Abstract 2614: Elucidation of the molecular mechanisms mediating sorafenib-resistance in Hepatocellular carcinoma

Abstract

Abstract Sorafenib is the only first line and FDA-approved drug available for the treatment of advanced hepatocellular carcinoma (HCC). Despite an increased overall patient survival of about 3-5 months with sorafenib, majority of the HCC patients acquire sorafenib-resistance, thus making sorafenib treatment ineffective in long-term. Therefore, there is an urgent need to explore the mechanisms underlying sorafenib-resistance to develop better treatment strategies to combat this deadly disease. To achieve this goal, we first established sorafenib-resistant cells by repeated treatment of different HCC cell lines (Huh7, Hep3B and HepG2) with increasing concentrations of sorafenib up to 6µM. MTT assays confirmed that all three cell types treated with sorafenib achieved resistance and were termed soraR to distinguish them from their sorafenib-sensitive (soraS) counterparts. Further characterization of the soraR cells showed increased expression of mesenchymal markers including Slug, Snail, Zeb2, vimentin, and reduced expression of epithelial marker (E-cadherin), indicative of Epithelial-Mesenchymal Transition (EMT). Moreover soraR cells were found to have high migratory potential in wound healing assay and higher sphere-forming capability compared to the soraS cells. To find out the signaling pathways which could be responsible for promoting sorafenib-resistance, RT2-profiler PCR Array analysis for cytokine and chemokines (PAHS-150Z from Qiagen) were performed, which revealed an increase in the expression of BMP6 and BMP7 mRNA levels in the soraR cells. The results were validated by qPCR. In addition, western blot analyses showed an increase in pSmad1, 5, 9 levels in the soraR cells, suggesting a potential activation of BMP6, 7 axis in sorafenib-resistance. Effect of inhibition of BMP6 and BMP7 signaling in re-sensitizing the SoraR cells towards sorafenib was determined next. Flow cytometric analysis of Hep3B-SoraR cells treated with DMH1, a small molecule inhibitor of BMP receptors in combination with sorafenib showed increased cell death which was accompanied with increased expression of cleaved PARP and cleaved caspase 3 in western blot. BMP6 and BMP7 inhibition also reduced migration capacity of soraR cell types as examined by wound healing assay. The results were further confirmed by treating soraR cells with recombinant BMP6 and BMP7 and examining their effect on migration and sorafenib-mediated apoptosis. Interestingly, exogenous BMP6 and BMP7 treatment was found to enhance migration capacity and rescue the soraS cells from sorafenib-mediated cell death. These findings indicated an important role of BMP6 and BMP7 pathways in mediating sorafenib-resistance in HCC and suggested that pharmacological targeting of BMP6 and BMP7 signaling could be an effective strategy to ameliorate sorafenib-resistance in advanced HCC. Citation Format: Kanchan Vishnoi, Rong Ke, Randhir Kumar, Navin Viswakarma, Ajay Rana, Basabi Rana. Elucidation of the molecular mechanisms mediating sorafenib-resistance in Hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2614.