Abstract 5442: Targeting PTB (polypyrimidine tract-binding protein): A promising therapeutic approach to the treatment of ovarian cancer

Abstract

Abstract Among women in the United States, ovarian cancer is the eighth most common cancer and the fifth leading cause of cancer death. In 2010, it is estimated that 21,880 women in the United States will be diagnosed with ovarian cancer and 13,850 (63%) of those women will die from this disease. Clearly, there is a need for more efficacious therapies and development of novel targets to treat ovarian cancer. Herein, we provide a proof-of-principle that a splicing factor, PTB, represents a class of novel therapeutic targets for cancer treatment, at least in epithelial ovarian cancer (EOC). PTB (also known as hnRNP I) is an RNA binding protein that participates in pre-mRNA splicing. PTB is upregulated in human ovarian tumors compared to adjacent normal tissues and knockdown of PTB expression by shRNA impairs ovarian tumor cell growth and malignant properties (He et al., Oncogene 26:4961-4968; 2007). Knock down of PTB in a xenograft mouse model of ovarian cancer slowed tumor growth. These data suggest that PTB is a novel therapeutic target for ovarian cancer and possibly other cancers. Our research tests the hypothesis that PTB activity in live cells can be monitored by measuring the splicing of a PTB target gene. First, we constructed a minigene reporter system to detect PTB activity. Our experimental approach is based on differential splicing of a PTB target gene that we identified by microarray and splicing-array analyses of cells with different levels of PTB expression. We have subcloned three exons, with the spanning introns of a PTB target gene, upstream of two fluorescent markers. Second, we validated the capability of the reporter system by using cells in which PTB expression was depleted by shRNA. We further verified that the splicing of the minigene is dependent on the PTB level in the cells by using flowcytometry and fluorescence microscopic techniques. We have now established stable reporter cell lines expressing this minigene, and we are presently validating their compatibility with both a high-throughput screening (HTS) assay and high-content analysis. Assays include measuring the stability of fluorescence protein expression over time and response to known PTB inhibitory stimuli (e.g. PTBshRNA) to determine the optimal readout system. Further 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 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 5442. doi:10.1158/1538-7445.AM2011-5442