Cindy Lobe1, Marie Vallette1, Javier Vaquero1,2,3,4, Laura Fouassier1, Ander Arbelaiz1, Ester Gonzalez-Sanchez1,2,3, Laura Izquierdo3,5, Anna Pellat1, Nathalie Guedj6, Corentin Louis7, Valérie Paradis6,7, Jesus M Banales3,5,8, Cédric Coulouarn9, Chantal Housset1,10. 1. Sorbonne Université, Inserm, Centre de Recherche Saint-Antoine, Paris, France. 2. TGF-β and Cancer Group, Oncobell Program, Bellvitge Biomedical Research Institute, Barcelona, Spain. 3. National Biomedical Research Institute on Liver and Gastrointestinal Diseases (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain. 4. LPP, Sorbonne Université, CNRS, Ecole Polytechnique, Université Paris-Sud, Observatoire de Paris, Université Paris-Saclay, PSL Research University, Paris, France. 5. Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute-Donostia University Hospital, University of the Basque Country, San Sebastián, Spain. 6. Service d'Anatomie Pathologique Hôpital Beaujon, Clichy, France. 7. INSERM, UMR 1149, Centre de Recherche sur l'Inflammation, Paris, France. 8. IKERBASQUE, Basque Foundation for Science, Bilbao, Spain. 9. Inserm UMR 1241, Université Rennes 1, Rennes, France. 10. Reference Center for Inflammatory Biliary Diseases and Autoimmune Hepatitis, Assistance Publique-Hôpitaux de Paris, Saint-Antoine Hospital, ERN Rare-Liver, Paris, France.
Abstract
BACKGROUND AND AIMS: Zinc finger E-box binding homeobox 1 (ZEB1) is a transcription factor that promotes metastatic and stem cell features, which has been associated with poor prognosis in cholangiocarcinoma (CCA), a desmoplastic cancer enriched in cancer-associated fibroblasts (CAFs). We aimed to define ZEB1 regulatory functions in malignant and stromal compartments of CCA. APPROACH AND RESULTS: Bioinformatic and immunohistochemical analyses were performed to determine correlations between ZEB1 and markers of progressiveness in human intrahepatic CCA (iCCA). Gain-of-function and loss-of-function models were generated in CCA cells and liver myofibroblasts as a model of CAFs. Conditioned media (CM) was used to unravel tumor-stroma interplay. In vivo experiments were performed using a xenograft CCA model. ZEB1 expression in tumor cells of human iCCA was associated with undifferentiated tumor and vascular invasion. In vitro, ZEB1 promoted epithelial-mesenchymal transition and stemness in tumor cells, leading to cell migration and spheroid formation. In vivo, ZEB1-overexpressing CCA cells formed larger tumors with more abundant stroma. Expression of cellular communication network factor 2 (CCN2, encoding connective tissue growth factor [CTGF]) was increased in tumor cells from ZEB1-overexpressing xenografts and correlated with ZEB1 expression in human tumors. In vitro, CM from ZEB1-overexpressing tumor cells or recombinant CTGF induced myofibroblast proliferation. ZEB1 was also expressed by CAFs in human CCA, and its expression correlated with CCN2 in myofibroblasts and CCA stroma. In mice, cotransplantation of CCA cells with ZEB1-depleted myofibroblasts reduced CCA progressiveness compared to CCA cells/ZEB1-expressing myofibroblasts. Furthermore, ZEB1 controls the expression of paracrine signals (i.e., HGF and IL6) in tumor cells and myofibroblasts. CONCLUSIONS: ZEB1 plays a key role in CCA progression by regulating tumor cell-CAF crosstalk, leading to tumor dedifferentiation and CAF activation.
BACKGROUND AND AIMS: Zinc finger E-box binding homeobox 1 (ZEB1) is a transcription factor that promotes metastatic and stem cell features, which has been associated with poor prognosis in cholangiocarcinoma (CCA), a desmoplastic cancer enriched in cancer-associated fibroblasts (CAFs). We aimed to define ZEB1 regulatory functions in malignant and stromal compartments of CCA. APPROACH AND RESULTS: Bioinformatic and immunohistochemical analyses were performed to determine correlations between ZEB1 and markers of progressiveness in human intrahepatic CCA (iCCA). Gain-of-function and loss-of-function models were generated in CCA cells and liver myofibroblasts as a model of CAFs. Conditioned media (CM) was used to unravel tumor-stroma interplay. In vivo experiments were performed using a xenograft CCA model. ZEB1 expression in tumor cells of human iCCA was associated with undifferentiated tumor and vascular invasion. In vitro, ZEB1 promoted epithelial-mesenchymal transition and stemness in tumor cells, leading to cell migration and spheroid formation. In vivo, ZEB1-overexpressing CCA cells formed larger tumors with more abundant stroma. Expression of cellular communication network factor 2 (CCN2, encoding connective tissue growth factor [CTGF]) was increased in tumor cells from ZEB1-overexpressing xenografts and correlated with ZEB1 expression in human tumors. In vitro, CM from ZEB1-overexpressing tumor cells or recombinant CTGF induced myofibroblast proliferation. ZEB1 was also expressed by CAFs in human CCA, and its expression correlated with CCN2 in myofibroblasts and CCA stroma. In mice, cotransplantation of CCA cells with ZEB1-depleted myofibroblasts reduced CCA progressiveness compared to CCA cells/ZEB1-expressing myofibroblasts. Furthermore, ZEB1 controls the expression of paracrine signals (i.e., HGF and IL6) in tumor cells and myofibroblasts. CONCLUSIONS: ZEB1 plays a key role in CCA progression by regulating tumor cell-CAF crosstalk, leading to tumor dedifferentiation and CAF activation.