Id1 - 11 Simposio Oncología Traslacional

Transcription

Prognostic, predictive and prometastatic implication of
Id genes in cancer:
The importance of tumor microenvironment
Dr. Ignacio Gil-Bazo, MD, PhD
Departamento de Oncología
Id proteins inhibit bHLH transcriptional factors
Helix-Loop-Helix
b
bHLH
Id bHLH
(a) Id proteins present
Ubiquito
us
E
proteins
Id
CANNTG
E-box
(b) Id proteins absent
Ubiquitous
E proteins
CANNTG
Tissuespecific
bHLH
proteins
No transcription
Tissue-specific
bHLH proteins
Transcription
Perk, Iavarone & Benezra, Nat Rev Cancer, 2005
Id1 (20q11.21)
Catañón and Gil-Bazo, Atlas of Gen and Cytogen in Oncol and Haematol, 2012
Wong et al, Apoptosis, 2004
Wong et al, Apoptosis, 2004
Id1 is expressed during embryogenesis
Id1 or Id3 are required for embryonic viability
E10.5
E11.5
E12.5
E13.5
WT
Id1-/-Id3-/-
Id1 expression (H-Score) during embriogenesis
Organ/Structure
Stage of development
E-12.5
E-13
E-14
E-15
E-16
E-17
E-18
E-19
P-1
Brain
Eye
N.D.
Ear
N.D.
N.D.
N.D.
N.D.
Oropharyngeal cavity
N.D.
Vibrissae
N.D.
Submaxilary gland
N.D.
Heart
N.D.
Lung
Liver
Kidney
N.D.
N.D.
N.D.
Intestine
Pancreas
N.D.
N.D.
N.D.
Skin
Cartilage
Stomach
Nostrils
N.D.
N.D.
N.D.
N.D.
Muscular tissue
Redrado et al. Histology and Histopathology, 2013
N.D.
Vibrissae, mouse embryo E18
Basal strata of skin, mouse embryo E17
Vibrissae, mouse embryo E18
Developing eye, mouse embryo E16
Cartilage, mouse embryo E16
Adult mammary gland
Cartilage, mouse embryo E18
Adult MMTV-Myc mammary tumor
Id1 is specifically expressed in the endothelium of tumor vessels
CD31
Id1
Id1 wild type
Id1
endothelium
Id1 KO
Role of Id proteins in peripheral and bone-marrow-derived endothelial cells
Li et al, Cancer Research, 2004
Li et al, Cancer Research, 2004
Id wild type
De Candia, PNAS 2003
Id deficient
viable
rim of
tumor
cells
cystic
core
Sorafenib inhibits CRAF, VEGFR-2, VEGFR-3, PDGFR-ß, BRAF, V600E BRAF,
KIT and FLT-3
March 15
May 15
Ab-37-2
Ab-195-14 Ab-SC-488
Perk J & Gil-Bazo I et al, Cancer Research 2006
B
TC
Id1, Triple Neg. Mammary Ca. (630X)
D
C
TC
TC
Id1, Metaplastic Mammary Ca. (200X)
Id1, Metaplastic Mammary Ca. (630X)
Perk J & Gil-Bazo I, Cancer Research 2006
B
A V
V
TG
TG
TG
Id1, Prostate Ca. (200X)
Id1, Prostate Ca. (630X)
C
D
D
TC
CIS
U
V
Id1, Bladder CIS (630X)
Id1, Bladder Carcinoma (200X)
Perk J & Gil-Bazo I et al, Cancer Research 2006
Prostate Cancer
and Id1
3% of Low Risk Express Id1
*
39% of High Risk Express Id1
*
Ponz-Sarvisé M, et al. Clinical Genitourinary Cancer, 2014
Prostate Cancer Metastatic Tissue Samples Also Express Id1
Ponz-Sarvisé M, et al. Clinical Genitourinary Cancer, 2014
Can We Target Id1?
F3 peptide
Fluorescein
5’
3’
2’-Deoxy-phosphorothioate DNA
Id1-PCAOs co-localize with Nucleolin
Id1-PCAO-FITC Nucleolin
Id1-AO-FITC
Hoechst 33342
Hoechst 33342
Nucleolin
Henke E et al., Nature Biotechnology 2008
Inhibition of Id1-expression with F3-ASO conjugates
-
100 200 - 50
100 200
Id1-ASO /nM
F3-Id1-ASO /nM
Actin
Id1
Id1-levels in HUVEC after 72 h
treatment with Id1-ASO or F3-Id1-ASO
conjugate.
