SOX9 - Transcriptomics

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SLUG and SOX9 Cooperatively
Regulate Tumor Initiating Niche
Factors in Breast Cancer
Babak Behnam, MD, PhD
Department of Genetics and Molecular Biology,
Iran University of Medical Sciences
behnam.b@iums.ac.ir
3
Introduction
• Metastasis is the major cause of cancer mortality
• Metastasis is a multistage process
• Successful initiation of metastatic growth, ‘metastatic
colonization’, accomplished only by a minority of cancer
cells that reach distant sites
4
Gupta & Massague 2006, and Valastyan & Weinberg 2011
Introduction
• A small population of cancer stem cells is critical for
metastatic colonization
• 1889 Stephan Paget’s “Seed and Soil” hypothesis,
today’s “Metastatic Tropism”
- Seed: Cancer Stem Cells (CSCs)
- Soil: Metastatic niche
5
Valastyan & Weinberg 2011, and Fidler I.J. 2003
Cancer Stem Cells
• CSCs: Minority proportion of tumor mass
•
•
•
•
•
•
Self-renewal
Tumorigenicity
Multilineage differentiation
Migration
Invasiveness
Apoptosis resistance
6
Jordan C.T. et al. 2006
CSCs and Metastatic Colonization
7
Fazilaty, H. et al., Tumor Biol 2013
EMT, CSCs and Metastasis
• Epithelial to Mesenchymal Transition
• Embryonic morphogenesis
• In adult:
• Wound healing
• Most pivotal escape mechanism of tumor cells
•
•
•
•
Migration
Invasiveness
Apoptosis resistance
Expression of Extracellular matrix (ECM) components
8
Thiery J.P. et al. 2009
EMT Inducers
• EMT transcription factors (EMT-TFs)
• SNAIL, SLUG, TWIST1, TWIST2, ZEB-1, ZEB-2, ….
• Signaling pathways
• Transforming growth factor-b (TGF-b)
• WNT/b-catenin
• …..
9
Craene BD & Berx G 2013
SLUG and SOX9 Master TFs
10
SLUG and SOX9 Master TFs
• Actions of key transcriptional regulators suffice to convert
differentiated epithelial cells into long-term repopulating
epithelial stem cells (SCs)
• Autoregulatory program activated by Slug and Sox9 is
involved in inducing and sustaining the SC state
• Adult SCs/CSCs, similar to ES cells, maintain their SC state
via master regulator-mediated autoregulatory networks
11
Guo et al., Cell 2012
Survival Rate
Patients with primary tumors
expressing high levels of both
SLUG and SOX9 had a
significantly lower overall
survival rate than the rest of
patients
12
Guo et al., Cell 2012
Metastatic Niche
• Fertile microenvironment
• Supports stem cell maintenance and manages cell
function and proliferation
• Cells, vascular networks, soluble factors, nutrients,
metabolic components and extracellular matrix (ECM)
• VEGF, MMPs, LOX
13
Psaila B. & Lyden D. 2009, and Hanahan D. & Weinberg R.A. 2011
ECM and Metastasis
• Regulatory signals
• Sophisticated organization
• POSTN and TNC, as metastatic niche components for CSCs
14
Malanchi I. et al. 2012, Oskarsson T. et al. 2011
Periostin (POSTN)
15
Tenascin C (TNC )
16
Link Between CSCs and Metastatic Niche
Several leading edge studies confirm a connection
between CSCs and metastatic niche
Hypoxia inducible factors (HIFs)
Transforming growth factor (TGF)-b
17
18
19
Fazilaty, H. et al., Tumor Biol 2013
20
Neovascular tips comprise micrometastatic
niches enriched for POSTN and TGF-b1
21
Ghajar et al. Nature Cell Biology 2013
22
23
Fazilaty and Behnam, Cell Biology International 2014
Hypothesis
Overexpression of SLUG and SOX9
transcription factors increases the
expression of POSTN and TNC genes
24
Methodology
• Cell culture
• MDA-MB-231 cell line
• MCF7 cell line
• Lentiviral vectors and transduction containing SLUG and
SOX9 genes & their knock down
• Quantitative Real-time PCR
• Check the expression of SLUG, SOX9, TNC and POSTN before
and after transduction and knock down
25
Lentiviral constructs
26
Lentiviral constructs
pWPXL-SOX9
27
Results
28
Overexpression of SLUG and SOX9
upregulates POSTN and TNC
29
MCF7 versus MDA-MB-231
MDA231 vs MCF7
100000
Relative Expression
80000
MCF-7
MDA-MB-231
60000
40000
20000
3
2
1
0
SLUG
SOX9
POSTN
30
TNC
Quantitative gene expression analysis
MCF7
SLUG
SOX9
POSTN
TNC
***
300000
Relative Expression
200000
100000
***
***
**
600
***
400
**
200
**
**
6
4
2
0
empty
MCF7-SLUG
P-Value 0.0001 = ***, P-Value 0.001 = **, P-Value 0.01 = *
MCF7-SOX9
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MCF7-SLUG-SOX9
Knock-down of SLUG and SOX9
downrgulates POSTN and TNC
32
Sh-RNA Construct
33
Quantitative gene expression analysis
MDA231
SLUG
SOX9
POSTN
TNC
Relative Expression
1.5
1.0
**
0.5
**
**
**
***
** *** ***
***
0.0
empty
MDA231-Sh-SLUG
34
MDA231-Sh-SOX9
***
*** ***
MDA231-Sh-SLUG-SOX9
Discussion
35
Coexpression of SLUG-SOX9 and POSTN
• SLUG and SOX9 expression may have synergistic effects
• SLUG and SOX9 coexpression is required for POSTN and
TNC upmost expression
• POSTN is likely one of the most important factors
regulated by SLUG and SOX9
36
Acknowledgment
• Hassan Fazilaty, PhD student
• IUMS Research deputy; Grant No. 17856
37
References:
1.
