Physiological and pathological properties of EMTassociated

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1 Physiological and pathological properties of EMTassociated plasticity Stéphane Ansieau Inserm UMR 1050, CNRS UMR 8256 Centre de Recherche en Cancérologie de Lyon, France

2 EMT: a reversible phenotypic and functional switch EMT Primary epithelium Loss of cell polarity Loss of cell to cell contact Cytoskeleton reorganization Disruption of the basal membrane Gain in motility / invasion Resistance to anoikis Phenotypic switch Functional consequences E Cell plasticity M External signals MET EMT is a transient, reversible, and highly regulated transdifferentiation process Secondary epithelium The phenotypic switch is associated with a profound genetic reprogramming

3 EMT: a prerequisite for both morphogenesis and organogenesis during the embryonic development First wave Second waves Cell plasticity Third waves Thiery JP et al., Cell 2009

4 EMT and wound-healing Arnoux V. et al. Rise and fall of Epithelial Phenotype: Concepts of Epithelial-Mesenchymal Transition edited by P. Savagner Eurekah. Com and Kluwer Academic / Plenum Publishers

5 Pathological EMT: the metastatic cascade initiation Polyak K & RA Weinberg, Nature Reviews 2009

6 EMT, MET & the metastatic cascade Primary tumor Secondary tumor 1 5 EMT 3 MET 2 EMT 4 EMT 6 Differentiated tumor cell Cancer cell in EMT Stromal cell Immune cell 1. Dissemination 2. Intravasation 3. Migration in the flux 4. Extravasation 5. Colonisation

7 Schematic representation of the EMT process Wnt, TGFβ, Hdg, Notch mechanical constraint, hypoxia Zeb Snail Twist Epithelial markers (E-cadherin) Attached cells Non-motile Proliferative Mesenchymal markers (N-cadherin /vimentin) Individual cells Motile and invasive (MMP) Poorly proliferative

8 A complex mirna-emt-tf interactome TGFβ Mir-200 ZEB1 Targets Diaz-Lopez A. et al. Cancer Manadgment & Res. 2014

9 EMT is regulated by four interconnected regulatory networks De Craene B. & G. Berx, Nature Reviews Cancer, 2013

10 Observation of EMT figures EMT-TF & permissive environment Protein expression analysis E M Epithelial markers E-cadherin vimentin Mesenchymal markers Acquisition of migratory properties Wound healing (scratch) assay

11 Acquisition of migratory and invasive properties (Boyden chamber) EMT features Crystal violet Chick Chorioallantoic Membrane (CAM) Assay Bio-luminescence xenografted or injected in the caudal vein

Experimental demonstration of the biological

cells Cytokeratin + Fibronectin- T T: tumor ST:

12 Experimental demonstration of the biological relevance of EMT in vivo LacZ Myc WAP Cre Mammary epithelial cells Experimental control Lox-STOP-Lox LacZ LacZ ST LacZ+ epithelial cells Cytokeratin + Fibronectin- T T: tumor ST: stroma Breast carcinoma Trimboli AJ et al. Cancer Res. 2008

Twist1 (Topical Dox) EMT transiently induced (Oral Dox) Primary tumor volume control topical

13 Reversion to an epithelial phenotype is a prerequisite for the second site colonization Healthy keratenocytes Non invasive carcinoma Invasive carcinoma K5 rtta Twist1 DMBA +TPA Twist1 + Dox Twist1 (Topical Dox) EMT transiently induced (Oral Dox) Primary tumor volume control topical oral Secondary tumors Constitutive EMT Tsai JH et al., Cancer Cell 2012 temps O T O T Control Twist1

14 Schematic representation of the EMT process Wnt, TGFβ, Hdg, Notch mechanical constraint, hypoxia Zeb Snail Twist Self-renewal potential Secondary tumor formation Cancer stem cell properties Tumorigenic potential Differential potential Intratumoral heterogeneity

KRASG12D; TWIST1 MaSC Bipotent progenitor Mesenchymal Claudin-low Basal Progenitors Differentiated cells

15 EMT associates with a gain in stem-like cell properties epithelial cells EMT (even partial) EMT-TF + Oncogene + EMT permissive environment Tumorigenic potential Stemness properties Tumor subtype WAP: KRASG12D; TWIST1 MaSC Bipotent progenitor Mesenchymal Claudin-low Basal Progenitors Differentiated cells Luminal Luminal Myoepithelial Luminal Mani et al., Cell 2008, Morel et al. PLos ONE 2008; Morel et al., PLoS Genet. 2012

