Concomitant Gain of Function of Notch and Loss of p53 Signalling trigger an EMT Like Programme Driving Metastatic Intestinal Cancers

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8 AVRIl 2015 8 Avril 2015 Concomitant Gain

Daniel LOUVARD UMR 144 / CNRS - Institut

1 8 AVRIl Avril 2015 Concomitant Gain of Function of Notch and Loss of p53 Signalling trigger an EMT Like Programme Driving Metastatic Intestinal Cancers Daniel LOUVARD UMR 144 / CNRS - Institut Curie Laboratoire de Morphogénèse et Signalisation Cellulaires

Notch signaling in intestinal Cancers Somatic

Src? p53 other alterations cmet, Src, Fascin?

early adenoma intermediate adenoma late adenoma

mutation unlike other tumors Images from Dr. C.

3 Notch signaling in intestinal Cancers Somatic mutation APC, b-catenin ADN methylation K-ras DCC Src? p53 other alterations cmet, Src, Fascin? normal epithelium Hyperproliferative epithelium early adenoma intermediate adenoma late adenoma carcinoma metastasis APC Notch activation but no mutation unlike other tumors Images from Dr. C. Rosty, Institut Curie modified from Fearon and Vogelstein, 1990

4 A simplified view of the NOTCH signaling pathway Delta Jagged NOTCH RBP-Jk HES, HERT differentiation g-secretase proliferation apoptosis

Notch signaling in normal gut development Constitutive Activation of Notch, Nic (gain of fonction) Inhibition of Notch (loss of fonction) increased proliferation severe reduction of all three

5 Notch signaling in normal gut development Constitutive Activation of Notch, Nic (gain of fonction) Inhibition of Notch (loss of fonction) increased proliferation severe reduction of all three secretory types no proliferation the intestinal epithelium is almost exclusively composed of goblet cells Differentiation Gain of function Nic Loss of function Notch Fre et al, Nature, 2005 Proliferation Van Es et al, Nature, 2005

6 Construction of mouse models of intestinal cancers Inducible and tissue specific expression of transgene(s) in the proliferative compartment of the adult intestinal mucosa with the Villin gene promoter

Bundles, nucleates, caps and severs actin microfilaments Remains expressed during

7 Villin, an actin bundler in intestinal microvilli Villin Restricted cellular expression: Intestines, kidney proximal tubule Concentrated in the brush border Bundles, nucleates, caps and severs actin microfilaments Remains expressed during intestinal carcinogenesis Genomic structure and gene regulatory sequences identified 100µm

8 Inducible transgene expression in intestinal stem cells pvill/creer T2 /Rosa26 + TAM In absence of Tam Tam j + 5 Tam j + 60 pvill Cre X b-gal OFF Tamoxifen Cre pvill Cre b-gal ON b-gal Targeting to the stem cell compartment: b-gal staining maintained longer than 12 months

9 Crosstalks between major signaling pathways in CCR Notch Wnt Ras Nic/Apc +/1638N Apc +/1638N /K-ras V12G Fre et al. PNAS, 2009 Janssen et al Gastroenterology, 2006 These 2 models lead to tumor initiation but incomplete tumor progression, in particular no metastasis to distant organs

10 Can we built a mouse model of intestinal cancer with invasive properties? M.Chanrion et al Nature Comm We hypothesized that EMT is a prerequisite to initiate the onset of metastasis 2-We develop a system biology approach to reconstruct signalling networks allowing to predict the onset of an EMT-like phenotype. In collaboration with the SysBio team. Department of Bioinformatic, Institut Curie

Epithelial Mesenchymal Transition EMT is a process during development by which polarized epithelial cell acquire mesenchymal cell phenotype : enhanced migratory capacity, invasiveness, elevated

11 Epithelial Mesenchymal Transition EMT is a process during development by which polarized epithelial cell acquire mesenchymal cell phenotype : enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, increased production of ECM components EMT is involved in normal embryological processes: Gastrulation Neural crest formation Heart morphogenesis EMT-like is induced in adult pathological processes: Fibrosis Inflammation Cancer Kalluri et al., 2009

12 Building an Atlas of Cancer signaling networks D.Hannahan,R.Weinberg Assemble Formalize Visualize Exchange Analyze

13 NOTCH-p53-Wnt interactome etwork Notch-p53-Wnt map : 10 mirnas 13 phenotypes 77 RNAs 86 genes 122 proteins 397 reactions 406 species 135curated references

14 Main predictions from the System Biology analysis: In Notch activated mice, * activation of EMT-like program is prevented by p53 In p53 null mice, * activation of EMT-like program is partially prevented by p63. In Notch/p53 mice * activation of an EMT-like program is on EMT program is activated only when the 5 key EMT inducers (Snail1, Slug, Zeb1, Zeb2, and Twist1) are turned ON p53 loss of function and Notch gain of function have a synergistic effect on the onset of an EMT-like phenotype EMT inducers stimulate Wnt pathway through b-catenin activation

16 ?

