausal women.
Material and Method: Human breast cancer CAMA-1 cell along with
aromatase-transfected MCF-7 (MCF7/Aro) and ZR75-1 (ZR75-1/Aro/CL10)
cells were cultured in low attached plate to mimic circulating tumor cells.
Quantitative real-time PCR and western blot were used to detect the
expression level of aromatase in each cell lines cultured in different conditions.
The apoptosis rate of tumor cells under different treatment were measured by
cell death detection ELISA. Orthotopic tumor formation and bone metastasis
progression of nude mice intracardiacally inoculated with tumor cells were
detected by whole mouse fluorescence imaging.
Result and Discussion: The aromatase expression was found to be
increased when ERa positive breast cancer cells were cultured in suspension.
Furthermore, treatment with the aromatase substrate, testosterone, inhibited
suspension culture-induced apoptosis whereas an aromatase inhibitor
attenuated the effect of testosterone suggesting that suspended circulating
ERa positive breast cancer cells may up-regulate intracrine estrogen activity
for survival. Consistent with this notion, a moderate level of ectopic aromatase
expression rendered a non-tumorigenic ERa positive breast cancer cell line not
only tumorigenic but also metastatic in female nude mice without exogenous
estrogen supplementation. The increased malignant phenotype was confirmed
S57
to be due to aromatase expression as the growth of orthotopic tumors
regressed with systemic administration of an aromatase inhibitor.
Conclusion: This study provides experimental evidence to suggest that
circulating hormone dependent breast cancer cells may utilize intracrine
estrogen signaling for their survival and dissemination to distant metastasis
sites.
245 CD9 is required for stromal cell invasion of breast cancer cells
G. Rappa1 , T. Green2 , A. Lorico2 . 1 Roseman University of Health Sciences,
Las Vegas, USA, 2 Roseman University of Health Sciences, Cancer Research
Center, Las Vegas, USA
Introduction: The formation of breast cancer metastases requires an interplay
between malignant epithelia and local stroma, including bone marrow-derived
multipotent stromal cells (MSC), in the tumor microenvironment. To spread and
reach distant sites, cancer cells not only move through the extracellular matrix,
but also invade stromal cells, in some cases determining the formation of
heterologous hybrids. We previously reported that spontaneous breast cancerMSC hybrids acquired increased malignancy and maintained mixed genetic
backgrounds in vivo (Rappa et al., Am J Pathol. 180(6): 2504, 2012). Here,
we investigated the role of CD9, a protein required for egg-sperm and muscle
cell fusion and involved in the metastatic process, in the invasion and fusion
of breast cancer cells with MSC.
Materials and Methods: We down-regulated CD9 expression in three different
human breast cancer cell lines by shRNA. By dynamic total internal reflection
fluorescence (TIRF) and confocal imaging, we investigated the role of CD9
in breast cancer-MSC invasion and fusion by comparing parental and CD9knockdown breast cancer cells. Then, the role of CD9 in breast cancer cell
growth and metastasis was evaluated in mice orthotopically implanted with
CD9-knockdown breast cancer cells.
Results and Discussion: Both CD9 knock-down and two different antiCD9 monoclonal antibodies significantly decreased the invasiveness of breast
cancer cells into MSC. Invasion was associated with spontaneous fusion of
MDA and MA-11 with MSC, which amounted to 0.68% and 0.12% of the
originally plated breast cancer cells, respectively. CD9 knock-down suppressed
and CD9 blocking antibodies reduced heterologous fusion; no invasion or
fusion events between human mammary epithelial cells and MSC were
observed. In immunodeficient mice, CD9-knockdown suppressed local tumor
growth of MDA-MB-231 breast cancer cells upon orthotopic implant in immunedeficient hosts and resulted in partial loss of metastatic capacity.
Conclusions: We have here demonstrated that CD9 has an important role in
the process of breast cancer cell invasion and fusion with MSC. CD9 targeting
is a potential strategy against the development of breast cancer metastases.
246 Effects of the potential energy restriction mimetic agent
delta2-troglitazone in breast cancer cells
I. Grillier-Vuissoz1 , C. Colin-Cassin2 , X. Yao1 , C. Cerella3 , A. Berthe1 ,
S. Mazerbourg1 , M. Boisbrun4 , S. Kuntz1 , M. Diederich5 , S. Flament1 .
1
Université de Lorraine, CRAN UMR 7039, Vandoeuvre les Nancy, France,
2
CNRS, CRAN UMR 7039, Vandoeuvre les Nancy, France, 3 Hôpital
Kirchberg, Laboratoire de Biologie Moléculaire et Cellulaire du Cancer,
Luxembourg, Luxembourg, 4 Université de Lorraine, SRSMC UMR 7565,
Vandoeuvre les Nancy, France, 5 Seoul National University, Department of
Pharmacy College of Pharmacy, Seoul, Korea
Background: The development of de novo and acquired resistance to
anticancer therapies and the absence of targeted therapy for some types
of tumors are strong motivations for discovering new therapeutic agents.
Thiazolidinediones (TZD) are studied in this context although they were initially
designed for the treatment of type II diabetes due to their PPAR gamma agonist
activity. They display anticancer effects that are independent of PPAR gamma,
but that are not well understood. In prostate cancer cells, TZD derivatives
were shown to act as energy restriction mimetic agents. Our team studies
the effects of delta2 troglitazone (delta2-TGZ), a TZD devoid of PPAR gamma
agonist activity, on breast cancer cell lines. The aim of this work was to better
characterize delta2-TGZ-induced cell death and to understand the underlying
molecular events.
Material and Methods: Delta2-TGZ was obtained by chemical synthesis.
We used hormone-dependent (MCF-7) and hormone-independent (MDAMB-231) breast cancer cell lines. Gene expression was studied by RT-PCR,
western blotting and immunocytochemistry. RNA interference was used for
gene silencing.
Results and Discussion: Delta2-TGZ induced endoplasmic reticulum (ER)
stress in MCF-7 cells as shown by phosphorylation of Pancreatic endoplasmic
reticulum kinase-like Endoplasmic Reticulum Kinase (PERK) detected after 1.5
hours, splicing of XBP1 (X-box binding protein 1) after 3 hours, accumulation
of the chaperone BiP (Binding immunogloblulin protein) and the pro-apoptotic
protein CHOP (CCAAT-enhancer-binding protein homologous protein) after
S58
6 hours. CHOP was located in the nucleus of treated cells. Similar events
were observed in MDA-MB-231 cells exposed to delta2-TGZ. In both cell
lines cleavage of PARP-1 and caspase-7 revealed apoptosis. Moreover, the
compound induces mitochondrial potential loss, caspase-7 cleavage and
PARP cleavage (as estimated after 24 and 48 h) similarly to the apoptogenic
inducer staurosporine. Incubation with the pan-caspase inhibitor z-VAD
confirmed the relevance of caspase activation for the cell demise. In MCF-7
cells, knock-down of CHOP or inhibition of c-Jun N-terminal kinase (JNK) did
not impair apoptosis. Preliminary results also indicated that AMP-dependent
kinase (AMPK) is activated early in MCF-7 cells after delta2-TGZ treatment.
Conclusions: This work contributes to a better understanding of the
PPARgamma-independent effects of TZD in breast cancer cells. ER stress
is an early response to delta2-TGZ, occurring prior to, but not causative of
apoptosis. We have now to confirm whether AMPK phosphorylation is a marker
of the energy restriction mimetic action of delta2-TGZ and if this is responsible
for ER stress and apoptosis.
247 Ovarian cancer cells treated with leptin contribute with a
pro-tumoral phenotype in macrophages in vitro
L. Abarzua-Catalan1 , S. Kato1 , A.M. Delpiano1 , G.I. Owen2 , M.A. Cuello1 .
1
Departamento de Obstetricia y Ginecologı́a, Centro de Investigaciones
Médicas Facultad de Medicina Pontificia Universidad Católica de Chile,
Santiago, Chile, 2 Departamento de Fisiologı́a, Facultad de Ciencias
Biológicas Pontificia Universidad Católica de Chile, Santiago, Chile
Background: Tumor-associated macrophages (TAMs) are the dominant
leukocyte population found in the tumor microenvironment. TAMs are derived
from circulating monocytes or resident tissue macrophages, and ‘re-educated’
by cancer cells promoting tumor initiation, growth, and development. In ovarian
cancer different signals may contribute with changes in TAM phenotype.
Leptin is a pro-inflammatory cytokine secreted by adipose tissue involved in
proliferation and tumorigenesis in ovarian cancer cells. Here we explored if
ovarian cancer cells treated with leptin modulate function and phenotype of
macrophages to TAM in-vitro.
Methods: Ovarian cancer cell line HEY was treated with or without leptin
(100 ng/ml) for 6, 12 and 24 hours and the conditioned medium (CM) was
harvested for each condition. A monocyte cell line THP-1 was differentiated
to macrophage and cells were incubated with each collected CM from HEY
cells during 24 hours. We measured IL-6Ra, IL-6, Vegf and Tgf-b mRNA
expression levels by Real-time PCR and IL-6 and IL-1 production (ELISA) in
macrophages.
Results: In macrophages treated with CM from HEY cells with leptin, we found
significant increase in Tgf-b mRNA levels at 6 and 24 hours and a decrease
in Vegf mRNA levels at 12 h, but we did not observe changes in IL-6Ra and
IL-6 mRNA expression levels (folds respect to control). Preliminary result from
ELISA assay shows that incubation of CM (HEY cells treated with leptin) with
macrophages increase IL-6 and IL-1 secretion compared with direct effect of
leptin in differentiated THP1 cells.
Conclusion: Here we have shown that leptin treatment in HEY cancer cells
induce ‘factors’ release that contribute with an increase in mRNA expression
levels of Tgf-b, IL-6 and IL-1 secretion by macrophages. These results suggest
a change in macrophages phenotype similar to TAMs which secrete many
different cytokines, chemokines and proteases involved in cancer progression
and immunosupression.
Grants: Fondecyt 1120292, Fondecyt Postdoctorado 3140335, BMRC
CTU06.
248 Radiotherapy response of breast cancer stem cells
P. Cordeiro1 , T. Costa1 , M.J. Carvalho2 , M. Laranjo3 , P. Rachinhas4 ,
J.E. Casalta-Lopes5 , A.M. Abrantes3 , M. Botelho6 . 1 Faculty of Medicine
University of Coimbra, Biophysics Unit, Coimbra, Portugal, 2 Coimbra Hospital
and Universitary Centre, Gynaecology A Service, Coimbra, Portugal, 3 Faculty
of Medicine of University of Coimbra, Biophysics Unit CIMAGO IBILI,
Coimbra, Portugal, 4 Coimbra Hospital and Universitary Centre, Radiation
Oncology Department, Coimbra, Portugal, 5 Faculty of Medicine of University
of Coimbra, Biophysics Unit, Coimbra, Portugal, 6 Faculty of Medicine
University of Coimbra, Biophysics Unit IBILI CIMAGO, Coimbra, Portugal
Introduction: In clinical practice, breast cancer (BC) is stratified into 3 groups:
tumors that express hormonal receptors (HR), tumors overexpressing Her2,
and those which do not express HR nor Her2, the triple negative (TN) BC.
The recent theory of cancer stem cells (CSC), refer to a small tumor cell
population that has ability of differentiation and self-renewal. It is believed that
CSC are responsible for tumor progression, recurrence as well as resistance
to therapy. With this work we intend to evaluate response of radiotherapy of
CSC of these tree types of BC.
Materials and Methods: BC cell lines MCF-7 that express HR, HCC1954 that
overexpress HER-2 and HCC1806, TN, were submitted to mammosphere (MS)
forming protocol. The first MS generation (MS1) was cultured in adherent
conditions. This procedure was repeated in order to obtain successive
generations of MS (MS1, MS2, MS3) and MS-derived cells in adherent
conditions (G1, G2, G3). These cell populations were irradiated with 0, 0.5,
15 and 30 Gy in a Clinac 600 linear accelerator with a 4MV photon beam.
Response to radiotherapy was evaluated 24, 48 and 72 h later using MTT,
Alamar-Blue and clonogenic assay (CA).
Results: Irradiation with 0.5 Gy did not show differences between the 3
cell lines and for the times considered, except for 48 hours. In this time,
proliferation of HCC1954 was inferior to HCC1806 (p = 0.002). Considering
15 Gy, at 48 hours of evaluation, there were differences between the three cell
lines, with a decrease in proliferation for HCC1806 (p < 0.001) and the highest
proliferation for MCF7 (p = 0.019) in comparison with HCC1954. At 72 hours,
there was a higher proliferation rate for MCF7 than HCC1806 (p = 0.027) and
HCC1954 (p = 0.002). After irradiation of cell lines with 30 Gy, MCF7 presents
a proliferation superior to HCC1806, either for 48 hours (p < 0.001) or for 72
hours (p = 0.002). Nevertheless, comparing MCF7 with HCC1954, there were
only differences considering evaluation at 72 hours (p = 0.021). Comparing
HCC1954 with HCC1806, there were only differences in 48 hours proliferation,
which was superior in the first cell line (p = 0.001). CA revealed an increase
in proliferation inhibition with dose increase, reaching statistical significance
between 0.5 Gy and 30 Gy (p = 0.007). Comparing HCC1806 and the MS derived adherent cells, there was a significant result for 30 Gy between HCC1806
and HCC1806-G1 (p = 0.002). Comparing MCF7 and MS-derived adherent
cells (MCF7-G1 and MCF7-G3), there was a significant difference comparing
15 Gy for MCF7 and MCF7-G1 (p < 0.001), being the survival fraction inferior
in MCF7-G1. It is apparent a resistance to irradiation in MCF7-G3.
Conclusion: There is a differential response to radiation considering the
different cell lines and MS and MS-derived adherent cells. Tumor cells
population with stem cells properties seem more resistant to radiotherapy,
particularly considering subsequent generations isolated.
249 Radiosensitive and radioresistant colorectal cancer cells
A. Ferreira1 , J.E. Casalta-Lopes1 , M. Laranjo2 , A.M. Abrantes2 , A. Cavaco3 ,
M. Borrego3 , P. Soares3 , M. Botelho4 . 1 Faculty of Medicine University of
Coimbra, Biophysics Unit, Coimbra, Portugal, 2 Faculty of Medicine University
of Coimbra, Biophysics Unit CIMAGO IBILI, Coimbra, Portugal, 3 Coimbra
Hospital and Universitary Centre, Radiation Oncology Department, Coimbra,
Portugal, 4 Faculty of Medicine University of Coimbra, Biophysics Unit IBILI
CIMAGO, Coimbra, Portugal
Introduction: Radiochemotherapy is used as neoadjuvant treatment for locally
advanced rectal cancer, allowing tumor downstaging and improving local
control. However, not all patients have therapeutic benefit from treatment
because cancer cells may be refractory. A desirable focus of research is to
understand which factors predict sensitivity or resistance to the neoadjuvant
therapy. We aimed to establish a radioresistant colorectal cell line and to
compare metabolic activity and viability of both sensitive and radioresistant
colorectal cancer cells, after treatment with 5-fluorouracil (5-FU), radiation
alone and combined therapy.
Material and Method: WiDr cell line was used to study radioresistance. Cells
were irradiated in 10 cumulative fractions of 2 Gy using a Varian Clinac 600
linear accelerator with a 4MV photon beam. To measure in vitro response to
ionizing radiation the clonogenic assay was used and survival curves were
plotted according to the linear-quadratic model.
Parental WiDr cell line and one derivative were submitted to chemotherapy,
radiation alone, and chemoradiotherapy. Cells were submitted to 20 mM of
5-FU for chemotherapy and irradiated with 0, 2 and 10 Gy for radiation alone.
Cells treated with combined therapy were exposed to 20 mM of 5-FU three
hours prior irradiation and then irradiated with the same doses of radiation.
Metabolic activity was assessed by the MTT assay 24 and 96 hours after
treatment and comparisons between both cell lines were made. Flow cytometry
using annexin-V/Propidium iodine double labeling was performed 96 h after
treatment in order to assess cell viability.
Results and Discussion: Resistance to radiotherapy was observed in cell
lines irradiated from 6 to 10 times (WiDr/6r and WiDr/10r, respectively).
Statistically significant differences were observed in surviving fraction (SF)
between WiDr, WiDr/6r and WiDr/10r cell lines. Pairwise comparisons showed
a higher SF for WiDr/10r (p < 0.001) and WiDr/6r (p 0.002) when comparing
to WiDr. However, no differences between WiDr/6r and WiDr/10r were
observed.
Comparing both cell lines after treatment, we verified differences at 24 hours
with 10 Gy, 5-FU + 2 Gy and 5-FU + 10 Gy, since WiDr/6r cells showed superior
metabolic activity than WiDr cells. When it comes to 96 hours, despite both
cell lines exhibited lower metabolic activity than at 24 hours, the WiDr/6r cells
showed significantly superior metabolic activity than the WiDr ones with 5-FU
alone, 5-FU + 2 Gy and 5-FU + 10 Gy. Regarding cell viability, WiDr/6r cells
showed higher cell viability when treated with 10 Gy, related to WiDr.
Conclusions: We were able to establish radioresistant cell lines from WiDr
cell line. The resistant phenotype was observed as soon as after 6 cumulative
fractions of 2 Gy. The results obtained by the MTT assay suggest that
radioresistant WiDr/6r cell line shows resistance to chemoradiotherapy, as well
as to chemotherapy alone.
250 Apoptosis and proliferation in micropapillary structures of
colorectal polyps and carcinomas
M. Patankar1 , S. Sajanti1 , A. Tuomisto1 , M. Mäkinen1 , T. Karttunen1 .
1
Institute of Diagnostics, Department of Pathology, Oulu, Finland
Background: Micropapillary structures (MIP) can be defined as focal piles
of epithelial cells in columnar epithelium. They are found in several cancer
types, but they are characteristic for serrated colorectal carcinomas and their
precursor lesions. However, in lesser amounts they can be found in nonserrated (conventional) colorectal polyps and carcinomas. Suprabasal cells
of MIP have no contact with the extracellular matrix (unpublished). We have
assessed apoptosis and cell proliferation rates in MIP in colorectal polyps and
carcinomas to evaluate whether this subpopulation of tumor cells would show
evidence for anoikis inhibition.
Materials and Methods: We stained sections from human colorectal
lesions including conventional adenomas (n = 15), serrated polyps (n = 29),
conventional adenocarcinomas (n = 32), and serrated adenocarcinomas
(n = 30) with antibodies to Ki67 and M30. Two independent observers counted
positive cells separately in the epithelial cells of micropapillary structures (MIP)
and non-micropapillary epithelium (non-MIP).
Results: In carcinomas, apoptosis rate was lower in the cells of MIP
(M30; median 0, range 0−40) than in the non-MIP cells (median 2, range
0−40; p < 0.001, Wilcoxon). Similarly, proliferation index was lower in the
MIP cells (Ki67; MIP: median 17, range 0−93; non-MIP: median 30, range
1−93; p < 0.001). Similar differences between MIP and non-MIP cells were
evident in subsets of serrated lesions, in both carcinomas (p < 0.001) and
polyps (p < 0.001), and in non-serrated carcinomas (p < 0.004). In nonserrated adenomas only M30 index was lower in the MIP (p < 0.001).
Apoptosis/proliferation ratio was lower in the MIP cells than in the non-MIP
epithelium in both cancers and their precursor lesions (P < 0.01).
Conclusion: Apoptosis rate was lower in micropapillary structures than
in other subpopulations of tumor epithelium indicating that the cells in
these structures are able to survive without matrix contact, i.e. anoikis is
inhibited in them. Lower cell proliferation rate could suggest that cells in
the MIP are in some kind of quiescent stage. However, significantly lower
apoptosis/proliferation ratio in the MIP infers that these structures may form
a fast growing subpopulation of tumor cells. Further studies are needed to
dissect the mechanism of formation of MIP and those related with matrix
independent survival of tumor cells in them.
251 Polo-like kinase 4 (plk4) modulates cancer cell polarity and
invasion
K. Kazazian1 , R. Xu1 , O. Brashavitskaya1 , F. Zih1 , C.J. Swallow1 . 1 Lunenfeld
Tanenbaum Research Institute, Research, Toronto, Canada
Background: High expression of Plk4 has been identified as a molecular
predictor of aggressive behaviour and resistance to therapy in breast,
pancreas and colorectal cancers. During a genome-wide screen for secondary
alterations in Plk4-related tumours, we identified an unexpected correlation
between Plk4 status and motility gene expression.
Our hypothesis is that Plk4 functions as an oncogene, enhancing cancer cell
invasion by modulating cytoskeletal dynamics and cell polarity. Our present
objectives are to explore the role of Plk4 in cancer cell migration and
invasion, in order to better understand the pathways and networks that facilitate
metastatic capacity.
Methods and Results: Flag-Plk4 transfected HeLa cells showed increased
protrusion formation and spreading compared to Flag-Plk4 K41M (kinasedead) and control (p < 0.001 vs. Flag and Flag-Plk4 K41M, n = 6
independent experiments). Plk4 knockdown using siRNA (pool of 4 and
individual constructs) decreased HeLa spreading and caused a rounded
morphology (p < 0.01 vs. siLuciferase, n = 4 independent experiments). Rescue
experiments confirmed phenotype specificity for the siRNA target gene.
Scratch wound healing was delayed with Plk4 knockdown (p < 0.01 vs.
siLuciferase, n = 4 independent experiments), demonstrating an effect of
Plk4 on 2D migration. Following a scratch wound, an appropriately oriented
Golgi apparatus was detected in 50% of cells that had been depleted of
Plk4, compared with 70% in siLuciferase control (p < 0.05, n = 3 independent
experiments). Furthermore, Plk4 siRNA transfected HeLa cells showed
decreased invasion through Matrigel in an automated transwell invasion assay;
this was true for the pool as well as 3 of 4 individual constructs. In all assays,
the observed effects were not attributable to altered viability or proliferation.
A HEK293 cell line stably expressing Flag-Plk4 under tetracycline control was
S59
generated and AP-Mass Spec performed. Interaction proteomics identified
novel Plk4-interacting proteins, including a RhoA GEF, in keeping with potential
involvement in the regulation of cytoskeletal dynamics.
Conclusions: Plk4 enhances HeLa cancer cell spreading, migration, invasion
and development of cell polarity. Motility-related Plk4 interacting proteins may
mediate these effects. These results support the pursuit of Plk4 inhibitors for
clinical use in patients who experience cancer progression on conventional
chemotherapy.
252 RhoGTPase-based regulation of cell spreading by Plk4
V. Brashavitskaya1 , K. Kazazian1 , R. Bagshaw1 , C.O. Rosario1 , F.S.W. Zih1 ,
Y. Haffani1 , J.W. Dennis1 , T. Pawson1 , C.J. Swallow1 . 1 Lunenfeld Tanenbaum
Research Institute, Research, Toronto, Canada
Background: Polo-like kinase 4 (Plk4) is a serine-threonine kinase that
localizes to centrioles and is essential for centriole duplication. Plk4 expression
is increased in colorectal, pancreas and breast cancers, and predicts
resistance to therapy and poor survival. While centriolar overduplication and
multipolar spindle formation is one mechanism by which dysregulated Plk4 can
facilitate oncogenesis, our laboratory has found that Plk4 promotes spreading,
migration and invasion of fibroblasts and of cancer cells by mechanisms
currently under investigation. Haploid levels of Plk4 are associated with an
increased incidence of improper cleavage furrow positioning during mitotic
division, and we previously showed that RhoA is not adequately activated to
effect proper actomyosin ring placement and contraction in Plk4 heterozygous
murine embryonic fibroblasts (MEFs). We showed that the effect of Plk4 on
RhoA activation is mediated by phosphorylation of the GEF Ect2 by Plk4
(Rosario et al., PNAS 2010). The small Rho GTPase RhoA is known to
regulate cell spreading and motility.
We hypothesize that Plk4 promotes cancer cell spreading by altering the
activation of RhoA through one or more RhoA GEFs or GAPs.
Methods and Results: To determine the mechanism of the effect of Plk4
on RhoA activation we investigated the upstream regulators that may be
affected by Plk4, aside from Ect2. We scanned a library of 148 GEFs and
GAPs cloned into the Creator system and identified 12 potential interactors
(all GEFs) that contain the Plk4 consensus phosphorylation motif; 5 of the
potential substrates are RhoA GEFs. We are evaluating these 5 candidates
for physical interaction with Plk4 by co-transfection and co-immunoprecipitation
from HeLa cells. By this method, we find that Plk4 physically interacts with the
RhoA GEF ARHGEF1. To establish functional validation of this interaction,
we are examining the phenotype of spreading HeLa cells. As expected,
depletion of Plk4 from HeLa cells (siPlk4 for 48 h) resulted in a decrease in
spreading, as measured by cell area. Transient transfection with ARHGEF1 for
18 h decreased HeLa spreading. Decreased spreading was similarly observed
in control cells that were transiently transfected with constitutively active
RhoA. These results suggest that Plk4 might be regulating the activation of
RhoA during cell spreading by inhibiting ARHGEF1. Ongoing experiments
will determine the effect of ARHGEF1 and Plk4 on RhoA activation during
spreading and test for dependence on the Plk4–ARHGEF1 interaction.
Conclusions: We demonstrate a physical interaction of Plk4 with the RhoA
GEF ARHGEF1. Spreading assays suggest a functional relevance of this
interaction. Regulation of cancer cell spreading and motility via this pathway
may contribute to promotion of invasion and metastasis by Plk4.
253 Expression of TRF2 and its prognostic relevance in advanced
stage cervical cancer patients
O. Orun1 , S. Ozden2 , P. Mega Tiber1 , N. Serakinci3 , Z. Ozgen4 , H. Ozyurt2 .
1
Marmara University, Biophysics, Istanbul, Turkey, 2 Dr. Lutfi Kirdar Kartal
Education and Research Hospital, Clinic of Radiation Oncology, Istanbul,
Turkey, 3 Near East University, Medical Genetics Department, Istanbul, Turkey,
4
Marmara University, Department of Radiation Oncology, Istanbul, Turkey
Introduction: Telomeres are protective caps consisting of specific tandem
repeats (5 -TTAGGG-3 ). Shortening of telomeres at each cell division is
known as ‘mitotic clock’ of the cells, which renders telomeres as important
regulators of lifespan. TRF2 is one of the critical members of shelterin
complex, which is a protein complex responsible for the preservation of cap
structure, and loss or mutation of TRF2 results in DNA damage, senescence
or apoptosis. Since cancer is frequently associated with aberrant cell cycle
progression, defective DNA repair or apoptosis pathways, TRF2 could be one
likely candidate for cancer therapy.
Materials and Methods: Here we investigated prognostic role of TRF2 levels
in cervical cancer patients. TRF2 expression was determined in cervical cancer
patients attending the Dr. Lutfi Kirdar Kartal Education and Research Hospital
between 2005 and 2006. Mean follow up time was 71 months, median age
of patients was 56 (range 39−75). Fold-induction rates were evaluated and
assessed as ‘high’ or ‘low’ levels with respect to median values after real-time
S60
PCR analysis. Overall survival, distant disease free- and local recurrence-free
survival rates were calculated using Kaplan–Meier log rank test.
Results: Both five year overall- and disease-free survival rates were longer
in patients with higher TRF2 expression compared to lower expression, but
results were not statistically significant (60% vs 50%, respectively). Local
recurrence-free survivals were also very similar but lower expression group
showed better profile (mean values and calculated 95% confidence intervals
were 70 months [95% CI 43.6–97.3] vs 75 months [95% CI 52.9–97.9] for high
and low expressions, respectively). Survival was closely correlated with BclXL and p53 as expected, but not with TRF2 (P = 0.001, 0.055 and 0.389,
respectively).
Conclusion: Our results show that even though TRF2 is an important factor
in chromatin stability and higher expression levels of TRF2 resulted in better
survival rates, it is not a suitable prognostic factor for cervical cancer.
Acknowledgements: This study was supported by the following grants:
TUBITAK 111S165 and Marmara University Research Fund SAG-D-1004130119.
254 Role of prominin-1 (CD133)-exosomes released by melanoma
cells in intercellular communication
A. Lorico1 , T. Green1 , F. Anzanello1 , G. Rappa1 . 1 Roseman University of
Health Sciences, Cancer Research Center, Las Vegas, USA
Introduction: Tumor-derived exosomes are implicated in neoplastic growth
and in the metastatic process. We previously reported that CD133 had a
pro-metastatic role in melanoma cells (Stem Cells 26: 3008, 2008) and
that microvesicles and exosomes released from metastatic melanoma cells
expressed high levels of CD133 (Exp Cell Res 319: 810, 2013; Mol Cancer
12:62, 2013). We have here characterized the interaction of CD133-exosomes
released by human FEMX-I metastatic melanoma cells with other cells in the
microenvironment.
Materials and Methods: We employed CD133-based immunomagnetic
selection in combination with filtration and ultracentrifugation to purify CD133expressing exosomes from melanoma cells. Dynamic total internal reflection
fluorescence (TIRF) and confocal imaging were used to image live exosomal
entry into target cells.
Results and Discussion: By mass spectrometry, we observed that the
tetraspanin CD9 was among the ten most expressed proteins in CD133immunomagnetically purified melanoma exosomes. Upon stable expression
of the CD9-GFP fusion proteins in melanoma cells, cells were cultured
under serum-free conditions and GFP+ exosomes were isolated. Progressive
accumulation of exosomes was observed during a 3 h time period after their
addition to cultures of melanoma and human bone marrow-derived stromal
cells (MSC). While melanoma cells accumulated exosomes in a perinuclear
region, in MSC exosomes were scattered throughout the cytoplasm. Shortterm co-culture of melanoma cells and MSC resulted in heterologous
CD133 transfer. Exposure of MSC to melanoma exosomes increased their
invasiveness.
Conclusions: Our study supports the concept that a specific CD133+
population of cancer exosomes contains determinants of the metastatic
potential of the cells from which they are derived.
255 Apoptosis-inducing effect of Usnea filipendula Stirt. in breast
cancer cells in vitro
M. Sarimahmut1 , S. Celikler1 , F. Ari1 , S. Oran1 , N. Aztopal1 , S. Ozturk1 ,
E. Ulukaya2 . 1 Uludag University, Department of Biology, Bursa, Turkey,
2
Uludag University, Department of Medical Biochemistry, Bursa, Turkey
Background: Successful cancer treatment still needs novel compounds
from any sources (e.g. lichens). Lichens are complex organisms and
their metabolites may have diverse biological activities such as antiviral,
antimutagenic, antioxidant, antiinflammatory, antiproliferative and cytotoxic
effects. Thus, we investigated cytotoxic and genotoxic activities of the methanol
extract of U. filipendula (UFE) in MCF-7 and MDA-MB-231 breast cancer cell
lines and human lymphocytes, respectively.
Material and Methods: Anti-growth effect was assayed by the MTT and
ATP viability assays. The mode of cell death was evaluated morphologically
after nuclear stainings with fluorescent probes. Apoptosis was evaluated
biochemically by the measurement of caspase-cleaved cytokeratin 18 (M30),
caspase-3 activity and of poly-(ADP-ribose) polymerase (PARP) cleavage by
ELISA and Western blotting. Genotoxic activity was studied using chromosome
aberration, micronuclei and Comet assays in human lymphocytes culture.
Results: Usnea filipendula had anti-growth effect in a dose dependent manner.
IC50 values were determined as 23 and 44.7 mg/ml for MCF-7 and MDAMB-231 cells, respectively. Cell death occurred by apoptosis as evidenced
by the pyknotic nucleus in both cells. Cleavage of PARP and increased M30
levels further support apoptotic cell death in MCF-7 cells. In addition to these
two, caspase-3 activation was detected in MDA-MB-231 cells. It also showed
genotoxic activity at the doses of 125 and 250 mg/ml.
