Abstract 1652: Global proteomics analysis by SILAC (stable isotope labelling with amino acid in cell culture) in hepatocellular carcinoma treated by sorafenib: identification of phospho-AKT activation as a resistant mechanism

Abstract

Sorafenib, a multikinase inhibitor against Raf kinase and vascular endothelial growth factor receptor (VEGFR), was recently approved for the treatment of advanced hepatocellular carcinoma (HCC). However, the majority of HCC are inherently resistant to sorafenib. A global proteomic approach was applied to delineate the molecular mechanisms affecting the effectiveness of sorafenib in HCC cells. HCC cells (Huh7, SKHep1, and PLC5) were treated with sorafenib and analyzed by the SILAC (Stable Isotope Labeling with Amino acids in Cell culture) methodology. A total of 354 phosphoproteins were identified from untreated Huh7 cells (labeled with light amino acid, designated as L) and sorafenib-treated cells (labeled with heavy amino acid, designated as H). Forty-five proteins with high L/H ratio, indicating decreased expression after sorafenib treatment, were recognized. Among them, downstream proteins of Raf kinase, which are expected to be downregulated by sorafenib, were found. On the other hand, 30 proteins with low L/H ratio, indicating increased expression after sorafenib treatment, were recognized. Biological interaction network analysis of the phosphoproteome using IPA (ingenuity pathway analysis) method revealed that several signaling pathways including PI3K/AKT were significantly altered in sorafenib-treated Huh7 cells. To validate the relevance of these pathway alterations, the expression of p-AKT (Ser 473) was examined and was found significantly upregulated by sorafenib. Downregulation of AKT by siRNA attenuated the upregulation of p-AKT induced by sorafenib and enhanced the cytotoxicity of sorafenib in Huh7 cells. Combination of MK-2206, an allosteric inhibitor of AKT, abolished sorafenib-induced p-AKT upregulation, and resulted in a synergistic anti-proliferative effect in HCC cells. In conclusion, an unbiased and global phosphoproteomic approach by SILAC proves useful in identifying molecules altered by sorafenib in HCC cells; and by using this approach we showed that targeting AKT is a potentially useful strategy to improve the efficacy of sorafenib in advanced HCC. (The study was supported by the grant NSC 97-2628-B-002-004-MY3 from National Science Council of Taiwan and a research grant from Merck & Co., Inc.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1652.