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Hybrid Epithelial-Mesenchymal Phenotypes Are Controlled By Author Manuscript Published OnlineFirst on March 26, 2020; DOI: 10.1158/0008-5472.CAN-19-3147 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 2 Hybrid epithelial-mesenchymal phenotypes are controlled by 3 microenvironmental factors 4 5 Gianluca Selvaggio (1, 2) ±, Sara Canato (1, 3, 4) ±, Archana Pawar (1, 5), Pedro T. Monteiro 6 (6, 7), Patrícia S. Guerreiro (1,3, 4), M. Manuela Brás (3, 8, 9), Florence Janody* (1, 3, 4) 7 and Claudine Chaouiya* (1, 10) 8 9 (1) Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, P-2780-156 Oeiras, Portugal 10 (2) Fondazione The Microsoft Research - University of Trento Centre for Computational and 11 Systems Biology (COSBI), Rovereto (TN), Italy 12 (3) i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo 13 Allen, 208, 4200-135 Porto, Portugal. 14 (4) IPATIMUP - Instituto de Patologia e Imunologia Molecular da Universidade do Porto, 15 Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal. 16 (5) Haffkine Institute of Training Research and Testing, Acharya Donde Marg, Mumbai 17 400012, India 18 (6) Department of Computer Science and Engineering, Instituto Superior Técnico (IST), 19 Universidade de Lisboa, Lisbon, Portugal 20 (7) Instituto de Engenharia de Sistemas e Computadores, Investigação e Desenvolvimento 21 (INESC-ID), Lisbon, Portugal 22 (8) INEB – Instituto de Engenharia Biomédica, Universidade do Porto, 4200 – 135 Porto, 23 Portugal 24 (9) FEUP – Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias s/n, 25 4200-465 Porto, Portugal 26 (10) Aix Marseille Univ, CNRS, Centrale Marseille, I2M, Marseille, France 27 28 Running Title: Microenvironment driving EMT plasticity 29 30 ± These authors contributed equally to this work. 31 *corresponding authors: 32 Claudine Chaouiya 33 Institute of Mathematics of Marseille (I2M UMR7373) 34 Avenue de Luminy - Case 907 1 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 26, 2020; DOI: 10.1158/0008-5472.CAN-19-3147 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 35 13288 MARSEILLE Cedex 9 FRANCE- 36 phone: +33 491269614 37 email: [email protected] 38 39 Florence Janody 40 i3S Rua Alfredo Allen, 208, 4200-135 Porto, Portugal 41 phone: +351 42 email: [email protected] 43 44 45 Conflict of interest statement 46 All the authors of this manuscript declare that they have no conflict of interest and no 47 competing financial interests in relation to the work described. 2 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 26, 2020; DOI: 10.1158/0008-5472.CAN-19-3147 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 48 Abstract 49 50 Epithelial-to-mesenchymal transition (EMT) has been associated with cancer cell 51 heterogeneity, plasticity, and metastasis. However, the extrinsic signals supervising these 52 phenotypic transitions remain elusive. To assess how selected microenvironmental signals 53 control cancer-associated phenotypes along the EMT continuum, we defined a logical model 54 of the EMT cellular network that yields qualitative degrees of cell adhesions by adherens 55 junctions and focal adhesions, two features affected during EMT. The model attractors 56 recovered epithelial, mesenchymal, and hybrid phenotypes. Simulations showed that hybrid 57 phenotypes may arise through independent molecular paths involving stringent extrinsic 58 signals. Of particular interest, model predictions and their experimental validations indicated 59 that: 1) stiffening of the ExtraCellular Matrix (ECM) was a prerequisite for cells 60 overactivating FAK_SRC to upregulate SNAIL and acquire a mesenchymal phenotype, and 61 2) FAK_SRC inhibition of cell-cell contacts through the Receptor-type tyrosine-protein 62 phosphatases kappa led to acquisition of a full mesenchymal, rather than a hybrid, phenotype. 63 Altogether, these computational and experimental approaches allow assessment of critical 64 microenvironmental signals controlling hybrid EMT phenotypes and indicate that EMT 65 involves multiple molecular programs. 66 67 Statement of significance: A multi-disciplinary study sheds light on microenvironmental 68 signals controlling cancer cell plasticity along epithelial-to-mesenchymal transition and 69 suggests that hybrid and mesenchymal phenotypes arise through independent molecular 70 paths. 