Conclusions: As a result, U. filipendula has a cytotoxic effect on breast
cancer cell lines while exhibiting genotoxic effects at relatively higher doses.
To the best of our knowledge, this is the first report showing the cytotoxic and
genotoxic activity of U. filipendula in breast cancer cells, which warrants further
in vivo experiments.
257 TXNRD2 and DCBLD2 are novel targets of osteosarcoma
metastasis
E. Topkas1 , N. Cai1 , A. Cumming1 , N. Saunders1 , L. Endo-Munoz1 .
1
Translation Research Institute, Diamantina Insitute, Woolloongabba,
Australia
Background: Osteosarcoma (OS) accounts for 56% of malignant bone
cancers in children and adolescents. Pulmonary metastasis occurs in
approximately 50% of patients with a 5 year survival rate of only 20%.
Therefore it is crucial to identify genes and pathways that drive the metastatic
behavior of OS for the identification of therapeutic targets.
Material and Methods: To identify markers that may define inherent metastatic
OS we conducted microarray-based comparative profiling analysis of clonal
variants from an inherently metastatic cell line, KHOS. Two highly metastatic
(C1 and C6) and two poorly metastatic clones (C4 and C5) were compared in
the transcriptomic screen.
Results: Vascular endothelial growth factor A (VEGFA), discoidin CUB and
LCCL domain containing 2 (DCBLD2) and thioredoxin reductase 2 (TXNRD2)
were identified as potential markers for OS metastasis with 2−4 fold increased
expression in highly metastatic clonal variants. The transcriptomic expression
of VEGFA, DCBLD2 and TXNRD2 was also investigated in non-malignant
bone (NB), OS patients with non-metastatic (NM) and metastatic (M) disease.
All three markers were found to be highly expressed in 29−42% of M-OS with
little to no expression seen in NB and NM-OS. Knockdown of DCBLD2 using
shRNA showed a significant decrease pulmonary metastasis in vivo. Targeting
TXNRD2 with a commercially available drug specific inhibitor, auranofin, we
showed significantly reduced migration and invasion in vitro. In an orthotopic
mouse model of OS, auranofin treatment significant decrease pulmonary
metastasis in a number of metastatic OS cell lines.
Conclusions: This transcriptomic screen identified TXNRD2 and DCBLD2
which are strong and novel therapeutic targets of OS metastasis.
258 Stimulated prostatic angiogenesis in senescence resembles
the microenvironment of neoplastic lesions and can be reversed
by antiangiogenic therapy
F. Montico1 , L.A. Kido1 , A.C. Hetzl1 , V.H.A. Cagnon1 . 1 State University of
Campinas, Structural and Functional Biology Department, Campinas SP,
Brazil
Introduction: Angiogenesis is essential for the progression of malignant
disorders. Antiangiogenic drugs represent promising alternatives in the
treatment of prostate cancer, an age-associated disease. Finasteride also
interferes with prostatic angiogenesis due to its anti-androgenic action. Thus,
the aim herewith was to characterize prostatic angiogenic responses during
senescence and after antiangiogenic and/or hormone blocking therapies,
comparing them to the transgenic adenocarcinoma of mouse prostate
(TRAMP) model.
Material and Methods: Aged male mice (52-week-old) were treated with
SU5416 and/or TNP-470. Finasteride was given alone or associated to both
inhibitors. Dorsolateral prostate was collected for immunohistochemical and
Western blotting analyses of the pro-angiogenic vascular endothelial growth
factor (VEGF), fibroblast growth factor-2 (FGF-2), hypoxia-inducible factor
1-alpha (HIF-1a) and transforming growth factor beta (TGF-b) as well as of the
antiangiogenic factor Endostatin. Microvessel density (MVD) was determined
for the different experimental groups by means of CD31 immunolabeling to
evaluate the prostatic angiogenic status.
Results: Prostatic microenvironment in senescence showed increased VEGF
and FGF-2, as well as raised MVD, resembling the features in the TRAMP
model. However, while elderly mice’s prostate characterized increased HIF-1a,
TGF-b and Endostatin levels, progressive decline of these molecules was
observed during cancer progression in TRAMP. Antiangiogenic therapies
resulted in reduction of pro-angiogenic factors and MVD whereas the combined
treatment with SU5416 and TNP-470 was able to simultaneously maintain high
levels of Endostatin. In contrast, androgen ablation led to higher TGF-b levels,
although decreased MVD was also verified in the finasteride-treated groups.
Conclusions: Thus, prostatic angiogenesis is stimulated in senescence,
resembling the microenvironment during prostate cancer progression and
therefore predisposing the gland to malignancy. HIF-1a and TGF-b may be
involved in the early steps of angiogenesis induction during tumorigenesis,
considering their increase in preneoplastic lesions. Antiangiogenic drugs are
efficient to reverse the pro-angiogenic stimuli and the neovascularization in
senescence, showing enhanced effects when administered in combination.
Androgen ablation as an antiangiogenic approach must be looked carefully,
since finasteride has positive effects on TGF-b stimulatory pathways.
259 Procathepsin B and endogenous inhibitors of cysteine proteases
as possible biomarkers of ovarian cancer
E. Gashenko1 , V. Lebedeva2 , E. Tsykalenko3 , G. Russkikh4 , K. Loktev5 ,
I. Brak5 , T. Korolenko1 . 1 Institute of Physiology and Fundamental Medicine
Siberian Branch of Russian, Cellular Physiology & Biochemistry, Novosibirsk,
Russian Federation, 2 Novosibirsk Medical University, Department of
Oncology, Novosibirsk, Russian Federation, 3 Novosibirsk Medical University,
Department of Clinical Biochemistry, Novosibirsk, Russian Federation,
4
Institute of Biochemistry Siberian Branch of Russian, Department of Clinical
Biochemistry, Novosibirsk, Russian Federation, 5 Institute of Physiology
and Fundamental Medicine Siberian Branch of Russian, Department of
Neurophysiology, Novosibirsk, Russian Federation
Background: The search of new biomarkers in human tumors is important,
especially for early diagnostic of ovarian cancer. Tumor cells as well as
tumor-associated macrophages have been shown to secrete active forms
of proteases and their inhibitors; however, their roles, especially those of
proenzymes as markers of malignancy, are still under investigation.
Aim: To evaluate procathepsin B and endogenous inhibitor of cysteine
proteases (cystatins B and C) as possible tumor biomarkers in ovarian tumors
compared to the commonly used tumor biomarker CA-125.
Material and Method: Serum and ascetic fluid of 38 patients with ovarian
cancer (among them 15 patients after treatment) and benign ovarian
tumors (n = 9) from Department of Gynecology of Regional Oncology
Center, Novosibirsk, were under investigation. Serum of practically healthy
women aged 18−80 (n = 82), from Regional Diagnostic Center, Novosibirsk
was used as a control group. Serum procathepsin B concentration was
measured by ELISA commercial kit for human (R&D); cystatin C using Bio
Vendor commercial kits (Czechia), cystatin B − with help of ELISA kits
for human (USCN Life Science Inc., Wuhan, China). Common biomarker
of ovarian cancer, CA-125, was assayed by using a commercial kit
(Vector, Koltsovo, Novosibirsk Region, Russia). Statistical analysis was
performed by one-way ANOVA Statistic 10 Program with help of Kruskal–
Wallis test.
Results and Discussion: In patients with benign ovarian tumors increased
serum common tumor marker CA-125 (p = 0.005 vs healthy controls) was
shown without any changes in serum level of cystatin C, cystatin B and
procathepsin B. However, in serum of patients with ovarian cancer significant
increase in procathepsin B (p < 0.001), cystatin B (p = 0.024) and CA-125
(p < 0.001) were noted. In serum (p < 0.05) and ascetic fluid (p < 0.01) more
significant increase in procathepsin B was shown in ovarian cancer vs benign
ovarian tumors.
Conclusion: One can conclude that serum procathepsin B is perspective
as a new tumor biomarker in ovarian cancer. Procathepsin B and cystatin B
seem important in differential diagnostic of ovarian cancer and benign ovarian
tumors.
260 A novel MYC directed apoptosis pathway controls NOXA and
BIM transcription
M. Wirth1 , N. Stojanovic1 , R. Schmid1 , O.H. Krämer2 , D. Saur1 ,
G. Schneider1 . 1 Klinikum rechts der Isar, II. Department of Internal Medicine,
München, Germany, 2 University of Mainz, Institute of Toxicology, Mainz,
Germany
Introduction: The MYC oncogene is an important driver of human cancers.
However, MYC can also induce apoptosis of tumor cells, although this MYC
function warrants further clarification.
Materials and Methods: We used proteasome inhibitor (bortezomib) induced
apoptosis in pancreatic cancer, colon cancer and genetically defined mouse
embryonic fibroblasts (MEFs) to investigate MYC regulated death pathways.
Several gain and loss of function experiments were used to define the role of
MYC, EGR1, BIM and NOXA for bortezomib-induced apoptosis. Quantitative
promoter-scanning chromatin immunoprecipitations (qChIP) was performed to
investigate transcriptional regulation and promoter binding.
Results and Discussion: We show that bortezomib significantly increased
MYC protein levels, thereby triggering apoptosis. MYC-induced cell death is
mediated by enhanced expression of the pro-apoptotic BCL2 family members
NOXA and BIM. Promoter-Scanning qChIP revealed binding of MYC to both
BH3-only gene promoters upon proteasome inhibition, leading to increased
transcription. We demonstrate that recruitment of MYC to the NOXA as well
as the BIM gene promoter depends on its interaction with the transcription
factor EGR1, defining a novel pathway of MYC directed death.
Conclusion: Our study uncovers a novel molecular mechanism, which shows
that bortezomib-induced apoptosis depends on functional cooperation of MYC
S61
with EGR1. This observation may be important for novel therapeutic strategies
dependent on the inherent pro-death function of MYC.
261 Cancer-associated fibroblast (CAF)-derived exosome may mediate
breast cancer progression by reducing exosomal microRNAs
J.E. Kim1 , N.H. Cho1 . 1 Yonsei University College of Medicine, Brain Korea 21
PLUS Project for Medical Science Department of Pathology, Seoul, Korea
Cancer-associated fibroblasts (CAFs) are one of the main populations in tumor
microenvironment. As widely accepted, CAFs enhance tumor growth, invasion
and metastasis, while normal fibroblasts possibly function as a barrier at the
early stage of tumorigenesis. Recently, it has been found that CAFs secrete
small vesicles called ‘exosome’ to communicate with adjacent cancer cells.
Exosomes contain various microRNAs, mRNA, DNA and proteins, which can
be transferred to other cells and affect tumor progression. To elucidate whether
the levels of microRNAs differ in exosomes derived from normal fibroblasts and
CAFs, and also function actively in the recipient cancer cells, we investigated
the contents of exosomes in two conditions of fibroblasts and the effects
of exosomal microRNAs on breast cancer cells. Fibroblasts were isolated
from human breast cancer tissues and activated by MDA-MB-231 conditioned
media (CM) to mimic the nature of CAFs in human body. Normal fibroblasts
were isolated from human tissue as well. The contents of exosomes derived
from normal fibroblasts and CAFs were analyzed by microRNA microarray.
Six microRNAs (miR-1253, miR-144-3p, miR-1915-3p, miR-29b-3p, miR-30e,
and miR-4516) were significantly downregulated in CAF-derived exosomes.
The expression level of target microRNAs in breast cancer cells, CAFs and
normal fibroblasts were determined by RT-qPCR. We treated CAF-derived
exosomes and normal fibroblast exosomes to MCF7 breast cancer cells and
observed the uptake of microRNAs in cancers. The target microRNA in cancer
cells decreased significantly with CAF-derived exosome treatment. These data
support that cancer cells and fibroblasts in tumor microenvironment interact in
both ways via exosomal microRNAs, which may impact on tumor progression.
262 STAT3-induced WDR1 expression is associated with breast cancer
cell migration
J.H. Lee1 , N.H. Cho1 . 1 Yonsei University College of Medicine, Brain Korea 21
PLUS Project for Medical Science Department of Pathology, Seoul, Korea
WDR1 was identified as a major co-factor that collaborates with cofilin to
disassemble F-actin. WDR1 can bind cofilin/F-actin complex and strongly
enhance the severing activity of cofilin on filamentous actin by capping
the barbed ends of the severed filaments, resulting in acceleration of actin
depolymerization.
In our previous study, WDR1 is overexpressed in human breast cancer. Its
function in human breast cancer was not been well understood. However,
STAT3, a member of STATs (signal transducers and activators of transcription)
family, regulates wide range of cellular processes, including oncogenesis, cell
proliferation and cancer metastasis. It is well known that STAT3 is activated
in breast cancer and promotes tumor progression. These evidences suggest
that WDR1 expression is regulated by STAT3 as a transcription factor and
associated with breast cancer progression. We examined to see whether
STAT3 regulates WDR1 promoter activity and searched for predicted STAT3
binding sites in up-stream 10Kb region of WDR1 gene. We found total three
candidates. Two sites are located far from TSS (Transcriptional start site), so
the nearest site from TSS (named SITE1) was chosen. SITE1 is located at
−1931 to −1924 and the sequence is AAGTCCTT. We performed transient
transfection studies with a Dual-luciferase assay driven by the human WDR1.
SITE1-WT luciferase activity was higher than negative control up to nearly
2-fold, also overtook the positive control. We observed lower luciferase activity
at SITE1 mutant (deletion) form.
Furthermore, STAT3 directly binds to the WDR1 promoter in response to IL-6
treatment is confirmed by ChIP (Chromatin Immuno-precipitation) assays with
phosphorylated STAT3 antibody and SITE1 PCR primers. In the breast cancer
cells treated with IL-6, there was a significant increase STAT3 binding to the
WDR1 promoter compared with untreated cells. Taken together, these data
provide evidences that STAT3 may directly binds to the WDR1 promoter to
regulate WDR1 transcription in response to IL-6 treatment.
STAT3-induced WDR1 overexpression may be associated with breast cancer
cell motility.
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263 Mimicking the tumour microenvironment (TME): Angiogenesis
in tumour progression
U.H. Bonda1 , L.J. Bray2 , N. Lister3 , S. Ellem3 , G. Risbridger3 ,
U. Freudenberg2 , C.C. Werner2 , D.W. Hutmacher1 , E.M. De-Juan-Pardo1 ,
D. Loessner1 . 1 Institute of Health and Biomedical Innovation, Queensland
University of Technology, Brisbane, Australia, 2 Leibniz Institute of Polymer
Research Dresden, Max Bergmann Center of Biomaterials Dresden, Dresden,
Germany, 3 Prostate Cancer Research Centre, Department of Anatomy
and Developmental Biology School of Biomedical Sciences, Melbourne,
Australia
Introduction: Two-dimensional (2D) substrates cannot accurately mimic the
complex matrix of native TME, whereas 3D models can recapitulate the natural
tumour progression in vitro. As part of the tumour stroma, fibroblasts and
endothelial cells (ECs) are well-known to not only support tumour growth but
also to reduce the efficacy of anti-cancer drugs. Particularly, ECs are involved
in the process of tumour vascularisation which represents a crucial step in the
progression of cancer. Most of the previous studies are carried out in animal
models or 2D cultures; hence, a detailed evaluation of experimental data is
poor. To address this issue, we aim to develop a novel 3D in vitro approach, to
mimic native tumour angiogenesis in 3D and to quantify the developed vascular
network.
Materials and Methods: hECs were embedded in protease-cleavable starshaped polyethylene glycol-heparin hydrogels of different stiffness. Gels
were either unloaded or pre-loaded with VEGF. The vascular network was
characterised by branching points, length and density after 7 days. To develop
a 3D model of tumour angiogenesis, the breast cancer, MCF-7 and MDAMB-231, and prostate cancer cell lines, LNCaP and PC3, were embedded
within hydrogels allowing spheroid formation. Cancer cells were pre-grown
in hydrogels for 3−5 days and then re-seeded into functionalised hydrogels
as spheroids with hECs and bone marrow-derived mesenchymal stem cells
(hMSCs) and grown for 10 days as a triple-culture. Cultures were fixed and
analysed via immunostaining and confocal microscopy.
Results and Discussion: Capillary-like structures made of hEC monocultures
embedded in softer gels (~200 Pa) were denser and longer than those in stiffer
gels (~600 Pa). Tubular structures in unloaded hydrogels collapsed after day 5.
Cancer cells formed spheroids when embedded within hydrogels in contrast to
adherent growth on tissue culture plastic. The 3D culture angiogenesis model
displayed evidence of tumour/vascular interactions.
Conclusion: We confirmed the suitability of this hydrogel approach for
investigations of the process of angiogenesis and to study cell–cell and cell–
matrix interactions of hECs and hMSCs co-cultured with cancer cell lines in
a bioengineered 3D microenvironment. Outcomes represent a step forward in
the development of 3D technology platforms to study the pathomechanisms
of breast and prostate cancer.
264 Histone deacetylase inhibitors resensitize glioblastoma cells
to EGFR-directed therapy with tyrosine kinase inhibitors after
primary treatment failure
K. Liffers1 , K. Kolbe1 , M. Westphal1 , K. Lamszus1 , A. Schulte1 .
1
Universitatsklinikum Eppendorf, Department of Neurosurgery, Hamburg,
Germany
Background: Glioblastoma multiforme (GBM) is the most common and
most malignant primary brain tumor and is characterized by a variety of
genomic rearrangements and mutations. One of the most common alteration
is the overexpression/amplification of the epidermal growth factor receptor
(EGFR) gene, which is present in 40−60% of all GBM. Therefore, EGFR
is a promising target in GBM therapy, although EGFR-directed antibodies
as well as tyrosine kinase inhibitors had not yet the desired success.
Because chromatin modification, especially histone deacetylases (HDACs),
might control EGFR expression, we combined anti-EGFR and anti-HDAC
approaches to investigate the benefit of combinatorial therapy in GBM cells.
Methods: We treated a large panel of highly representative in vitro
model systems of EGFR-amplified glioblastoma (glioblastoma stem-like cells
and EGFRvIII-positive glioblastoma cell lines) (N = 8) and non-amplified
glioblastoma (N = 2) either with HDAC-inhibitors alone or in combination with
the EGFR tyrosine kinase inhibitor Erlotinib and determined proliferation,
migration and EGFR-dependent downstream signaling.
Results: Inhibition of HDAC activity reduced proliferation of GBM cell lines
irrespective of their EGFR status. The combined inhibition of HDACs and
EGFR had an additive effect and led to enhanced inhibition of proliferation
significantly more pronounced in EGFR-amplified cells. Interestingly, even
in Erlotinib resistant GBM cells, HDAC inhibition significantly decreased
proliferation and partially restored sensitivity to Erlotinib. Importantly, combined
treatment of Erlotinib-sensitive GBM cells with Erlotinib and HDAC inhibitor did
not cause the development of Erlotinib resistance as treatment with Erlotinib
alone did. While inhibition of HDAC had no significant effect on either random
migration of GBM cell lines or migration towards EGF, treatment with HDAC
inhibitors enhanced expression of pro-apoptotic genes like FASL. Furthermore,
the inhibition of HDACs significantly decreased expression of wtEGFR as well
as of EGFRvIII without affecting phosphorylation of EGFR.
Conclusions: The combined inhibition of HDACs and EGFR shows additive
anti-proliferative potential in a preclinical model for EGFR-amplified GBM.
Importantly, HDAC inhibition can overcome Erlotinib resistance in EGFRamplified GBM. In summary, HDAC inhibitors might serve as a new class
of potential therapeutics for newly diagnosed tumors or treatment-refractory
GBM, especially in combination with anti-EGFR therapy approaches.
265 The effects of INhibitor of Growth 3 (ING3) on prostate cancer
cell survival and invasion
A. Nabbi1 , A. Almami1 , T. Bismar1 , K. Riabowol1 . 1 Southern Alberta Cancer
Research Institute, Department of Biochemistry and Molecular Biology,
Calgary Alberta, Canada
Introduction: Prostate cancer (PCa) is the most common malignancy among
men worldwide. Like other types of cancer, metastasis in patients with
advanced PCa is highly correlated with disease mortality. INhibitor of Growth
(ING) proteins are epigenetic regulators that, via their plant homeodomains
(PHD), recognize H3K4me3 and recruit histone acetyltransferase (HAT) or
histone deacetylase (HDAC) complexes leading to alteration of histone
acetylation and subsequent regulation of gene expression. ING3 is the most
conserved member of this family and is a stoichiometric member of the
TIP60 HAT complex. TIP60 is recently reported to be involved in metastasis
by acetylating Twist transcription factor. In addition, ING3 has been shown
to regulate the mammalian target of rapamycin (mTOR) pathway, which is
also known to regulate metastasis-promoting genes in PCa. We, therefore,
hypothesize that ING3, by virtue of being a member of the TIP60 HAT complex,
can play a role in progression of PCa.
Material and Methods: Data mining has been performed using the NCBI
GEO database and Oncomine platforms to ask whether there is differential
expression of ING3 in PCa versus normal prostate. RWPE-1, PC3 and DU145
cells were used for in vitro studies. Two days after transfection of either
scrambled siRNA or siRNA against ING3 (siING3), cells were replated for
MTT survival assays, clonogenic assays, wound healing assays and invasion
assays.
Results and Discussion: Datamining of various publicly available datasets
showed that ING3 is overexpressed in PCa by an average of two-fold. Knockdown of ING3 resulted in less cell proliferation and formation of fewer and
smaller colonies. Compared to control, colonies appeared to be in close
proximity suggesting less cell mobility. Scratches on cell monolayers healed
slower in siING3-transfected cells. DU145 cells that were transfected with
siING3 exhibited less invasion ability when compared to control siRNA.
Conclusion: Our preliminary results show that ING3 is involved in the PCa
proliferation and migration processes. Our ongoing work using tissue samples
and established cell lines aims to investigate the molecular mechanism
underlying the effects of ING3, which may provide us with novel therapeutic
targets for the treatment of metastatic PCa.
266 EphB4 negatively regulates blood vessel network formation and
perfusion in human A375 melanoma xenografts
C. Neuber1 , F. Hofheinz1 , R. Bergmann1 , S. Meister1 , J. Steinbach1 ,
B. Mosch1 , J. Pietzsch1 . 1 Helmholtz-Zentrum Dresden-Rossendorf, Institute
of Radiopharmaceutical Cancer Research, Dresden, Germany
Background: Since receptor tyrosine kinase EphB4 and its ligand ephrinB2
play a key role during angiogenesis, we hypothesized EphB4 to be also a
regulator of tumor angiogenesis and lymphangiogenesis. Using a multi positron
emission tomography (PET) tracer approach and immunohistochemistry we
investigated the influence of EphB4 on lymphangiogenesis, angiogenesis,
perfusion, and oxygenation in a recently established EphB4 overexpressing
melanoma xenograft model.
Material and Methods: Human A375 melanoma cells transfected with EphB4
(A375-EphB4) or mock (A375-pIRES) were xenotransplanted into the right
and left hind leg, respectively, of athymic nude mice. In addition to tumor
growth; glucose metabolism, perfusion, and oxygenation of tumors were
studied by dynamic small animal PET using the established radiotracers
2-[18 F]fluoro-2-desoxy-D-glucose ([18 F]FDG), H2 [15 O]O ([15 O]H2 O), and
[18 F]fluoromisonidazol ([18 F]FMISO). Furthermore, perfusion, oxygenation,
and vascularization of tumors were investigated ex vivo by autoradiography,
immunohistochemistry, and fluorescence microscopy.
Results: A375-EphB4 tumors grew significantly faster than A375-pIRES
tumors. Overexpression of EphB4 was confirmed by western blot and
immunohistochemistry. Semi quantitative analysis of the amount of CD31+
blood vessels and LYVE-1+ lymph vessels in tumors revealed that EphB4
significantly diminished blood vessel network (A375-pIRES vs. A375-EphB4:
1.12±0.20 vs. 0.88±0.20, p < 0.001) whereas amount of lymph vessels was
not affected. In line with the former, perfusion (1.24±0.28 vs. 0.76±0.28,
p < 0.001) and by trend also oxygenation of A375-EphB4 tumors were
decreased. Metabolic uptake rate (Km ) of [18 F]FDG was significantly reduced
in A375-EphB4 tumors (1.03±0.08 vs. 0.97±0.08, p < 0.05), whereas Km
of hypoxia tracer [18 F]FMISO was unaffected. PET with [15 O]H2 O, the
gold standard technique for measuring perfusion, revealed that EphB4
significantly reduced tumor perfusion (k1) in melanoma xenografts (1.19±0.07
vs. 0.81±0.07, p < 0.001).
Conclusion: Using a multi PET tracer approach and immunohistochemistry
we demonstrated a diminished blood vessel network formation as well as
a lowered perfusion in an EphB4 overexpressing human melanoma model
compared to mock transfected control tumors. The results indicate EphB4 to
be a negative regulator of tumor angiogenesis. This could be of importance
for, e.g., metastasis and drug delivery in melanoma. By contrast, tumor
lymphangiogenesis was not influenced by EphB4.
267 Mesenchymal cells regulate growth of intestinal crypts by
a Wnt independent mechanism in 3D culture system
A. Pastula1 , S. Hauck2 , K.P. Janssen3 , R.M. Schmid1 , M. Quante1 . 1 Klinikum
rechts der Isar − Technische Universität München, Gastroenterologie II,
München, Germany, 2 Helmholtz Zentrum München, Research Unit Protein
Science, München, Germany, 3 Klinikum rechts der Isar − Technische
Universität München, Department of Surgery, München, Germany
Intestinal stem cells (ISC) reside at the bottom of each crypt and give
rise to all lineages of the intestinal epithelium. Crypts are surrounded
by pericryptal fibroblasts, which are believed to create a stem cell niche
for the ISC. The mechanisms, by which local environment regulates ISC
proliferation and differentiation are unknown so far. In order to investigate
it, we established several 3D culture systems, which involve intestinal crypts
and mesenchymal cells. The effects of different types of mesenchymal cells:
primary murine intestinal fibroblasts (pFB), murine carcinoma associated
fibroblasts (CAF), as well as human esophageal fibroblasts (FEF) and human
primary fibroblasts (hpFB) were analysed.
Co-culture studies revealed that different types of mesenchymal cells (pFB,
CAF, FEF, hpFB) induce sphere-like phenotype in intestinal organoids.
Analysis of mRNA levels by RT-PCR showed that mesenchymal cells
express ligands for Wnt, Notch, Hedgehog and BMP pathways. Mesenchymal
cells increased proliferation and decreased differentiation in crypts, as
shown by Ki-67 and PAS staining. Similar effects were observed when
fibroblast conditioned medium was used. Intestinal organoids derived from
a tumor tissue of the Apc+/1638N mouse phenotypically resembled crypts cocultured with mesenchymal cells. However, Wnt inhibition studies revealed
that mesenchymal cells regulate growth of intestinal organoids by a Wnt
independent mechanism. Mass spectrometry analysis of the conditioned
medium from the co-culture uncovered activation of extracellular matrixreceptor and focal adhesion pathways.
Our studies show that mesenchymal cells contribute to the phenotype
of epithelial cells. We conclude that mesenchymal cells are a regulatory
component of the intestinal stem cell niche in 3D culture system and influence
proliferation and differentiation of crypt cells by Wnt independent mechanism.
Our findings have implications for understanding the contribution of niche
environment to tumorigenesis, where cellular proliferation and differentiation
are altered.
268 Comparison of membrane fatty acid compositions of
mesenchymal stem cells and mature mesenchymal cell lines
using GC-FID method
T. Ozgurtas1 , A. Tas1 , F. Yesildal1 , F. Avcu1 . 1 GATA Faculty of Medicine,
Biochemistry, Ankara, Turkey
Introduction: The aim of this study was to investigate whether there
was a significant difference between membrane fatty acid compositions of
mesenchymal stem cell lines and mature mesenchymal cell lines.
Material and Method: Mesenchymal stem cell, gingival fibroblast, dental
pulp cell lines were used which were prepared by passaging each cell
line in GATA Research and Development Centre, Cancer and Stem cell
Research Laboratory. Each sample was prepared at a concentration of 10
million cells/mL. The cells were administered with methanolic hydrochloric
acid to create methyl esters of fatty acids and analyzed. Analyses were
performed twice using Gas Chromatography Flame Ionization Detector
(GC-FID) (Thermo-Finnigan Trace GC Ultra).
Results and Discussion: In all groups, existence of 21 fatty acids (C10-C24:1)
was evaluated using this method. C22:1n9 could not be detected in all samples
so it was excluded. Other fatty acids were detected in all samples. There were
statistically significant differences between the gingival fibroblast cell line and
dental pulpa cell line comparing both C16 (p = 0.017) and C18:2n6c (p = 0.029)
fatty acids.
S63
Conclusion: There was no statistically significant difference between
mesenchymal stem cell line and mature mesenchymal cell line. However there
were statistically significant differences between the two mature mesenchymal
cell lines comparing both C16 and C18:2n6c fatty acids.
269 Multidrug resistance gene MDR1 is transactivated by Oct4 to
increase chemotherapy resistance
C.L. Wu1 , C.S. Lu1 , A.L. Shiau2 . 1 National Cheng Kung University,
Department of Biochemistry and Molecular Biology, Tainan City, Taiwan,
2
National Cheng Kung University, Department of Microbiology and
Immunology, Tainan City, Taiwan
Background: Platinum-based drug like cisplatin is the standard first-line
drug to treat patients with bladder cancer. However, tumors may recur
and develop into multiple-drug resistant characteristics. The acquisition of
resistance to conventional chemotherapy is a challenge in the treatment of
bladder cancer relapse. Cancer stem cells can resist therapeutic assaults,
giving rise to multiple drug resistance and promotion of tumor relapse and
metastasis. It has been shown that the fraction of cancer cells expressing
MDR1 that functions as an energy-dependent drug efflux pump may be
associated with Oct4 expression. We have demonstrated that Oct4 expression
in bladder cancer predicts tumor progression. Here we investigated whether
Oct4 expression in bladder cancer increases MDR1 gene expression and
thereby results in multiple drug resistance.
Material and Methods: We examined immunohistochemically the expression
of Oct4 and MDR1 in human and mouse bladder tumor tissues. RT-PCR
analysis and immunofluorescence microscopy were used to determine
the expression levels of Oct4 and MDR1 in human bladder transitional
cell carcinoma cells after treatment with various chemotherapeutic agents.
Sensitivity to continuous exposure of cisplatin was assessed for bladder cancer
cells by determining their IC50 values with the colorimetric WST-8 assay.
Transactivation of the MDR1 promoter by Oct4 was analyzed by luciferase
reporter and chromatin immunoprecipitation assays. We also evaluated the
impact of Oct4 expression on MDR1 expression and treatment outcome in
bladder tumor-bearing mice after treatment with cisplatin.
Results: We found a positive correlation between Oct4 and MDR1 expression
levels in clinical samples of bladder cancer. Furthermore, overexpression of
Oct4 increased MDR1 expression, which resulted in poor response to cisplatin
in human bladder cancer cells. Conversely, knockdown of Oct4 reduced MDR1
expression and rendered cells hypersensitive to cisplatin. We also verified that
Oct4 transactivated the MDR1 gene promoter by binding to the Oct4 response
element (ORE). More importantly, Oct4 and MDR1 gene expression could be
induced by treatment with cisplatin. Our data can explain, in part, the high
recurrence rate and drug resistance of bladder cancer. For clinical implication,
we demonstrated that reduction of Oct4 expression by all-trans retinoic acid, a
derivative of vitamin A, improved sensitivity of bladder cancer cells to cisplatin
and gemcitabine. Furthermore, we found that high expression levels of Oct4
and MDR1 were associated with high tumor recurrence in human bladder
cancer.
Conclusions: Our results provide evidence that Oct4 plays an important role
in cisplatin-acquired resistance in bladder cancer and implicate Oct4 as a
therapeutic target.