2.
3.
4.
5.
Gupta GP and Massague´ J. Cancer metastasis: building a framework. Cell 2006; 127(4):679-95.
Valastyan S and Weinberg RA. Tumor Metastasis: Molecular Insights and Evolving Paradigms. Cell 2011; 147(2):275-92.
Fidler I J. The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited. Nature Reviews Cancer2003; 3(6):453-8.
Jordan CT, Guzman ML, and Noble M. Cancer Stem Cells. N Engl J Med 2006; 355(12):1253-61.
Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009 Nov 25;139(5):87190.
6. Craene BD and Berx G. Regulatory networks defining EMT during cancer initiation and progression. Nat Rev Cancer. 2012 Dec 21;13(2):97110.
7. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts
by Defined Factors. Cell 2007; 131(5):861-72.
8. Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, et al. Twist, a Master Regulator of Morphogenesis, Plays an Essential
Role in Tumor Metastasis. Cell 2004; 117(7):927-39.
9. Lu X, Kang Y. Hypoxia apnd Hypoxia-Inducible Factors: Master Regulators of Metastasis. Clin Cancer Res. 2010;16(24):5928-35
10. Nieto MA The snail superfamily of zinc-finger transcription factors. Nat Rev Mol Cell Biol. 2002;3(3):155-66.
11. Guo W, Keckesova Z, Donaher JL, Shibue T, Tischler V, Reinhardt F, et al. Slug and Sox9 Cooperatively Determine the Mammary Stem Cell
State. Cell 2012; 148(5):1015-28.
12. Psaila B, and Lyden D. The metastatic niche: adapting the foreign soil. Nat Rev Cancer 2009; 9(4):285-93.
13. Hanahan D, and Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell 2011; 144(5):646-74.
14. Malanchi I, Santamaria-Martı´nez A, Susanto E, Peng H, Lehr HA, et al. Interactions between cancer stem cells and their niche govern
metastatic colonization. Nature 2012; 481: 85–89.
15. Oskarsson T, Acharyya S, Zhang XH, Vanharanta S, Tavazoie SF, Morris PG, et al. Breast cancer cells produce tenascin C as a metastatic
niche component to colonize the lungs. Nat Med 2011; 17(7):867-74.
16. Oskarsson T and Massague J. Extracellular matrix players in metastatic niches. EMBO J 2012; 31(2):254-6.
17. Serrano I, McDonald PC, Lock FE, Dedhar S. Role of the integrin-linked kinase (ILK)/Rictor complex in TGFβ-1-induced epithelialmesenchymal transition (EMT). Oncogene. 2013 Jan 3;32(1):50-60.
18. Ghajar CM, et al. (2013) The perivascular niche regulates breast tumour dormancy. Nat Cell Biol 15: 807–17.
19. Fazilaty H, Gardaneh M, Bahrami T, Salmaninejad A, Behnam B (2013) Crosstalk between breast cancer stem cells and metastatic
niche: emerging molecular metastasis pathway? Tumour biology: the journal of the International Society for. Oncodev Biol Med
34: 2019–30.
20. Fazilaty, H., and Behnam, B. (2014). The perivascular niche governs
an autoregulatory network to support breast cancer
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metastasis. Cell biology international 38, 691-694.
Thanks for your attention
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