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MRP3-Mediated Chemoresistance in Cholangiocarcinoma: Target for Chemosensitization Through Restoring SOX17 Expression
Elisa Lozano1,2, Maitane Asensio1, Laura Perez-Silva1
1Experimental Hepatology and Drug Targeting, IBSAL, University of Salamanca, Salamanca, Spain.
Background And Aims:
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 (SRY-box containing gene 17), 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 is investigated in this study.
Approach And Results:
SOX17 expression in human CCA cells (EGI-1 and TFK-1) selectively potentiated cytotoxicity of SN-38, 5-fluorouracil and mitoxantrone, but not that of gemcitabine, capecitabine, cisplatin, or oxaliplatin. The analysis of the resistome by TaqMan low-density arrays revealed changes affecting primarily ABC pump expression. Single-gene quantitative real-time PCR, immunoblot, and immunofluorescence analyses confirmed that MRP3 (multidrug resistance associated protein 3), which was highly expressed in CCA human tumors, was down-regulated in SOX17-transduced CCA cells. The substrate specificity of this pump matched that of 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-fluorouracil. 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 down-regulation and subsequent intracellular drug accumulation.
Insights
Restoring SOX17 expression in cholangiocarcinoma (CCA) selectively enhances chemotherapy effectiveness by down-regulating multidrug resistance-associated protein 3 (MRP3) pumps. This increases intracellular drug accumulation, improving treatment outcomes for CCA patients.
Area of Science:
- Molecular Biology
- Oncology
- Cancer Therapeutics
Background:
- Unresectable cholangiocarcinoma (CCA) exhibits poor response to chemotherapy.
- Reduced intracellular drug levels due to ATP-binding cassette (ABC) pumps contribute to chemoresistance.
- Low SOX17 expression is linked to cholangiocyte malignant transformation.
Purpose of the Study:
- To investigate the role of SOX17 in CCA chemoresistance.
- To determine if SOX17 influences the expression of ABC pumps.
- To evaluate the therapeutic potential of SOX17 restoration in CCA.
Main Methods:
- SOX17 expression was modulated in human CCA cells (EGI-1, TFK-1).
- Chemosensitivity assays were performed with various anticancer drugs.
- Resistome analysis, quantitative PCR, immunoblotting, immunofluorescence, and reporter assays were used to study gene expression and protein activity.
- In vivo studies involved implanting CCA tumors into immunodeficient mice and assessing treatment efficacy.
Main Results:
- SOX17 expression selectively potentiated the cytotoxicity of SN-38, 5-fluorouracil, and mitoxantrone.
- SOX17 down-regulated the expression of multidrug resistance-associated protein 3 (MRP3), a key ABC pump highly expressed in CCA.
- SOX17 inhibited MRP3 promoter activity, suggesting transcriptional regulation.
- Restored SOX17 expression enhanced the anti-tumor effect of 5-fluorouracil in vivo.
Conclusions:
- SOX17, SOX2, and SOX9 are implicated in MRP3-mediated CCA chemoresistance.
- Restoring SOX17 expression induces selective chemosensitization by down-regulating MRP3.
- SOX17 exhibits tumor suppressive effects and enhances intracellular drug accumulation, offering a promising therapeutic strategy for CCA.
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