MRP3-mediated chemoresistance in cholangiocarcinoma: Target for chemosensitization through restoring SOX17 expression.

A limitation for the treatment of unresectable cholangiocarcinoma (CCA) is its poor response to chemotherapy, which is partly due to reduction of intracellular levels of anticancer drugs through ATP-binding cassette (ABC) pumps. Low expression of SOX17, a transcription factor that promotes biliary differentiation and phenotype maintenance, has been associated with cholangiocyte malignant transformation. Whether SOX17 is also involved in CCA chemoresistance has been investigated. SOX17 expression in human CCA cells (EGI-1 and TFK-1) selectively potentiated cytotoxicity of SN-38, 5-fluorouracil (5-FU) and mitoxantrone, but not that of gemcitabine, capecitabine, cisplatin or oxaliplatin. The analysis of the resistome by TLDAs revealed changes mainly affecting ABC pumps expression. Single-gene RT-qPCR, immunoblot, and immunofluorescence analyses confirmed that MRP3, which was highly expressed in CCA human tumors, was downregulated in SOX17-transduced CCA cells. The substrate specificity of this pump matched SOX17-induced in vitro selective chemosensitization. Functional studies showed lower ability of SOX17-expressing CCA cells to extrude specific MRP3 substrates. Reporter assay of MRP3 promoter (ABCC3pr) revealed that ABCC3pr activity was inhibited by SOX17 expression and SOX2/SOX9 silencing. The latter was highly expressed in CCA. Moreover, SOX2/9, but not SOX17, induced altered electrophoretic mobility of ABCC3pr, which was prevented by SOX17. The growth of CCA tumors subcutaneously implanted into immunodeficient mice was inhibited by 5-FU. This effect was enhanced by co-treatment with adenoviral vectors encoding SOX17. Conclusions: SOX9/2/17 are involved in MRP3-mediated CCA chemoresistance. Restored SOX17 expression, in addition to its tumor suppression effect, induces selective chemosensitization due to MRP3 downregulation and subsequent intracellular drug accumulation.