Standard growth media EGM-2
supplemented with the compounds at the
indicated concentrations.
Media exchange every 24 h.
F3-Id1AS delivered I.V. inhibits Id1 expression in
endothelial cells of transgenic tumors
Id1AS
F3-Id1AS
Treatment with Id1-PCAO inhibits tumor growth
Tumor growth
600
*
500
Id1-PCAO
F3 + Id1-AO
Control
400
300
200
100
Tumor Mass (Day 12)
0
0
1
2
3
*
5
6
7
8
9 10 11 12
Day
*
1400
YDneu Id1-/- allografts in NCR nu mice
5x106 cells, intradermal
Treatment with 10 nmol Id1-PCAO i.v. qed
*
1200
1000
800
600
400
200
F3-conj
F3 + ASO
Control
Treatment with Id1-PCAO leads to
increased hemorrhage
Control
F3 + Id1-AO
Id1-PCAO
**
Combination treatment Id1-PCAO + 17-AAG
Treatment with Id1-PCAO and Metastatic Spread
Lung Cancer is a leading cause of cancer and death
Siegel et al. CA Cancer J Clin, 2014
Ponz et al. Clin Cancer Res 2011
Patients Characteristics
Total n= 347
Age-yr (median-interquartil range)
Sex-n (%)
Male
Female
Stage-n (%)
Stage I
Stage II
Stage III
Stage IV
Histology-n (%)
Adenocarcinoma
Squamous cell carcinoma
Other
Smoking status-n (%)
Current smoker
Former smoker
Non-smoker
Unknown
Cohort A
(n= 101)
68 (60-76)
Cohort B
(n=212)
66 (60-75)
Cohort C
(n=34)
59 (53-63)
85 (84%)
16 (16%)
102 (48%)
110 (52%)
27 (79%)
7(21%)
61 (60%)
22 (22%)
13 (13%)
5 (5%)
141 (67%)
42 (20%)
24 (11%)
5 (2%)
34 (100%)
41 (41%)
53 (53%)
7 (6%)
128 (61%)
79 (38%)
3 (1%)
24 (70%)
6 (18%)
4 (12 %)
59 (58%)
34 (34%)
8 (8%)
0 (0%)
72 (34%)
100 (47%)
40 (19%)
0 (0%)
15 (44%)
12 (35%)
3 (9%)
4 (12%)
NSCLC samples express Id1 compared to non-tumor tissue
Normal
SCC
Adenocarcinoma
High expression
Low expression
NSCLC samples express Id1 compared to non-tumor tissue.
SCC of the lung express higher levels
Normal vs. Tumor tissue
p<0.001
65 patients
with
matched
samples
Adenocarcinoma versus SCC
Cohort
Cohorte A A
Cohort
Cohorte BB
150
250
80
200
p<0.001
100
50
H-score
60
H-score
H-score
p<0.001
40
20
0
SCC
AC
CohorteC
C
Cohort
p=0,03
p=0.03
150
100
50
0
0
SCC
AC
SCC
AC
High Id1 levels correlate with lower DFS and OS in Stage I-III NSCLC patients
Global, DFS
Global, OS
_Low Id1
_Low Id1
--High Id1
--High Id1
Median DFS Low 90 m.
Median DFS High 51 m.
p=0.025
Ponz et al. Clin
Cancer Res 2011
Median OS Low 122 m.
Median OS High 63 m.
p=0.001
Adenocarcinoma, DFS
Adenocarcinoma, OS
_Low Id1
_Low Id1
--High Id1
--High Id1
Median DFS Low 139 m.
Median OS Low NR
Median DFS High 49 m.
Median OS High 67 m.
