Loss of Raf Kinase Inhibitor Protein Promotes Cell Proliferation and Migration of Human Hepatoma Cells

Background & Aims: The Raf kinase inhibitor protein (RKIP) has been identified as a suppressor of the mitogen-activated protein kinase (MAPK) pathway. Loss of RKIP function promotes tumor metastasis in prostate cancer and melanoma. The insulin-like growth factor I (IGF-I)–mediated MAPK cascade is often activated in hepatocellular carcinoma (HCC), but the role of RKIP in the molecular pathogenesis of these tumors is unknown. This study was performed to evaluate the role of RKIP in the development of HCC. Methods: The levels of RKIP expression in HCC tumor and corresponding peritumoral tissues were determined by immunohistochemistry and Western blot analysis. The underlying mechanisms of RKIP were assessed with immunoblot analysis, Raf kinase activity assay, cell proliferation, and migration assays after either overexpression or knockdown of RKIP expression in HCC cell lines. Results: RKIP expression is down-regulated in human HCC compared with adjacent peritumoral tissues. Low RKIP levels were correlated with enhanced extracellular signal–regulated-kinase (ERK)/MAPK pathway activation. Reconstitution experiments antagonized IGF-I–mediated MAPK pathway activation, resulting in reduced nuclear accumulation of phospho-ERK. In contrast, knockdown of RKIP expression using small interfering RNA induced activation of the ERK/MAPK pathway. Ectopic expression of RKIP altered HCC cell proliferation and migration. Conclusions: Our findings indicate that down-regulation of RKIP expression is a major factor in activation of the IGF-I/ERK/MAPK pathway during human hepatocarcinogenesis.