1551P - Epithelial to mesenchymal transition induces autophagy in renal cell carcinoma: Implications in cancer therapy

Abstract

Epithelial to mesenchymal transition (EMT) contributes to the metastatic and invasive potential of tumors including renal-cell carcinoma (RCC). Various preclinical and clinical results have indicated that dysregulated elements leading to EMT can be a potential target in RCC. We assessed the expression profile of EMT associated genes in surgically resected tumor tissue and targeted the survival mechanism of EMT-induced cells. We studied the expression of epithelial marker (E-cadherin), mesenchymal markers (Snail, Slug, Vimentin, Twist) and cancer stem cell marker (ALDH1) in 71 patients of histologically proven RCC. These were analysed in both tumor section and the adjoining normal looking parenchyma by real time PCR and Western immunoblot. In vitro studies were carried out in primary cultures and RCC cell line (A498) where EMT was induced pharmacologically using tumor growth factor-beta (TGF-&bgr;, 10ng/ml). Autophagy was assessed in EMT induced cells by acridine orange staining. Tetrazolium dye, (MTT) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and propidium iodide (PI)-Annexin staining were done to evaluate post-treatment cell survival in EMT cells with temsirolimus and autophagy inhibitor, choloroquine. Epithelial marker, E-cadherin was significantly down-regulated in tumor tissue while expression of mesenchymal and cancer stem cell markers increased in tumor tissue as compared to adjoining normal tissue. EMT signature proteins showed an increase in in vitro culture of primary cells from tumor tissue as well as in EMT- induced A498 cells. We also observed increased invasiveness and autophagy in EMT induced A498 cells and primary tumor cells as compared to the normal cells. It was observed that addition of autophagy inhibitor (chloroquine) with temsirolimus to EMT-induced cells decrease their viability. Annexin-PI assay showed a significant cell death in combination of chloroquine and temsirolomus on EMT induced cells. Our study shows that the process of EMT is involved in the metastatic spread of RCC and autophagy helps in survival of the EMT-induced cells. Thus, inhibition of autophagy might represent a future therapeutic option.