270 TPRG1L is a novel microRNA-21 target: a possible linkage
between miR-21 and cellular senescence in liver fluke-associated
cholangiocarcinoma
P. Chusorn1 , N. Namwat1 , W. Loilome1 , A. Techasen2 , C. Pairojkul3 ,
N. Khuntikeo4 , B. Tean Teh5 , I. Lee6 , P. Yongvanit1 . 1 Department of
Biochemistry Faculty of Medicine, Khon Kaen University, Khon Kaen,
Thailand, 2 The center for research and development of Medical diagnostic
laboratories Faculty of Associated Medical Sciences, Khon Kaen University,
Khon Kaen, Thailand, 3 Department of Pathology Faculty of Medicine, Khon
Kaen University, Khon Kaen, Thailand, 4 Department of Surgery Faculty
of Medicine, Khon Kaen University, Khon Kaen, Thailand, 5 Translational
Research Laboratory, National Cancer Centre, Singapore, Singapore,
6
miRcore, Ann Arbor, Michigan, USA
Background: Cholangiocarcinoma (CCA), a common bile duct tumor in
northeast Thailand, is primarily associated with chronic infection of the liver
fluke, Opisthorchis viverrini (Ov) that causes long-standing inflammation,
the hallmark of carcinogenesis. There is strong evidence supporting that
microRNA-21 (miR-21) acts as an upregulated oncomiR upon the inflammatory
stimuli and it functions to suppress the set of tumor suppressor mRNAs in many
types of cancer. This study aimed to discover the novel miR-21 targets that
contribute functionally to control CCA genesis.
Material and Methods: Knockdown of miR-21 by RNAi in two human CCA cell
lines was performed prior the identification of mRNA expression profile. Data
was normalized and analyzed by miBridge, TargetScan, PITA and miRTarBase
S64
to predict miR-21 targets. Candidate mRNA was confirmed by qRT-PCR and
western blotting. Immunohistochemistry staining was used to detect protein
targets in CCA tissues. The 3 UTR binding sites (wild type and mutant) of
validated target were designed and co-transfected with miR-21 antagomir or
agomir for luciferase assay. The molecular function of candidates was studied
by siRNA for cellular proliferation and senescence in CCA cell lines.
Results: MiR-21 depleting CCA cells showed an increase in mRNA
expressions of various genes including ANKRD46, DDAH1, CDC25A, YOD1,
GLT8D3 and TPRG1L. Of those targets, TPRG1L, a tumor protein P63
regulated 1-like with a little known function, had a negative correlation with
miR-21 expression (P = 0.049) in CCA tissues. Luciferase assay confirmed
that TPRG1L was suppressed by miR-21. Upon TPRG1L suppression, we
discovered that TPRG1L was involved in cellular senescence in CCA cells.
Conclusion: Our data support that TPRG1L, the novel miR-21 target possibly
plays roles in the genesis of Ov-associated CCA. Our findings suggests that
TPRG1L may be involved in oncogene-induced senescence in CCA. The
underlying mechanism by which miR-21 induces cellular senescence through
suppression of TPRG1L needs to be further investigated.
271 AR-associated p21 induces apoptosis by neutralizing anti-apoptotic
Bcl-2 protein in androgen-dependent prostate cancer LNCaP cells
K. Choi1 , H. Suh1 , C.H. Lee1 . 1 Hanyang University, Pharmacy, Ansan,
Korea
Introduction: Prostate cancer, the most frequent type of cancer, is a leading
cause of cancer death in males in the US, and dysregulation of androgen
signaling is known to be a key factor of this disease. Androgen is essential
for prostate development and homeostasis, and exerts its biological effects by
binding to androgen receptor (AR). Androgen regulates not only a series of
androgen target genes, but also genes for cell cycle- and apoptosis-regulatory
molecules, such as p53 or 21CIP1 , in prostate epithelial cells.
We have found that MCS-C3, a novel pyrrolo-pyrimidine analogue, showed
anti-cancer activity by cell cycle inhibition and apoptosis induction. MCS-C3
induced conventional apoptosis in several types of cancer cells, including
human prostate cancer LNCaP cells. In this study, we investigate the molecular
mechanisms underlying apoptosis-inducing activity of MCS-C3 in androgendependent prostate cancer LNCaP cells.
Material and Method: We tested MCS-C3 (6 mM) on AR-positive LNCaP cells
to evaluating cell cycle arrest and apoptosis induction by FACS scans and
Western blot assays. To investigate the molecular mechanisms, we performed
knock-down of p53 and AR gene by specific siRNA, inhibition of AR activation
using AR antagonist (Flutamide® ), and co-immunoprecipitiation of p21CIP1 and
Bcl-2.
Results and Discussion: In our study, MCS-C3 induced caspase-dependent
apoptosis with increased transcriptional activity of p53 in LNCaP cells.
During this apoptotic induction, significant increase of p21CIP1 expression and
Bax/Bcl-2 ratio was detected. Interestingly, we confirmed that expression of the
p21CIP1 is associated with AR through AR blockade with Flutamide® inhibited
p21CIP1 expression and apoptosis by 6 mM of MCS-C3. And also, knock-down
of AR using siRNA down-regulated p21CIP1 protein level. Therefore, AR may
play a role in apoptosis through p21CIP1 . In addition, we found that p21CIP1
bound to Bcl-2. p21CIP1 has been known as one of CDK inhibitor which
expression is regulated by p53. But recently, several studies implicate that
p21CIP1 as a pro-apoptotic factor. In addition, emerging evidence suggests a
role of p53 as non-transcriptional pro-apoptotic factor at the mitochondria, such
as neutralization of anti-apoptotic Bcl-2 and Bcl-XL. Therefore, we assume that
p21CIP1 may play a role via preventing anti-apoptotic function of Bcl-2 protein
through protein–protein interaction.
Conclusion: Taken together, our results suggest that AR-associated p21CIP1
induces apoptosis by interacting with Bcl-2 protein in androgen-dependent
prostate cancer cell line LNCaP.
272 TWIST1 and ZEB1 EMT inducers contribute to melanoma
development through regulating MITF
G. Richard1 , M. Houang1 , A. De la Fouchardière2 , R. Marais3 , L. Larue4 ,
S. Dalle5 , E. Tulchinsky6 , S. Ansieau1 , A. Puisieux1 , J. Caramel1 . 1 Centre
de Recherche en Cancérologie de Lyon (CRCL), Tumoral escape, Lyon,
France, 2 Centre Léon Bérard, Anatomopathology, Lyon, France, 3 Paterson
Institute for Cancer Research, Molecular Oncology, Manchester, United
Kingdom, 4 Institut Curie, Normal and pathological signaling: From the embryo
to the innovative therapy of cancers, Paris, France, 5 Hopitaux Lyon Sud,
Dermatology, Lyon, France, 6 University of Leicester, Department of Cancer
Studies and Molecular Medicine, Leicester, United Kingdom
Introduction: Embryonic transcription factors inducers of Epithelial to
Mesenchymal Transition (EMT-TFs) are frequently reactivated during tumorigenesis. In addition to promoting metastasis in carcinoma, they favor neoplastic
transformation of epithelial cells by enabling escape from oncogene-induced
senescence and apoptosis and providing cells with stem-like properties. We
recently unveiled different regulation and function of EMT-TFs in melanoma,
a highly metastatic neural crest-derived cancer. We observed a switch in
expression from SNAIL2 and ZEB2, which are expressed in melanocytes
and behave as oncosuppressive proteins to TWIST1 and ZEB1, which are
aberrantly reactivated in melanoma and cooperate with BRAFV600E oncogene
to induce neoplastic transformation of melanocytes. This switch in EMTTF expression represents a novel independent factor of poor prognosis
in patients with malignant melanoma (Caramel et al, Cancer Cell, 2013).
We further investigated the oncogenic properties of TWIST1 and ZEB1 in
melanoma development and analyzed the crosstalk with the master regulator
of melanoma phenotypic plasticity, the microphthalmia-associated transcription
factor MITF.
Methods: We first studied the role of TWIST1 in melanoma progression in vivo
by crossing conditional Twist1 transgenic mice with BRAFV600E /TyrCreERT2
mice. We then analysed the expression of MITF and TWIST1/ZEB1 in
melanoma cell lines and human melanoma specimens.
Results and Discussion: We show that Twist1 enables escape from
BRAFV600E -induced senescence in primary mouse melanocytes. BRAFV600E /
Twist1 melanomas are more aggressive and exhibit dedifferentiation features
compared to BRAFV600E melanomas. Moreover, TWIST1 and ZEB1 regulate
MITF, the master regulator of melanoma phenotypic plasticity. TWIST1
and ZEB1 cooperate with BRAFV600 in down-regulating MITF expression,
concomitantly with the induction of invasive/stem-like associated gene
signatures. Finally, a correlation of MITF expression with TWIST1 and ZEB1
is observed in human melanoma specimens at the intratumoral level.
Conclusion: Overall, by regulating MITF-dependent phenotype switching,
TWIST1/ZEB1 contribute to malignant progression of melanoma. Therefore,
targeting the EMT-TF network represents an attractive strategy for metastatic
melanomas that invariably develop resistance to BRAFV600 targeted therapy.
273 Androgen receptor interacting protein HSPBAP1 facilitates growth
of prostate cancer cells in androgen-deficient conditions
P. Ostling1 , K. Saeed1 , M. Björkman2 , T. Mirtti3 , T. Vesterinen1 , J. Lundin1 ,
A. Rannikko3 , J.P. Mpindi1 , O. Kallioniemi1 , J. Rantala2 . 1 University of
Helsinki, Institute for Molecular Medicine Finland FIMM, Helsinki, Finland,
2
VTT Technical Research Centre of Finland, Medical Biotechnology, Turku,
Finland, 3 University of Helsinki and HUSLAB, Haartman Institute Department
of Pahtology, Helsinki, Finland
Introduction: Androgen-deprivation or chemical castration therapy is routine
practice in the treatment of advanced PCa, but the treatment is not curative
and most cancers relapse to a lethal castrate-resistant state. Improved
understanding of the cellular events during androgen-deprivation would help
to identify survival and stress pathways whose inhibition could synergize with
androgen-deprivation. Towards this aim, we performed an RNAi screen on
2068 genes, including kinases, phosphatases, epigenetic enzymes and other
druggable gene targets in VCaP cells using cell spot microarray screening
(CSMA).
Material and Method: The CSMA screen, with a total of 4136 siRNA per
array, was performed in VCaP cells in the presence and absence of androgens
with detection of Ki67 (proliferation) and cleaved ADP-ribose polymerase
(cPARP, apoptosis) by high content imaging using Olympus ScanR. DNA
counterstaining was used in order for image segmentation of nuclei and
analysis of nuclear Ki-67 and cPARP signal intensities with automated
image analysis. 39 candidate genes were identified, whose silencing inhibited
proliferation or induced apoptosis of VCaP cells exclusively under androgendeprived conditions.
Results and Discussion: One of the candidates, HSPB (heat shock 27
kDa) associated protein 1 (HSPBAP1) was confirmed to be highly expressed
in tumor samples and its mRNA expression levels increased with the
Gleason grade. We found that strong HSPBAP1 immuno-histochemical
staining (IHC) was associated with shorter disease-specific survival of PCa
patients compared with negative to moderate staining in a cohort of 371
primary prostate cancer patients. Furthermore, we demonstrate that HSPBAP1
interacts with AR in the nucleus of PCa cells specifically during androgendeprived conditions. This interaction seems to be important as knock down
of HSPBAP1 further sensitized prostate cancer cells to androgen-deprivation
and decreased expression of AR target genes PSA and TMPRSS2.
Conclusions: Our data suggest a novel role and a possible link for HSPBAP1
in promoting prostate cancer cell survival in androgen-deficient conditions
by maintaining basal level AR mediated transcription, and highlights the
critical importance of better understanding context-specific gene and protein
interactions for the development of novel therapeutic interventions in prostate
cancer.
274 Tumor metabolism and docetaxel resistance in prostate cancer
M. Taddei1 , A. Marini2 , L. Ippolito1 , A. Morandi1 , E. Giannoni1 , P. Chiarugi1 .
1
University of Firenze, Dipartimento di Scienze Biomediche Sperimentali
e Cliniche, Firenze, Italy, 2 University of Sassari, Dipartimento di Scienze
Biomediche, Sassari, Italy
Background: Drug resistance is recognized as the primary cause of failure
of chemotherapeutic treatment in most human cancers. Mounting evidences
support the idea that deregulated cellular metabolism is linked to drug
resistance in cancer therapy. Indeed, both components of the glycolytic
and mitochondrial pathways are involved in altered metabolism linked to
chemoresistance in several cancers. In this context, we characterized a PCa
cell line resistant to docetaxel and we evaluated the phenotypic and metabolic
behavior of this cell line, in order to identify the drug-induced metabolic
adaptations conferring advantages in resistant cells.
Material and Methods: A PC3 cell line resistant to docetaxel (Doce Res) was
obtained by treating sensitive PC3 cells with increased doses of drug, until the
final concentration of 10 nM. Western blot analysis was used to investigate the
levels of EMT markers and key metabolic players in the PC3 and PC3 Doce
Res cells. Reactive oxygen species were evaluated using the redox sensitive
probe: 2 ,7 -dichlorofluorescein diacetate (DCF-DA), selectively responsive to
hydrogen peroxide. Boyden assay was used to assess the invasive properties
of PC3 and Doce Res cells. Radioactive assays were used to determine
the levels of glucose, glutamine and lactate uptake and to monitor OXPHOS
activity. Metformin, an inhibitor of mitochondrial respiratory chain complex I,
was used to assess the dependence on mitochondrial respiration of the two
different cell lines.
Results and Discussion: Doce Res cells acquire a pro-invasive behavior and
a down-regulation of E-cadherin, consistent with the acquisition of an EMT
program and a pro-metastatic phenotype. Moreover, DoceRes cells show a
decrease of intracellular ROS, NADPH level and proliferation compared to
sensitive cells. These features are not linked to an induction of the pentose
phosphate pathway, but are associated with an enhancement in the antioxidant
response, mainly driven by increased expression of the transcription factor
Nrf2 (Nuclear factor erythroid 2 related factor 2). Metabolic analysis in Doce
Res cells reveals a shift toward OXPHOS, with a greater utilization of glucose,
glutamine and lactate by mitochondrial respiration. In agreement, metformin,
impairing mitochondrial complex I function, selectively decreases proliferation
and invasiveness in resistant cells. Furthermore, stromal cancer associated
fibroblasts, which cause a ‘reverse Warburg’ phenotype in prostate cancer
cells, are able to protect sensitive and resistant cell lines from docetaxel toxicity.
In keeping, an approach based on re-expression of miR-205, able to shift
metabolism from OXPHOS to a Warburg phenotype, induces an increase of
docetaxel toxicity in prostate cancer cells.
Conclusions: Taken together, these findings suggest that chemoresistance
to docetaxel induces an escape from Warburg metabolism with a potential
involvement of OXPHOS to confer a metabolic advantage to these cells. We
hypothesize that the impairment of mitochondrial function could be an attractive
adjuvant therapy for several anticancer regimens.
275 MiR-136 targeting Notch3 is involved in chemoresistance and
angiogenesis in ovarian cancer cells
H. An1 , J. Jeong2 , M. Lee2 , J. Song2 , Y. Jung2 , Y. Kim2 , A. Kwon1 , J. Huh1 ,
K. Kim1 , H. Kang1 . 1 CHA University, Pathology, Gyeonggi-do, Korea, 2 CHA
University, Institute for Clinical Research, Gyeonggi-do, Korea
Introduction: The Notch signaling pathway plays a key role in the proliferation
and differentiation of many tissues. Recent data and our preliminary study
indicated that Notch3 amplification was related with poor prognosis and
chemoresistance in ovarian cancer patients. MicroRNAs (miRNAs) are
noncoding regulatory RNAs, which are involved in various regulatory cellular
processes, such as cell cycle, apoptosis, and human tumorigenesis by
targeting multiple protein-coding genes through partial base pairing to
the 3 untranslated region of the target gene. We, therefore, sought to
identify miRNAs targeting Notch3, and to investigate whether regulation
of these miRNAs induces downregulation of Notch3, and overcomes the
chemoresistance in ovarian carcinoma, the most lethal cancer among
gynecological malignancies.
Material and Methods: We searched miRNAs targeting Notch3 using
computer program, such as Target Scan or PicTar. As for the candidate
miRNAs, the expression of miRNAs was examined in 38 high grade ovarian
serous carcinoma samples using qRT-PCR, and correlated with the expression
of Notch3, and chemoresponse of these clinical samples. We validated that
these miRNAs directly target Notch3 in paclitaxel (PTX)-resistant SKpac
sublines by immunoblotting and luciferase assay. The function of candidate
miRNAs in ovarian cancer cells was further evaluated by apoptosis (TUNEL)
and angiogenesis assays.
Results: We found that miR-136 directly targets Notch3 through computer
program, luciferase assay, and immunoblotting. The expression of miR-
S65
136 was inversely correlated with Notch3 expression in 38 high grade
ovarian serous carcinomas. Down-regulation of miR-136 was associated with
chemoresistance and poor overall survival in these patients. Transfection
with pre-miR-136 enhanced apoptosis (30%) and decreased angiogenesis
(30−50%) compared to cells treated with PTX only in PTX-resistant SKpac
cells.
Conclusion: MiR-136 targets Notch3 responsible for poor prognosis and
chemoresistance in ovarian cancers. Modulation of miR-136 resensitizes
PTX-resistant ovarian cancer cells and reduces angiogenesis. Our results
suggest that miR-136 is a potential target for new strategy to overcome
chemoresistance.
277 COX-2/PGE2 promotes lung cancer invasion/metastasis via MIG-7
and phosphorylated prohibitin
S.M. Liang1 , M.Y. Ho2 , C.M. Liang2 . 1 Academia Sinica, ABRC, Taipei City,
Taiwan, 2 Academia Sinica, GRC, Taipei City, Taiwan
Background: The mechanism of promoting lung cancer invasion and
metastasis by high levels of cyclooxygenases-2 (COX-2) and prostaglandin
E2 (PGE2) is largely unclear. Migration inducting gene-7 (MIG-7) protein, a
potential mediator of metastasis, is functionally associated with COX-2/PGE2induced lung cancer metastasis. Another potential mediator of lung cancer
metastasis is phosphorylated prohibitin (phospho-PHB) in the raft domain
of cell membrane. The involvement of phospho-PHBT258 in COX-2/PGE2mediated effects and functional association between phospho-PHBT258 and
MIG-7 in lung cancer invasion/metastasis remains, nonetheless, largely
unexamined. Here, we show that phospho-PHBT258 and MIG-7protein play a
critical role in the induction and sustainment of PGE2 effects, respectively.
Material and Methods: We undertook this study with the following
technologies and methods: Transfection of lung cancer cell lines with
cDNA, siRNAs and shRNAs; immunoblotting, immunoprecipitation and gelatin
zymographic analysis; migration and invasion assays; generation of biotinlabeled or plasma membrane bound active PHB; generation of lung cancer
cells stably expressing not only green fluorescent protein and luciferase but
also MIG-7 shRNA, dominant negative phospho-PHB mutants; experimental
xenograft murine metastasis model; immunohistochemistry and histopathology
examination.
Results: We found that PGE2 initiated its metastatic effects by interacting with
prostaglandin E receptor 4 to transiently increase phosphatidylinositol-(3,4,5)triphosphate (PIP3) resulting in elevation of cellular COX-2 and generation
of more cellular PGE2 after 12 hours. This self-reinforcement of PGE2 was
phospho-PHBT258 dependent. PGE2/PIP3 also induced MIG-7 but the increase
in MIG-7 was phospho-PHBT258 independent. The PGE2/PIP3-mediated
increase in cancer invasion was functionally associated with elevation of
E-cadherin suppressors, notably ZEB1, Snail and Twist. The increase in
Snail was dependent on phospho-PHBT258 , whereas ZEB-1 and Twist were
dependent on MIG-7 that also sustains PGE2 effects via positive feedback
on PIP3/Akt signaling pathway. Downregulating phospho-PHBT258 and MIG-7
had an additive effect on attenuating lung cancer invasion/metastasis and
prolonging the survival of lung cancer xenograft mice.
Conclusion: The findings suggest the potential of a combination therapy
targeting phospho-PHBT258 and MIG-7 to block the initiation and sustainment
of COX-2/PGE2 effects on cancer metastasis.
278 The kallikrein-related serine peptidase, KLK4, regulates the TGFb1
pathway in the tumour–stroma microenvironment in prostate
cancer
J. Clements1 , R. Fuhrman-Luck1 , S. Stansfield1 , M. Hastie2 , T. Stoll2 ,
C. Stephens1 , D. Loessner1 , M. Lehman1 , C. Nelson1 , J. Gorman2 .
1
Translational Research Institute Queensland University of Technology,
Cancer Program, Brisbane, Australia, 2 QIMR Berghofer Medical Research
Institute, Protein Discovery Centre, Brisbane, Australia
Background: Prostate cancer cells reside in a complex stromal microenvironment often referred to as ‘reactive’ stroma which is a critical component
of prostate cancer initiation and progression. The mounting evidence for its
critical nature has led to increased interest in this niche as a target for new
therapeutic approaches. Cancer associated fibroblasts (CAFs) play a key role
in this niche regulating the tumour microenvironment. Factors secreted by
prostate cancer cells can ‘activate’ non-malignant associated fibroblasts to
become CAFs. KLK4 is over-expressed in both localised and bone metastatic
prostate cancer and so has the capacity to act as a paracrine factor on the
surrounding stroma.
Materials and Methods: To elucidate the role of KLK4 in tumour–stroma
cross-talk, its substrates were identified from the prostate cancer lines, LNCaP
and PC3 (also derived from a bone metastasis), and the prostate fibroblast
line WPMY-1, utilising the ‘PROtein TOpography Migration Analysis Platform’
S66
(Dix et al., Cell, 2008). Gene expression changes following KLK4 treatment
were assessed by gene microarray analysis.
Results: We identified 50 putative novel KLK4 substrates, 11 of which directly
interact with TGFb1. Strikingly, the most enriched pathway (DAVID analysis,
p < 0.01) on transcriptome analysis of the KLK4 treated cells was the TGFb1
pathway. KLK4-treated fibroblasts also expressed elevated levels of a number
of genes consistent with a CAF genotype.
Conclusions: These findings suggest that KLK4 is a critical regulator of
the reactive stromal niche, via the TGFb1 pathway, and a potential novel
therapeutic target for prostate cancer.
279 Alterations of hepatocyte metabolic identity are triggered by
the hepatitis C virus through systemic wnt/b-catenin signalling
M. Moreau1 , B. Rivière2 , S. Vegna3 , J. Ramos2 , E. Assenat3 , U. Hibner1 .
IGMM Institut de Génétique Moléculaire de Montpellier, Montpellier cedex
05, France, 2 Hôpital Saint Eloi—Gui de Chauliac, Montpellier, France,
3
Institut de Génétique Moléculaire, Montpellier, France
1
Introduction: Hepatitis C virus (HCV) is a major causative agent of
hepatocellular carcinoma (HCC), the fifth most frequent cancer and the third
cause of cancer-related deaths worldwide. The aetiology of HCV-associated
HCC comprises direct effects of the virus on the infected cell and global
alterations of liver physiology.
Various molecular mechanisms account for aberrant activation of the
wnt/b-catenin pathway that occurs in up to 40% of HCC, including those
associated with HCV. The same pathway operates in a healthy liver to maintain
metabolic zonation of the hepatic lobule, thus determining the metabolic
identity of a hepatocyte as a function of its position along the porto-centrilobular
axis. Here we report that physiological levels of HCV proteins give rise to
profound alterations of liver metabolic zonation, likely to constitute a risk factor
for subsequent tumorigenesis.
Material and Methods: We have used tumour-prone transgenic mice with
hepatocyte-targeted expression of all HCV proteins and needle biopsies from
early-stage hepatitis C patients. Alterations of metabolic zonation of the liver
lobule were investigated by biochemistry and IHC.
Results and Discussion: We studied HCV-driven lipogenesis because this
cancer-related alteration is also essential for the viral replication and spread.
We show that viral proteins drive striking redistribution of expression of fatty
acid synthase, a major lipogenic enzyme, both in mouse and in human
livers. Interestingly, changes of zonation pattern are associated with systemic
signalling by the conventional wnt/b-catenin pathway and in consequence are
not limited to lipogenesis; for example expression of enzymes involved in
glutamine metabolism is also profoundly altered. Careful analysis of a large
cohort of patients suggests that perturbed metabolic zonation occurs at early
stages of human disease, preceding other pathological alterations of the liver.
Conclusion: Our results rationalize systemic effects on liver metabolism
triggered by a minority of infected cells. Infection with HCV, a major agent
of hepatocellular carcinoma, gives rise to an early chronic deregulation of wnt
signalling in the liver and in consequence to alterations of metabolic functions
that are also typically deregulated in hepatic tumorigenesis.
280 Silencing of mitochondrial Lon protease deeply alters mitochondrial
proteome and functionality in RKO colorectal carcinoma cells
L. Gibellini1 , M. Pinti2 , F. Boraldi2 , V. Giorgio3 , P. Bernardi3 , M. Nasi4 ,
S. De Biasi4 , P. Pinton5 , D. Quaglino6 , A. Cossarizza4 . 1 University of Modena
and Reggio Emilia, Surgery Medicine Dentistry and Morphological Sciences,
Modena, Italy, 2 University of Modena and Reggio Emilia, Department of Life
Sciences, Modena, Italy, 3 University of Padova, Department of Biomedical
Sciences, Padova, Italy, 4 University of Modena, Department of Surgery
Medicine Dentistry and Morphological Sciences, Modena, Italy, 5 University
of Ferrara, Department of Morphology Surgery and Experimental Medicine,
Ferrara, Italy, 6 University of Modena, Department of Life Sciences, Modena,
Italy
Background: Lon is a nuclear-encoded, mitochondrial ATP-dependent
protease that assists protein folding, degrades oxidized/damaged proteins and
participates in maintaining mitochondrial DNA (mtDNA) levels. Here we show
that Lon is upregulated in several human cancers, including the colon cancer
derived cell line RKO, and that its silencing in these cells causes profound
alterations of mitochondrial proteome and massive cell death.
Materials and Methods: Lon mRNA was downregulated by RNA interference
using two vectors for constitutive and inducible (doxycycline-regulated)
expression of Lon short hairpin RNA. Mitochondrial proteome was analysed
by 2-dimensional gel electrophoresis and mass spectrometry. Mitochondrial
DNA and RNA were quantified by real time PCR. Apoptosis, content
of mitochondrial anion superoxide and cellular hydrogen peroxide, and
mitochondrial membrane potential were measured by flow cytometry.
Mitochondrial morphology was analysed by confocal and transmission electron
microscopy.
Results and Discussion: Mitochondria of Lon-silenced cells displayed low
levels of mtDNA transcripts, reduced levels of several subunits of oxidative
phosphorylation complexes (Complex I being the most affected), a marked
reduction of oxygen consumption rate, and impaired capability to synthetize
ATP. Higher levels of hydrogen peroxide and mitochondrial superoxide
anion were also observed. Mitochondrial network appeared fragmented, with
mitochondria heterogeneous in size and shape, dilated cristae, vacuoles
and electron-dense inclusions. The triterpenoid 2-cyano-3,12-dioxooleana-1,9dien-28-oic acid, an inhibitor of Lon proteolytic activity, was able to partially
mimic Lon silencing.
Conclusion: Lon is essential for maintaining mitochondrial shape and
functions, and for the survival of RKO colon cancer cells.
281 A panel of 20 genes involved in cellular adhesion and ECM
remodelling distinguishes renal cancer and control samples
J. Boguslawska1 , H. Kedzierska1 , B. Rybicka1 , P. Poplawski1 , Z. Tanski2 ,
A. Nauman1 , A. Piekielko-Witkowska1 . 1 Centre of Postgraduate Medical
Education, Department of Biochemistry and Molecular Biology, Warsaw,
Poland, 2 Regional Hospital Ostroleka, Department of Urology, Warsaw,
Poland
Background: Each year in Europe about 30,000 cases of renal cell cancer
(RCC) are diagnosed. Approx. 80% of RCC cases are classified as clear
cell renal cell carcinoma (ccRCC). RCC is highly resistant to conventional
therapies. Up to 30% of patients present metastasis at the time of diagnosis
when treatment options are limited. Thus, there is an urgent need for
identification of new molecular markers and therapeutic targets. Recent
studies have shown that analysis of networks of genes contributing to cancer
pathogenesis can offer more significant results than analysis of individual gene
markers. Key processes disturbed during tumoural progression are cellular
adhesion and ECM remodelling. Thus, we analysed the expression of genes
involved in these processes in ccRCC tumours.
Material and Methods: 20 pairs of ccRCC tumours and matched control
samples were used for analysis of expression of 84 genes involved in
cellular adhesion and ECM remodelling using RT2 Profiler™ PCR Arrays
(SABioscience). 21 differentially expressed genes were selected for further
validation using 54 pairs of ccRCC and control samples and Real-Time
Custom Panels (Roche Diagnostics). Reference genes were analysed using
NormFinder. Statistical analysis was performed using GraphPad Prism. The
study was approved by the institutional Bioethics Committee.
Results: We have found concomitant and statistically significant (P < 0.05)
changes of expression of 20 genes. CNTN1 was the only one with expression
decreased, while the expression of residual 19 genes (COL1A1, COL5A1,
COL8A1, COL11A1, COL15A1, FN1, ICAM1, ITGAL, ITGAM, ITGA5, ITGB2,
LAMA3, MMP1, MMP9, MMP16, NCAM1, TGFB1, THBS2, and TIMP1) was
upregulated in tumours. Correlation matrix revealed clusters of genes whose
expression was strongly correlated (P < 0.001), including the group of integrins
whose expression correlated with collagens. Notably, some of the correlations
were lost in tumour samples (eg. LAMA3 vs COL8A1, FN1, MMP16, TGFB1).
Conclusion: We have identified a panel of 20 genes involved in cellular
adhesion and ECM remodelling whose expression is concomitantly disturbed
in ccRCC. Correlated expression of groups of genes suggests that their
expression may be coregulated by a common pathway. To our knowledge
this is the first study aiming in the targeted concomitant analysis of multiple
adhesion and ECM-related molecules in renal cancer.
Financially supported by National Science Centre grant no. 2012/05/B/NZ5/
01541.
282 p53-directed translational control can shape and expand the
universe of p53 target genes
S. Zaccara1 , C. Martinez Bolado1 , C. Pederiva1 , T. Tebaldi1 , Y. Ciribilli1 ,
A. Bisio1 , A. Inga1 . 1 Centre for Integrative Biology CIBIO, University of Trento,
Trento, Italy
Background: Several genome-wide transcriptome analyses that focused on
p53-induced cellular responses in many cellular contexts have continued to
expand the already vast p53-regulated transcriptional networks.
Material and Methods: To investigate post-transcriptional controls as an
additional dimension of p53-directed gene expression responses we performed
translatome analysis by polysomal profiling on MCF7 cells treated with
Doxorubicin and Nutlin-3a.
Results: A comparison between the transcriptome and the translatome
revealed a considerable level of uncoupling meaning genes whose
transcription did not correlate with translation. Interestingly, this genes category
was significantly associated to apoptosis, DNA and RNA metabolism as
well as cell cycle functions, suggesting that post-transcriptional control can
modulate classical p53-regulated responses. Furthermore, even for wellestablished p53 targets that were differentially expressed both at transcriptional
and translational levels, quantitative differences between transcriptome,
subpolysomal and polysomal RNAs were evident. This observation, confirmed
by qPCR assays, led us to identify processes of fine-tuning mRNA fate
that we classify as translational-thrust or -drag that can act on up to
25% of the ~170 known p53 target genes. Seeking mechanisms underlying
gene expression uncoupling, we identified p53-dependent modulation of
five RNA binding proteins where hnRNPD (AUF1) and CPEB4 are direct
p53 transcriptional targets and SRSF1, DDX17, YBX1 are indirect targets
modulated at translational level in a p53-directed manner. In particular, YBX1
translation appeared to be reduced by p53 via two different mechanisms, one
related to mTOR inhibition and the second to miR-34a expression.
