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 isenriched of cancer stem cells associated with aggressiveness anddrug resistance. FXR, involved in neoplastic transformation of stemcells and/or cholangiocytes, is down-regulated in human CCA. OurAIM was to evaluate, in primary cultures of human intrahepatic CCA(iCCA) the effects of the FXR agonist, obeticholic acid (OCA), on thecancerogenic potential of human CCA cells.Method: Primary human cell cultures were prepared from specimensof iCCA obtained from patients submitted to surgical resection andclassified into mucin- or mixed-iCCA subtypes by morphologic andimmunohistochemical 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 cancerogenicpotential (spheroid formation, clonogenic assay, colony formationcapacity) were evaluated.Results: FXR was downregulated (RT-qPCR) in iCCA cells vs normalhuman biliary tree stem cells (p < 0.001) and in mucin-iCCA vsmixed-iCCA (p < 0.05). OCA significantly (p < 0.05) inhibited proliferation of both mucin-iCCA and mixed-iCCA cells starting at aconcentration as low as 0.05 μM (IC50 = 0.38 μM in mixed- and 2.1 μMin mucin-iCCA). Also CDCA (but not UDCA) inhibited cell proliferation, although to a much lower extent than OCA, consistent with thedifferent potency in FXR activation (i.e. OCA > CDCA, no agonisticeffect for UDCA). OCA significantly induced apoptosis of both iCCAsubtypes and decreased the in vitro cancerogenic potential of iCCAcells as evaluated by impairment of colony and spheroid formationcapacity and delayed wound healing and matrigel invasion. Ingeneral, these effects were more evident against mixed- thanmucin-iCCA cell. When tested together with gemcitabine andcisplatin, OCA potentiated the anti-proliferative and pro-apoptoticeffects of these chemotherapeutics but mainly on mixed-iCCA. OCAabolished the capacity of both mucin- and mixed-iCCA cells to formcolonies when administered together with gemcitabine and cisplatin.