Abstract
Abstract Sorafenib, a multikinase inhibitor targeting Raf kinase and vascular endothelial growth factor receptor, is currently the only approved agent for hepatocellular carcinoma (HCC). However, the majority of HCC patients are inherently resistant to sorafenib treatment. To study molecular mechanisms underlying the resistance to sorafenib in vivo, we studied immunocompromised mice implanted with xenografts of human HCC cells subcutaneously, and identified tumors that showed primary resistance to sorafenib. Primary cultured cells were established from sorafenib-resistant xenografts, and verified for in vitro and in vivo sensitivity to sorafenib. The differentially expressed genes of the resistant cells versus control cells were analyzed by cDNA microarray. The expressions of candidate markers were confirmed by Western blotting, and their significances were verified by specific pathway inhibitors. A subline of Huh7 cells was established by primary culture from a sorafenib-resistant HCC-xenograft (designated as Huh7.1.SR). Compared to Huh7 cells primarily cultured from HCC-xenograft treated with vehicle alone (designated as Huh7.1.v), Huh7.1.SR had a moderate increase of resistance to sorafenib in vitro, with 50%-inhibitory concentration (IC50) increased from 5 to 10 μM. Subcutaneous xenograft studies confirmed that Huh7.1.SR retained the phenotype of in vivo resistance to sorafenib. The IPA (ingenuity pathway analysis) of differentially expressed genes of Huh7.1.SR versus Huh7.1.v cells revealed several pathway networks centering at transforming growth factor beta receptor (TGFBR), hepatocyte nuclear factors, NOTCH, pro-inflammatory cytokines, and nuclear factor kappa B. Huh7.1.SR had an increased expression of TGF-beta and p-Smad2, indicating that the TGF-beta pathway was activated. Huh7.1.SR cells expressed phenotypic features of epithelial to mesenchymal transition (EMT), including higher expression of vimentin, lower expression of E-cadherin, and increased cell migration. The expressions of Slug and Twist, two well-known transcriptional factors of inducing EMT, also increased in Huh7.1.SR cells. Treatment of SD-208, an inhibitor of TGF-beta receptor, partially reversed the phenotypic features of EMT in Huh7.1.SR cells. Moreover, concomitant treatment of SD-208 improved the sensitivity to sorafenib in Huh7.1.SR cells. Our data indicate that activation of TGF-beta signaling pathway is an important mechanism mediating the resistance to sorafenib in HCC in vivo. (The study was supported by the grant NSC 100-2325-B-002-043- from National Science Council of Taiwan) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1904. doi:1538-7445.AM2012-1904
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