Conclusions: Overall, we establish p53 as a master regulator of translational
control and identify new p53 target genes affecting translation that can
contribute to p53-dependent cellular responses. Based on the functions of
these target genes, it becomes apparent that selectivity at the level of mRNA
translation, next to transcriptional selectivity, provides an important contribution
to shape p53-directed responses.
283 Disruption of cortical tension patterning drives the outgrowth
of oncogenic cells
S. Wu1 , G.A. Gomez1 , M. Michael1 , S. Verma1 , H. Cox1 , J.G. Lefevre1 ,
R.G. Parton1 , N.A. Hamilton1 , A.S. Yap1 . 1 The University of Queensland,
Institute for Molecular Bioscience, Brisbane Qld, Australia
Background: Oncogenic cell extrusion is a recently discovered process
where transformed cells are physically expelled from their growth suppressive
epithelial environment. Exciting advances in the past 2−3 years implicate
oncogenic extrusion as a mechanism for cells that have acquired new
mutations to escape the suppressive environment of their parental tumor
during clonal proliferation and initiating cancer cells dissemination program.
Elucidating the cellular mechanisms responsible for oncogenic extrusion thus
has immediate implications for understanding tumor dissemination, diagnosis
and therapeutics.
Material and Methods: Oncogenic extrusion was tested in adenocarcinoma
cell cultures that mosaically express H-RasV12 (at low concentrations, where
single or small groups of H-RasV12 expressing cells are surrounded by control
cells). Mosaic cultures were achieved either by mixing control and H-RasV12
expressing cells or by controlling the rate of H-RasV12 transfection.
To uncover the core mechanisms mediating oncogenic extrusion, a variety
of photonic methods including laser nanoablation and photoactivation were
employed to probe the molecular and biophysical properties of extruding
transformed cells. This is combined with genetic, growth factor and
pharmacological perturbations to achieved a systems level understanding of
how oncogenic signaling can translate into physical forces that initiates the
cancer cells dissemination program via cellular extrusion.
Results: In particular, the study by Leung and Brugge at Havard Medical
School in 2012 showed that de novo transformation of normal cells has
increased proliferative potential only when they were extruded from the growth
suppressive normal epithelium. My work has extended this finding by showing
that even within a transformed population, further mosaic overexpression of the
H-RasV12 oncogene within an adenocarcinoma epithelium lead to the escape
of these severely transformed cells. As cancer is a multigenic disease that
develops from a multitude of genetic mutations, the ability for transformed
cells to colonize specific tissues most likely will only develop in response to
the selective pressure on already disseminated cancer cells (Hanahan and
Weinberg, 2011). Collectively, these findings suggest a multistage invasion
program that relies on extrusion as a selection mechanism for driving
metastasis of ‘fitter’ malignant cells.
Conclusions: Transformed cells were extruded from monolayers when the
apicolateral patterning of junctional contractility was altered; either when
N-WASP (actin cytoskeleton regulator) redistributes from apical to lateral
E-cadherin junctions in cells expressing oncogenic H-RasV12 . As studies
investigating the impact of oncogenic transformation on the spatial control
of cellular mechanics are still relatively limited, this finding provides novel
mechanistic insights into the emerging role of tension in the regulation of
tumour progression.
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284 Evidence of a correlation between bcl-2 protein and miR-211
expression in melanoma cell lines
T. De Luca1 , A. Pelosi2 , D. Trisciuoglio1 , S. D’Aguanno1 , A. Felsani3 ,
A. Urbani4 , M.G. Rizzo2 , D. Del Bufalo1 . 1 Regina Elena National Cancer
Institute, Experimental Chemotherapy Laboratory, Rome, Italy, 2 Regina
Elena National Cancer Institute, Molecular Oncology, Rome, Italy, 3 Santa
Lucia Foundation-IRCCS Rome Italy, CNR-Institute of Cell Biology
and Neurobiology, Rome, Italy, 4 University of Tor Vergata Santa Lucia
Foundation-IRCCS Rome Italy, Department of Internal Medicine Laboratory
of Proteomics, Rome, Italy
Background: Bcl-2 is a proto-oncogene often associated with poor prognosis
in melanoma. In this context, we previously demonstrated the ability of
bcl-2 protein to increase tumorigenic and metastatic potential in melanoma
cells. The molecular mechanisms behind invasive melanoma are poorly
understood. Recent studies implicate microRNAs (miRNAs) as important
agents in melanoma and other cancers. miRNAs regulate several molecular
pathways, such invasion and metastasis, by targeting various oncogenes
and tumour suppressors. Among these, miR-211, that is located within
TRPM1 gene, is prevalently expressed in the melanocyte lineage and acts
as oncosuppressor.
Material and Methods: Whole Transcriptome Analysis (WTA) was performed
to analyze change in gene expression in M14 human melanoma cell line
and its derivative bcl-2 overexpressing clone (M14/bcl-2). Ingenuity Pathway
Analysis (IPA) software was used to identify the most relevant signaling
pathways and biological functions of differentially regulated proteins. miR-211
expression and its target genes changes were examinated by RT-qPCR. A375SC human melanoma cell line and its derivative bcl-2 overexpressing clone
(A375-SC/bcl-2) were also used.
Results: To identify new bcl-2-related gene networks, we performed analysis
of gene expression followed by IPA. Interestingly, the top functional network
identified by IPA was cellular movement, with 50 molecules signed. Notably,
we found that most significative nodes of this network were miR-211 predicted
targets, and some of them were upregulated in bcl-2 overexpressing cells
respect to the control. These results lead us to investigate if there is a
correlation between the expression of bcl-2 and mirR-211. According with
bioinformatics results, we found that expression of miR-211 in M14 parental
cells is higher than in bcl-2 overexpressing cells, revealing that miR-211
is modulated by bcl-2. Similar results were also obtained in A375-SC and
its derivative bcl-2 overexpressing clones, despite the difference in miR211
expression between the two cell lines. Analysis of pri-miR-211 and TRPM1
levels in M14 and bcl-2overexpressing cells indicates that bcl-2 regulates miR211 at transcriptional level. Finally, mRNA levels of two known mir-211 targets,
IGF2R and TGFBR2 were analyzed, but only TGFBR2 mRNA was found to be
upregulated in bcl-2 overexpressing cells when compared to control cells.
Conclusions: Our results demonstrated a correlation between the expression
of bcl-2 protein and miR-211 in melanoma cell lines. Further investigations are
necessary to elucidate the mechanism underlying this evidence.
285 MicroRNA expression in triple-negative versus other subtypes
of breast cancer
D. Kalniete1 , M. Nakazawa-Miklasevica1 , I. Strumfa1 , A. Abolins1 , A. Irmejs1 ,
G. Trofimovics1 , J. Gardovskis1 , E. Miklasevics1 . 1 Riga Stradins University,
Institute of Oncology, Riga, Latvia
Background: Breast cancer is a clinically, morphologically, and genetically
heterogeneous disease thus requiring a personalized approach to the
treatment. Currently used biomarkers are not enough informative to predict
the pace or the outcome of the disease, therefore new markers are required.
One of such potential biomarker is microRNA: a class of small, non-coding
molecules that regulate gene expression at a post-transcriptional level. In
malignancies, expression of the microRNAs is altered and correlates with
the clinical features of the disease. This study attempted to explore some
microRNA expression differences in a more aggressive subtype (triplenegative) compared to other breast cancer subtypes (luminal-A, luminal-B,
and HER2+).
Material and Methods: The study group consisted of 13 LA, 7 LB, 2 HER2+,
and 50 TN breast cancer tissues. MicroRNAs were extracted from the formalinfixed and paraffin embedded tumor tissues. A quantitative analysis of miR-10b,
miR-21, miR-29a, miR-31, and miR-214 in cancer tissues was performed by
real-time PCR. RNU6B was used as an internal control. All cancer tissues
contained more than 50% of cancer cells per sample. A disease-specific
survival was calculated from the date of the diagnosis to the date of the death
from the cancer. The median follow-up period of breast cancer patients was 47
months. The disease-specific survival was analyzed using a Log-rank (MantelCox) test. Statistical significance was set at the 95% level (p < 0.05).
Results: Expression of miR-21, miR-31, and miR-214 was higher in TN
than in other tumor tissues (p = 0.0027; p = 0.0012; p = 0.0230, respectively).
No statistically significant differences in miR-10b and miR-29a expressions
between TN and other subtypes were observed (p = 0.1902 and p = 0.1707,
S68
respectively). Breast cancer patients with the high expression of miR-31 and
miR-214 showed a non-significant trend for a worse disease-free survival than
patients with the low expression (p = 0.1707 and p = 0.2220, respectively). No
statistically significant difference in regard of high and low miR-21 expression
was observed (p = 0.3170).
Conclusion: Different microRNA expression in distinct subtypes of breast
cancer reflects genetic heterogeneity of breast cancer and indicates outcome
of the disease; breast cancer patients with the high expression of miR-31 and
miR-214 have a non-significantly worse disease-free survival than patients with
the low expression.
286 Expression of markers of epithelial–mesenchymal transition
E-cadherin and vimentin in different immunohistochemical
subtypes of breast cancer
Y.M. Zasadkevich1 , S.V. Sazonov1 , A.A. Brilliant1 . 1 Institute of Medical
Cell Technologies, Laboratory of Pathomorphology, Ekaterinburg, Russian
Federation
Background: For realization of invasion and intravasation in metastasis it
is necessary for tumor cells to change their phenotype from epithelial to
mesenchymal. Herewith the mechanism known as epithelial–mesenchymal
trasition (EMT) starts. The first step of it is the decrease of expression of
epithelial tissue markers such as E-cadherin and the increase of expression
of mesenchymal tissue markers such as vimentin.
The aim of the research was to study features of expression of E-cadherin and
vimentin in different immunohistochemical subtypes of breast cancer (BC).
Material and Methods: 152 cases of infiltrative lobular BC were studied.
E-cadherin expression was detected with use of Monoclonal Mouse AntiHuman E-cadherin Clone NCH-38 (DAKO, Denmark). Vimentin expression
was detected with use of Monoclonal Mouse Anti-Swine Vimentin Clone V9
(DAKO, Denmark). Expression of E-cadherin was evaluated as positive when
70% tumor cells were stained, vimentin − in any positive cytoplasmic staining
of tumor cells.
Results: The decrease of E-cadherin expression was found in 22 (14%) of
all the cases while the appearance of vimentin expression was found in 78
(51%) of the general group. The decrease of E-cadherin expression was
identified in 12 (21%) of the cases in Luminal A subtype, in 6 (8%) − in
Luminal B subtype, in 4 (8%) − in Basal-like subtype. None of cases in Her2overexpression subtype showed the decrease of E-cadherin expression. In
comparison with the general group, the significant increase of % of the cases
with decreased level of E-cadherin was determined in 1.5 times (p < 0.05)
in Luminal A subtype, in 1.3 times (p < 0.05) in Luminal B subtype and the
decrease of % of the cases with decreased level of E-cadherin in 1.75 times
(p < 0.05) in Basal-like subtype.
The appearance of vimentin expression was found in 38 (76%) of the cases in
Luminal A subtype, in 22 (65%) − in Luminal B subtype, in 6 (60%) − in Her2overexpression subtype and in 12 (24%) − in Basal-like subtype. The significant
increase of % of the cases with positive expression of vimentin was identified
in 1.5 times (p < 0.05) in Luminal A subtype, in 1.3 times (p < 0.05) in Luminal B
subtype and in 1.75 times (p < 0.05) in Basal-like subtype in comparison with
the general group. The decrease of % of the cases with positive expression of
vimentin was found in 2.1 times (p < 0.05) in Her2-overexpression subtype.
Besides, the strong positive correlation between proliferation level, detected by
Ki67 expression, and vimentin expression in Her2-overexpression and Basallike subtypes was found (r = 0.85, p < 0.05; r = 0.87, p < 0.05 respectively).
Conclusions: The decrease of E-cadherin expression connects with the high
expression of ER and PR that suggests the existence of ER-associated
pathway which downregulates E-cadherin in ER-positive BC.
EMT mostly arises in cases of BC with the high level of proliferation and
dedifferentiation of tumor cells that reflects in the link between the appearance
of vimentin expression and the high expression of Ki67, which is common for
Basal-like and her2-overexpression subtypes.
to the pathophysiological in vivo situation. We have developed a novel method
to grow multiple homogenous spheroids per well in 96 well plate format.
Because the spheroids are attached to the well bottom, medium changes are
easy to handle and spheroids can be fixed/stained without spheroid loss. The
spheroids are directly compatible with High Content Imaging and Analysis with
no troublesome plate transfer.
Material and Methods: Using biochemical HTS readouts (viability) and complementary image analysis (diameter/area/roundness), basic characteristics
of the micropatterned spheroids were validated including: (1) growth as a
function of time, (2) kinetics of necrotic center formation, (3) response to
standard chemotherapeutic drugs. Flexibility of the micropatterned system
was demonstrated using cancer cell lines from different origins including lung,
breast and colon (HT29, MCF7, A549).
Results: Our method allows formation of up to 15 cancer cell spheroids
per well. Kinetic studies show that the spheroids are uniform (CV% <10%)
and size can be tightly controlled over time reaching maximal diameters of
600 mm. Functional asymmetry was demonstrated with a proliferative region
at the periphery together with a necrotic region in the center. Dose response
experiments with anti-cancer agents demonstrate cytotoxic effects with higher
significance and statistical confidence than single spheroids per well.
Conclusions: Overall, our results showed that multiple highly reproducible
spheroids per well can be obtained in 96 well plate format contributing to
higher reliability and robustness compared to standard methods. Spheroids
are metabolically relevant and precisely localized in the same optical plane
making this approach ideally suited to HCA approaches that provide access
to a higher quality of information.
288 Effect of boric acid on head and neck cancer cell lines
M. Gunduz1 , M. Acar2 , K. Fakioglu2 , B. Dogan2 , M. Oznur2 , E. Gunduz2 .
1
Turgut Ozal University Faculty of Medicine, Medical Genetics and
Otolaryngology, Ankara, Turkey, 2 Turgut Ozal University Faculty of Medicine,
Medical Genetics, Ankara, Turkey
Background: About 15% of all head and neck cancers are laryngeal and
pharyngeal. Alcohol and tobacco use and exposure to irritants play an
important role in the etiology of these cancers. They are not easily diagnosed at
early stages, with about 60−70% of cases escaping early diagnosis. Treatment
typically consists of radiation and chemotherapy. As the world’s richest source
of boron, we aimed to investigate the use boron in the treatment of head and
neck cancer. The raw material borax is one of the richest resources in Turkey.
Studies have shown that boron is essential for the immune and endocrine
systems as well as the metabolism of bones, minerals, and lipids. In recent
years, the idea of using boron in medicine has gained interest. Initially, the
use of boron in cancer therapy came in the form of Boron Neutron Capture
Therapy (BNCT), which was developed over 50 years ago and has been used
primarily for the treatment of brain and head and neck cancers. The advantage
of this system is that it is relatively specific to cancer cells, causing minimal
damage to normal cells. The direct effect of boron on cancer cells has also
been investigated.
Material and Methods: In this study we have examined the effect of boric acid
on head and neck cancer cell lines on cell proliferation. We have examined
UT-SCC 6A, 6B, 9A, 16A, 16B, 24A, 54C, 74A and 74B via treatment of 200–
2000 ug/mL of boric acid by XTT assay.
Results: We have detected that the most effective dosage of boric acid which
inhibited cell proliferation of the cell lines were as follows: 6B 1800 ug/mL,
24A 1600 ug/mL, 54C and 6A 1200 ug/mL, 16B 1000 ug/mL, 74B, 74A,
9A and 16A 800 ug/mL.
Conclusions: Our results displayed that 800–1600 ug/mL of boric acid inhibits
cell proliferation of head and neck cancer cell lines. We will also confirm
whether if this inhibition is through the tumor suppressor genes by examining
the expression of very well-known tumor suppressors P53, RB and ING1 after
treatment with boric acid.
287 Improved robustness for fully automated 3D spheroid HCA
screening
290 Examination of role of ING1 splicing variant (p33ING1) in
carcinogenesis and metastasis of head and neck carcinomas
S. Degot1 , J. Young1 , E. Duchemin-Pelletier1 , F. Monjaret1 . 1 CYTOO, R&D,
Grenoble, France
E. Gunduz1 , O.F. Hatipoglu1 , K.O. Yaykasli2 , K. Erdogan1 , E.N. Cetin1 ,
G. Nas1 , M. Gunduz1 . 1 Turgut Özal University Institute of Health Sciences
Faculty of Medicine, Department of Medical Genetics, Ankara, Turkey,
2
Kahramanmaras Sutcu Imam University Medical Faculty, Department of
Medical Biology, Kahramanmaras, Turkey
Introduction: For drug screening of tumorigenic cell lines, the most widely
used method involves culturing of malignant cells in conditions simulating
anchorage-independent growth. The result is a single multicellular spheroid per
well that mimics the initial avascular stages of solid tumours in vivo. Depending
on cell type, spheroids with a diameter above 400–500 mm develop hypoxia and
subsequent necrosis in their centre due to limited inward and outward diffusion
of nutrients and waste. Due to the presence of quiescent cells, spheroids are
often more resistant to compounds compared to the same cells grown as
conventional 2D monolayers. With regard to overall 3D cytoarchitecture and the
effect of diffusion gradients on drug penetration, spheroids are therefore closer
Introduction: Head and Neck Squamous Cell Carcinoma (HNSCC) occurs
in the Oral Cavity, oropharynx, larynx or hypopharynx and is the sixth most
frequent cancer worldwide. ING tumor suppressor family has recently been
identifed by us and other research groups and mentioned as important genes
similar to p53 and RB1 tumor suppressors. In the current work, we have
examined the role of splicing variant of ING1 (p33ING1) in primary head
and neck cancer tissues as well as cell lines. In conclusion, final aim of this
project is to open a gate for defining novel molecular diagnostic and thrapeutic
methods by elucidating the functions of p33ING1 ING1 splicing variant in
carcinogenesis and metastasis of head and neck cancer.
Materials and Methods: We have used head and neck cancer cell lines as
material derived from the primary tumor and their lymph node metastasis,
which belong to our collaborator Prof. Grenman from Turku University, Finland.
First expressions of the ING1 splicing variants and p53 mutation status will be
examined in these cells. Then expression vector of the splicing variants will
be prepared and overexpressed in the cells by transfecting each of them,
followed by analysis of apoptosis, cell cycle and cell growth. Moreover their
roles in metastasis will also be investigated with specific tests.
Results: We have shown that splice variant of p33ING1 suppresses the
proliferation of primary and metastatic cells and also it restrains the cancer
cell movement via suppressing the migration of head and neck cells.
Conclusion: Splice variant of p33ING1 has oncogenic function in head and
neck cancer cells. It suppresses the proliferation and migration of head and
neck cancer cells.
291 The 5 -untranslated region of p16INK4a acts as a cellular IRES,
controls mRNA translation during hypoxic and energetic stresses,
and is a target of YBX1
A. Bisio1 , E. Latorre2 , V. Andreotti3 , P. Ghiorzo3 , B. Bressac-de Paillerets4 ,
R.C. Spitale5 , A. Provenzani2 , A. Inga1 . 1 Laboratory of Transcriptional
Networks Centre for Integrative Biology CIBIO, University of Trento, Trento,
Italy, 2 Laboratory of Genomics ScreeningsCentre for Integrative Biology
CIBIO, University of Trento, Trento, Italy, 3 Laboratory of Genetics of Rare
Hereditary Cancers, DiMI University of Genoa, Genoa, Italy, 4 Institut
Gustave Rousy, Villejuif, France, 5 Howard Hughes Medical Institute, Stanford
University School of Medicine, Stanford CA, USA
Background: In mammalian cells, controlled progression through the cell
cycle is essential for normal proliferation and its loss is a hallmark of
malignancy. p16INK4a is a well known tumor suppressor gene acting as an
inhibitor of cell cycle progression and its deregulation is often associated with
many types of cancer, including melanoma. Here we report that p16INK4a
belongs to the expanding group of proteins whose translation is influenced
by sequence/structural features of the 5 UTR mRNA that are endowed of socalled cellular Internal Ribosome Entry Site (IRES) activity.
Material and Methods: To study the potential for p16INK4a 5 UTR to drive capindependent translation we developed a dual-luciferase assay using bicistronic
vectors, where wild type or deletion mutants of the p16INK4a 5 UTRs can be
studied.
Results: Quantification of reporters’ relative activities coupled to control
analyses for actual bicistronic mRNA transcription, indicated that the wild
type p16INK4a 5 UTR could stimulate cap-independent translation. Notably,
hypoxic stress in particular, but also the treatment with mTOR inhibitors,
enhanced the translation-stimulating property of the wild type p16INK4a 5 UTR.
RNA immuno-precipitation (RIP) assays performed in the p16INK4a -positive
melanoma-derived cell line SK-Mel-28, and in melanoma patients-derived
lymphoblastoid cell lines, indicated that the RNA-binding protein YBX1, known
to act in translation control, can target wild type p16INK4a mRNA and enhance
its translation, particularly during hypoxic stress. Experiments where YBX1 was
over-expressed or knocked-down confirmed its involvement in p16INK4a capindependent translational regulation. The p16INK4a c.−42T>A sequence-variant
was instead no longer influenced by changes in YBX1 protein level, consistent
with predictions of the binding site of this RBP to the p16INK4a 5 UTR and with
results based on RNA SHAPE assays.
Conclusions: Taken collectively, our results suggest that the p16INK4a 5 UTR
region acts as cellular IRES that can modulate mRNA translation efficiency
and can be positively regulated by YBX1.
292 Cytotoxic effect and apoptosis induction by phytohemagglutinin
erythroagglutinating on lung cancer cells
K.Y. Chen1 , W.T. Kuo2 , Y.J. Ho3 , C.H. Yao3 . 1 National Yunlin University of
Science and Technology, Department of Chemical and Materials Engineering,
Yunlin, Taiwan, 2 China Medical University, Graduate Institute of Clinical
Medical Science, Taichung, Taiwan, 3 China Medical University, Department
of Biomedical Imaging and Radiological Science, Taichung, Taiwan
Introduction: Lung cancer is currently the leading cause of cancer deaths in
the world. Therefore, it is critical to study new and effective drug treatments
for lung cancer. Phytohemagglutinin, a lectin derived from red kidney beans,
has been reported to inhibit the growth of cancer cells. In this study, the
anticancer effects of phytohemagglutinin erythroagglutinating, one of isoforms
of phytohemagglutinin, on lung cancer cell A549 were evaluated.
Material and Method: Human A549 lung cancer cells were treated with various concentrations of phytohemagglutinin erythroagglutinating. After 2 days of
culture, the cytotoxicity and apoptosis-inducing potential of phytohemagglutinin
S69
erythroagglutinating were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay, glucose-6-phosphate dehydrogenase)
release assay and flow cytometry.
Results and Discussion: Phytohemagglutinin erythroagglutinating caused
a dose-dependent increase in cell growth inhibition and cell death of A549
cells. The percentage of apoptotic cells was increased with increasing
phytohemagglutinin erythroagglutinating concentrations. Moreover, dead cells
and live cells were a dose-dependent increase and decrease. These results
suggest that phytohemagglutinin erythroagglutinating induced growth inhibition
and cytotoxicity of A549 cells is mediated through an induction of apoptosis.
Conclusion: Phytohemagglutinin erythroagglutinating effectively inhibited
the growth of A549 cells and induced their apoptosis. Therefore,
phytohemagglutinin erythroagglutinating could be developed into an effective
anti-lung cancer drug.
293 MNT roles and expression in the absence of MAX
M.C. Lafita1 , A. Quintanilla1 , J. Rodrı́guez2 , I. Varela1 , A. Von Kriegsheim2 ,
J. León1 . 1 IBBTEC Universidad de Cantabria, Biologı́a Molecular, Santander,
Spain, 2 Conway Institute, System Biology Ireland, Dublin, Ireland
Introduction: MNT is a transcription factor of the MXD family. MXDs
proteins take part in MYC/MAX/MXD network regulating genes involved in cell
proliferation, differentiation, metabolism, cell growth and apoptosis. Alterations
in MYC/MAX/MXD network have been found to be responsible of cancer
development. We wanted to explore whether MNT has biological functions
independent from MAX.
Material and Methods: UR61 cells derivated from rat pheochromocytoma
cell line PC12 that lacks MAX wt protein; UR61MAX cells, UR61 expressing
a zinc-inducible human MAX gene; K562 human chronic myeloid leukemia
cells and 293T human embryonic kidney cell line. siRNA against human MAX
and rat MNT to downregulate their expression. RT-qPCR and WB to study
gene expression at mRNA and protein levels. High-throughput sequencing
techniques to analyse chromatin protein binding (ChIPseq). High-throughput
proteomic techniques, IP-mass spectrometry, to look for new interacting
partners. IP and ChIP to confirm the results obtained.
Results and Discussion: To study the role of MNT in cells that lack MAX
protein we silenced MNT protein in UR61 with siRNA and observed that it
became lethal for the cells, suggesting a pro-survival role of MNT in these cells
lacking MAX protein. Therefore, MNT down-regulation provokes cell growth
inhibition in a MAX independent manner. We analysed MNT expression in
UR61 and UR61MAX. In UR61, MAX expression leads to a downregulation of
MNT at mRNA and protein levels. In K562 cells, silencing of MAX provoked
MNT upregulation, confirming the results obtained in UR61. Bioinformatic
analysis of the promoter of MNT gene showed 2 Eboxes within −1Kb from
the transcription start site in rat and human MNT genes. ChIP assays for MNT
and MAX proteins indicates that MNT and MAX are bound to MNT promoter
in UR61MAX cells but not in UR61 cells. This suggests that MNT binds to its
own promoter and regulates its own expression only when there is MAX in
the cell. To elucidate whether MNT have different target genes when MAX is
not present we did a ChIPseq assay in UR61 and UR61MAX and we found
differences in MNT-DNA binding depending on MAX presence. To look for
new possible partners of MNT, IP-mass spectrometry assay was performed in
UR61 and UR61MAX. At least 70 protein interactions were detected. However,
MNT interacting partners are different when MAX is present.
Conclusion: We can conclude that MNT down-regulation impairs cell growth
in a MAX-independent manner and that MAX induces a decrease in MNT
expression. We have revealed new MNT interacting partners that might be
involved in a pro-survival role of MNT in the cell. We think it is worthy to take
into a count our work since understanding cancer development mechanisms
can help design new treatments for cancer therapies.
294 In vitro modulation of CITED4 gene expression in a colorectal
cancer cell line
M.A. Rogers1 , V. Kalter1 , G. Marcias1 , M. Zapatka1 , S. Barbus1 ,
B. Radlwimmer1 , P. Lichter1 . 1 German Cancer Research Center, Division of
Molecular Genetics (B060), Heidelberg, Germany
Background: CITED4 is one member of the CITED family of transcriptional
cofactors. Several of the CITED family members are deregulated in a variety of
tumors. Analysis of data from the literature points to a possible role of CITED4
in colon carcinogenesis. In this study we deregulate CITED4 expression,
in vitro, in a human colorectal cancer cell line, and analyze the phenotypic
and gene expression changes induced by modulation of CITED4.
Methods: CITED4 overexpressing- and shRNA-mediated knockdown cell
lines, as well as control cell lines, were established in the colorectal cancer
(CRC) cell line SW480. The cells were analyzed phenotypically, in vitro,
for changes in proliferation, apoptosis/cell cycle, migration, invasion, colony
formation and adhesion. Changes in mRNA expression were determined by
S70
Agilent 4 x 44k microarray analysis, and subsets of the deregulated genes were
validated by qRT-PCR and Western blotting. In addition, pathway analysis was
performed on the knockout cell line.
Results: The CITED4 overexpressing cell line showed only moderate changes
in adhesiveness to the basement membrane proteins collagen IV and
vitronectin. Microarray analysis identified a small number of deregulated
genes, including several G-protein coupled receptors (GPR64, GPR110,
LGR6). Phenotypic analysis of the CITED4 shRNA knockdown cell line
demonstrated a decrease in cell proliferation accompanied by a moderate G2
cell cycle block. Microarray analysis identified a large number of deregulated
genes, and pathway analysis pointed to the involvement of several genes found
in actin-associated adherens junctions/tight junctions (claudin-4, claudin-7,
ezrin, MET, b-catenin). These genes were validated independently by qRTPCR and, partially, by western blotting.
Conclusion: CITED4 shRNA mediated knockdown leads to decreased cellular
proliferation associated with the modulation of a large number of genes,
including the c-MET tyrosine kinase, as well as deregulation of several actin
associated adherens junctions/tight junction genes. The status of the actin
cytoskeleton and adherens/tight junctions in the knockdown cell line is currently
being further evaluated.
proliferation, the migration and the tumor development in vivo of glioblastoma
rat (C6) and human (U373MG) cell lines (C. Nasarre, et al. Oncogene 2010).
To further validate the anti-tumoral activity of MTP-NRP1, we evaluated its
therapeutic potential using another glioblastoma cell line (U118MG highly
expressing NRP1) in heterotopic and orthotopic xenografting models. Our
results show that MTP-NRP1 significantly reduces tumor development in
the heterotopic model as exemplified by the determination of the RECIST
criteria identifying 10% of mice presenting stable disease while 90% exhibited
partial responses (>30% diminution of the tumor volume increase). Strikingly,
intraperitoneal delivery of MTP-NRP1 every three days for a period of three
weeks had a beneficial impact on the tumor development in the orthotopic
model as it reduced the tumor volume by 50%. This effect related to the
inhibition of the proliferative index (−38%; p = 0.023) and to a mild but
significant inhibition of the vascular density (−11%; p = 0.0394). Hence, the
inhibition of tumor growth in vivo can be explained by an anti-proliferative and
an anti-angiogenic effect of MTP-NRP1 mirroring the results obtained with
in vitro assays.
J. Kolodziejski11 , U. Hibner1 , P. Lassus1 . 1 IGMM Institut de Génétique
Moléculaire de Montpellier, CNRS, Montpellier, France
297 The metabolic cooperation between prostate carcinoma cells
and cancer associated fibroblasts: pyruvate kinase M2 at the
crossroads
E. Giannoni1 , M.L. Taddei1 , A. Morandi1 , G. Comito1 , P. Chiarugi1 . 1 University
of Florence, Department of Experimental and Clinical Biomedical Sciences,
Florence, Italy
Background: Twist proteins are bHLH transcription factors essential for proper
embryonic development that are over-expressed in many human tumors.
Among their oncogenic activities, induction of epithelial to mesenchymal
transition leads to increased invasion and might confer to an epithelial cell a
cancer stem cell phenotype. In addition, Twist factors override two oncogeneinduced failsafe programs: senescence and apoptosis, thereby promoting the
malignant conversion.
Current knowledge of the pleiotropic activities of Twist prompts us to consider
these factors as major regulators of stress response. Cancer cells survive and
grow within a continuously changing environment that creates multiple stresses
to which they must adapt in order to survive and strive. Such adaptations can
give rise to the acquisition of an aggressive phenotype. Consistent with this
hypothesis, we recently unveiled a new activity of Twist proteins: we have
shown that they are regulators of oxidative stress. Of note, cancer cells suffer
from exacerbated oxidative stress caused by stimuli such as inflammation,
increased cellular metabolism or changes in oxygenation.
