Abstract 2193: Application of a novel cell-based dual luciferase assay to identify inducers of the tumor suppressor RhoB

Abstract

Abstract RhoB is a small GTPase tumor suppressor whose expression is commonly repressed during tumorigenesis by mechanisms such as Akt activation, HDAC1-mediated histone modification and/or specific microRNAs such as miR-21 overexpression. Forced expression of RhoB inhibits cell proliferation, survival, invasion and metastasis. Because RhoB is rarely mutated or deleted in human tumors, its restoration may have therapeutic benefit. Therefore, a dual-luciferase high throughput assay was developed to monitor induction of RhoB transcription. Four different cancer cell lines were stably transfected with a single vector in which firefly luciferase and a fusion protein of renilla luciferase/neomycin resistant gene were driven by RhoB or CMV promoters, respectively. These cell lines were screened in 96-well format with the NCI Diversity Set II library (1364 compounds). Induction of RhoB activity was compared to that caused by a known RhoB inducer (NSC126188), an HDAC inhibitor (TSA), a farnesyltransferase inhibitor (L744832), and an Akt inhibitor (PIA23). Seven compounds were identified, and induction of RhoB transcription and translation was validated under different serum conditions at several time points. They were also screened in secondary assays that included inhibition of Akt, induction of protein acetylation, and screening in a cancer cell line that cannot induce endogenous RhoB due to a homozygous deletion of chromosome region 2p24. One compound caused sustained induction of endogenous RhoB in multiple cell lines under different conditions, and inhibited cellular proliferation and migration. These studies show that this assay system can identify specific RhoB inducers with potential anticancer activity, and suggest that it can be expanded to screen larger compound libraries, or be adapted to monitor transcriptional activation of other tumor suppressor genes. 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 2193. doi:10.1158/1538-7445.AM2011-2193