ZEB1 promotes cholangiocarcinoma progression through tumor dedifferentiation and tumor-stroma paracrine signaling

BACKGROUND and AIMS: 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 (CAF). Here, we aimed to define ZEB1 regulatory functions in malignant and stroma 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/loss of function models were generated in CCA cells, and liver myofibroblasts, as a model of CAF. Conditioned media (CM) was used to unravel tumor-stroma interplay. In vivo experiments were performed using 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. CCN2/CTGF expression 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 CAF in human CCA and its expression correlated with CCN2 in myofibroblast and CCA stroma. In mice, co-transplantation 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 cross-talk, leading to tumor dedifferentiation and CAF activation.