Obeticholic acid, a FXR agonist, inhibits the cancerogenic potential of primary human cholangiocarcinoma (CCA) cells cultures

Background and Aims: Cholangiocarcinoma is an aggressive cancer, resistant to chemotherapeutics. We demonstrated that CCA is enriched of cancer stem cells associated with aggressiveness and drug resistance. FXR, involved in neoplastic transformation of stem cells and/or cholangiocytes, is down-regulated in human CCA. Our AIM was to evaluate, in primary cultures of human intrahepatic CCA (iCCA) the effects of the FXR agonist, obeticholic acid (OCA), on the cancerogenic potential of human CCA cells. Method: Primary human cell cultures were prepared from specimens of iCCA obtained from patients submitted to surgical resection and classified into mucin- or mixed-iCCA subtypes by morphologic and immunohistochemical criteria. Increasing concentrations (0–5 μM) of OCAwere added to culture media and, after 3–10 days, the effect on proliferation (MTS assay, cell population doubling time), apoptosis (annexin V-FITC / propidium iodide), cell migration and invasion (wound healing and matrigel invasion assay) and cancerogenic potential (spheroid formation, clonogenic assay, colony formation capacity) were evaluated. Results: FXR was downregulated (RT-qPCR) in iCCA cells vs normal human biliary tree stem cells (p < 0.001) and in mucin-iCCA vs mixed-iCCA (p < 0.05). OCA significantly (p < 0.05) inhibited proliferation of both mucin-iCCA and mixed-iCCA cells starting at a concentration as low as 0.05 μM (IC50 = 0.38 μM in mixed- and 2.1 μM in mucin-iCCA). Also CDCA (but not UDCA) inhibited cell proliferation, although to a much lower extent than OCA, consistent with the different potency in FXR activation (i.e. OCA > CDCA, no agonistic effect for UDCA). OCA significantly induced apoptosis of both iCCA subtypes and decreased the in vitro cancerogenic potential of iCCA cells as evaluated by impairment of colony and spheroid formation capacity and delayed wound healing and matrigel invasion. In general, these effects were more evident against mixed- than mucin-iCCA cell. When tested together with gemcitabine and cisplatin, OCA potentiated the anti-proliferative and pro-apoptotic effects of these chemotherapeutics but mainly on mixed-iCCA. OCA abolished the capacity of both mucin- and mixed-iCCA cells to form colonies when administered together with gemcitabine and cisplatin.