We now have characterized the molecular mechanisms controlling Twist antioxidant activity. Following this work we recently investigated links between
Twist and another type of cellular stress: hypoxia. Our results suggest that
Twist oncoproteins play a major role in cancer cell adaptation to environmental
stress, further defining their crucial role in tumor progression.
Material and Methods: Expression of genes of interest was modulated by
siRNA, shRNA or retroviral infection in several primary and immortalized cell
types. Expression levels were analyzed by immunoblotting or RTq PCR. ROS
levels were assessed by DHE and CM-H2DCFDA staining and subsequent
FACS analysis. Apoptosis was measured by Annexin V-Cy3 labelling followed
by FACS analysis.
Results and Discussion: To unravel the molecular mechanism involved
in Twist anti-oxidant activity, we performed a micro-array analysis of cells
expressing Twist. This approach identified several possible targets, previously
described as being involved in modulation of reactive oxygen species
(ROS). Further investigation led us to discover that Twist controls a specific
transcriptional program that regulates oxidative stress. Importantly, we also
showed that this program is required for Twist anti-apoptotic activity. Following
this study, we found that Twist also protects cells from hypoxia, which is another
type of cellular stress commonly found in tumors. Interestingly, preliminary
data suggest that Twist plays a role in hypoxic adaptation by regulating Hif1-a
through a direct interaction.
Conclusion: Overall, our results suggest that Twist could play a major role in
cellular stress response and thus could be important for cancer cell adaptation
during carcinogenesis.
Introduction: The ability of cancer cells to invade and metastasize is
influenced by the surrounding tumor microenvironment. It is established
that cancer associated fibroblasts (CAFs) can promote tumor progression
by enhancing cancer cell invasiveness and stemness. In addition the
reciprocal interaction between CAFs and prostate cancer (PCa) cells has
been demonstrated to induce their metabolic reprogramming. Notably, both
tumor microenvironment and metabolic reprogramming have been included
in the revised model of the Hallmarks of Cancer. Interestingly, upon tumor–
stroma interaction, CAFs undergo Warburg metabolism (i.e. increased glucose
consumption and lactate extrusion), while PCa cells undergo a ‘reverse
Warburg metabolism’. This metabolic switch allows PCa cells to reactivate
OXPHOS and exploit CAF-derived lactate to drive anabolic pathways, thereby
supporting cell growth.
Material and Method: Prostate carcinoma cell lines (PC3, DU145) and
prostate fibroblasts isolated from intratumoral regions of aggressive prostate
carcinoma (CAFs) were used. Conditioned media from CAFs were used to
mimic the effect of stromal cells on PCa cells. Motility and metabolism of
PCa cells was evaluate by invasion assays as well as western blot analysis,
respectively. Radioactive assays were used to establish the levels of glucose
and lactate uptake and to evaluate mitochondrial respiration.
Results and Discussion: We demonstrate that the metabolic reprogramming
of PCa cells is strictly dependent on a CAF-mediated inactivation of the M2
isoform of the pyruvate kinase (PK-M2), an enzyme largely expressed by
cancer cells. In particular, we observed that CAFs conditioned media induce
in PCa cells (i) PK-M2 phosphorylation mediated by Src tyrosine kinase and
(ii) PK-M2 oxidation mediated by the CAF-induced pro-oxidant environment.
These events lead to PK-M2 inactivation, granting for its nuclear migration and
association with hypoxia-inducible factor (HIF-1). The complex PK-M2/HIF-1
is responsible for the recruitment of the transcriptional repressor Differentially
Expressed in Chondrocytes-1 (DEC-1), which in turn promotes the downregulation of miR-205, a mandatory event for the execution of the epithelial–
mesenchymal transition (EMT) program. Treatment of PCa cells with DASA58 (a chemical activator of PK-M2) or Metformin (an inhibitor of mitochondrial
respiratory chain complex I) interferes with PK-M2 nuclear translocation and
association with HIF-1/DEC-1, ultimately abrogating the EMT and the proinvasive spur in PCa cells, as well as their ‘reverse Warburg metabolism’.
Conclusion: Our data suggest an intriguing role for PK-M2 in coupling the
motile and the metabolic programs, proposing a direct connection between
the EMT program and the metabolic switch. Finally, targeting PK-M2 could be
a potential therapeutic strategy that allows to simultaneously impair the motile
and the metabolic advantages of cancer cells.
296 Preclinical validation of the therapeutic potential of neuropilin-1
targeting transmembrane peptide in glioblastoma
298 Induction of HIF1a influences estrogen receptor expression in
ex-vivo culture of tumour tissue
E. Davies1 , A. Rahi1 , M. Cumberbatch1 , C. Eberlein1 , E. Anderson2 ,
S. Wedge3 , M. Smalley4 , S. Barry1 . 1 AstraZeneca, Oncology iMed, Cheshire,
United Kingdom, 2 Boehringer-Ingelheim RCV, Oncology, Vienna, Austria,
3
Newcastle University, Northern Institute for Cancer Research, Newcastle,
United Kingdom, 4 Cardiff University, European Cancer Stem Cell Research
Institute, Cardiff, United Kingdom
295 Twist oncoproteins are modulators of cellular stress
J. Fritz1 , L. Jacob1 , A. Fernandez1 , D. Bagnard1 . 1 INSERM U1109,
Strasbourg, France
The median survival of patients with glioblastoma multiform, the most severe
brain tumor, is not exceeding 15 months. This poor prognosis indicates
the inefficiency of the current therapeutic arsenal. We have developed in
the lab a novel strategy based on the use of a peptide (MTP-NRP1)
disrupting the neuropilin-1 receptor (NRP1) signaling platform by antagonizing
its transmembrane domain. As previously described, this peptide inhibits the
Background: Using an established ER+ breast cancer model, MCF-7
xenografted tumours, we have optimised a method of ex vivo tumour tissue
slice culture. This technique offers the potential to validate new targets ex-vivo
in tumour material derived from both animal models and from patients.
Material and Methods: We have established that 250 mm thick MCF-7 tumour
slices can be cultured for up to 72 h maintaining both tumour cell viability and
retaining the tumour stroma derived from the mouse.
Results: The MCF-7 tumour slices respond to ER modulators in a dosedependent manner, with downregulation of ER. ER levels and function
can be modulated by many different signaling pathways which co-operate
with estrogen to regulate signaling. We have found that in MCF-7 tumour
slices, hypoxia driven stabilisation of HIF1a causes downregulation of ER.
Furthermore, inhibition of AKT with AZD5363 reduces HIF1a accumulation,
and causes an increase in the number of ER+ cells in tumour slices.
Conclusions: These data suggest that HIF1a–AKT cross talk may play a
critical role in regulating ER levels, potentially changing the dependency of
the tumour cell on ER mediated signaling.
299 Metformin induces apoptosis and dowregulates pyruvate kinase
M2 in MCF7 breast cancer cells only when grown in nutrient-poor
conditions
A. Silvestri1 , I. Rasi1 , F. Palumbo1 , D. Posca1 , L. Castagnoli1 , G. Cesareni1 .
1
Tor Vergata University, Department of Biology, Rome, Italy
Introduction: Metformin has been proposed as adjuvant treatment for anticancer therapy because of its ability to dampen the PI3K/AKT/mTOR pathway.
Aside from inhibiting cell proliferation metformin can also induce apoptosis in
cancer cells. Since the molecular mechanism underlying this second effect,
which may contribute to the anticancer activity of metformin, is still poorly
characterized, we investigated the culture conditions that modulate metformininduced apoptosis in MCF7 breast cancer cells. More specifically we asked
whether the alteration of the glycolytic pathway is implicated in this process.
Material and Method: MCF7 cells were grown in MEM 5.5 mM glucose +
0.01 mg/mL insulin. Cells were then plated in different culture media containing
diverse concentrations of glucose or amino acids and treated with 10 mM
Metformin for 24 or 48 hours. Cell viability was monitored by Trypan Blue
assay and the effect of the treatment on the Akt/mTOR pathway and on the
expression of glycolytic enzymes was analyzed by Western Blot.
Results and Discussion: Our data demonstrated that metformin is able to
induce apoptosis in MCF7 cells only when plated at high density and that an
increase in glucose concentration causes a reduction of metformin-induced
apoptosis as well as a decrease in its anti-proliferative effect. Moreover,
apoptosis was completely inhibited when cells were grown in high glucose/high
amino acid medium demonstrating that not only glucose but also amino acid
availability plays a key role in MCF7 resistance to metformin. Finally, we
demonstrated that in nutrient poor conditions metformin is able to alter the
intracellular glycolytic equilibrium downregulating pyruvate kinase M2 (PKM2)
expression and that this mechanism is mediated by AMPK activation.
Conclusion: Metformin is able to induce breast cancer cell apoptosis and
PKM2 downregulation only in nutrient-poor conditions. Not only high glucose
levels but also high amino acid concentration can influence metformin antiproliferative and pro-apoptotic effects. These data demonstrated that the
reduction of nutrient supply in tumors can increase metformin efficacy and that
metformin is able to affect the glycolytic pathway at PKM2 level. Therefore,
the modulation of PKM2 expression/activity to reduce upstream glycolytic
intermediates could be a promising strategy to boost the metformin anti-cancer
effect.
300 Loss of PI3K-C2a promotes tumorigenesis and aneuploidy in
breast cancer
M. Martini1 , F. Gulluni1 , M.C. De Santis1 , A. Ghigo1 , J.P. Margaria1 ,
E. Ciraolo1 , U. Ala1 , F. Cavallo1 , R. Chiarle2 , E. Hirsch1 . 1 MBC − Molecular
Biotechnology Center, Department of Molecular Biotechnology and Health
Sciences, Torino, Italy, 2 Center for Experimental Research and Medical
Studies (CERMS), Department of Molecular Biotechnology and Health
Sciences, Torino, Italy
Background: PI3K signaling axis is one of the most frequently deregulated
pathways in human cancer impacting on cell growth, survival and metabolism.
Whereas the majority of efforts have so far focused on class I PI3K, increasing
evidence is pointing to the importance of class II enzymes in cell proliferation
and survival.
Material and Methods: We generated a mouse strain lacking PI3K-C2a
expression and found that the mutation is embryonic lethal. Pik3c2a+/− mice
were intercrossed with a transgenic strain that specifically expresses the
activated HER-2/Neu oncogene in the mammary gland. Mice were weekly
followed for survival, tumor appearance and growth. We derived mouse
embryonic fibroblast (MEF) and Primary Murine Mammary Epithelial Tumor
cells (MMET). Effects of heterozygous loss of Pik3c2a were evaluated by cell
proliferation, immunofluorescence, karyotype and CGH analysis. Efficacy of
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chemotherapeutic and anti-mitotic cancer drugs were examined in MMET cells
and in mouse models.
Results: We generated MEFs from Pik3c2a+/+ , Pik3c2a+/− and Pik3c2a−/−
embryos, and their ability to proliferate was assessed. While Pik3c2a+/+ MEFs
readily proliferated in culture, Pik3c2a−/− MEFs displayed a strongly reduced
proliferative capacity, accompanied by increased apoptosis. Heterozygous
MEFs also displayed haploinsufficiency and gene dosage dependency. Time
lapse analysis of cell-cycle in Pik3c2a−/− MEFs showed a significant delay
in the progress from prophase to anaphase. PI3K-C2a was specifically
enriched at the metaphase spindle interacting with transforming acidic coiledcoil protein 3 (TACC3)/colonic, hepatic tumor overexpressed gene (ch-TOG)/
clathrin complex to stabilize K-fibres during early mitosis. Loss of PI3K-C2a
resulted in reduced spindle length, altered microtubule (MT) stability and
increased metaphase plate width. Karyotype analysis revealed high levels
of aneuploidy in Pik3c2a−/− cells compared to wt controls. The effects of
PI3KC2A down-regulation were also investigated in a mouse model of breast
cancer. Heterozygous loss of PI3K-C2a resulted in an initially delayed tumor
onset followed by a faster growth rate in Pik3c2a+/− mice compared to wt.
The ability of tumors with low PI3K-C2a to grow faster appeared to correlate
with increased sensitivity to anti-microtubule agents like Paclitaxel. Gene
expression profiles of breast cancer patients showed that reduced levels
of PIK3C2A transcripts correlated with high grade tumors, indicating that
reduction in PI3K-C2a expression provides a growth advantage in mice as
well as in patients.
Conclusions: We demonstrated that loss of PI3K-C2a plays a crucial role in
promoting genomic instability, altering chromosome congression/segregation
during cell division. These findings will eventually validate PI3K-C2a as a
new diagnostic/prognostic tool that can be exploited to tailor more effective
therapies for aggressive breast cancers.
302 Normal and oncogenic proliferation under control of microRNAs:
A functional high content screening
D. Sastre1 , I.M.S. Lima2 , J.F. Guerreiro1 , D.T. Covas3 , M.A. Zago3 ,
R.A. Panepucci3 . 1 Federal University of Pará, Institute of Biological Sciences,
Belém, Brazil, 2 University of São Paulo, Department of Genetics, Ribeirão
Preto, Brazil, 3 University of São Paulo, Blood Center of Ribeirão Preto,
Ribeirão Preto, Brazil
Background: Cancer cells share several characteristics with normal stem cells
especially those concerning cell cycle regulation. Therefore it is believed that
cancer cells might arise from cells possessing or abnormally acquiring selfrenewal capabilities. MicroRNAs (miRNAs) are small non-coding RNAs that
act regulating gene expression post-transcriptionally by targeting hundreds of
mRNAs simultaneously, many of which can be involved in the same cellular
process such as the cell cycle. With this in mind, we hypothesized that
significant changes in the proliferation and cell cycle could be good tools to
identify miRNAs capable of regulating normal and oncogenic proliferation in
normal (fibroblasts) and cancer cell (HCT-116) lines.
Material and Methods: In the first phase of the screening, BJ foreskin
fibroblasts (2,000 cells) were transfected with 50nM of 28 miRNAs mimics (premiR) and inhibitors (anti-miR) in 96-well microplates. After 5 days, proliferation
was measured by XTT assay. Cell cycle classification (Click-it EdU® assay) and
viability (Sytox Green® staining) following miRNA transfection were performed
in a High-Content Screening platform. qPCR was used to evaluate key mRNA
targets.
Results: Preliminary data for BJ cells has shown nineteen treatments that
significantly altered cell proliferation. Interestingly, while transfection of premiRs of miR-20b, miR-101, and miR-181d decreased the proliferation, the
corresponding anti-miRs had the opposite effect as expected for our approach.
Pre-miR-181d, pre-miR-20b and pre-miR-101 had the most cytotoxic effect.
Pre-miR-181d and pre-miR-101 induced a significant reduction in the
percentage of cells in S phase. Cyclin D1 expression was elevated by antimiR-101 and pre-miR-24, indicating that this cell cycle regulator might be
the mediator of these miRNA’s effects. Among the predicted targets of miR20b and miR-101 we identified and validated EZH2 and SUZ12. Both targets
are core components of the polycomb repressor complex 2 (PRC2), which
contributes for maintaining the undifferentiated proliferative state of embryonic
stem cells.
Conclusions: These results indicate that miRNAs identified here can be good
candidates for inducing alterations of interest in the cell cycle, permitting
a better understanding of normal and oncogenic proliferation mechanisms.
It is known that aberrant expression of miRNAs occurs in many if not all
malignancies. Therefore, next step in this work includes screening these
miRNAs in colorectal cancer cells (HCT-116 cell line).
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303 A role for senescent cell-derived IL6 in HER2+ breast cancer
progression
M. Zacarias Fluck1 , B. Morancho1 , P.D. Angelini1 , R. Vicario1 , L. Villarreal2 ,
C. Aura3 , P. Nuciforo3 , J. Villanueva2 , I.T. Rubio4 , J. Arribas5 . 1 Vall d’Hebron
Institute of Oncology, Growth Factors Laboratory, Barcelona, Spain, 2 Vall
d’Hebron Institute of Oncology, Preclinical Research Program, Barcelona,
Spain, 3 Vall d’Hebron Institute of Oncology, Clinical Research Program,
Barcelona, Spain, 4 Vall d’Hebron University Hospital, Breast Surgical
Oncology Breast Cancer Center, Barcelona, Spain, 5 Vall d’Hebron University
Hospital, Growth Factors Laboratory Preclinical Research Program,
Barcelona, Spain
Background: Senescence, an irreversible cell proliferation arrest, can be
triggered by an excessive number of cell divisions or a variety of stressors,
including oncogenes. Senescent cells are characterized by senescenceassociated beta-galactosidase (SA-bGal) staining along with nuclear p21
staining, sustained DNA damage response, heterochromatin foci and a
senescence-associated secretory phenotype (SASP). The receptor tyrosine
kinase HER2 is a proto-oncogene overexpressed in approximately 20% of
breast cancers and its overexpression is associated with poor patient outcome.
The aim of this study was to evaluate the presence of senescent cells in
HER2-overexpressing breast cancer cell lines and determine their secretory
phenotype and possible implications on tumor progression.
Material and Methods: MCF7/HER2, HCC1954 and SkBr3 cell lines were
used in this study, stained with CFSE (carboxyfluorescein succinimidyl
ester) or PKH26 cell tracers and label-retaining cells were sorted by flow
cytometry. Nuclear staining of gH2AX, 53BP1, p21 and Ki67 were evaluated
by immunofluorescence through confocal microscopy. SA-bGal activity was
evaluated at pH = 6. Conditioned media of these cells were analyzed by
label-free proteomics and/or ELISA. Tocilizumab (anti-IL6R) was used in vitro
whereas Siltuximab (anti-IL6) was used in vivo.
Results and Discussion: Label-retaining HCC1954, PDXD118 [HER2+ derived cell line from a patient derived xenograft (PDX118)], MCF7/HER2
and SkBr3 cells showed an increase in the percentage of senescent cells,
characterized by SA-bGal and p21 nuclear staining. When the secretory
phenotype of these cells was evaluated, neither MCF7/HER2 or SkBr3
senescent cells showed a distinct secretory phenotype. However, HCC1954
cells and PDXD118 showed a significant increase in prototypical senescenceassociated cytokines IL6 and IL8 along with MMP1. Moreover, IL6 expression
was detectable preferentially on p21+ and Ki67− cells in both parental cell
lines. Notably, when PDX118 is treated in vivo with anti-IL6 blocking antibody,
tumor growth is significantly impaired.
Conclusions: Here we show that in HER2+ breast cancer models, IL6 is
produced mainly by spontaneous senescent cells and in a patient derived
xenograft this cytokine is beneficial for tumor growth in vivo.
304 Myc mediates the phosphorylation and degradation of p27
through activation of Cyclin A/CDK1
L. Garcı́a-Gutiérrez1 , G. Bretones1 , I. Arechaga1 , D. Santamarı́a2 ,
M. Barbacid2 , J. León1 . 1 IBBTEC, Molecular Biology, Santander, Spain,
2
CNIO, Madrid, Spain
Introduction: p27KIP1 (p27 herein after), member of the KIP/CIP family of
CDK inhibitors, accumulates in the nucleus of quiescent cells provoking cell
cycle arrest at the G1 phase by inactivating Cyclin/CDK complexes. The best
characterized pathway leading to p27 downregulation involves phosphorylation
at threonine 187 targeting p27 for SCFSKP2 -mediated ubiquitination and
degradation. The only kinase known so far to mediate this phosphorylation is
the Cyclin E/CDK2 complex. There is a correlation between high levels of Myc
expression with low levels of p27 in many human tumors. We have previously
shown that Myc induces the expression of SKP2 as well as the pT187p27.
Material and Method: We used the Kp27MER cell line, a K562 derivative
cell line carrying a ZnSO4 -inducible p27 construct and the chimerical MycER
protein which can be activated by 4-hydroxy-tamoxifen, and three mouse
embryonic fibroblast derived cell lines lacking functional CDK genes: CDK2−/− ,
Cyclin E−/− and TKO (CDK2−/− ; CDK4−/− ; CDK6−/− ). Overexpressing Mycstable cell lines generated for the three mouse derived cell lines by lentiviral
transduction. RT-qPCR and WB to study gene expression at mRNA and protein
levels. In vitro kinase assays to study pT187p27 and phosphorylation detected
by WB using phospho-specific antibodies. Purvalanol A and CAN508 (CDK1
and CDK9 inhibitors respectively) were tried in vitro to study the specificity of
CDK1 kinase activity over pT187p27.
Results and Discussion: Induction of p27 in Kp27MER cell line inhibits less
efficiently the kinase activity of CDK1 whereas it completely inhibits CDK2. Myc
activation leads to an increase of pT187p27 and Cyclin A induction. Besides,
it increases the in vitro kinase activity of CDK1 and CDK2. Interestingly,
CDK1 complexes from cells overexpressing p27 were able to phosphorylate
p27 in vitro upon Myc activation, but CDK2 complexes were not. Cyclin
B/CDK1 was reported to phosphorylate p27 in vitro, but the involvement
of Cyclin A is unknown. As cyclin A is induced by Myc in our model, we
asked for the role of Cyclin A/CDK1 in p27 phosphorylation. In vitro kinase
assays with immunoprecipitated Cyclin A complexes showed the same p27
phosphorylation pattern as the observed with CDK1 complexes.
CDK1 and Cyclin A complexes from CDK2−/− MEFs showed increased
pT187p27 levels when Myc was overexpressed. Similarly, CDK1, CDK2 and
Cyclin A complexes from Cyclin E−/− MEFs showed increased pT187p27 when
Myc was overexpressed. CDK1 complexes from TKO MEFs were unable to
phosphorylate p27 in vitro while overexpression of Myc induced it. Purvalanol
A abolished pT187p27 but not CDK9 inhibitors. Consistent with the in vitro
data, extracts from CDK2−/− and TKO MEFs showed higher levels of pT187p27
levels when Myc was activated.
Conclusion: Myc promotes p27 degradation by inducing its phosphorylation
at Thr187, which is mediated not only by Cyclin E/CDK2, but also by Cyclin
A/CDK1.
305 Natural and synthetic inhibitors of mTOR blunt the p53 response
to low concentrations of actinomycin D but not nutlin-3
K.M. Goudarzi1 , M. Nistér1 , M.S. Lindström1 . 1 Karolinska Institutet,
Department of Oncology-Pathology, Stockholm, Sweden
Background: Mechanistic target of rapamycin (mTOR) is a master regulator
of cell growth through its ability to stimulate ribosome biogenesis and mRNA
translation. In contrast, the p53 tumor suppressor negatively controls cell
growth and is activated by a wide range of insults to the cell. A better
understanding of how mTOR and p53 pathways are intertwined is needed
in order to increase the benefit of using mTOR inhibitors in anti-cancer
therapy. Inhibition of ribosome biogenesis causes nucleolar stress and leads
to p53 stabilization and activation. Nucleolar stress is often triggered by
chemotherapeutic agents and may contribute to their therapeutic efficacy. p53
protein stabilization requires that ribosomal protein L11 (RPL11) binds to and
inhibits the p53 master regulatory protein MDM2. Treatment of cells with mTOR
inhibitors may lead to reduced synthesis of ribosomal proteins including RPL11
and thereby destabilize p53 by reduced inhibition of MDM2. Here we have
investigated how the p53 response to nucleolar stress is affected by mTOR
inhibition.
Material and Methods: We tested a wide range of natural and synthetic
compounds that inhibit the mTOR pathway and combined them with low
concentrations of actinomycin D in the osteosarcoma cell line U2OS and the
glioma cell line U343MGa Cl2:6 cultured in vitro. As a comparison the p53
activating compound nutlin-3 was used. Nutlin-3 activates p53 independently of
RPL11. We used phosphorylation of S6K1 as an indicator of mTOR inhibition.
Levels of p53, p21, MDM2 and RPL11 were analyzed by immunoblotting.
Results: We found that inhibitors of the mTOR pathway including rapamycin,
wortmannin and caffeine blunted the p53 response to nucleolar stress.
Similarly, synthetic inhibitors of mTOR including temsirolimus, LY294.002 and
PP242 impaired p53 stabilization and subsequent p21 induction. Rapamycin
mimicked the effect of RPL11 depletion in terms of blunting the p53 response
to nucleolar stress. In contrast, the p53 response to nutlin-3, when combined
with mTOR inhibitors, was much less affected.
Conclusions: mTOR inhibitors interfere with the p53 response and this could
be of relevance in different settings. Our study reinforces the notion that a
careful consideration of chemotherapy dosages, timing of mTOR inhibition,
p53 status and cell type is important for a successful outcome of combination
chemotherapy regimens.
306 Chemotherapy sensitizes p95HER2-positive breast cancers to
trastuzumab
B. Morancho1 , J.L. Parra-Palau1 , V. Peg2 , R. Vicario1 , M. Zacarias-Fluck1 ,
K. Pedersen1 , C.M. Perou3 , A. Prat4 , I.T. Rubio5 , J. Arribas1 . 1 Vall d’Hebron
Institute of Oncology, Preclinical Research Program, Barcelona, Spain, 2 Vall
d’Hebron University Hospital, Pathology Department, Barcelona, Spain,
3
Lineberger Comprehensive Cancer Center, Chapel Hill, USA, 4 Vall d’Hebron
Institute of Oncology, Clinical Research Program, Barcelona, Spain, 5 Vall
d’Hebron University Hospital, Breast Surgical Oncology, Barcelona, Spain
Introduction: HER2-positive breast cancers are currently treated with
trastuzumab, an anti-HER2 antibody. Early reports indicated that resistance
to trastuzumab monotherapy could be associated with the expression of
the HER2 fragment p95HER2, which occurs in ~30% of these tumors. In
apparent contrast, recent preliminary results show that p95HER2-positive
tumors respond to trastuzumab plus chemotherapy.
The aim of this work is to clarify the role of p95HER2 in response to these
treatments.
Material and Methods: p95HER2-positive breast cancers were determined
by immunohistochemistry and their intrinsic molecular subtype defined using
Counter platform. The effect of trastuzumab, lapatinib, doxorubicin, paclitaxel
or combinations on cell proliferation, viability, receptor levels and localization
was studied using MCF10A cell lines stably expressing HER2, p95HER2 or
both. HER2-positive patient-derived xenografts expressing or not p95HER2
were treated with doxorubicin or/and trastuzumab.
Results: Here we show p95HER2-positive tumors belong to the HER2enriched subtype and have a high proliferation rate. Expression of p95HER2
in tumor cells increases cell death in response to chemotherapy. Furthermore,
the DNA-damaging agent doxorubicin sensitizes a p95HER2-positive patientderived xenograft to trastuzumab. This chemotherapeutic drug stabilizes
HER2 in p95HER2-positive cells and, thus, increases trastuzumab-dependent
cell-mediated cytotoxicity. Similar results were observed with paclitaxel, a
chemotherapy that disrupts the cytoskeleton.
Conclusion: Chemotherapy is effective to treat p95HER2-positive breast
tumors and, in addition, sensitizes them to trastuzumab.
307 Gelsolin promotes the survival of cancer cells under stress by
modulating autophagy
S. Deng1 , L. Tochhawng1 , T.D. Dinh1 , H.M. Shen1 , C.T. Yap1 . 1 National
University of Singapore, Physiology, Singapore, Singapore
Background: The ability of cancer cells to survive from various stresses not
only facilitates cancer development in nutrient-deficient microenvironment, but
also impedes the effectiveness of chemotherapeutic agents. Accumulating
evidences suggest that autophagy, a lysosome-mediated degradation of the
cell’s own components, plays a major role in promoting cancer cell survival
under stress. Recent studies have revealed that actin cytoskeleton could
regulate autophagy and cell survival. Meanwhile, gelsolin, an actin-binding
protein, is well known for its functions in regulating actin dynamics. While
gelsolin has been reported to promote cell survival by inhibiting apoptosis, it
is not known whether gelsolin can also modulate other survival mechanisms
such as autophagy in cancer cells. In the present study, we aim to investigate
the influence of gelsolin on autophagy, and their roles in cancer cell survival
under stress.
Material and Methods: To study the effect of gelsolin on cell survival, we
modulate cellular gelsolin levels via overexpression and siRNA knockdown in
the human colorectal cancer cell lines HCT116 and RKO, and the cervical
cancer cell line HeLa. Starvation and 5-Fluorouracil (5-FU) were used to
induce autophagy and cell death. To measure autophagy levels, immunoblots
of LC3-II and p62 were used, and autophagic vesicles were visualized by
immunofluorescence microscopy. Cell death was quantified by propidium
iodide exclusion assay in live cells and sub-G1 analysis of fixed cells using flow
cytometry. Apoptosis was assessed by caspase-3 and PARP-1 cleavage.
Results: We found that gelsolin overexpression reduced cell death induced
by starvation and 5-FU treatment; whilst knockdown of gelsolin sensitized
cells to these treatments. Moreover, our data showed that apoptosis was
significantly inhibited by gelsolin under these stresses, indicating that gelsolin
confers resistance to apoptosis induced by stress. We also observed
that starvation- and 5-FU-induced autophagy were enhanced by gelsolin
overexpression, and autophagy level was reduced by siRNA knockdown
of gelsolin. Furthermore, inhibition of autophagy, either by silencing the
autophagic protein Atg7 or treating with lysosome inhibitor chloroquine,
attenuated the protective effect of gelsolin. These results suggest that
autophagy may have a potential role in gelsolin-mediated cell survival under
stress conditions.
Conclusions: In summary, our study suggests that gelsolin is a novel mediator
of autophagy and promotes survival under nutrient deprivation and 5-FU
treatment via modulating autophagy levels.
308 Hybrid peptide tat-Ram13-induced necrosis-like cell death
depends on expression of PTEN in human leukemia cell lines
A. Kuniyasu1 , M. Kurogi2 , M. Setoguchi2 , M. Makise1 . 1 Sojo University,
Molecular Cell Pharmacology, Kumamoto City, Japan, 2 Kumamoto University,
Pharmaceutical Biochemisty, Kumamoto City, Japan
Introduction: Necrosis is an important physiological process. Potent tumorspecific inducers of necrotic cell death promise to be a valuable tool for
development of novel chemotherapeutic agents. We previously developed a
hybrid 25mer peptide (termed tat-Ram13) that induces a necrosis-like cell
death in multiple human leukemic cell lines. Interestingly, this peptide did not
affect human normal T-cells and leukemic cell line TALL-1 at all. To understand
the molecular mechanisms underlying the necrosis-like cell death by the hybrid
peptide, we determined key amino acid residues important for triggering cell
death and examined the differently expressed proteins between tat-Ram13sensitive leukemia cell lines and the insensitive cells.
Materials and Methods: All peptides were synthesized by Fmoc chemistry
using the PSSM-8 peptide synthesizer (Shimadzu Co.). Human monocytic cells
and leukemic cell lines were maintained in RPMI-1640 medium supplemented
with 10% fetal calf serum. Cells were plated in 96-well multiplates and
incubated with the peptides for 12 h. Cell viability was estimated with WST-8
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reagent (Dojin Chemical). Stable Jurkat-T cells over-expressing PTEN were
selected with G418 after being transfected with pCMFlag-hsPTEN.
Results: Tat-Ram13 peptide killed almost the leukemia cells (Jurkat-T, CCRFCEM, Molt-4 etc.) except T-ALL1 cells in a tumor cell-specific manner. We
searched an active core of the cytotoxicity in the Ram13 sequence. Alaninescanning analysis using both Jurkat-T and CCRF-CEM leukemia cells revealed
that the Leu-Trp-Phe motif plays critical roles in the cytotoxic effect. On the
other hands, western blot analysis revealed that the loss of PTEN protein
is observed in all the tat-Ram13-sensitive cell lines, although TALL-1 cells
constitutively expresses the PTEN protein. Correspondingly, phosphorylation
of AKT-1, a downstream signaling of PTEN was activated in the sensitive ones.
