TGF-beta signaling is an effective target to block proliferation and induce apoptosis of human cholangiocarcinoma (CCA) cells: a study on human primary cell cultures

CCA and its subtypes (mucin- and mixed-CCA) arise from the neoplastic transformation of cholangiocytes, the epithelial cells lining the biliary tree CCA has high mortality owing to its aggressiveness, late diagnosis and high resistance to radiotherapy and to chemotherapeutics We have demonstrated that CCA is enriched of cancer stem cells expressing epithelial to mesenchymal transition (EMT) traits, these features being associated with aggressiveness and drug resistance TGF-β signaling is upregulated in CCA and involved in EMT We have recently established primary cell cultures from human mucinand mixed-intrahepatic CCA In human CCA primary cultures with different levels of EMT trait expression, we evaluated the anticancer effects of: (i) CX-4945, a casein kinase-2 (CK2) inhibitor that blocks TGF-β1-induced EMT; and (ii) LY2157299, a TGF-β receptor I kinase inhibitor We tested primary cell lines expressing EMT trait markers (vimentin, N-cadherin and nuclear β-catenin) but negative for epithelial markers and cell lines expressing epithelial markers (CK19-positive) in association with EMT trait Cell proliferation was evaluated by MTS assays, apoptosis by Annexin V FITC and cell migration by wound-healing assay Results: at the low dose of 10 mM, CX4945 significantly inhibited cell proliferation of primary human cell cultures from both mucin- and mixed CCA, whereas in CK19-positive cell cultures, the anti-proliferative effect of CX4945 required higher concentrations (>30mM) At the same concentrations, CX4945 also induced apoptosis (3- fold increase vs controls) that correlated with the expression level of CK2 in the different CCA cell lines (mucin- and mixed-CCA) Indeed, no apoptotic effects were observed in CK19-positive cells expressing lower CK2 levels The anti-proliferative and pro-apoptotic effects of CX4945 were associated with an increased number of γ-H2ax (biomarker for DNA double-strand breaks) foci, suggesting an active role of CK2 as repair mechanism in CCAs LY2157299 showed no inhibitory effect on cell proliferation and apoptosis but significantly inhibited cell migration At 50 mM concentration, in fact, the wound-healing of primary cell cultures from both mucin-and mixed-CCA In conclusion, we demonstrated that CX4945 and LY2157299 exerted relevant but distinct anticancer effects against human CCA cells; CX4945 acting on cell proliferation and apoptosis, LY2157299 impairing cell migration These results suggest that targeting the TGF-β signaling with a combination of CX-4945 and LY2157299 could have potential benefits in the treatment of human CCA.