Abstract 3690: Development of a cell based high-throughput assay for drug discovery to treat ovarian cancer

Abstract

Abstract PTBP1 (polypyrimidine tract-binding protein, PTB) is a 59.6-kD nuclear protein involved in pre-mRNA splicing. Its molecular functions include regulation of alternative splicing (AS) and internal ribosomal entry site (IRES)-mediated translation. Our previous work revealed that PTB was overexpressed in human ovarian tumors and that knockdown of PTB expression by shRNA impaired ovarian tumor cell growth and malignant properties in vitro (Oncogene 26:4961-68, 2007). We have also observed slower tumor growth in a xenograft mouse model of ovarian cancer when PTB was knocked down (unpublished). These data suggest that PTB is a novel therapeutic target. To attempt to identify small molecules that have potential to act as shRNA and inhibit PTB, we have designed a cell-based high-throughput screening (HTS) system that allows fast and reliable detection of small molecule inhibitors of PTB protein activity that have potential to alter mRNA splicing. Our approach is based on differential splicing of a PTB target gene (identified by microarray analysis) at different levels of PTB activity in the cell. We have subcloned exons 14 - 16 of one of these PTB targets, Gamma-aminobutyric acid type B receptor 1 (GABBR1, upstream of two different fluorescent markers; the resulting two minigenes were designated “Gred” [short form], and “Ggreen” [long form]). We further verified that the splicing of the minigenes is dependent on the PTB level in the cells. We are presently establishing stable reporter cell lines expressing both minigenes, and validating their compatibility for HTS assay by measuring the stability of fluorescence protein expression over time and response to known PTB inhibitory stimuli (e.g. PTBshRNA). Optimization for HTS includes testing cell seeding density, DMSO tolerance, pilot experiments with specific chemical libraries, data analyses, and verification of “hits” in vitro, followed by pilot HTS with small libraries of chemical diversity to determine if any of these agents will block the activity of PTB. Since overexpressed PTB plays an integral part in maintaining ovarian tumor cell growth and malignant properties, identification of small molecule PTB inhibitors by this approach may uncover novel drugs for the treatment of ovarian and possibly other cancers. (Support in part by grants RO1 CA40570 and RO1 CA138762 (to WTB), by OCRF (to XH), and by UIC. 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 3690.