Abstract 2564: High-throughput siRNA library screen for discovery and identification of new melanoma therapeutic targets in combination with antioxidant pretreatment

Abstract

Abstract Melanoma is one of the most prevalent cancers in the United States, having the fifth highest cancer incidence in males and sixth highest incidence in females. Primary cutaneous melanoma can often be cured by surgery, but for those patients in whom metastasis has occurred, mortality is high in part due to numerous chemoresistance mechanisms. Thus, there is an urgent need to discover and identify novel therapeutic targets for the efficient treatment of this lethal disease. Gene silencing by RNA interference (RNAi) is a powerful tool to identify novel genes involved in specific biological processes. In our study, we used high-throughput druggable siRNA library (Dharmacon) targeting 6028 human genes to screen metastatic melanoma cell lines and found that knockdown of gene expression by siRNA pools targeting 885 genes significantly inhibited melanoma growth (p<0.005). Among them, 67 siRNA pools resulted in >50% inhibition of cell viability in the melanoma A375 cells which we employed for the screening analysis. We validated the top-six cancer-related genes (RRM2, BCAR3, PSMA2, UBB, RRM1, TCP1) as potential melanoma therapeutic target candidates, as knockdown of these 6 genes significantly inhibited cell growth and induced apoptosis in multiple melanoma cell lines. We have hypothesized that antioxidant treatment relieves resistance mechanisms, and showed that among the screened >6000 siRNA pools, treatment with 20 pools significantly increased the growth inhibition in the presence of nontoxic levels of bardoxolone methyl (p<0.005), a novel semi-synthetic triterpenoid (previously known as CDDO-Me) with potent antioxidant activity generously provided by Reata Pharmaceuticals. We also selected 5 additional cancer-related genes (GNPAT, SUMO1, SPINT2, FLI1 and SSX1) for further analysis and validated them as additional candidates for therapeutic targeting in the presence of antioxidant. Our results thus demonstrate the feasibility of the high-throughput siRNA library screen system in discovering new melanoma therapeutic targets and shows that these new candidate genes are promising for the development of novel therapeutic targets for melanoma treatment, many of which are revealed by antioxidant pretreatment. Results from our study provide the basis for significant advances in identifying targets for the treatment of melanoma patients. (This study was partially supported by the grant from the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation and the NIH Melanoma SPORE grant P50, CA093459). 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 2564.