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Cholangiocarcinoma (CCA) and its subtypes (mucin- and mixed-CCA) arise from the neoplastic transformation of cholangiocytes, the epithelial cells lining the biliary tree. CCA has a high mortality rate owing to its aggressiveness, late diagnosis and high resistance to radiotherapy and chemotherapeutics. We have demonstrated that CCA is enriched for cancer stem cells which express epithelial to mesenchymal transition (EMT) traits, with these features being associated with aggressiveness and drug resistance. TGF-β signaling is upregu-lated in CCA and involved in EMT. We have recently established primary cell cultures from human mucin- and 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 catenin) but negative for epithelial markers, and cell lines expressing epithelial markers (CK19-positive) in association with EMT traits. Cell viability was evaluated by MTS assays, apoptosis by Annexin V FITC and cell migration by wound-healing assay. Results: at a dose of 10 μM, CX4945 significantly decreased cell viability of primary human cell cultures from both mucin and mixed CCA, whereas in CK19-positive cell cultures, the effect of CX4945 on cell viability required higher concentrations (>30μM). At the same concentrations, CX4945 also induced apoptosis (3- fold increase vs controls) which 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 effects of CX4945 on viability and apoptosis were associated with an increased number of γ-H2ax (biomarker for DNA double-strand breaks) foci, suggesting the active role of CK2 as a repair mechanism in CCAs. LY2157299 failed to influence cell proliferation or apoptosis but significantly inhibited cell migration. At a 50 μM concentration, in fact, LY2157299 significantly impaired (at 24, 48 and 120 hrs) the wound-healing of primary cell cultures from both mucin-and mixed-CCA. In conclusion, we demonstrated that CX4945 and LY2157299 exert relevant but distinct anticancer effects against human CCA cells, with CX4945 acting on cell viability and apoptosis, and 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.

We investigated the sensitivity of intrahepatic cholangiocarcinoma (IHCCA) subtypes to chemotherapeutics and molecular targeted agents. Primary cultures of mucin- and mixed-IHCCA were prepared from surgical specimens (N. 18 IHCCA patients) and evaluated for cell proliferation (MTS assay) and apoptosis (Caspase 3) after incubation (72 hours) with increasing concentrations of different drugs. In vivo, subcutaneous human tumor xenografts were evaluated. Primary cultures of mucin- and mixed-IHCCA were characterized by a different pattern of expression of cancer stem cell markers, and by a different drug sensitivity. Gemcitabine and the Gemcitabine-Cisplatin combination were more active in inhibiting cell proliferation in mixed-IHCCA while Cisplatin or Abraxane were more effective against mucin-IHCCA, where Abraxane also enhances apoptosis. 5-Fluoracil showed a slight inhibitory effect on cell proliferation that was more significant in mixed- than mucin-IHCCA primary cultures and, induced apoptosis only in mucin-IHCCA. Among Hg inhibitors, LY2940680 and Vismodegib showed slight effects on proliferation of both IHCCA subtypes. The tyrosine kinase inhibitors, Imatinib Mesylate and Sorafenib showed significant inhibitory effects on proliferation of both mucin- and mixed-IHCCA. The MEK 1/2 inhibitor, Selumetinib, inhibited proliferation of only mucin-IHCCA while the aminopeptidase-N inhibitor, Bestatin was more active against mixed-IHCCA. The c-erbB2 blocking antibody was more active against mixed-IHCCA while, the Wnt inhibitor, LGK974, similarly inhibited proliferation of mucin- and mixed-IHCCA. Either mucin- or mixed-IHCCA showed high sensitivity to nanomolar concentrations of the dual PI3-kinase/mTOR inhibitor, NVP-BEZ235. In vivo, in subcutaneous xenografts, either NVP-BEZ235 or Abraxane, blocked tumor growth. In conclusion, mucin- and mixed-IHCCA are characterized by a different drug sensitivity. Cisplatin, Abraxane and the MEK 1/2 inhibitor, Selumetinib were more active against mucin-IHCCA while, Gemcitabine, Gemcitabine-Cisplatin combination, the c-erbB2 blocking antibody and bestatin worked better against mixed-IHCCA. Remarkably, we identified a dual PI3-kinase/mTOR inhibitor that both in vitro and in vivo, exerts dramatic antiproliferative effects against both mucin- and mixed-IHCCA.