Competitive mammary fad pat reconstitution

progenitors) dsred dsred - SNAIL2 + SNAIL2

tree Luminal differentiated cells - SNAIL2 +

SNAIL2 + SOX9 Cooperation with endogenous

16 EMT associates with a gain in stem-like cell properties Organoid-forming efficiency Competitive mammary fad pat reconstitution assay GFP SNAIL2-GFP MEC (Myoepithelial progenitors) dsred dsred - SNAIL2 + SNAIL2 Rudimentary structures Elaborated ductal tree Luminal differentiated cells - SNAIL2 + SNAIL2 GFP dsred SNAIL2/Sox9-GFP dsred SOX9 SNAIL2 + SOX9 Cooperation with endogenous stemness program Organoid-forming efficiency Guo W. et al. Cell 2012

based Therapy) Mesenchymal Intermediate Epithelial chemoresistance chemoresistance Tan et al.

17 EMT and chemoresistance Genotoxic or cytotoxic stress Definition of an universal EMT signature Epithelial Mesenchymal M Breast (doxorubicin + cyclophosmamide) Ovary (Platinium based Therapy) Mesenchymal Intermediate Epithelial chemoresistance chemoresistance Tan et al., EMBO Mol Med 2014 CR: complete response; PR partial response; SD stable disease; PD progressive disease

18 Gain in migratory and invasive properties Cell commitment to EMT Gain in stemness Gain in chemoresitance As easy as that?...

Complete EMT is dispensable for cancer cell dissemination Breast carcinomagenesis model

(independently of their outcome) Epithelial (RFP,Red), mesenchymal (GFP,Green) No epithelial

Tumor-derived RFP cells Orthotopic tumors RFP+-cells Primary tumor Metastasis These few GFP

19 Complete EMT is dispensable for cancer cell dissemination Breast carcinomagenesis model (MMTV-PyMT) with lung metastasis Tracing experiments to follow fully EMT-committed cells (independently of their outcome) Epithelial (RFP,Red), mesenchymal (GFP,Green) No epithelial GFP+ cells in primary and secondary tumors (Same results with MMTv-Neu and vimentin-cre-ert2) Tumor-derived RFP cells Orthotopic tumors RFP+-cells Primary tumor Metastasis These few GFP cells do not contribute to lung metastasis (not detectable in lung) Few positive GFP cells No positive GFP cells

Removal of the primary tumor Tumor-derived RFP+ cells CTX: cyclophosmamide EMT

Nature 2015 Lineage tracing to mark carcinoma cells need to fill two criteria: -

activation - When activated the Cre/Cre-ER protein needs to activate the GFP reporter in

20 Removal of the primary tumor Tumor-derived RFP+ cells CTX: cyclophosmamide EMT associates with chemoresistance Enrichment in EMT cells Fischer KR et al. Nature 2015 Lineage tracing to mark carcinoma cells need to fill two criteria: - Cre/Cre-ER driver needs to be expressed in all cells that transiently undergo EMT activation - When activated the Cre/Cre-ER protein needs to activate the GFP reporter in all cells where Cre/Cre-ER cells are expressed. Fsp1 Snail1-YFP; MMTV-PyMT Vim Fsp1 Vim Snail1 + Zeb1 + Ye X et al. Nature 2017

21 Interfering with EMT plasticity pertubs its contribution to metastasis? Murine pancreatic carcinomagenesis model (activated KRAS and loss of p53 function): KPC Compare KPC with KPC Twist1 KO or with Twist1 Snail1 KO Carcinoma cell tracing with YFP. In absence of Twist/Snail, reduction of YFP+/α-SMA+ cells Similar primary tumor growth Similar local invasion Similar number of DTCs Similar metastatic potential Zheng et al. Nature 2015

22 Inhibition of EMT sensitizes KPC tumors to gentamycin Treatment with gentamycin Zheng et al. Nature α-sma is not a good EMT marker -Reduction of snail2/zeb1 following Twist/Snail depletion is very partial. Aiello NM et al. Nature Difficulty to trace EMT-committed cells: no universal marker - Not a single EMT but EMTs - A highly dynamic process, needs more flexible tools

