IL-6/STAT3 Signaling Contributes to Sorafenib Resistance in Hepatocellular Carcinoma Through Targeting Cancer Stem Cells

Yu Li,1 Gang Chen,1 Zhijian Han,2 Huijuan Cheng,2 Liang Qiao,3 Yumin Li1,2 1General Surgery Department, Lanzhou University Second Hospital, Lanzhou, Gansu, People’s Republic of China; 2Key Laboratory of Digestive System Tumors of Gansu Province, Lanzhou University Second Hospital, Lanzhou, Gansu, People’s Republic of China; 3Storr Liver Unit at the Westmead Millennium Institute, The University of Sydney at Westmead Hospital, Sydney, NSW 2145, AustraliaCorrespondence: Yumin LiLanzhou University Second Hospital, Lanzhou 730030, People’s Republic of ChinaTel +86-13893615421Fax +86-0931-8458109Email liym@lzu.edu.cnLiang QiaoStorr Liver Unit at the Westmead Millennium Institute, The University of Sydney at Westmead Hospital, Westmead, Sydney, NSW 2145, AustraliaTel +86-17820019796Email liang.qiao@sydney.edu.auBackground: Sorafenib is the standard first-line treatment for advanced hepatocellular carcinoma (HCC), even though acquired resistance to sorafenib has been found in many HCC patients, resulting in poor prognosis. Accumulating evidence demonstrates that liver cancer stem cells (LCSCs) contribute to sorafenib resistance in HCC. The inflammatory factor interleukin 6 (IL-6) plays a role in sorafenib resistance in HCC. However, the mechanism by which IL-6 in LCSCs is involved in the process of HCC sorafenib resistance remains elusive.Methods: In this study, the sorafenib-resistant cell line PLC/PRF/5-R was generated by the concentration gradient method, and cell viability was determined by the CCK-8 assay. LCSCs were isolated from the PLC/PRF/5-R cell line by flow cytometry, and tumorigenesis was confirmed in nude mice. Blockade of IL-6 cells was achieved by lentiviral-mediated interference. The protein levels of stem cell markers (EpCAM, CD44), stemness markers (Oct3/4, β-catenin), and hepatocyte differentiation markers (glucose-6-phosphate, AFP) were measured by Western blotting analysis. Finally, a xenograft model was used to evaluate the function of IL-6 in the sorafenib resistance of HCC.Results: The stable sorafenib-resistant HCC cell line PLC/PRF/5-R was established and showed significant epithelial–mesenchymal transition (EMT) characteristics; the isolated resistant LCSCs from PLC/PRF/5-R were more tumorigenic than the control LCSCs. We showed that IL-6, IL-6R, STAT3 and GP130 expression were dramatically increased in resistant LCSCs compared to control LCSCs. Downregulation of IL-6 expression with short hairpin RNA (shRNA) restored sorafenib sensitivity in resistant LCSCs, suggesting the critical roles of IL-6/STAT3 in inducing sorafenib resistance. Furthermore, a xenograft tumor model showed that IL-6 downregulation improved the antitumor effect of sorafenib.Conclusion: LCSCs play an important role in sorafenib-resistant HCC, and inhibition of the IL-6/STAT3 signaling pathway improves the antitumor effects of sorafenib against HCC in vitro and in vivo. These findings demonstrate that IL-6 in LCSCs may function as a novel target for combating sorafenib resistance in HCC.Keywords: IL-6/STAT3 signaling, cancer stem cells, hepatocellular carcinoma, drug resistance, sorafenib