p=0.008
p=0.003
High Id1 levels correlate with lower DFS and OS in Stage IV NSCLC patients
High Id1 mRNA levels correlate with a shorter OS in stage I-III
AP and IHC features of primary cells
Gon8
Gon8
Gon8
TTF-1 +
CK 7 +
10x
20x
CK 20 -
Mesotelin -
AP and IHC features of primary cells
Mai9
Mai9
Mai9
TTF-1 +
CK 7 +
10x
20x
CK 20 -
Mesotelin -
Our primary cells are resistant to chemotherapy and radiotherapy
A549
125
100
125
100
75
100
75
50
75
50
25
50
25
0
25
0
0
0
1
0
0
1
1
Gon8
A549
Gon8
A549
A549
Gon8
Gon8
125
% de absorbancia
absorbancia
dede
de absorbancia
%%
% absorbancia
125
100
75
50
25
0
0
2
Gy
2
2
4
Mai9
Mai9
A549
A549
0
25
3
3
3
Carboplatin
(µg/mL)
0
1
2
3
Basal Id1 levels and after silencing
Basal Id1 levels
Id1 levels after silencing
Gon8
Mai9
Id1
Id1
GAPDH
GAPDH
72.80
50
50
0
29.77
0
Mock
100
% of Id1
expressi
on
expression
Densitometric
Quantification
% of Id1
100
shId1
Gon8
Mock
Mai9
shId1
Clonogenic assays after Id1 shRNA
Gon8
100
80
60
*
40
% of Colonies
% of Colonies
120
120
100
80
60
20
0
0
shRNA Id1
*
40
20
Mock
Mai9
Ctrl
shRNA Id1
Id1 silencing restores sensitivity
to RT in MTT assays
.
n.s
*
% Absorbance
.
100
***
*
75
*** ***
50
25
0
0
4
8
Gon8
Pup8Ctrl
Ctrl
Gon8
Ctrl
0
4
8
0
Gon8
Pup8shId1
shId1
Gon8
shId1
Gy
4
A549
8
Id1 silencing restores sensitivity to Carboplatin in MTT assays
.
% Absorbance
n.s.
*
100
*
75
***
50
***
***
25
***
0
0
25
200
0
25 200
0
25
200
Mai9 Ctrl Mai9 shId1
A549
Carboplatin (µg/mL)
Id1 and Id3 Co-expression in N2 NSCLC
Castañón et al. Journal of Translational Medicine, 2013
Id1 and Id3 Correlation in N2 NSCLC
Id1
Id3
10
R=0.579, p=0.015
8
20x
20x
Id1
6
4
2
0
20x
20x
0
1
2
3
Id3
4
5
6
Id1 and Id3 Co-expression as a predictor of Overall Survival
The role of Id1 and Id3 genes in the development of liver metastasis from NSCLC
Id1-/-
C57BL/6
N=10
N=10
LLC Go Mai
Id1
8 week-old
females
Gil-Bazo et al. Journal of
Thoracic Oncology, 2013
Weekly micro-PET
Id1 and Id3 expression and lung cancer
metastasis to the liver
C57BL6
Id1-/- Id3+/+
Gil-Bazo et al. Journal of Thoracic Oncology, 2013
Id1 and Id3 expression and lung cancer metastasis to the liver
Id1-/- Id3+/+
C57BL6
FDG microPET: an interesting tool
for in vivo metastasis
surveillance
Id1 absence delays liver
C57 BL6
ID1KO
metastasis formation
Week 1
4
SUV
Week 2
0
Week 3
Gil-Bazo et al. Journal of
Thoracic Oncology, 2013
Id1 absence in tumor microenvironment
delays liver metastasis formation
Liver background SUV
SUV week 1
SUV week 2
SUV week 3
1.035 (0.82-1.24)
1.045 (0.64-1.44)
1.35 (1.3-1.39)
ID1KO
0.7 (0.63-0.77)
0.81 (0.72-0.89)
0.8 (0.67-0.92)
P value
0.001
0.005
0.02
C57 BL6
Id1 absence in tumor microenvironment partially
impairs liver metastasis formation
Liver metastatic rate
Mets. rate
Mets. rate
C57 BL6
70%
100%
ID1KO
10%
20%
P value
0.02
0.015
Id1 absence in tumor microenvironment regulates
gene expression in liver metastasis
-Departament of Oncology
Dr. Mariano Ponz
Dr. José M. López-Picazo
-Departament of Pathology
Dra. Lola Lozano
-Department of Lung Medical Oncology
Dr. Ignacio Wistuba
Dr. Carmen Behrens
-Division of Oncology
Dr. Eduardo Castañón
Dr. Paul Nguewa
Dra. Jaqueline Agorreta
Dra. María J. Pajares
Prof. Luis Montuenga
Inés López
Miriam Redrado
Prof. Rubén Pío
Dr. Alfonso Calvo
-Department of Histology and Cell Biology
Prof. Fernando Vidal-Vanaclocha
e-mail: igbazo@unav.es

Id1 - 11 Simposio Oncología Traslacional

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