Abstract 4969: RNA interference screens to identify genes essential for ovarian tumor growth and maintenance
Authors: Sethi Geetika , Pathak Harsh B. , Zhang Hong , Zhou Yan , Einarson Margaret , Godwin Andrew K. 
Published: June 2012

BACKGROUND: Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the U.S. Although most women afflicted with EOC respond to primary therapy, the relapse rate is 85%. It is essential that we better understand the genetic and epigenetic factors that contribute to EOC in order to improve prevailing conventional therapies. We have used a functional genomic approach to help identify potential molecular targets that represent key points of therapeutic intervention.

METHODS: Building on the concept of “oncogenic addiction” we employed a small interfering RNA (siRNA) screen against 6022 druggable genes using high-throughput cell-based, one-well/one-gene screening approach and tested for effects on growth and/or survival of A1847 EOC cells. From primary lethality screens, we identified 300 genes that were considered essential for tumor cell growth and/or survival. We derived a custom siRNA library consisting of 1,200 siRNAs (pooled 4 siRNAs/gene) targeting these 300 genes. We optimized high-throughput transfection conditions for six additional EOC cell lines (OVCAR5, SKOV3, A2780, C30, CP70 and UPN275) and three non-tumorigenic, immortalized human ovarian surface epithelial (HOSE) cell lines (HIO-80, HIO-117, HIO-120) and screened our custom library. We identified lead candidates and eliminated potential off-target effects by rescreening individual siRNAs following deconvolution of siRNAs pools and by quantitative RT-PCR (qRT-PCR). We examined gene expression of the validated hits in all the EOC cell lines under two different growth conditions (monolayer and 3D spheroids) by qRT-PCR (Fluidigm instrument) and in primary serous adenocarcinoma tumors by microarrays (Affymetrix).

RESULTS: A hit was defined as a gene that reduced cell viability by &gt;15% with a false discovery rate of ≤5%. In the 300-gene library screen, each of the ten cell lines exhibited a unique number of hits ranging from 27-94% of the total siRNAs screened. We validated the top priority candidates and ultimately identified four genes (HSPA5, NDC80, NUF2 and PTN) that when suppressed, significantly reduced the viability of all the EOC cell lines tested but none of the non-tumorigenic HOSE lines. Expression of two of the genes was significantly (p=0.1) down-regulated (&gt;1.5-fold) in cells grown as 3D spheroids versus monolayers. Expression of all four genes was significantly higher in primary serous tumors (n=53) compared to normal HOSE samples (n=10) (p&lt; 0.005).

CONCLUSION: We identified four druggable candidates that could contribute to ovarian tumorigenesis and form the basis of targeted combination therapy to block the multiple oncogenic pathways activated in EOC. This work has the potential to contribute to the next-generation approach for cancer therapies: combining targeted drugs in coherently designed multi-agent combinations useful in the clinical treatment of EOC to improve patient survival.

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 4969. doi:10.1158/1538-7445.AM2011-4969