71 72 Key words: Epithelial to Mesenchymal Transition / Microenvironmental signals / Cell 73 adhesions / Computational modeling / In vitro human cell models 3 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 26, 2020; DOI: 10.1158/0008-5472.CAN-19-3147 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 74 Introduction 75 76 Metastasis is a hallmark of cancer and the leading cause of mortality among cancer patients. 77 Despite intensive effort in basic and clinical research, metastatic cancers still present a major 78 barrier to favorable clinical outcomes (1). Therefore, the fight against cancer calls for a better 79 understanding of the involved cellular mechanisms. 80 81 The progression from carcinoma to metastatic cancer has been proposed to involve a shift 82 from an epithelial to a mesenchymal phenotype, in a highly plastic and dynamic process 83 referred to as Epithelial to Mesenchymal Transition (EMT). During this transition, epithelial 84 cells downregulate epithelial markers, lose their connections with neighboring cells through 85 the breakdown of E-Cadherin (ECad)-mediated adherens junction (AJs), upregulate 86 mesenchymal markers and acquire a marked migratory capacity mediated by the dynamic 87 remodeling of focal adhesions (FAs). Colonization at distant sites and metastatic outgrowth 88 may require that disseminated cancer cells lose their migratory capacities and re-establish AJs 89 through a Mesenchymal to Epithelial Transition (MET). However, cancer cells are rarely 90 purely mesenchymal nor purely epithelial. They often exhibit both epithelial and 91 mesenchymal features. Cells with such hybrid phenotypes appear to reside at intermediate 92 states along the epithelial to mesenchymal continuum. Unlike mesenchymal phenotypes, 93 these hybrid phenotypes may bear multiple advantages to cancer cells, including drug 94 resistance and tumor-initiating potential (2). 95 96 The cancer-associated EMT program is hardly activated in a cell autonomous manner. 97 Indeed, the tumor microenvironment, induces EMT in carcinoma cells by releasing paracrine 98 cell-cell signaling molecules, (WNT - the NOTCH ligand DELTA), growth factors 99 (Epidermal Growth Factor - EGF, Hepatocyte Growth Factor - HGF) and inflammatory 100 signals (Interleukin-6 - IL-6, Reactive Oxygen Species – ROS, Transforming Growth Factor 101 - TGFB) (2). In addition, recent evidence suggests that direct physical interactions of tumor 102 cells with their neighbors or with the ExtraCellular Matrix (ECM) greatly affect the 103 EMT/MET program. In particular, ECM stiffening can induce EMT and promote tumor 104 invasion and metastasis (3). In contrast, the cell adhesion molecule FAT4, which 105 heterophilically interacts with its ligand on adjacent cells, prevents EMT and metastasis of 106 gastric cancer cells (4). Other cell adhesion molecules, such as Receptor-type tyrosine-protein 107 phosphatases (RPTPs) of the R2B sub-family, that display homophilic cell-cell adhesion 4 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 26, 2020; DOI: 10.1158/0008-5472.CAN-19-3147 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 108 capabilities, could also have major impact on the EMT/MET program. Indeed, they are 109 regulators of AJs, are frequently mutated in solid cancers and appear to display tumor 110 suppressive capabilities (5). These microenvironmental signals cooperate to induce a group of 111 EMT-inducing transcription factors (EMT-TFs), including the zinc-finger proteins SNAIL 112 (SNAIL, SLUG) and the E-box-binding protein ZEB1. These EMT-TFs regulate each other 113 and, in different combinations, control the expression of genes associated with the epithelial 114 and mesenchymal phenotypes, respectively (2). However, it remains unclear which 115 combinations of external signals can stabilize carcinoma cells into hybrid phenotypes. 116 The complexity of the molecular networks involved in EMT has prompted numerous 117 modeling studies, recently reviewed in (6). Ordinary differential equation models focused on 118 core regulatory circuitries, and demonstrated the existence of stable, hybrid phenotypes (7,8). 119 In contrast to these continuous models, discrete logical models were developed that 120 encompass numerous players and signaling pathways (9–12). Steinway et al.’s model was 121 built around the TGFB pathway in the context of hepatocellular carcinoma, hybrid 122 phenotypes being obtained through model perturbations (10). Other models considered the 123 regulatory control of cell fate decisions between cell cycle arrest,
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