Abstract 2039: Applying genomic approaches to identify novel targets for human hepatocellular carcinoma

Abstract

Abstract Hepatocellular Carcinoma (HCC), a primary malignant cancer of the liver, is the 5th most common cancer worldwide and the 3rd most common cause of cancer mortality. This aggressive cancer has a poor prognosis with a median survival of 6 months, and overall 5 year survival rate of about 14%, which falls to 2% for metastatic disease. Currently only one drug, Sorafenib, is FDA approved for the treatment of HCC, and has been shown to prolong survival approximately 3 months. However, the adverse side effects of the drug significantly diminish patient quality of life. Consequently, there is an urgent need for the discovery of novel gene targets selective for HCC, which can guide the development of new therapies in HCC. High-throughput RNAi technology was applied in order to identify novel gene targets. Small interfering RNA (siRNA) inhibits gene expression at a post transcriptional level by preventing mRNA translation of protein products. Through systematic knockdown of individual genes, potential drug targets that inhibit cellular proliferation can be identified. This approach was applied to a panel of 8 HCC cell lines (SNU449, SNU182, SNU387, SNU475, SNU423, Hep3B, HepG2, and PLC/PRF/5). Initially, assay development was extensively conducted in order to optimize conditions specific for each cell line including doubling time, seeding density, FBS concentration, DMSO toxicity, and transfection reagent concentration (≥95% transfection efficiency and ≤20% toxicity). Following assay optimization, each cell line was screened against 953 genes (711 validated kinase, 36 MAPK, and 206 Phosphatase) containing 4 unique siRNA sequences per gene. Cell lines were run in duplicate plates containing siRNA, negative controls (Buffer, Non-Silencing, All Star Non-Silencing, and GFP), and positive controls (UBB and ACDC) generating over 70,000 data points. Quality control was conducted following data collection to evaluate cell viability, toxicity, transfection efficiency, coefficient of variation (CV) of controls, Z’ factor, and screen reproducibility prior to moving onto hit selection. “Hits” will then be selected as siRNA that induce over 60% cell death/inhibition of growth relative to negative controls. In order to reduce false positive rates from siRNA “off-target” effects, targets will then be prioritized as genes with 2 or more siRNA sequences showing phenotypic inhibition of cell proliferation. Eight HCC cell lines have been screened against this custom siRNA set thus far with hits currently being identified. Ultimately, targets will be evaluated in follow up studies, assigning priority to hits where lethality is observed across multiple HCC cell lines while not affecting non-liver and normal liver cell lines, thereby validating liver-specific targeting. Therefore, the discovery of potential HCC targets for novel therapies would potentially impact a broader population of patients with HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2039. doi:10.1158/1538-7445.AM2011-2039