Then we over-expressed PTEN in Jurkat-T cells and examined the cytotoxic
activity of tat-Ram13. Jurkat-T cells over-expressing PTEN was significantly
reduced the sensitivity of cell death rather than parent cells.
Conclusion: The hybrid peptide tat-Ram13 may serve as a useful tool for
studying the molecular mechanism of necrosis-like cell death and the present
data suggested that PI3K-AKT signaling is a potential targeting pathway for
the tat-Ram13-induced leukemia cell death.
309 Clonal succession of transiently active TIC clones in human
pancreatic cancer
C.R. Ball1 , F. Oppel1 , R. Ehrenberg2 , J. Weitz3 , J. Werner4 , F. Bergmann5 ,
N. Ishaque6 , B. Brors7 , C. Von Kalle1 , H. Glimm1 . 1 National Center for Tumor
Diseases (NCT) and German Cancer Research Center (DKFZ), Translational
Oncology, Heidelberg, Germany, 2 National Center for Tumor Diseases,
Medical Oncology, Heidelberg, Germany, 3 University Hospital Dresden,
Visceral Thoracic and Vascular Surgery, Dresden, Germany, 4 University of
Heidelberg, Department of Surgery, Heidelberg, Germany, 5 University of
Heidelberg, Institute of Pathology, Heidelberg, Germany, 6 German Cancer
Research Center (DKFZ), Theoretical Bioinformatics and Heidelberg Center
for Personalized Oncology DKFZ-HIPO, Heidelberg, Germany, 7 German
Cancer Research Center (DKFZ), Theoretical Bioinformatics, Heidelberg,
Germany
Introduction: Although tumor-initiating cell (TIC) self-renewal has been
postulated to play a major role in tumor progression and metastases formation
of human pancreatic adenocarcinoma (PDAC), the clonal dynamics of TIC
within PDAC tumors in vivo are yet unknown. To monitor clonal dynamics and
self-renewal activity of TIC during tumor formation we used a genetic labeling
strategy in a serial xenotransplantation model.
Material and Methods: We enriched and lentivirally marked PDAC TIC in
serum-free adherent culture conditions as 3-dimensional epithelial colonies
with tight cell-to-cell contacts. Upon subcutaneous transplantation into NSG
mice they reliably form serially transplantable tumors (1º, 2º, 3º).
Results and discussion: To evaluate the clonal in vivo kinetics of individual
self-renewing PDAC TIC, lentivirally marked patient derived (n = 3) cultures
were serially transplanted. Clone specific integration loci of the lentiviral
vector within established tumors were identified by highly sensitive LAM-PCR
and high throughput sequencing. Whereas in 1º mice 0.003–0.113% of all
transduced cells actively contributed to tumor formation, subsequent serial
tumors were mainly driven by distinct TIC clones not detectable in previous
but recruited to tumor formation in later generations. In addition, pairs of
2º mice from the same 1º tumor showed very little overlap of active clones.
Mathematical modeling indicates substantial changes in the proliferative
activity of individual, otherwise homogenous TIC, which predominantly produce
non-tumorigenic progeny with very limited self-renewal. Exome sequencing
demonstrated remarkable genetic stability of pancreatic cancer xenografts
during serial transplantation with only few acquired additional mutations in
coding genes thereby supporting that the observed clonal dynamics were
caused by changes in the functional activity state of TIC and not genetic
instability.
To investigate whether TIC function is tightly linked to a stem celllike phenotype, differentiation was induced by FBS addition and cytokine
withdrawal, resulting in monolayer formation and down regulation of described
TIC or normal progenitor markers. Interestingly, this did neither systematically
alter tumor initiation and self-renewal nor TIC frequency in vivo. Moreover,
sorted CD133+ and CD133− fractions efficiently formed tumors containing
similar numbers of CD133+ cells, again indicating an unexpected phenotypic
plasticity of PDAC TIC.
Conclusion: We demonstrate that a succession of transiently active TIC
generating tumor cells in temporally restricted bursts drives long-term
progression of serially transplanted PDAC. The recruitment of inactive
TIC clones to tumor formation after serial transplantation indicates an
unprecedented functional and phenotypic plasticity of PDAC TIC in vivo. These
finding points out the need to develop targeting strategies directed against
functional TIC activity in human PDAC.
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310 The novel use of BAMLET in the treatment of oral squamous
cell carcinoma
N. Sinevici1 , N. Harte2 , K. Hun Mok2 , Y. Xie2 , J. O’Sullivan1 . 1 Trinity College
Dublin, Dental Science, Dublin, Ireland, 2 Trinity College Dublin, School of
Biochemistry and Immunology TCD, Dublin, Ireland
Introduction: Oral Cancer (OC) remains in the top most common cancers
worldwide with no realistic prospect of overcoming the occurrence of the
disease since the main culprits responsible for its onset are tabacco smoking
and alcohol drinking. These factors cause genomic, transcriptomic, proteomic
and metabolomic changes which culminate in dysplatic/cancerous lesions.
The geno/phenotype of patients is of major importance with ~50% of OCs
displaying a p53 mutation which is directly correlated to the aggressiveness
of the disease. Fortunately, Bovine a-lactalbumin made lethal to tumour
(BAMLET) cells is an attractive potential anti-cancer agent. The complex is
formed by partial unfolding of a-lactalbumin with oleic acid. The prospective
of a novel therapeutic strategy is of particular interest in OC as surgery
remains the gold treatment standard. Despite advances in both surgery and
radiation therapy with/without chemotherapy, late diagnosis and cumulative
side effects are the major patient/doctor concerns. Furthermore life expectancy
remains low at ~50% after diagnosis. This study explored the potential of
BAMLET to disrupt dysplastic/cancerous oral cell lines and also investigated
the mechanism through which cell death is achieved.
Materials and Method: BAMLET was prepared using anion exchange
chromatography and its activity was tested using two OSCC cell lines
(+/− p53 mutation). For all experiments cells were seeded at the required
density and serum starved in 1% FBS overnight prior to BAMLET treatment.
Cells were maintained in FBS free medium for the duration of the treatment.
Cell viability was investigated using Alamar blue and analysed using a
microplate spectrophotometer. The mechanism of cellular death was examined
by propidium iodide using flow cytometry, testing for apoptosis-inducing activity.
Autophagy was investigated using acridine orange coupled with confocal
microscopy to determine lysosomal activity in cells. Localisation of BAMLET
after cellular administration was investigated using live imaging in confocal
microscopy.
Results and Discussion: We found the complex to be cytotoxic to both
of the cell lines tested although the dose was dependant on p53 status.
Cells displaying the p53 wildtype gene were more sensitive to treatment
compared to the mutated p53 cancer cell line. Interplay between apoptotic
and autophagic mechanisms appear to be the mechanism through which cell
death is achieved.
Conclusion: BAMLET presents itself as the ideal anti-cancer agent with the
advantage of selectively killing transformed cells while normal differentiated
cells are unaffected. However further studies must be undertaken before
translating these results into clinical practice. With respect to OC, a
topical treatment may provide a convenient strategy for patients without the
undesirable/numerous side effects that significantly diminish the patients’
quality of life.
311 A CXCR4-positive subpopulation exhibits cancer stem cell
properties in renal cell carcinoma
W. Zimmermann1 , M. Gassenmaier1 , D. Chen2 , A. Buchner2 , H. Pohla1 .
1
University of Munich, Tumor Immunology Laboratory LIFE Center, München,
Germany, 2 University of Munich, Department of Urology, München,
Germany
Background: Over the last decade, the development of targeted molecular
therapies directed against signaling pathways that foster angiogenesis has
substantially improved the prognosis for patients with metastatic renal cell
carcinoma (RCC). Although some tumors show regression, most patients
develop therapy resistance over time. There is increasing evidence that tumor
growth, metastasis and drug resistance are mediated by a small subpopulation
of cells, termed tumor-initiating or cancer stem cells (CSC). These cells are
characterized by their ability for self-renewal and their capacity to form serially
transplantable tumors which recapitulate the heterogeneous tumor phenotype
in immunodeficient mice.
Material and Methods: Expression of stem cell markers was studied
by flow cytometry, reverse transcription followed by quantitative PCR and
immunohistology. CSC properties of cells were assessed by sphere formation
under serum-free/non-adherent conditions and growth in immunodeficient
NOD/SCID mice. Transfection of RCC cell lines with CXCR4-specific siRNA
was used to reduce CXCR4 mRNA levels.
Results: We found two RCC cell lines differing widely in their capacity to
form spheres and to establish tumors in mice, potentially reflecting differences
in their CSC content. A subpopulation expressing the CXC chemokine
receptor 4 (CXCR4) was present only in the more tumorigenic cell line.
When grown as spheres, most of these cells were CXCR4+ , and expressed
stem cell-associated transcription factors at elevated levels. Sorted CXCR4+
cells exhibited greater tumor growth-inducing potential than CXCR4− cells.
Significantly, higher CXCR4 mRNA levels in primary RCC tumors from
patients with localized but not disseminated disease predicted shorter survival.
Downregulation of CXCR4 expression by siRNA or inhibition with AMD3100
compromised sphere formation, viability of CXCR4+ cells, and increased their
responsiveness toward tyrosine kinase inhibitors commonly used in RCC
patients.
Conclusion: CXCR4 identifies a subpopulation of tumor-initiating cells in RCC
cell lines and CXCR4 signaling appears to be important for their maintenance.
The relative insensitivity of RCC stem cells to tyrosine kinase inhibitors
indicates that such cells contribute to the development of therapy resistance
in RCC patients. Combined blockade of CXCR4 signaling and angiogenesis
could lead to a more effective treatment of metastatic RCC in the future.
312 Zoledronic acid impairs stromal reactivity by inhibiting
M2-macrophages polarization and prostate cancer-associated
fibroblasts
G. Comito1 , C. Pons Segura1 , K. Sobierajska2 , L. Ippolito3 , M.L. Taddei3 ,
E. Giannoni3 , P. Chiarugi3 . 1 University of Firenze, Department of Experimental
and Clinical Biomedical Sciences, Firenze, Italy, 2 Medical University of Lodz,
Department of Molecular and Medical Biophysics, Lodz, Poland, 3 University
of Fienze, Department of Experimental and Clinical Biomedical Sciences,
Firenze, Italy
Introduction: It is established that cancer associated fibroblasts (CAFs),
the major cellular components of tumor microenvironment, promote epithelial
mesenchymal transition (EMT) and acquistion of stemness traits in prostate
cancer cells. Of note, prostate CAFs are active players in promoting
monocyte recruitment to tumor site. CAFs secretion of stromal derived
growth factor-1 (SDF-1) delivery promote macrophage trans-differentiation
to the M2-macrophages phenotype. Moreover, M2 macrophages are able
to induce mesenchymal–mesenchymal transition of fibroblasts, leading to
their enhanced reactivity and highlighting the mutual relationship between
these cell compartments. This complex interplay among cancer cells, CAFs
and M2-macrophages, leads to (i) an increase in tumor cell motility, that
promotes cancer cells escape from primary tumor and metastatic spread,
and (ii) an activation of both endothelial cells and their bone-marrow-derived
precursors to drive de novo angiogenesis.
Zoledronic acid (ZA), an amino-bisphosphonate compound in use for the
treatment of symptomatic skeletal events, has recently been shown to
have immunomodulatory properties that need to be exploited in cancer
immunotherapy. There is in vivo evidence showing that ZA can reduce the
tumorigenic phenotype of M2-polarized macrophages.
Methods: Human Monocytes were obtained from normal donor buffy coat by
gradient centrifugation using Ficoll, fibroblasts were isolated from aggressive
carcinoma (CAFs) or from benign prostate hyperplasia (HPFs). Invasiveness
was evaluated using a Boyden chamber coated with matrigel.
Results: Here we show the key effects of ZA on M1/M2 macrophages and
CAFs in prostate carcinoma progression. First, we analyzed the phenotype
of the differentiated macrophages treated with ZA evaluating their M1 and M2
phenotype, by looking at the expression of IL-12 or IL-10, respectively. Our data
revealed that ZA treatment impairs M2-macrophages polarization, while it is
ineffective on M1-macrophages polarization. As a consequence, ZA-treated
M2-polarized macrophages lose their ability to foster the motility of prostate
cancer cells. We also investigated the effect of ZA on fibroblasts activation
and found that this molecule is able to reduce stromal fibroblasts reactivity,
as well as their ability to elicit EMT in prostate carcinoma cells. Finally, we
demonstrated that CAFs treated with ZA lack their ability to protect prostate
cancer cell to docetaxel toxicity.
Conclusion: Our data suggest that the benefit of ZA in the therapy of
prostate carcinoma patients, potentially goes beyond the simple skeletal/bone
symptoms treatment, but is enlarged to regulation of stromal inflammatory
events.
313 Breast cancer stem cells are more resistant than parental cells
to a novel palladium (II) saccharinate complex
D. Karakas1 , N. Aztopal1 , B. Cevatemre1 , F. Ari1 , A. Yilmaztepe Oral2 ,
V. Turan Yilmaz3 , E. Ulukaya2 . 1 Uludag University Science Faculty, Biology,
Bursa, Turkey, 2 Uludag University Medical Faculty, Clinical Biochemistry,
Bursa, Turkey, 3 Uludag University Science Faculty, Chemistry, Bursa,
Turkey
Background: Breast cancer is still a very significant health problem and there
is not enough success in its treatment despite new chemotherapy regimens.
It is considered that the most important reason for the poor success rate is
the theory of cancer stem cell, which is postulated in recent years. It has been
showed that the cancer stem cells are resistant to the treatment thereby they
play a role in recurrencies. Therefore, killing the cancer stem cells along with
the other cancer cells is gaining an importance. In the present study, it was
aimed to evaluate the cytotoxic and apoptotic effects of a novel palladium
complex {[PdCl(terpy)](sac)·2H2 O} on breast cancer stem cells, which is
synthesized by the chemistry department at the University of Uludag.
Material and Methods: To determine cytotoxic and apoptotic effects of Pd(II)
complex, breast cancer stem cells (CD44+ /CD24− cells) were propagated
from MCF-7 cell line (parental) and mammosphere structure formation was
induced. Characteristic cell surface markers (CD44+ /CD24− ) were determined
via flow cytometry in mammosphere. Also results were supported with CD44FITC staining. Cytotoxic and apoptotic analyses were performed on these
cells, which have cancer stem cell properties and mammosphere forming
ability. The cytotoxic activity of Pd(II) complex on MCF-7 cell line and MCF-7derived spheres (mammospheres) was investigated via the ATP viability assay.
Caspase-cleaved cytokeratin 18, which is a marker for apoptosis, (M30 ELISA)
assay was used to determine the cell death mechanism (apoptosis/necrosis).
The results were also confirmed with Hoechst 43332 staining for nuclear
morphology.
Results: It was shown that although different concentrations of Pd(II) complex
(3.13–100 mM) inhibited the growth of the cells in a dose-dependent manner,
mammospheres were more resistant to the treatment. We observed that M30
levels increased in MCF-7 parental cells at relatively higher doses (25, 50 and
100 mM). Increments in M30 values were also present in mammospheres at
the same doses but they were clearly lower, compared to MCF-7 parental
cells, implying that mammospheres were more resistant (less apoptosis) than
MCF-7 parental cells. Pd(II) complex resulted in pyknotic nucleus (a marker of
apoptosis) in MCF-7 parental cells and in correspondent with the M30 levels,
the number of pyknotic cells in mammospheres were also lower than those in
MCF-7 parental cells.
Conclusions: In conclusion, mammospheres (cancer stem cell-enriched
population) are more resistant to Pd(II) complex, compared to the parental
line.
314 BCLAF1; a multi-faceted protein involved DNA repair, apoptosis
and autophagy
E. Barros1 , K.S. Savage1 , D.P. Harkin1 . 1 Queen’s University of Belfast,
CCRCB, Belfast, United Kingdom
Introduction: Despite having been identified in several cellular processes
BCLAF1’s exact molecular function remains unclear. Evidence suggests that
BCLAF1 has a role in cell death, either through autophagy or apoptosis,
but some findings remain controversial. BCLAF1 has also been implicated
in mRNA splicing and transcript export.
More recently, our group has identified BCLAF1 as part of a novel
DNA damage induced complex, consisting of BRCA1, BCLAF1 and other
components of the mRNA splicing machinery. This complex regulates the
pre-mRNA splicing of a large subgroup of genes, mainly involved in DNA
damage signalling and repair. Disruption of the complex results in sensitivity
to DNA damage and defective DNA repair, promoting genomic instability that
may ultimately contribute to cancer development. Phosphorylation of BRCA1
is required for the assembly of this complex, however the functional importance
of BCLAF1 phosphorylation in this complex is unknown. Furthermore, cancer
associated mutations have been found in several genes of this complex,
including BCLAF1, suggesting these may have a role in carcinogenesis.
Our aim was to further investigate BCLAF1’s impact in these distinct
pathways.
Material and Methods: Using site directed mutagenesis, we generated
BCLAF1 phosphomutant and cancer associated mutant constructs and
assessed their role within the DNA damage response and cellular survival.
We performed Western Blot and immunocytochemical analysis using wellknown markers of DNA repair, autophagy and apoptosis and evaluated the
impact of wild-type and mutant BCLAF1 expression on these processes.
Results and Discussion: Our experiments showed that BCLAF1, like BRCA1,
is phosphorylated in response to DNA damage and that this modification
may influence its function within the BRCA1/BCLAF1 splicing complex. In
particular, phosphorylation at Ser122 seems to be, at least partially, required
for effective DNA damage repair. Furthermore, a similar functional defect was
observed with a cancer associated mutation within BCLAF1, suggesting that
the defective DNA repair may be capable of driving genomic instability and/or
tumourigenesis.
Our data also supports a role for BCLAF1 in autophagy and apoptosis,
suggesting there is interplay between the pathways that culminates in cell
death after BCLAF1 overexpression, independently of DNA damage.
Conclusion: Our results demonstrate BCLAF1 is a multi-functional protein
involved in distinct pathways, such as autophagy, apoptosis and mRNA
splicing, which can be triggered by different protein expression levels, posttranslational modifications or external stimuli. BCLAF1’s role in carcinogenesis
requires further investigation, but our study highlights the importance of this
protein in a new mRNA splicing complex, required for maintaining genomic
stability.
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315 Elovl6 overexpression promotes liver carcinogenesis
A.L. Shiau1 , Y.C. Su2 , Y.H. Feng3 , H.T. Wu4 , Y.S. Huang2 , P. Wu5 ,
C.J. Chang4 , C.L. Wu2 . 1 National Cheng Kung University, Department
of Microbiology and Immunology, Tainan, Taiwan, 2 National Cheng Kung
University, Department of Biochemistry and Molecular Biology, Tainan,
Taiwan, 3 Chi-Mei Medical Center, Division of Hematology and Oncology
Department of Internal Medicine, Tainan, Taiwan, 4 National Cheng Kung
University, Department of Family Medicine, Tainan, Taiwan, 5 Keele University,
Institute for Science & Technology in Medicine, Keele, United Kingdom
Background: The elongation of long-chain fatty acids family member 6
(Elovl6) is a rate-limiting enzyme for the elongation of saturated and
monounsaturated long-chain fatty acids. It has been shown that Elovl6
knockout mice are resistant to diet-induced insulin resistance, suggesting that
overexpression of Elovl6 may contribute to insulin resistance. Accumulating
evidence has revealed that insulin resistance is linked to obesity-related
malignancy, including hepatocellular carcinoma (HCC). Here we investigated
the role of Elovl6 in liver carcinogenesis.
Material and Methods: Sixty-one clinical specimens of resected HCC
were examined immunohistochemically for Elovl6 expression. The correlation
between Elovl6 expression and clinicopathological parameters of patients were
analyzed. We suppressed Elovl6 expression in ML-1 murine HCC cells by
adenovirus-mediated gene transfer of Elov6 shRNA or control shRNA and
examined their lipogenesis, cell proliferation, and Akt downstream pathway.
We also evaluated whether knockdown of Elov6 expression could retard tumor
growth and enhance survival in BALB/c mice bearing subcutaneous ML-1
tumors.
Results: High expression levels of Elovl6 in HCC were correlated with higher
incidence of cancer recurrence and worse survival outcome in patients who
received curative HCC operation. Knockdown of Elovl6 expression in HCC
cell lines decreased cell proliferation, Akt activation, and sensitivity to fatty
acids. Intra-tumoral injection of adenoviral vectors encoding Elovl6 shRNA
could reduce tumor growth and prolong survival time in ML-1 tumor-bearing
mice.
Conclusions: Elovl6 enhances oncogenic activity of liver cancer and is
associated with poor prognosis in patients with HCC. Thus, Elovl6 may be
further explored as a therapeutic target for HCC.
316 Influence of cancer stem cells on drug resistance in prostate
cancer
E.A. Castellón1 , V. Castillo1 , R. Valenzuela1 , H.R. Contreras1 . 1 University of
Chile, Faculty of Medicine, Physiology and Biophysics Program, Institute of
Biomedical Sciences, Santiago, Chile
Background: Prostate cancer (PCa) is one of the most diagnosed male
malignancies worldwide. Cancer stem cells (CSCs), tumor cells able to selfrenew and differentiate giving rise to cell heterogeneity of tumors, have been
identified in several cancers including PCa. CSCs are thought to be involved
in metastasis, relapse and therapy resistance.
The aim of this work is to evaluate functional features and drug resistance of
an isolated CSCs population from PCa.
Material and Methods: Tumor spheres were obtained by inducing primary
cultures from PCa explants to grow in non-adherent conditions. Adherent PCa
cell cultures were used as control. Resulting prostatospheres were assayed
for stemness and epithelial marker, clonogenic capacity, holoclone-forming
ability, colony-forming capacity in soft agar and proliferative and apoptotic
rate, using immunocytochemistry, immunofluorescence and corresponding
functional assays. Drug resistance was evaluated by daunorubicin and
topotecan treatments.
Results: Prostatospheres were positive for ABCG2 transporter, CD133, CD44,
cytokeratins 5 and 18, and negative for prostate specific antigen and androgen
receptor, showed higher clonogenic capacity, holoclone-forming ability and
self-renewal capacity, and lower proliferative and apoptotic rate than control
adherent cell cultures. Furthermore, exposing prostatospheres to ABCG2substrate drugs daunorubicin and topotecan, resulted in a high survival rate
compared with control PCa cells. This high drug resistance was decreased
using a selective inhibitor of ABCG2.
Conclusions: Prostatospheres from PCa explants showed a functional
stem phenotype and a marked drug resistance, probably mediated by high
expression of the ABCG2 transporter. Therefore, PCa CSCs may have an
important influence in the high intrinsic multidrug resistance exhibited by this
cancer and ABCG2 transporter might be considered as a suitable target for
selective therapy.
Funded by FONDECYT 1100183 grant.
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317 Analysis of HER2 amplification and IGF-IR expression in CETCs
and its possible association with resistance to trastuzumab in
HER2 positive breast cancers
D. Zimon1 , M. Pizon2 , U. Pachmann3 , K. Pachmann3 . 1 Simfo GmbH,
Bayreuth, Germany, 2 Simfo GmbH, Simfo GmbH, Bayreuth, Germany,
3
Transfusion Center, Labor Dr. Pachmann, Bayreuth, Germany
Background: HER2 overexpression or amplification appears in 20−30%
of invasive breast carcinomas and is associated with increased metastatic
potential and decreased overall survival. Trastuzumab (Herceptin® ) is a
recombinant humanized monoclonal antibody directed against the extracellular
domain of HER2 and has shown activity both as a single agent and in
combination with chemotherapy in HER2-overexpressing breast cancers.
Nevertheless, 70% of patients with HER2-positive breast cancers develop
intrinsic or secondary resistance to trastuzumab. This resistance has been
associated with overexpression of the insulin-like growth factor receptor-I
(IGF-IR). Therefore identification of circulating epithelial tumor cells (CETCs)
expressing HER2 can be regarded as an adverse prognostic marker. We
therefore investigated the expression of IGF-IR on the CETCs in addition
to HER2 amplification in breast cancer patients to identify patients who
might benefit from a combined targeted therapy against HER2 and IGFIR.
Methods: CETCs were determined from blood of 30 non-metastatic and
metastatic breast cancer patients. The number of vital CETCs and the
expression of IGF-IR were investigated using the maintrac® approach.
Fluorescence in situ hybridisation was used for analysis of HER2 amplification
in CETCs.
Results: CETCs could be detected in all breast cancer patients. The number
of CETCs ranged from 4 to 163 in 100 ml of cell suspension. IGF-IR expression
on the surface of CETCs was detected in all patients, whereas HER2 positive
CETCs were observed in 93.3% of patients. A statistically high correlation was
found between the percentage of IGF-IR positive and HER2 positive CETCs.
No statistically significant correlations were observed between the number of
CETCs and IGF-IR or HER2 in CETCs.
Conclusion: Our results demonstrate a parallel expression of IGF-IR and
HER2 in CETCs. Therefore, IGF-IR might be an important potential therapeutic
target in breast cancers resistant to trastuzumab. Targeting IGF-IR in addition
to HER2 may be a rational approach to improve response to trastuzumab in
the sub-group of CETCs that express both, HER2 and IGF-IR.
318 Chemosensitivity differs between CETCs and spheroids grown
from the CETCs in cancer patients with solid tumors
M. Pizon1 , D. Zimon1 , U. Pachmann2 , K. Pachmann2 . 1 Simfo GmbH, Simfo
GmbH, Bayreuth, Germany, 2 Transfusion Center, Labor Dr. Pachmann,
Bayreuth, Germany
Background: In vitro chemosensitivity testing of circulating epithelial
tumor cells (CETCs) provides real-time information about the sensitivity
of the tumor cells present in the patient and correlates with treatment
success. Nevertheless, a fraction of CETCs can survive after conventional
chemotherapy and grow into distant metastasis. A subpopulation of CETCs
with proliferation activity has the ability to form spheroids in suspension culture.
Spheroids exhibit stem cell-like properties and may be responsible for chemo
therapeutic resistance. Therefore, the aim of our study was to compare the
efficacy of chemo therapeutics on CETCs and on spheroids originated from
the same individuals.
Methods: The enumeration of CETCs collected from patients with solid
tumors in clinical stage 1−4 were performed using the maintrac® method.
Subsequently, CETCs in the context of the surrounding white blood cells were
cultured in a suspension culture system allowing for spheroid formation. To
evaluate the cytotoxic effect of drugs on CETCs and spheroids we exposed
them to anticancer drugs in short time culture in different concentrations and
for different periods of time.
Results: The response to chemotherapeutics differed between CETCs
and spheroids. In contrast to CETCs, spheroids of some of the same
patients were significantly more chemoresistant. Whereas active drugs led
to membrane permeability in single CETCs with subsequent staining of
the nuclei with propidium iodide, the same drugs led to disintegration of
tumorspheres with destruction of part of the cells but often part of the
cells in the spheres were able to survive. Epirubicin and, interestingly,
and especially salinomycin, a polyether ionophore antibiotic isolated from
Streptomyces albus, and Curcumin, a dietary pigment from the plant
Curcuma longa, showed the best effects. Docetaxel, cyclophosphamide
and 5-Fluoruracil showed almost no cytotoxic effects onto the cells in the
spheres.
Conclusion: Our results show, for the first time that stem cells circulating
in peripheral blood, capable of forming spheroids are way more resistant to
anticancer drugs than the remnant circulating tumor cells. We, furthermore,
demonstrate that salinomycin and Curcumin efficiently destroy spheroids
cultured from CETCs, strengthening their role as promising anti-cancer
therapeutic.
319 Expression profile of fibroblasts from tumor bearing prostate
exhibits significant differences compared to BPH-derived
fibroblasts
V. Jung1 , K. Schmitt2 , M. Saar1 , K. Junker1 , M. Stöckle1 , G. Unteregger1 .
1
University of the Saarland, Department of Urology, Homburg/Saar, Germany,
2
University of the Saarland, Department of Pathology, Homburg/Saar,
Germany
Introduction: Stromal environment is indispensable for the normal prostate
development and an important contribution of activated fibroblasts in
prostate cancer initiation and progression is well documented. Thus, a
comprehensive and comparative analysis of prostate stromal cells is
indispensable to understand their specific function and for an elucidation of
new prognostic markers and future therapeutic strategies. Whereas several
markers characteristic for tumor associated fibroblasts were described a
comparative analysis to normal BPH-derived fibroblasts is missing.
Material and Methods: Fibroblasts from 7 BPH (BPHF) and fibroblasts
from 7 prostate cancer patients (PNF = non-tumor-associated, PTF = tumorassociated) were selectively cultivated using patients-derived small specimens
without enzymatic digestion. Outgrowing cells were sub-cultivated for up
to six passages and genetic characterization (CGH) and gene expression
profile (qRT-PCR) were performed by standard techniques. We focused
on the expression of androgen receptor (AR), alpha smooth muscle actin
(aSMA), b-catenin, stromal derived factor 1 (SDF-1), fibroblast activating
protein (FAP), TGF-b, platelet derived growth factor receptor beta (PDGFRb)
and E-cadherin.
Results: CGH analysis revealed normal karyotypes without any chromosomal
alterations in all fibroblasts. PNF and PTF differ only by an enhanced
expression of TGF-b and PDGFRb in PTF. In contrast, SDF-1, FAP, and TGF-b
which are assigned as markers for activated fibroblasts were significantly
reduced in BPHF as compared to PNF and PTF. Interestingly, expression of
androgen receptor (AR), alpha smooth muscle actin (aSMA) and PDGFRb
was significantly up regulated in BPHF.
Conclusion: The obviously higher similarity in the expression profile of
normal and tumor-associated fibroblasts from prostate cancer specimens as
compared to BPHF underlines a pivotal role of stromal cells in prostate
disease. Additionally the results of PNF and PTF seem to reflect rather
a ‘cancer specific’ function independently from the distance of these cell
population within the patients prostate. Differences to the BPHF-specific
pattern indicate on a specific function in epithelial and mesenchymal cell
proliferation in this disease. Further investigations are now warranted to
determine the molecular function and the clinical relevance together with
therapeutic potential of these alterations.
321 Expression and cytogenetic profile of intratumor morphological
heterogeneity in breast cancer
E. Denisov1 , T. Gerashchenko1 , N. Skryabin2 , S. Vasilyev2 , M. Zavyalova3 ,
N. Litviakov1 , V. Perelmuter3 , N. Cherdyntseva1 . 1 Cancer Research Institute,
Laboratory of Molecular Oncology and Immunology, Tomsk, Russian
Federation, 2 The Russian Institute of Medical Genetics, Laboratory of
Cytogenetics, Tomsk, Russian Federation, 3 Cancer Research Institute,
Pathological Anatomy and Cytology, Tomsk, Russian Federation
Background: Intratumor morphological heterogeneity represented by five
types of morphological structures has been described for invasive carcinoma
of no special type, the main histotype of breast cancer (BC). Data obtained to
date demonstrate the contribution of intratumor morphological heterogeneity
of BC to chemotherapy efficiency and cancer metastasis; however, there is
little information about the nature of such morphological diversity.
Materials and Methods: In this study, we studied the expression of cell
adhesion genes (cadherins, catenins, and integrins) in different morphological
structures of breast tumors (n = 4). In addition, morphological structures from
three different regions of one breast tumor and its lymphogenic metastases
were cytogenetically characterized using SurePrint G3 Cancer CGH+SNP
4×180K microarray (Agilent Technologies). Tubular, trabecular, alveolar, solid
structures, and discrete groups of tumor cells were isolated from breast
tumors by laser microdissection PALM (Carl Zeiss). RNA samples (average
RIN ≈ 6.8) were undergone reverse transcription, ligation, whole transcriptome
amplification (QuantiTect Whole Transcriptome Kit, Qiagen) and were used
in real-time quantitative TaqMan PCR. DNA samples were whole genome
amplified.
