Regulatory Role Of Scaffolding Protein RACK1in Gastric Cancer Invasiveness And Metastasis

Part I. Expression pattern and clinical significance of RACK1protein in gastric cancerAims Gastric cancer remains the second leading cause of cancer-related mortality worldwide, and the exact mechanism of invasion and metastasis is not clear. Receptor of activated C kinase1(RACK1) is regarded as a scaffolding protein in multiple intracellular signal transduction pathways. The aim of present study is to evaluate the expression pattern of RACK1protein in gastric cancer and its clinical significance.Methods RACK1protein was examined by Western blot and immunohistochemistry in gastric cancer after surgical resection. Fifteen fresh-frozen patient samples used in Western blot analysis were obtained in2011. Eighty-four patient samples used in tissue microarray studies were obtained from2000through2005. Correlations of RACK1expression and clinicopathological features, as well as survival outcomes were assessed.Results Western blot assay revealed that expression of RACK1protein down-regulated in tumor tissues compared to paired non-tumor tissues. Immunohistochemistry study revealed that there had significant increased of RACK1expression in intestinal type (Lauren’s classification), well or moderate differentiated, absent lymph node metastasis, and early TNM stage (Ⅰ-Ⅱ) patients (P<0.05). The mean survival time, calculated by the Kaplan-Meier method, was88.4±8.3months and119.0±7.6months, respectively (log-rank test P=0.022). However, Cox regression multivariate analysis revealed that RACK1expression was not an independent prognostic factor for the survival of gastric cancer (P=0.941).Conclusion RACK1may involve in progression of gastric cancer, and can be a criterion to predict the good prognosis for the patients after surgical resection. Part Ⅱ. RACK1restraints invasiveness by suppressing IL-8autocrine in gastric cancerAims Increasing evidences support the notion that cancer cells could influence their microenvironments by modulating pro-inflammatory cytokines in autocrine or paracrine manners, and facilitate their progression. Increased expression of chemokine interleukin8(IL-8) has been characterized in various cancer cells, and IL-8may function as a significant regulatory factor within the tumor microenvironment. The aim of this study is to explore the regulatory role of RACK1in IL-8autocrine, and which may be involved in invasion of gastric cancer.Methods RACK1knockdown gastric cell lines (HGC27, MGC80-3) were established by shRNA. Secretion of cytokines secretion, as well as interleukin8(IL-8) was measured by antibody arrays and enzyme-linked immunosorbent assay. Cell migration and invasion was compared between normal and RACK1knockdown cells. Pharmacological inhibitors were employed in searching specific signaling pathway. The correlation of RACK1and IL-8expression in gastric cancer samples was analyzed by Spearman’s p test.Results Expression of IL-8was substantially enhanced after knockdown of RACK1in vitro, which contributed to enhanced migration and invasiveness of gastric cancer cells in autocrine manner. Specific inhibitors of NF-κB (PDTC) and c-Src (PP2), as well as overexpression of dominant-negative c-Src mutant, could suppress the expression of IL-8. Furthermore, the enhanced invasiveness after RACK1knockdown was depended on expression of IL-8receptor CXCR2. Finally, an inverse correlation of RACK1and IL-8expression was confirmed in gastric cancer samples (r=-0.514, P=0.000).Conclusion Down-regulation of RACK1resulted in the up-regulation of IL-8in gastric cancer, which might enhance invasiveness via CXCR2and Src/NF-KB pathway partially.