Relative quantification of protein expression in metastatic renal cell carcinoma using iTRAQ LC-MS/MS analysis reveals galectin-1 as a potential prognostic marker and therapeutic target.

Abstract

412 Background: Metastatic renal cell carcinoma (RCC) is one of the most treatment-resistant cancers. Identification of proteins involved in tumor progression will help gain a better understanding of the disease and will form the basis for the identification of novel therapeutic targets. Methods: Using six fresh-frozen primary and six unmatched metastatic RCC tumors, we used iTRAQ labeling and LC-MS/MS analysis to identify proteins differentially expressed in metastatic versus primary RCC. We verified protein expression by western blot and immunohistochemical analyses and the measured the effect of dysregulated protein expression on biological processes with RCC cell line models. Results: After analysis, we identified 29 proteins differentially expressed in metastatic versus primary RCC. We verified expressions of profilin-1, 14-3-3 zeta/delta, and galectin-1 (Gal-1) on two independent tissue sets by western blot (10 primary and 10 metastatic RCC tissues) and immunohistochemistry (22 primary and 23 metastatic tissues). Overexpression of Gal-1 in CAKI-1 cells lead to decreased actin, increased vimentin expression, and increased cellular migration. Additionally, when Gal-1 was decreased via siRNA, cells showed decreased cellular migration. Protein array analysis showed expression of cell motility-related proteins HSP27, JNK, and RSK, were altered after siRNA transfection. We also showed that Gal-1 expression was increased in response to HIF-1alpha. Furthermore, we analyzed the expression of Gal-1 mRNA in 404 RCC patients using the Cancer Genome Anatomy Project, and found that patients who had higher Gal-1 expression in the primary RCC had significantly decreased overall survival (41 vs. 78 months; p < 0.01). Conclusions: Gal-1 is increased in metastatic RCC and can effect cell migration by targeting proteins involved in cell motility. This may be a downstream effect of HIF-1α dysregulation. Decreased Gal-1 significantly decreased cellular migration suggesting Gal-1 may serve as a potential therapeutic target. Additionally, we showed that increased Gal-1 expression was associated with decreased overall survival.