Results: Expression of catenin genes was presented in almost all structures.
Oppositely, activity of cadherin and integrin genes was significantly changed
from one type of structures to another one. Cadherin gene activity decreased in
the order solid − alveolar and trabecular structures − discrete groups of tumor
cells, whereas integrins in the order solid and alveolar − trabecular structures −
discrete groups of tumor cells (p < 0.05). Tubular structures, which are used
in histological grading of BC, had the lowest activity of cell adhesion genes.
Chromosome aberrations specific for each type of morphological structures
have not been found; however, discrete groups of tumor cells demonstrated
the lowest number of cytogenetic abnormalities. Solid structures from only one
region of breast tumor and lymph node metastases displayed the highest level
of chromosome aberrations and two common amplifications of chromosome
bands − 17p13.1 (part of TP53 gene) and 12q11q24.33 (including MDM2
gene), which were not been detected in all other structures.
Conclusions: Intratumor morphological heterogeneity in BC is not associated
with specific chromosome abnormalities and is probably a reflection of different
patterns of invasive growth.
322 Identification of GREB1 as a potential mediator of estrogen effects
on ovarian cancer progression in a mouse model
K.M. Hodgkinson1 , L.A. Laviolette1 , B.C. Vanderhyden1 . 1 Ottawa Hospital
Research Institute and University of Ottawa, Cellular and Molecular Medicine,
Ottawa, Canada
Background: Hormone replacement therapy containing estrogen increases
the risk of developing ovarian cancer, and 17b-estradiol (E2) promotes
growth and survival of ovarian cancer cell lines. We have shown previously
that E2 promotes ovarian cancer progression in transgenic and allograft
mouse models, and now investigate the mechanism of E2 action on tumour
progression.
Material and Methods: To identify genes altered by E2 treatment, we used
an allograft model in which SCID mice were implanted with E2 pellets and
injected with mouse ovarian cancer cells (MAS) derived from the ascites
of the tgCAG-LS-TAg transgenic mouse model of ovarian cancer. One E2upregulated gene of particular interest is Gene regulated by estrogen in breast
cancer-1 (Greb1), which is an estrogen receptor a (ESR1) target gene which
mediates the proliferative actions of hormones in breast and prostate cancer
cells. In order to investigate the function of GREB1, we used lentiviral vectors
to cause knockdown and overexpression in MAS cells.
Results: Survival of mice engrafted with MAS ovarian cancer cells was
shortened by E2 treatment and microarray analysis showed upregulation of
197 genes and downregulation of 55 genes in tumours from E2-treated mice.
QPCR confirmed upregulation of Greb1 and other genes of interest in tumours
from E2-treated mice and cultured MAS cells as well as two human ovarian
cancer cell lines. MAS cell proliferation was decreased by GREB1 knockdown
and increased by GREB1 overexpression. When injected into SCID mice, MAS
cells with GREB1 knocked down resulted in fewer metastases and mice had
prolonged survival relative to mice injected with control MAS cells. GREB1 is
highly expressed in human ovarian tumours of 4 histological subtypes relative
to normal ovarian epithelial cells (on average, 347-fold higher in tumours), and
correlates with ESR1 expression, suggesting that GREB1 may be regulated
by ESR1 in ovarian cancer (as shown in breast cancer).
Conclusions: This study is the first to examine GREB1 action in mouse
models and its expression in ovarian cancer cell lines and tumours.
Characterization of the function of E2-target genes will elucidate the
mechanisms by which E2 increases the risk of ovarian cancer and help clarify
the effects of estrogen antagonists on ESR1-positive ovarian cancers.
323 Simplifying high throughput 3D tumour spheroid growth and
shrinkage assays using live content imaging
T. Dale1 , K. Patel1 , B. O’Clair2 , T. O’Callaghan1 , D. Appledorn2 , D. Trezise1 .
1
Essen BioScience, Welwyn Garden City, United Kingdom, 2 Essen
BioScience, Ann Arbor, USA
Introduction: For several years, 3D tumour spheroid models have been used
in the study of cancer biology as these are believed to be more reflective
of the in vivo micro-cellular environment than 2D systems. However, the
routine application of 3D models within drug discovery has been limited by
complexities in methodology and quantification. Our aim was to build relevant,
kinetic tumour spheroid growth & shrinkage assays that are as technically
straightforward, robust and cost equivalent to 2D assays.
Method: In this study, a kinetic, live-cell imaging approach was used to
measure the growth or shrinkage of non-adherent tumour spheroids using
Ultra Low Attachment (ULA) 96-well and 384-well micro-titre plates (Corning® ).
These round-bottomed, hydrogel coated plates facilitate the formation of
spheroids by promoting cellular self-adherence as opposed to adherence
to the plate surface. Post spheroid formation, quantitative pharmacology
on compounds with a range of mechanisms was performed by monitoring
spheroid size over 10 days using the IncuCyte™ ZOOM Live Content
Imaging system. Spheroid parameters were quantified using fluorescence area
and fluorescence intensity metrics via a RFP-labelled non perturbing probe
(NucLight-Red™).
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Results: In a monoculture model, NucLight-red labelled A549 lung epithelial
carcinoma cells developed into a single spheroid in each well of 96- and 384well plates within 48−96 hours in culture. In both plate types, the resulting
spheroid size, ranging from 330 mm to 600 mm, was proportional to the number
of cells seeded (1000–5000 cells per well). Data was obtained for a set of
compounds yielding a range of pIC50 values. For example, the clinically used
cytostatic agent cycloheximide inhibited spheroid growth (pIC50 5.8) with no
substantial effect on spheroid shrinkage. In contrast, the chemo-therapeutic
paclitaxel (Taxol® ) both inhibited spheroid growth (pIC50 7.9) and at high
concentrations (0.1 mM) induced spheroid shrinkage.
Conclusion: The data shown here describes validation of a technically
straightforward, robust and economical kinetic, live-cell approach to measuring
spheroid size. Such facile 3D assays allow for adoption into early phase drug
discovery projects.
324 BRCA1 as a regulator of global cell metabolism − identifying
functional targets through integromic analysis
A. Powell1 , K.I. Savage1 , D.P. Harkin1 . 1 Queen’s University Belfast, CCRCB,
Belfast, United Kingdom
Introduction: Mutations within the BRCA1 tumour suppressor gene
predisposes carriers to a high risk of breast and ovarian cancers. BRCA1
primarily functions to maintain genomic stability through a role in multiple cell
processes including DNA repair, cell cycle arrest and transcriptional regulation.
Recent studies suggest a role for BRCA1 in regulating fundamental metabolic
processes. We performed a discovery metabolomic screen to explore the
role of BRCA1 in regulating cellular metabolism and its relevance to tumour
progression. These data were integrated with existing BRCA1 genetic and
transcriptional data to identify metabolically relevant BRCA1 transcriptional
targets.
Materials and Methods: BRCA1-deficient HCC1937 cells were virally
transduced with wild type BRCA1 or GFP and matched cell and medium
samples were analysed by Metabolon Inc. Metabolite fold-change data were
then functionally annotated. This dataset was analysed in parallel with BRCA1
gene expression microarray and ChIP-chip data using the IMPaLA integromic
analysis tool in order to identify metabolically relevant targets of BRCA1
transcriptional regulation.
Results: Of 347 unique metabolites in the screen, 50 were significantly
associated with BRCA1 status. Loss of BRCA1 results in a trend of general
upregulation in diverse classes of molecules. Highly desaturated and long
chain fatty acids were strongly represented in the dataset; loss of BRCA1
results in upregulated levels of C18 and C20 desaturates including arachidonic
acid (AA). Conversely, levels of antioxidant a-Tocopherol were found to be
reduced, suggesting a complex involvement of fatty acid metabolism and
oxidative balance.
Pathway-driven integromic co-analysis of BRCA1 metabolite and transcription
data identified 13 pathways among which lipid and lipoprotein metabolism,
signal transduction and small molecule transporters were enriched for BRCA1
regulated transcripts. The potential role of BRCA1 in regulating these
transcripts was cross-validated in publically available datasets in order to
produce a high-confidence list of functionally important BRCA1-regulated
transcripts that are likely to have phenotypic relevance.
Conclusion: Our data suggest that BRCA1 has a much more diverse role in
regulating metabolism than was previously appreciated. Through an integromic
approach we have identified BRCA1-regulated genes that may represent
targets with relevance to the phenotype of BRCA1 deficient cancers. In order to
assess the functional relevance of this data, secondary screening techniques
will be employed: a real-time based screen of genetic targets in our primary
model and a metabolic dependency screen, consisting of a panel of small
molecule inhibitors of key enzymes in identified pathways.
326 Expression analyses of long non-coding RNAs in breast cancer
E. Knutsen1 , T. Fiskaa1 , S.L. Figenschau1 , E.S. Brun1 , S. Fismen1 ,
O.M. Seternes2 , E.S. Mortensen1 , S.D. Johansen1 , M. Perander1 . 1 UiT The
Arctic University of Norway, Department of Medical Biology, Tromsø, Norway,
2
UiT The Arctic University of Norway, Department of Pharmacy, Tromsø,
Norway
Background: Long non-coding RNAs (lncRNAs) are regulatory transcripts
longer than 200 nucleotides, the majority being transcribed from RNA pol
II promoters and processed by 5 capping, polyadenylation, and splicing.
They have recently attained much attention as important regulators of gene
expression at different levels. The lncRNAs exert their function by mediating
interaction with DNA elements, other RNA molecules, or proteins either by
complementary base pairing or by adapting specific structures. Many of them
show tissue-specific expression and are expressed at specific time points
during embryonic development, indicating an important role in turning on and
off genes at specific circumstances. Deregulated expression of many lncRNAs
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is associated with human diseases including cancer. The aim of this study is to
identify lncRNAs that upon aberrant expression contribute to the development
of invasive breast cancer.
Materials and Methods: By using SOLiD next-generation RNA-Seq and
SAGE-Seq we generated more than 1.5 billion reads from samples from breast
epithelial cells induced to go through epithelial–mesenchymal-transition (EMT),
and fresh tissue from breast cancer patients. Sequencing reads were aligned
to the human reference genome and annotated using the Ensembl annotation
(Grh37.75). LncRNAs of particular interest were selected for further expression
analyses in paraffin-embedded breast tumor biopsies.
Results: We identified several hundred lncRNA candidates, both novel and
previously described, that change their expression pattern during the EMT
process. Some of them are differentially expressed in breast tumor samples
compared to normal tissue.
Conclusion: Aberrantly expressed breast cancer lncRNA candidates are
currently investigated at molecular and cellular levels in order to unravel novel
biological function and contribution in cancer development.
327 Paraffin embedded tissue as valuable and challenging resource
for studying intratumoral genetic heterogeneity of clear cell renal
cell carcinoma
P. Ferronika1 , J. Bergsma1 , J. Li1 , M.M. Terpstra1 , A.M. Lelieveld-Kors2 ,
R. Danarto3 , R.H. Sijmons1 , G. Kats-Ugurlu4 , A. Moeljono3 , K. Kok1 .
1
University Medical Center Groningen, Genetics, Groningen, Netherlands,
2
University Medical Center Groningen, Urology, Groningen, Netherlands,
3
Gadjah Mada University, Urology, Yogyakarta, Indonesia, 4 University Medical
Center Groningen, Pathology, Groningen, Netherlands
Background: Clear Cell Renal Cell Carcinoma (ccRCC) is well known for the
heterogeneity of its clinical, histologic, and genetic profiles. Paraffin embedded
tissue provides valuable but challenging material for studying the complexity of
this tumor, especially for high-throughput sequencing procedures. This project
aimed to optimize the preparation of DNA samples from paraffin material to
identify the genetic profile of different tumor areas of one case of ccRCC.
Material and Method: Samples were taken from a patient diagnosed with
ccRCC after total nephrectomy. DNA was isolated from paraffin sections of
four different tumor areas and normal kidney cortex. The morphological variety
among different areas of tumor has been documented. We performed preanalytical RT-PCR to measure the quality of amplifiable DNA input. Aliquotes of
100 ng of DNA were subjected to whole exome sequencing (Agilent SureSelect
All Exon V2® , Illumina HISEQ 2500® ).
Result and Discussion: Based on published data a minimum Quantitative
Functional Index (QFI) of 6−7% from RT-PCR is needed to achieve 90%
confirmed variants on sequencing data. The QFI of our FFPE DNA samples
showed values of 8.9–12.6%, indicating that the quality of our DNA samples
was acceptable for further analysis. The quality control report of sequencing
data showed a 20x coverage of 68−86%. Preliminary analyses of the sequence
data identified 66 somatic mutations, including 2 indels. In these preliminary
data we see a unique genetic profile shared by areas which have similar tumor
histological grade. The mutational spectrum among different tumor areas and
its validation will be discussed in more detail at the meeting. In addition we are
evaluating a cancer panel (Nugen Ovation® Cancer Panel Target Enrichment
System) as an alternative approach to obtain reliable sequence data from
FFPE material.
Conclusion: The heterogeneity of mutational spectrum present among
different areas of ccRCC may be important in cancer development. With
optimized protocols, FFPE material could be used as a reliable resource to
study this intratumoral heterogeneity.
328 Identification and quantification of BRCA1 splicing variants
F. Lhota1 , J. Hojny1 , P. Kleiblova1 , J. Sevcik1 , J. Soukupova1 , M. Janatova1 ,
P. Boudova1 , M. Borecka1 , P. Pohlreich1 , Z. Kleibl1 . 1 Charles University in
Prague First Faculty of Medicine, Inst. of Biochemistry and Experimental
Oncology, Prague, Czech Republic
Introduction: BRCA1 protein contributes to the maintenance of genome
integrity through regulation of DNA double strand break repair. The absence
of fully functioning protein predisposes for breast cancer (BC) in carriers of
BRCA1 hereditary mutations. Many of these alterations affect cis-regulatory
splicing sites resulting in aberrant splicing of BRCA1 pre-mRNA and synthesis
of the functionally defective BRCA1 protein isoforms. Moreover, several
alternative splicing variants (ASVs) has been described as a result of
alternative splicing of wt BRCA1; however, little is known about their tissue
distribution and dynamics upon DNA-damaging conditions.
Material and Method: We characterized the BRCA1 ASVs in studied cell
lines (MCF-7, HeLa, MB-231, and EM-G3 cells; including their dynamics post
g-irradiation (PI)) and in mammary and adipose tissue and lymphocytes in
female BC patients and controls. Using a set of multiplex PCR with the cDNA
template, we amplified all theoretically possible BRCA1 ASVs. All primers
were localized 5−7 bp apart from exon-exon boundary yielding short amplicon
just in case of alternatively spliced BRCA1 mRNA. The prevailing longer
amplicons generated from wt mRNA were size-excluded using magnetic beads
purification prior sequencing library preparation. Purified amplicons were
analyzed by SOLiD next-gen amplicon sequencing (NGS). Subsequently, a
qPCR quantification has been performed to confirm the presence of individual
variants and their changes in DNA-damaging conditions.
Results and Discussion: So far we have identified 10 and 8 in-frame and
19 and 7 frame-shift ASVs in analyzed cells and human tissues, respectively.
The most ASVs were presented in MCF-7 cells, while only five of them were
identified in ‘normal-like’ mammary EM-G3 cells. qPCR analysis showed that
wt BRCA1 represents 60−90% of all BRCA1 ASVs in cell lines and tissues
respectively. Expression of ASVs in cell lines changed in quantitative but not
qualitative manner PI. The most significant changes were detected at a time
of 15 min post irradiation. Presented study indicates that NGS represents a
reliable approach for analysis of ASVs even for genes like BRCA1 where
presence of low-abundant ASVs mRNAs accompany the dominant form of
WT BRCA1 mRNA.
Supported by grants IGA MZ 12280, GACR P301/12/1850.
329 Inhibition of thioredoxin-1 by small interfering RNA reduces
chemoresistance for doxorubicine in two diffuse large B-cell
lymphoma cell lines
M.E.L. Kuusisto1 , P. Honkavaara1 , A. Hakalahti1 , P. Karihtala1 ,
T. Turpeenniemi-Hujanen1 , O. Kuittinen1 . 1 Oulu University Hospital, Oncology,
Oulu, Finland
Introduction: The basis of chemoresistance in lymphoma treatment is still
under further investigation. Relapse and refractory disease of diffuse large
B-cell lymphomas (DLBCL) need good therapeutic agents, and overcoming
chemoresistance is crucial. The thioredoxin redox protein family may be one
of the key players in the development of chemoresistance.
Material and Method: Two commercial human DLBCL cell lines (ATCC®
CRL-2630™, ATCC® CRL-2632™), expressing high amounts of thioredoxin-1
(Txn-1), were cultured for evaluation. Txn-1 knockdown was performed
by small interfering RNA (siRNA), using electroporation as a transfection
method. The knockdown rate was measured by western blotting and
reverse transcription polymerase chain reaction. The success of transfection
was verified with fluorescence-activated cell sorting. Chemoresistance was
tested with several drugs used in clinical practice: doxorubicin, etoposide,
vincristine, prednisolone and carboplatin. Chemotherapy agents were given
48 h after transfection to both Txn-1 siRNA transfected cells and control cells,
transfected with negative control siRNA. The survival of cells was measured
by spectrophotometric analysis.
Results and Discussion: Results showed a good transfection rate, approximately 70% of cells were transfected successfully. The chemoresistance for
doxorubicin was reduced after Txn-1 knockdown in DLBCL cell line. On the
contrary, after etoposide treatment, cell survival was better in cells that were
transfected with Txn-1 siRNA. No significant differences were found when
treated with carboplatin, vincristine or prednisolone.
Conclusion: It appears that over-expression of Txn-1 increases drug
resistance for doxorubicin. Based on the current results, cells with Txn-1 overexpression, etoposide could be a more suitable chemotherapeutic agent for
clinical practice.
330 Only an EpCAM–claudin-7 complex acts as a cancer initiating
cell biomarker
F. Thuma1 , S. Heiler1 , R. Philip1 , M. Zöller1 . 1 Uniklinik Heidelberg, Tumor
Cell Biology, Heidelberg, Germany
Introduction: The transmembrane glycoprotein EpCAM was identified as
a cancer initiating cell (CIC) marker in breast, colorectal, pancreatic, and
hepatocellular carcinoma. Its functional activity as a CIC marker is still
disputed. We observed in several human colon and pancreatic tumor lines that
EpCAM is associated with the tight junction protein claudin-7 and hypothesized
that only the EpCAM–claudin-7 complex acts as a CIC biomarker.
Material and Methods: Experiments were performed with a rat pancreatic
adenocarcinoma (PACa) line (ASML) and were controlled in human PaCa.
EpCAM and claudin-7 were knocked down (ASML-EpCkd , ASML-cld7kd ) and
ASML-EpCkd clones were rescued with wt EpCAM (ASML-EpCresc ) or mutated
EpCAM prohibiting claudin-7 binding (ASML-mutEpCresc ). Stem cell features
were evaluated in vitro and in vivo according to standard protocols.
Results and Discussion: ASML-EpCkd and, more pronounced, ASMLcld7kd cells poorly metastasize. Reduced metastatic capacity of ASML-EpCkd
was mostly due to claudin-7 supporting tumorigenic features of EpCAM
by provoking EpCAM cleavage and thereby cleaved EpCAM transcriptional
activity. Instead, claudin-7, besides supporting EpCAM cleavage, associates
with the a6b4, which promotes metastasizing tumor cell motility and strongly
affects Pten expression, which is accompanied by a strong increase in
cytotoxic drug resistance due to activation of the PI3K/Akt pathway. To control,
whether these CIC-bioactivities of claudin-7 are EpCAM-independent, ASMLEpCresc and ASML-mutEpCresc clones were established, the EpCAM mutation
prohibiting claudin-7 binding. Expectedly, ASML-EpCresc regained metastatic
and stem cell features, but ASML-mutEpCresc did not. Ongoing experiments
indicate that only EpCAM-associated claudin-7 resides in glycolipid-enriched
membrane microdomains, which serve as signaling platform. Furthermore,
the EpCAM association is essentially required for phosphorylation and
palmitoylation of claudin-7, which provides the initial trigger for EpCAM
cleavage as well as for the transcriptional activity of claudin-7.
Conclusion: This is the first demonstration for only a complex of two CIC
markers exerting bioactivity, where cld7 acts as executioner that requires
EpCAM to receive the initial trigger.
331 Integrative analysis reveals extensive association between
microRNA expression and mRNA−protein translation
M.R. Aure1 , S. Jernström1 , M. Krohn1 , E. Due1 , Oslo Breast Cancer
Consortium2 , G.B. Mills3 , A.L. Børresen-Dale1 , K.K. Sahlberg1 ,
O.C. Lingjærde4 , V.N. Kristensen5 . 1 Institute for Cancer Research,
Department of Genetics, Oslo, Norway, 2 www.ous-research.no/home/
kgjebsen/home/14105, Department of Genetics, Oslo, Norway, 3 The
University of Texas M.D. Anderson Cancer Center, Department of Systems
Biology, Houston, USA, 4 Biomedical Informatics Research Group University
of Oslo, Department of Informatics, Oslo, Norway, 5 Division of Medicine
Akershus University Hospital, Department of Clinical Molecular Biology and
Laboratory Science (EpiGen), Akershus, Norway
Background: Protein expression depends on mRNA expression as well as the
rate at which those transcripts are translated to form protein. The translation
process is controlled in part by microRNAs (miRNAs), and altering the activity
of miRNAs is one mechanism by which the cancer cell may deregulate
the expression of key genes. The importance of miRNA related regulation
of protein translation and the extent to which multiple miRNAs coordinately
regulate key proteins in breast cancer is only partly understood.
Materials and Methods: We analyzed genome-wide miRNA expression, as
well as mRNA and protein expression for a panel of 105 selected cancer
related genes in breast carcinomas from 283 patients. The effect of each
miRNA on the translation of each mRNA into protein was studied by modeling
the protein expression as a joint function of mRNA and miRNA expression. The
change in protein level is assumed to be proportional to mRNA expression,
and the proportionality factor may depend on miRNA expression. The effect
on translation of a single miRNA, as well as all miRNAs simultaneously, were
considered.
Results and Discussion: A genome-wide landscape of associations was
found between miRNAs and mRNA–protein translation for a selected panel
of 105 cancer related proteins. This landscape reveals several features,
including the fact that groups of miRNAs coordinately interact with groups
of proteins, hence suggesting block-wise interaction as a mode of modulation
of protein modules in breast cancer. Studying the effects of miRNAs on protein
expression, through mRNA regulation, adds to our understanding of the role of
miRNAs in breast cancer. Using the predicted effects of miRNAs on proteins
and considering the patient-specific miRNA expression to cluster the patients,
a separate cluster of high grade and hormone receptor negative patients was
revealed. Our model predicted both true positive miRNA–protein associations
and new candidate miRNA targets, and was able to correctly predict protein
expression levels in an independent cohort.
Conclusion: The suggested approach captures both direct and indirect
effects of miRNAs on protein expression, and reveals extensive and blockwise associations between the expression of miRNAs and the translation of
proteins.
332 Stem cell fractions of extra- and intrahepatic cholangiocellular
carcinoma cell lines differ in size and phenotype
R. Schmuck1 , C.V. Fischer1 , A. Schirmeier1 , P. Neuhaus1 , M. Bahra1 .
1
Charité − Universitätsmedizin Berlin Berlin Germany, General Visceral and
Transplantation Surgery Experimental Surgery, Berlin, Germany
Introduction: Side Population (SP) of tumor cell lines shares characteristics
with tumor stem cells. Cells with stem cell properties are said to be responsible
for tumor formation as well as resistance against conventional chemotherapy
and are crucial for the malignant potential of the tumor. In this study we
characterized and compared the SP of intra- and extrahepatic cholangiocellular
carcinoma (CCCs) cell lines.
Material and Methods: SP cells were obtained from two intrahepatic (HuCCT1
and RBE) and extrahepatic (TFK1 and EGI1) CCC cell lines using Hoechst
33342 staining and fluorescence-activated cell sorting (FACS). Cells were
S79
additionally stained with antibodies against DC133, CD44 and CD90 and
separately cultured after sorting. For morphological evaluation, cells were
sorted in SP and Non-SP cells and stained with H&E after CytoSpin
centrifugation.
Results: All four cell lines showed a reproducible SP-fraction with a
characteristic pattern. Sp cells were smaller and rounder in shape. SP- and
Non-SP cells showed distinct phenotypes in culture regarding cell shape and
colony-formation. Extrahepatic cell lines showed a highly significant larger
proportion of SP cells (4.5% vs. 0.7%, p-value 0.00007). Extrahepatic SP
cells also showed significant less CD44 positive cells than extrahepatic SP
cells (37.9% vs. 62.1%, p-value 0.027) and a tendency of less CD133 positive
cells, this difference did not reach significance though. All four cell lines were
negative for CD90.
Discussion: Extrahepatic CCC cell lines have a higher proportion of the stem
cell enriched side population. With regards to a potential stem cell origin of
cancer and their high resistance to conventional chemotherapy, the disparity
of intra- and extrahepatic CCCs regarding their stem cell fraction might
have diagnostic and therapeutic implications. Furthermore, extrahepatic- and
intrahepatic CCCs originate from different embryological tissue-sources and
recent studies describe the similarity between neoplasms of the extrahepatic
bile ducts and their pancreatic counterparts. Functional analysis as well as
examination of clinical samples are warranted to further clarify the role of SP
cells in CCCs.
Conclusion: Intrahepatic and extrahepatic CCCs should not be seen as one
tumor entity as they differ notably regarding their macroscopic and microscopic
aspect, surgical therapy and clinical behaviour. This can also be seen in the
fact that SP fractions of extra- and intrahepatic CCC cell lines differ in size and
phenotype.
334 Long-term inhibition of miR-200c in pancreatic adenocarcinoma
cells changes CDH1 expression and cell migration
M. Stölting1 , J. Schwarze1 , M. Starke1 , J. Haier1 , N. Senninger1 , S.T. Mees1 .
1
University hospital Muenster, Department of General and Visceral Surgery,
Muenster, Germany
Background: With a dismal 5-year survival rate below 5% and a median
survival about 6 months the ductal adenocarcinoma of the pancreas (PDAC)
represents a very aggressive malignancy, mainly caused by early metastasis.
Epithelial–mesenchymal transition (EMT) is a major factor of metastasis. The
miR-200 family of microRNAs (miRs) was previously shown to participate
in EMT but there are contrary data regarding the precise function of these
miRNAs. The aim of this study is to clarify the role of miR-200 by evaluating
the effects of a stable miR-inhibition in PDAC cell lines.
Material and Methods: Functional inhibition of miRs in general is achieved
by introducing miRNA antagonists into cells. Lentiviral gentransfer of pmiRZip200c (BioCat, Heidelberg) was used to generate a stable inhibition of the miR200 family member miR-200c in the PDAC cell line AsPC-1 (AsPC-1 antimiR200c). A pmiRZip-scrambled vector was used as control (AsPC-1 MOCK).
Evaluation of stable inhibition and morphologic characteristics was performed
by bright field and fluorescence microscopy. qRT-PCR and Westernblot were
used for monitoring of mRNA and protein expression levels and, migration
analysis was assessed by a modified Boyden chamber assays.
Results and Discussion: Successful generation of AsPC-1 antimiR-200c and
AsPC-1 MOCK cells was confirmed by puromycin resistence and cop-GFP
expression. Bright field microscopy reveals that AsPC-1 antimiR-200c cells
exhibit an extended cell body and appear to grow in a closer connection to each
other compared to AsPC-1 MOCK and AsPC-1 wildtype cells. Additionally,
immunofluorescence studies show an altered actin distribution in AsPC-1
antimiR-200c cells. While the mRNA expression of the direct miR-200c target
ZEB-1 is not significantly changed, analysis of the ZEB-1 protein expression
shows a 2-fold reduction. Expression of the epithelial protein CDH1, which is
repressed by the transcription factor ZEB-1, is more than 2-fold up regulated
at the mRNA- and about 4-fold up regulated at the protein level due to miR200c inhibition. Functional analysis of cell migration capacity illustrates that
miR-200c inhibition reduces migration capacity of AsPC-1 cells round about
35% compared to MOCK and wildtype cells (p < 0.005). This goes along
with previous data showing that an elevated CDH1 expression lowers cell
migration.
Conclusion: These preliminary results suggest that a stable miR-200c
inhibition leads to a long-term effect on PDAC cells including changes of
the epithelial–mesenchymal steady state by altering morphology, protein
expression and functional properties. The underlying mechanisms of this effect
as well as a possible impact on invasion and metastasis properties of PDAC
cells in vivo will be elucidated in further studies.
S80
336 EDI3, a glycerophosphodiesterase linking metabolism to cellular
migration and attachment
R. Marchan1 , M.S. Lesjak1 , B. Buettner1 , J.D. Stewart2 , J. Lambert3 ,
H. Keun4 , J. Rahnenfuehrer5 , J.G. Hengstler1 . 1 Leibniz Research Centre
for Working Environment and Human Factors (IfaDo), Systems Toxicology,
Dortmund, Germany, 2 University of Southampton, Center for Biological
Sciences, Southampton, United Kingdom, 3 Leibniz-Institut für Analytische
Wissenschaften − ISAS − e.V., Interface Processes, Dortmund, Germany,
4
Imperial College London, Surgery and Cancer, London, United Kingdom,
5
Technische Universitaet Dortmund, Statistics, Dortmund, Germany
Introduction: Metastasis from the primary tumour remains a therapeutic
challenge and is a major cause of death in cancer patients. We identified
the glycerophosphodiesterase (GDE), EDI3 (GPCPD1; GDE5) in a study
aimed to find markers of endometrial cancer metastasis. Patients with tumours
expressing high EDI3 went on to develop metastasis and had worse prognosis.
EDI3 was shown to hydrolyse glycerophosphocholine (GPC) to choline and
glycerol-3-phosphate (G3P), with consequences in both lipid and choline
metabolism. EDI3 expression was also associated with cellular migration via
PKCa. Our work was the first to implicate a GDE in cancer, but the link
between EDI3’s enzymatic activity and its role in migration or other processes
important in cellular transformation remain unknown. Therefore, our aim was to
identify other processes where EDI3 may be involved and investigate pathways
downstream of EDI3 to determine which may be more critical for the observed
phenotypic changes.
Materials and Methods: Gene array analysis after EDI3 knockdown was
performed and gene ontology analysis used to identify relevant biological
motifs. RNA, protein and functional assays were performed after knockdown
and overexpression to characterize EDI3’s potential role in integrin-mediated
processes. Choline kinase alpha (CHKA), which phosphorylates choline
to phosphocholine − a first step in phosphatidylcholine synthesis; G3P
acyltransferase 1 (GPAT1), which adds an acyl group to G3P, producing the
signalling lipid lysophosphatidic acid (LPA); and EDI3 were downregulated
using siRNA and the effect on migration, key signalling proteins, and
metabolism compared. Confirmation was obtained by overexpressing the
respective proteins.
Results and Discussion: An enrichment of genes involved in integrinmediated signalling led to the characterization of EDI3 in cellular attachment
and spreading on a fibronectin matrix. Loss of EDI3 decreased expression of
integrin a5/b1 expression, and was associated with delayed attachment and
spreading, processes linked to migration. GPAT1 influenced cell migration,
and the combined knockdown of both EDI3 and GPAT1 did not exacerbate
the migration effect, suggesting that their influence on migration is linked.
Furthermore, knocking down GPAT1 in EDI3-overexpressing cells ‘rescued’
EDI3’s mediated increase in migration. Interestingly, in contrast to previous
reports, CHKA had no influence on migration or viability, even though loss of
EDI3 leads to decreased phosphocholine levels.