17 Crosstalks between major signaling pathways in CCR Notch Wnt Ras Nic/Apc +/1638N NO Metastasis K-ras V12G /Apc +/1638N Fre et al. PNAS, 2009 Janssen et al Gastroenterology, 2006 Adenoma p53 p53 Invasion Adenocarcinoma Nic/p53 Metastasis

18 Construction of the inducible Nic/p53 mouse model pvill/creer T2 / Nic/ p53 -/- loxp pvill Cre + Tamoxifen loxp p53- IRES NOTCH p53 -/- NIC GFP Does it lead to an invasive phenotype : onset of EMT-like? El Marjou et al., 2004 Murtaugh et al., 2003 Jonkers et al., 2001 Chanrion et al 2014

19 Survival Curves 1 survival (%) 0,8 0,6 0,4 0,2 Np53 P53 N Time after Tamoxifen induction (month)

% animals w/tumors Tumor Intake 120 Tumor intake after Tam injection Np53 p53 N 100 80 60

20 % animals w/tumors Tumor Intake 120 Tumor intake after Tam injection Np53 p53 N m 5-7m 7-9m 9-11m 11-13m 13-15m 15-18m + de 18m Time after Tam. injection (month)

21 Primary Tumors in the N/p53 -/- Mucosa Muscularis Mucosa Submucosa Muscularis Serosa

22 Ex Vivo analysis using two photons microscopy on slices of live tumor samples

23 H&E and GFP stainings on N/p53-/- tumors Invasive ADK 96% Lymph Node invasion 23% Liver metastasis 10% Peritoneum met. 50% Nuclear GFP staining allows tracking of epithelial tumor cell dissemination

24 Alteration of the Wnt pathway in N/p53 -/- Normal Gut Invasive ADK Lymph Node Liver Peritoneum Nuclear beta catenin staining

25 E-CAD Pan CK Vimentin SMA SNAIL SLUG TWIST ZEB1

26 Evidence for an EMT-like phenotype in desmoplasic tumor N/p53-/- ZEB1-GFP-ECAD--DAPI

27 Stroma Desmoplasic area Bulk Phenotypic changes induced in N/p53-/- primary tumors GFP DAPI ECAD ZEB Epithelial cell EMT-1 ph EMT-2 ph Mesenchymal cell

29 Characterization of Nic/p53 mouse model Induction of the EMT-like process in the NN;p53 Relevance for Human CCR

30 Notch activation and Zeb1 expression in the desmosplastic area of invasive human colon ADK Normal Bulk Desmoplastic area Nicd Zeb1

32 The activity scores computed for the Notch, p53 and Wnt pathways in human colon cancer samples from Tumour Cancer Genome Atlas data set.

33 Concluding remarks

SysBio team Emmanuel Barillot, Andrei Zynovyev Inna Kuperstein David Cohen Loredana Martignetti Curie Hospitals Daniel Louvard team Sylvie Robine Maia Chanrion Fatima El Marjou Cédric Barrière Lev

34 SysBio team Emmanuel Barillot, Andrei Zynovyev Inna Kuperstein David Cohen Loredana Martignetti Curie Hospitals Daniel Louvard team Sylvie Robine Maia Chanrion Fatima El Marjou Cédric Barrière Lev Stimmer Jeanne Netter Silvina Dos Reis Tavares Silvia Fre Mathilde Huyghe Danijela Vignjevic Wulfran Cacheux Didier Meseure (Institut Curie-St Cloud) Yvan Bieche (Institut Curie-St Cloud) Animal Facility Virginie Dangles-Marie Stéphanie Boissel Isabelle Grandjean Imaging Facility Olivier Renaud Olivier Leroy Tristan Piolot Lucie Sengmanivong

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