23 Direct evidence of EMT obtained in unpertubed breast tumors All cancer cells are YFP+ Epithelial cells are blue Ecad-mCFP high (Epi) Ecad-mCFP low (partial EMT) Ecad-mGFP low Specific signature Primary tumors Ecad-mGFP low cancer cells are similar to human mesenchymal CTCs

(Epi)/ orthotopic injection Multi-photon

24 Direct evidence of EMT obtained in unpertubed breast tumors Ecad-mCFP high (Epi)/ orthotopic injection Multi-photon microscopy YFP CFP Nonmigratory field YFP No difference in their ability to extravasate Shift back to an epithelial phenotype CFP Migratory field Similar self-renewal potential Finger-like projection Beerling et al., Cell Reports 2016

Cells partially committed to EMT display CSC properties E or

(E) Mesenchymal Cells (M) Mammospheres Single cell RNAseq

Mesenchymal signature EM signature Breast cancers

25 Cells partially committed to EMT display CSC properties E or M cells Adherent Mammospheres HMEC SV40/RAS Epithelial cells (E) Mesenchymal Cells (M) Mammospheres Single cell RNAseq Shift to an EM gene expression profile Epithelial signature Mesenchymal signature EM signature Breast cancers Coexpression of M and E genes predict poor outcomes Grosse-Wilde A et al., 2015

26 Integrin-β4 segregates partial to fully EMT-committed cells Fully committed to EMT Partially committed to EMT ZEB1 Bierie A et al., 2017

27 Conclusions EMT is a dynamic continuum between an epithelial and a mesenchymal phenotype The partial EMT-committed cells contribute to metastasis EMT-inducers are transiently induced and need to be studied as such. Permanently expressing or deleting them aberrantly fix the epithelial or mesenchymal phenotype.

28 Partial EMT as a driver of metastasis Epithelial cells Partially committed cells Fully committed cells Epithelial shape Epithelial shape Mesenchymal shape Associates with a collective migration rather than single cells (collective migration frequently observed in patients). A metastable phenotype? CTC clusters intra- and extravasate more efficiently than fully committed EMT CTCs and form 50x time more tumors. Coexpression of epithelial and mesenchymal markers is an hallmark of aggressive tumors. mir200 ZEB Higher tumor-initiating and stem-like properties than fully committed EMT cells. Phenotypic Stability Factors (OVOL, GRHL2) Watanabe K et al., Dev. Cell 2014; Jolly MK et al., Oncotarget 2016

29 Partial EMT reflects an underestimated level of regulation Nieto MA et al., Cell 2016

SNAIL2 + Differentiation Tumor per mouse WT +/- -/- Twist1 MITF rheostat - ZEB1, TWIST1 Survival Proliferation RAS + p53 KO

30 EMT-TFs but not only. Failsafe program inhibition (RB and p53 dependent) Modulation of differentiation programs Modification of the cell metabolism ZEB2, SNAIL2 + Differentiation Tumor per mouse WT +/- -/- Twist1 MITF rheostat - ZEB1, TWIST1 Survival Proliferation RAS + p53 KO Dose-dependent activity TSG/oncogenic properties (cell context dependent) Glycolyse induced (increased glucose uptake & macromolecule biosynthesis) Beck B. et al, Cell Stem Cell 2015;Caramel J.. et al. Cancer Cell 2013;Dong C et al. Cancer Cell 2013

31 Concomitant fail-safe program escape and EMT induction ErbB2 Twist1 ErbB2 + Twist1 Sénescence prématurée Cellules épithéliales Cellules mésenchymateuses Ansieau S et al., Cancer Cell 2008

(MET) Dormancy Secondary tumors Schardt JA et al.

32 Early metastatic dissemination MMTV-ErbB2 Premalignant lesions edccs Malignant progression Her2 +, Twist1 +, Ecad low, DCCs (30% Twist1 + ) (MET) Dormancy Secondary tumors Schardt JA et al. Cancer Cell 2004; Hüsemann Y et al. Cancer Cell 2008, Harper et al., Nature 2016; Hosseini et al., 2016

Cell Polarity and Cancer

Cell Polarity and Cancer Pr Jean-Paul Borg Email: jean-paul.borg@inserm.fr Features of malignant cells Steps in Malignant Progression Cell polarity, cell adhesion, morphogenesis and tumorigenesis pathways