Conclusion: EDI3’s influence on cell migration appears to be mediated via the
GPAT1 pathway. However no common signalling protein has been identified.
Further investigation into the proteins and products of this pathway must be
investigated to understand which of the several candidates are important for
EDI3-mediated migration.
337 STAT3, IL-17 and COX-2 features in the transgenic adenocarcinoma
of mouse prostate (TRAMP) model and in the aging mice (FVB)
submitted to goniothalamin therapy
Goniothalamin treatment led to decrease of both cellular proliferation and inflammation in TRAMP and senile mice. The molecular results showed that Goniothalamin treatment displayed a reduction to STAT-3 and IL-17 in both models, mainly in senile mice. On the other hand COX-2 reactivity was not altered.
Conclusions: Goniothalamin treatment demonstrated significant effect on the
pro-inflammatory cytokine and on a transcription factor signal transducer,
acting in many pro-oncogenic signals, possibly delaying prostate cancer
progression. Moreover, this treatment suggested a preventive effect on senile
prostatic microenvironment, considering the role of senescence in different
clinical disorders and the potential to elderly mice to develop prostatic cancer.
338 Smad ubiquitination regulatory factor 2 (Smurf2) regulates the
gap junction protein connexin43 during mitosis
T.A. Fykerud1 , S.D. Koirala1 , M.Z. Totland1 , L.M. Knudsen1 , Z. Yohannes1 ,
Y. Omori2 , A. Brech3 , E. Leithe1 . 1 The Norwegian Radium Hospital,
Department of Cancer Prevention, Oslo, Norway, 2 Akita University School of
Medicine, Department of Molecular and Tumor Pathology, Akita, Japan, 3 The
Norwegian Radium Hospital, Department of Biochemistry, Oslo, Norway
Background: The connexins are a family of integral membrane proteins
that form channels between adjacent cells, which assemble into distinct
plasma membrane domains called gap junctions. Gap junctions enable the
adjacent cells to directly communicate with each other, by exchanging ions
and small molecules between the cells. Gap junctions play important roles
in cell growth control and in the maintenance of tissue homeostasis. There
is significant evidence that aberrant regulation of gap junction intercellular
communication is involved in carcinogenesis. The connexin protein family
comprises 21 members in humans, of which connexin43 (Cx43) is the beststudied member. Cx43 has been shown to act as a tumor suppressor in
various tissues. Previous studies have shown that Cdk1 phosphorylates Cx43
during mitosis, which is associated with a reduction in gap junction intercellular
communication and relocalization of Cx43 from the plasma membrane to
intracellular structures. Here, we have investigated the molecular mechanisms
involved in the regulation of Cx43 during mitosis.
Materials and Methods: IAR20 cells and HeLa cells stably transfected
with Cx43 were used as model systems for studying Cx43 during mitosis,
where both unsynchronized cells and cells synchronized with RO-3306 were
analyzed. The subcellular localization of Cx43 in mitotic cells was determined
by confocal microscopy. The binding partners to Cx43 and the changes in
Cx43 phosphorylation and ubiquitination status during mitosis was determined
by immunoprecipitation and western blotting.
Results and Discussion: Cx43 was found to relocalize from the plasma
membrane to early endosomes during mitotic progression. The increased
endocytosis of Cx43 started in prophase and continued until the late mitotic
phases. The E3 ubiquitin ligase Smurf2 (smad ubiquitination regulatory
factor 2), which previously has been shown to regulate the mitotic spindle
checkpoint, was found to colocalize with Cx43 gap junctions in the early phases
of mitosis. Depletion of Smurf2 by siRNA counteracted gap junction endocytosis and trafficking of Cx43 to early endosomes in mitotic cells, resulting in
increased levels of gap junctions at the plasma membrane during mitosis.
Conclusions: This study identifies Smurf2 as a novel regulator of Cx43
gap junctions during mitosis. The data represent the first evidence that the
intracellular trafficking of Cx43 during mitosis is regulated by an E3 ubiquitin
ligase.
339 MALDI-MS peptide profiling of thyroid cancer cell lines to detect
peptide signatures changes with the PI3-K inhibitor GDC-0941,
in hypoxia and normoxia
L.A. Kido1 , F. Montico1 , D.B. Vendramini-Costa2 , J.E. Carvalho3 , R.A. Pilli2 ,
V.H.A. Cagnon1 . 1 Institute of Biology, Structural and Functional Biology,
Campinas São Paulo, Brazil, 2 Institute of Chemistry, Organic Chemistry,
Campinas São Paulo, Brazil, 3 Institute of Biology − CPQBA, Structural and
Functional Biology, Campinas São Paulo, Brazil
F. Henderson1 , K. Williams1 . 1 University of Manchester, Manchester, United
Kingdom
Background: Senescence is associated with significant changes in the
hormonal environment causing prostatic disorders, including inflammatory and
proliferative processes. TRAMP model has been widely used to study the
molecular events in the progression of prostate cancer, since this animal
develops prostate tumors that share many features with human cancer. The
aim herewith was to characterize and compare IL-17, COX-2, and STAT-3
signaling in the ventral prostate from TRAMP and senile (FVB) mice treated
with goniothalamin treatment, a promising synthetic substance which exhibits
anti-inflammatory and anti-proliferative properties.
Material and Methods: The animals were divided into: Control groups; Young
(18 week-old FVB), Senile (52 week-old FVB) and TRAMP: 8 and 12-week-old
mice. Treated groups; Senile-Goniothalamin (150 mg/kg orally) and TRAMP8Goniothalamin (150 mg/kg orally). The ventral lobe was collected after 4 weeks
for light microscopy, immunohistochemistry and western blotting.
Results: Structural analysis demonstrated that senescence led to changes in
prostatic microenvironment, such atrophy, inflammation, and especially, proliferative lesions (PIN), which were similar to those observed in TRAMP mice.
Introduction: Tumour hypoxia is a state of deprived oxygen due to cancer cells
over-proliferating with insufficient blood vessel formation, and is associated
with aggressive tumours and a poor prognosis. The transcription factor Hif-1a
is a central regulator for pathways involved in the tumour hypoxia. The PI3K/Akt pathway is commonly upregulated in a variety of cancers, and has been
associated with the activation of Hif-1a. Therefore, inhibiting the PI3-K/Akt
pathway offers a potential anti-cancer therapeutic target.
MALDI-MS is a soft ionization technique which uses an untargeted approach
to look at large number of peptides/proteins in a single experiment. MALDIMS will be used to detect protein changes in thyroid cancer cell lines with
and without the presence of the PI3-K inhibitor GDC-0941, in hypoxia and
normoxia. This will be carried out by generating peptide profiles of cell lysates,
and using MS/MS to identify these peptides and thus the protein from which
it’s derived.
Western blots will be carried out for Akt and Hif-1a to ensure the drug is
acting as expected, and to investigate the interactions of GDC-0941, hypoxia
and Hif-1a.
Materials and Methods: The cell line FTC-133 was used in all experiments.
Time course assays were set up in 6-well plates. Cells were treated with 1mM
GDC-0941 or with an equal volume of DMSO (used as the control). Plates
were left to incubate in normoxic conditions or hypoxic conditions (0.1% O2 )
for 48 hours. Cells were lysed by freeze-thaw using dH2O with added protease
inhibitors. Cell lysates were treated with ice-cold acetone and left at −20ºC
overnight. The resulting pellets were suspended in ammonium bicarbonate,
reduced by DTT, alkylated by IAA and underwent a tryptic digestion overnight.
Tryptic digests were analysed using a MALDI-TOF instrument (Kratos of
Shimadzu) using a mass range between 700 and 5000Da Peptides were
identified using MALDI-MS/MS and Mascot to search the NCBInr database.
Results: Hypoxic peptide profiles differed hugely from those of normoxia. Two
obvious differences were the presence of a 1046Da and 1905Da in hypoxic
conditions but not in normoxia. The peak at 1046Da was identified using
MS/MS as angiotensin II peptide. The 1905Da peak was identified as a peptide
from the protein p24k-1.
Peptide profiles of controls versus drug treated variants showed more subtle
differences. One such difference was the presence of a 1763Da peak in
hypoxic and normoxic controls, and 1mM treated cells in normoxia, but not in
1mM treated cells in hypoxia. The peak at 1763Da was identified as GAPDH.
Conclusions: Hypoxia causes a large change in the protein profile of FTC133 thryoid cancer cells for cells treated with GDC-0941 and controls. The
peptide angiotensin II can be seen in hypoxic conditions but not in in normoxia
indicating it may play a role in tumour hypoxia. Angiotensin II has been
associated with promoting cancer metastasis, however it is usually produced
by the RAS system. Further experiments will have to be carried out to confirm
its unpredicted presence in thyroid cells. P24k-1, a variant of heat shock 27kDa
protein 1 and angiotensin II may play key roles in tumour hypoxia.
GDC-0941 causes minimal changes in the protein profile of FTC-133 thyroid
cancer cells, which is a beneficial for GDC-0941 as a potential therapeutic; if
the drug causes minimal protein profile changes it is unlikely to cause many
unwanted effects.
Experiments will be repeated using thyroid cell lines 8505c and FaDu. Principal
component analysis be used to statistically analyse all peptide profiles and
identify further key peaks.
340 Resistin induces a disintegrin and metalloproteinase
with thrombospondin motifs-4 gene expression in human
chondrosarcoma cell line
O.F. Hatipoglu1 , K.O. Yaykasli2 , E. Yaykasli3 , E. Kaya4 , M. Ozsahin5 ,
M. Uslu6 , K. Yildirim1 , H.E. Gurses1 , E. Gunduz1 . 1 Turgut Özal University
Institute of Health Sciences Faculty of Medicine, Department of Medical
Genetics, Ankara, Turkey, 2 Kahramanmaras Sutcu Imam University Medical
Faculty, Department of Medical Biology, Kahramanmaras, Turkey, 3 Duzce
University Institute of Health Science, Department of Medical Biology and
Genetics, Duzce, Turkey, 4 Duzce University Medical Faculty, Department of
Medical Pharmacology, Duzce, Turkey, 5 Duzce University Medical Faculty,
Department of Physical Medicine and Rehabilitation, Duzce, Turkey, 6 Duzce
University Medical Faculty, Department of Orthopedics, Duzce, Turkey
Introduction: The irreversible destruction of extracellular matrix (ECM)
components is key process for inflammatory and autoimmune diseases. A
disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), a
family of metallopeptidase involve in several physiological and pathological
processes including degradation of matrix components. Resistin, an adipokine
was characterized in 2001. The involvement of resistin in several biological
processes was established before. However, the putative catabolic role of
resistin on the components of ECM was not elucidated in detail. The aim
of study was investigate to ADAMTS-4 gene expression level in SW1353
chondrosarcoma cell line after resistin induction.
Methods: The SW1353 chondrosarcoma cell line was plated at 37 o C in a
humidified atmosphere of 5% CO2. After confluent, the cell was stimulated
by resistin at 100 ng/mL dose, and incubated for 6, 12, 24, and 48 h. At the
end of the stimulation, the total RNA was isolated, and reverse transcribed
into cDNA using random primer. The gene expression level of ADAMTS4 was
determined by Real-Time Polymerase Chain Reaction.
Results: The expression level of ADAMTS-4 gene was found to increase
gradually and peak for 48 hours incubation.
The elucidation of underlying pathways for ECM components degradation
has pivotal importance to develop new therapy methods for inflammatory and
autoimmune diseases. So, our study was revealed that resistin has catabolic
effect on ECM degradation by increasing ADAMTS-4 expression level at
chondrosarcoma cell line. However, this finding should be supported by other
comprehensive studies.
Acknowledgements: This study was supported by TUBITAK (The Scientific and
Technical Research Council of Turkey), Project Number: SBAG/111S218.
S81
341 SLC22A18, a solute carrier transporter, acts on a tumor
suppressor in colorectal cancer via KRAS
Y. Jung1 , H.Y. Lee1 , J. Keum1 , Y. Jung1 , S. Kim1 , H.K. Chun2 , W.Y. Lee2 ,
S. Lee1 , J. Kim1 . 1 Ewha Womans University, Ewha Research Center for
Systems Biology Division of Life Sciences, Seoul, South Korea, 2 Samsung
Medical Center Sungkyunkwan University School of Medicine, Department of
Surgery, Seoul, South Korea
Background: Identification of novel cancer biomarkers is important for
improved diagnosis, prognosis, and therapeutic intervention. We previously
showed clinical validation results of colorectal cancer biomarkers identified
from bioinformatics analysis of public expression data. SLC22A18 is one of 34
meta-analyzed candidate marker genes of colorectal cancer (CRC).
Materials and Methods: Multiple case-matched normal and tumor tissues of
CRC were examined for differential expression by RT-PCR. We performed
colony forming assay in several CRC cell lines overexpressing SLC22A18
ectopically and KRAS-driven anchorage-independent growth assay with or
without SLC22A18 overexpression. FACS analysis and western blot analysis
of several cell cycle-related genes after SLC22A18 overexpression were
performed.
Results: SLC22A18 was significantly transcriptionally down-regulated in tumor
of CRC. Growth inhibitory results show that SLC22A18 might act as a
tumor suppressor in CRC. Importantly, SLC22A18 suppressed the KRASdriven anchorage-independent growth activity of NIH3T3 cells and induced
changes of cell cycle profiles in CRC cells. The results show that SLC22A18
plays a role on via a part of the KRAS signaling pathway and may reveal
potential value as a biomarker in diagnosis, prognosis, and treatment of
CRC.
Conclusions: We demonstrate that SLC22A18 regulates KRAS-driven
oncogenesis negatively in colorectal cancer.
All samples were collected with the written informed consent from patient and
prior approval of the Samsung Medical Center-Institutional Review Board.
This study was supported by the GIST Systems Biology Infrastructure
Establishment Grant and by the National Research Foundation of Korea (NRF)
grant funded by Korea government (MEST) (2013R1A1A2059405).
342 Modulated electrohyperthermia induced immunogenic cell death
specific signals in colorectal adenocarcinoma model
N. Meggyeshazi1 , G. Andocs2 , C. Kovago3 , L. Balogh4 , T. Krenacs1 .
1
Semmelweis University, 1st Department of Pathology and Experimental
Cancer Research, Budapest, Hungary, 2 Tottori University, Department of
Veterinary Clinical Medicine Faculty of Veterinary Science, Tottori, Japan,
3
Szent Istvan University, Department of Pharmacology and Toxicology Faculty
of Veterinary Science, Budapest, Hungary, 4 “Frederic Joliot Curie” National
Research Institute for Radiobiology and Radiohygiene, Budapest, Hungary
Objective: Electric field and the concomitant heat (modulated electrohyperthermia − mEHT) can synergistically provoke cell death in tumor
tissue. Due to elevated glycolysis (Warburg effect) a concomitant ion
concentration elevation occur leading to elevated permittivity in cancer
compared to non-malignant tissues. Immunogenic cell death (ICD) can
cause programmed cell death and provoke cell mediated anti-tumor immune
response. In overlap with the molecular signs of programmed cell death
the spatiotemporal occurrence of a so called damage associated molecular
pattern (DMAP) is required for professional antigen presenting cells for
inducing ICD.
Here we studied the molecular mechanism of cell death and damage
associated molecular patterns (DAMP) upon mEHT treatment in vivo in
colorectal cancer.
Methods: HT29 human colorectal carcinoma cells xenografted in BalbC
(nu/nu) mice, were treated with a single shot mEHT for 30 minutes. 3 parallel
samples were collected at 0, 1, 4, 8, 14, 24, 48, 72, 120, 168, 216 h posttreatment. 4 h post-treatment mRNA expression was compared to untreated
samples. Human Apoptosis Arrays were also used on 8, 14 and 24 h treated
samples. Histomorphologic, immunohistochemical and TUNEL assay results
were analyzed in tissue micro array (TMA) section using digital slides.
Results: Modulated electrohyperthermia treatment induced significant tumor
cell death linked with DNA fragmentation (24−48 h) using TUNEL assay, in line
with the mitochondrial translocation of Bax (8−14 h), cytochrome c release from
mitochondria to the cytoplasm (8−14 h) and concomitant nuclear translocation
of apoptosis inducing factor AIF (14−24 h). Activated caspase-3 was not
detected in tumor cells. In mRNA assay at 4 h, significant differential expression
of 48 genes including heat shock proteins was seen upon mEHT treatment.
Immunohistochemistry and apoptosis protein array confirmed elevated hsp70
expression (14−24 h and 72–216 h) and hsp90 expression (24–216 h) in the
morphologically intact peripheral parts of treated tumors. Furthermore, an early
(4 h) cytoplasmic to cell membrane exposure of carleticulin and later (48–
216 h) the release HMGB1 protein from cell nuclei was also revealed in the
treated samples.
S82
Conclusion: In our in vivo colorectal model mEHT caused a dominantly
caspase independent programmed cell death involving with the spatiotemporal
appearance of DAMP signals relevant to ICD.
343 Deciphering the role of ubiquitin specific peptidase 15 (USP15)
in human glioblastoma
M. Oikonomaki1 , M.E. Hegi2 . 1 Centre hospitalier Universitaire vaudois,
Lausanne Switzerland, Switzerland, 2 Centre hospitalier Universitaire vaudois,
Neurosurgery, Lausanne Switzerland, Switzerland
Background: Glioblastoma (GBM) is the most aggressive malignant brain
primary tumor. Despite aggressive therapy the median survival remains low
with 15 months. Previous gene expression analysis in human GBM samples
identified Ubiquitin Specific Peptidase 15 (USP15) as a potential tumor
suppressor gene. USP15 belongs to the ubiquitin-specific protease (USPs)
family of which the main role is the reversion of ubiquitination resulting in
stabilization of substrates. Previously, USP15 has been suggested to have a
tumor suppressor function via its substrates APC and Caspase 3. Therefore
the aim of this study is the investigation of the putative tumor suppressing role
of USP15 in human glioblastoma.
Material and Methods: To investigate the role of USP15 in GBM we
performed Mass spectrometry (MS) to identify its protein-binding partners.
Those interactions were confirmed by co-immunoprecipitation (co-IP). USP15
functional analysis was performed by in vivo ubiquitination assays and transient
knockdown in GBM cell lines. Furthermore, we evaluated the effect of USP15
in cell proliferation and migration by gene silencing in LN-229 GBM cell line.
Results: We indentified eight new proteins interacting with USP15 by MS, of
which we have confirmed three by co-IP in LN-229 cell line. One of those
is HECTD1 E3 ligase of which the murine homologue promotes the APC–
Axin interaction to negatively regulate the Wnt pathway. Furthermore, in vivo
ubiquitination assays in LN229 showed that USP15 deubiquitinates HECTD1
while depletion of USP15 lead to decrease of HECTD1 in GBM cell lines.
Moreover, preliminary data suggest that stable knock-down of USP15 has a
positive effect on cell proliferation and increases cell migration as determined
in a 24 h scratch assay.
Conclusion: All the above preliminary data supports the notion that USP15
may have a tumor suppressing function that needs to be evaluated in vivo.
Further investigations aim at uncovering the pathways that are affected by
USP15 and its binding partners.
344 Characterisation of RNA content of cancer-derived exosomes
and microvesicles
A. Line1 , D. Andrejeva1 , A. Cirulis1 , A. Abols1 , P. Zayakin1 . 1 Latvian
Biomedical Research and Study Centre, Riga, Latvia
Introduction: Exosomes and microvesicles (MVs) are the main two types
of extracellular vesicles secreted by live cells. Increasing evidence suggests
that they play a major role in intercellular communication, both locally and
systemically, by transferring various RNAs, enzymes, lipids and other signalling
molecules. They differ in the mode of biogenesis and have distinct structural
and biochemical properties. However, to the best of our knowledge, the RNA
content of exosomes and MVs has not been systematically compared. The
current study aims to compare the RNA content of exosomes and MVs
released from lung cancer cells by deep sequencing.
Material and Methods: Exosomes and MVs were isolated from the
supernatant of COR-L23 cells grown as multicellular spheroids by differential
centrifugation and gel filtration. The vesicles were characterised by Western
blot analysis of CD9, AGO2, ITGB5 and histone H3 expression, transmission
electron microscopy and Zetasizer Nano ZS. RNA size distribution was
analysed using Agilent Bioanalyzer Nano and Small RNA chips. RNA libraries
were constructed from exosomal, MV and cellular RNA and sequenced on Ion
Proton NGS system. The obtained reads were mapped to the human genome
with TopHat and analysed using Cufflinks and Cuffdiff software tools.
Results: The obtained vesicle fractions differed in their size, CD9 and AGO2
expression and RNA size distribution. Small RNA analysis revealed that
exosomes were enriched in RNAs corresponding by size to miRNAs whereas
MVs were enriched in RNAs ranging from 80 to 100 nt. RNA-seq data analysis
is still in progress, however the preliminary results show that mature miRNAs
are the most abundant RNA species in exosomes while MVs mostly contained
fragments of mRNAs and miRNA precursors.
Conclusions: Proportions of RNA species differ substantially in the
extracellular vesicles and in their cells of origin. Furthermore, different subsets
of RNAs are selectively sorted into exosomes and MVs. These findings
may serve as a solid basis for understanding the principles of intercellular
communication by extracellular vesicles.
345 In vivo model of dormancy in ER breast cancer to bone metastasis
S. Gawrzak1 , M. Guiu1 , J. Urosevic1 , E. Fernandez1 , M. Pavlovic1 ,
R.R. Gomis1 . 1 IRB Barcelona-Institute for Research in Biomedicine,
Oncology, Barcelona, Spain
Background: Breast cancer is the most frequently diagnosed cancer and
remains the second leading cause of death among women in Europe and
United States. Majority of cases of patient death are caused by metastatic
relapse. In the ER positive breast cancer, tumors are characterized by the late
appearance of metastasis, years or decades after primary tumor resection.
In this setting metastatic dormancy plays a relevant role. The identification
of mechanisms that allow dormant metastases to become active is essential
for understanding the biology of ER positive breast cancer metastatic latency
and has putative implications for clinical practice. Therefore, we aim to
establish xenograft model of dormancy in ER positive breast cancer to bone
metastasis.
Materials and Methods: ER positive ductal breast adenocarcinoma
(parental) cells labeled with luciferase and GFP were used to create a
derivative population of cells that shows dormant behavior (DBM cells) in
immunodeficient BALB/C nude mice.
Results: Using in vivo selection in mice we isolated the bone metastatic
derivative population from poorly aggressive parental cells. DBM cells survive
in bone as dissaminated tumor cells and after extended periods of time form
micrometastatic lesions which rarely progress into X-ray detectable osteolythic
macrometastasis. This frequent and prolonged bone residency is opposite to
the standard 3 weeks metastatic relapse that has been reported for breast
to bone metastasis models. Moreover, it mimics metastasis latency reported
in patients. Furthermore, single nucleotide polymorphism (SNP) and copy
number variation (CGH) analysis between parental and DBM cell line showed
that no major genetic alterations such as deletions and amplifications as well
as a non-significant number of different SNPs have been gained.
Conclusions: We have identified a fully transformed ER positive breast cancer
cell population that retains dormant properties in the bone upon lodging in the
mouse. This model will be used to study mechanisms of dormant residual
disease in mice xenografts.
347 Fibroblast crosstalk with anti-Her2 therapies breast cancer
resistant clones
P. Fernandez1 , G. Fuster2 , M. Mancino2 , A. Zubeldia2 , E. Ametller2 ,
E. Enreig2 , P. Gascón2 , H. Slovang3 , H. Russnes3 , V. Almendro4 .
1
Hospital Clinic de Barcelona/Universitat de Barcelona/IDIBAPS, Medical
Oncology/Faculty of Medicine, Barcelona, Spain, 2 Hospital Clinic de
Barcelona/IDIBAPS, Medical Oncology, Barcelona, Spain, 3 Institute for
Cancer Research Oslo University Hospital Radiumhospitalet, Department
of Genetics, Oslo, Norway, 4 Dana Farber Cancer Institute, Department
Oncology, Boston, USA
Background: Mechanisms underlying tumor progression after chemotherapy
are not well understood. Therapeutic treatments can favor the clonal
selection of cells with unique properties and different fitness for a given
microenvironment. Indeed, tumor cells can induce changes in the structure and
composition of the microenvironment to support their growth and spread. Our
aim is to study if clonal selection induced by target therapies like trastuzumab
and lapatinib favors the outgrowth of cells with different microenvironmental
crosstalk capability, and in particular the role of fibroblast in the selection of
these particular clones.
Material and Methods: In order to investigate if clonal selection induced
by target therapies like trastuzumab and lapatinib favors the outgrowth of
cells with different capability of microenvironmental crosstalk, we developed
different cell lines resistant to these drugs from the parental SKBR3, BT474
and MDA-MB-453 Her2+ cells lines. We determined their molecular profile and
investigated the changes in the expression of selected genes codifying soluble
factors known to induce stromal changes, like chemokines, cytokines, matrix
remodeling-related enzymes, angiogenic and neurogenic factors. To study the
role of fibroblast in the selection of the resistant clones, and the crosstalk
between these two populations, we developed fibroblast immortalized lines
derived from breast cancer tumors and normal breast tissue, and study the
effect of the fibroblasts in resistant induction, as well as the effect of resistant
clones in the fibroblast activation.
Results: We observed that the major changes in the genes investigated were
obtained in the Lapatinib resistant cell lines, suggesting that the acquisition
of Lapatinib resistance can provide the tumor with a higher capability of
stromal interaction. In vivo, the resistant cell lines showed an invasive growth
pattern and higher angiogenesis compared to the parental cell lines. We
observed a different pattern of fibroblast distribution in the tumors derived
from the resistant cell lines, which display a more infiltrative distribution.
The resistant tumors were also more fibroblast-enriched suggesting a higher
microenvironment crosstalk capacity. In vitro, we found that the supernatant
of breast cancer associated fibroblast was able to induce an increase in the
resistant phenotype of parental lines, and that the resistant cell lines were able
to induce a slightly activation of fibroblasts.
Conclusion: These results highlight the importance of microenvironment in
supporting tumor progression after chemotherapy.
348 BCAT1 promotes cell proliferation through amino acid catabolism
in gliomas carrying wild-type IDH1
B. Radlwimmer1 , M. Tönjes1 , S. Barbus1 , Y.J. Park2 , W. Wang1 ,
I. Weibrecht1 , S.M. Hutson3 , C. Plass2 , G. Reifenberger4 , P. Lichter1 .
1
Deutsches Krebsforschungszentrum, Molecular Genetics, Heidelberg,
Germany, 2 Deutsches Krebsforschungszentrum, Epigenomics and Cancer
Risk Factors, Heidelberg, Germany, 3 Virginia Tech, Human Nutrition Foods &
Exercise, Blacksburg VA, USA, 4 Heinrich Heine University, Neuropathology,
Düsseldorf, Germany
Background: The aggressive clinical phenotype of malignant gliomas, in
particular glioblastoma, is closely linked to characteristic adaptations of cellular
metabolism. Here we analyzed the role of branched chain amino acids
metabolism in sustaining glioblastoma growth.
Material and Methods: BCAT1 protein and RNA expression was analyzed
using various methods in a large number of glioma samples. MassArray
analysis was used to characterize BCAT1-promoter methylation and enzyme
activity assays were used to evaluate inhibition by the oncometabolite
2-hydroxyglutarate. Effects of BCAT1 inhibition by either inhibitor treatment
or stable lentiviral shRNA-knockdown on cellular phenotype were analyzed by
MS-MS, cell proliferation, apoptosis, migration and xenograft assays.
Summary of the results of the research: We show that glioblastoma
express high levels of branched-chain amino acid transaminase 1 (BCAT1), the
enzyme that initiates the catabolism of branched-chain amino acids (BCAAs).
Expression of BCAT1 was exclusive to tumors carrying wild-type isocitrate
dehydrogenase 1 (IDH1) and IDH2 genes and was highly correlated with
methylation patterns in the BCAT1 promoter region. BCAT1 expression was
dependent on the concentration of a-ketoglutarate substrate in glioma cell
lines and could be suppressed by ectopic overexpression of mutant IDH1
in immortalized human astrocytes, providing a link between IDH1 function
and BCAT1 expression. Suppression of BCAT1 in glioma cell lines blocked
the excretion of glutamate and led to reduced proliferation and invasiveness
in vitro, as well as significant decreases in tumor growth in a glioblastoma
xenograft model.
Conclusions: These findings suggest a central role for BCAT1 in glioma
pathogenesis, making BCAT1 and BCAA metabolism attractive targets for
the development of targeted therapeutic approaches to treat patients with
glioblastoma.
349 Potential interconnection between alternative splicing and
microRNA regulation of SRSF1 oncogene in renal cancer
J. Boguslawska1 , E. Sokol1 , H. Kedzierska1 , B. Rybicka1 , P. Poplawski1 ,
Z. Tanski2 , A. Nauman1 , A. Piekielko-Witkowska1 . 1 The Centre of
Postgraduate Medical Education, Biochemistry and Molecular Biology,
Warsaw, Poland, 2 Regional Hospital, Ostroleka, Poland
Introduction: SRSF1 (ASF/SF2) is SR-family splicing factor involved in
constitutive and alternative splicing reactions. This protein was revealed
to possess oncogenic properties. SRSF1 transcript undergoes alternative
splicing, resulting in two splice variants: var 1 (NM_001078166) and var 2
(NM_006924), that differ in 3 UTR length. Our previous studies showed
disturbances in expression of SRSF1 in clear cell renal cell carcinoma
(ccRCC), which is the most common type of renal cancer. ccRCC is
characterized by high mortality and frequent metastasis at the early stage
of the disease. Disturbed expression of SRSF1 in ccRCC may be caused by
different factors, one of them can be microRNAs (miRNAs). These short, noncoding RNA molecules regulate gene expression via binding to 3 UTRs of
mRNAs, leading to inhibition of translation or degradation of transcript. In this
work we hypothesize that SRSF1 expression may be regulated by miRNAs in
ccRCC.
Material and Methods: We designed primers detecting SRSF1 ORF (open
reading frame), as well as 3 UTRs variants var2 and var1+var2 (it was not
possible to design primers detecting sole var1) and measured their expression
using Real-Time PCR in 34 pairs of ccRCC and control samples. Using
Pick & Mix microRNA PCR Panels (Exiqon) we analysed expression of 10
miRNA molecules potentially binding to 3 UTRs of SRSF1. References genes
were analysed using NormFinder, whereas statistical analysis using GraphPad
Prism.
Results and Discussion: We observed statistically significant decrease in
SRSF1 ORF as well as var2 and var1+var2 in tumours. The expression of
ORF was lower than the expression of var2, and var1+var2 both in control and
tumour tissues. This suggests the presence of other SRSF1 transcripts, not
revealed by our primers detecting ORF. The expression of var2 was similar
S83
to the expression of var1+var2, suggesting that most of SRSF1 3 UTR is
expressed as a second, longer variant 2. Correlation matrix demonstrated that
var2 expression did not correlate with the expression of miRNAs potentially
regulating SRSF1 in control samples but negatively correlated with the
expression of miR-200c-3p, miR-203a, and miR-206 in tumours samples.
Additionally, we did not observe any correlation between the level of var1+var2
3 UTRs in controls, but in tumours this variants correlated negatively with the
expression of miR-206.
Conclusions: miR-206 is a potential regulator of SRSF1 expression in ccRCC.
This regulation seems to depend on alternative splicing of SRSF1 3 UTR.
Functional studies are needed to confirm interactions between miR-206 and
SRSF1 transcript. To our knowledge this is the first study describing the role
of miRNAs in the regulation of splicing factors in ccRCC.
This project is supported by the programme of Polish Science Foundation:
PARENT/BRIDGE co-financed from European Union funds under Innovative
Economy Operational Programme 2007–2013.
350 Recapitulation of non-sm

the Proceedings Book

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