Abstract 2036: Differential expression of the C-Terminal Binding Protien-2 highlight important protein pathways in ovarian carcinogenesis and suggest that its transcriptional function may be modulated by the novel nuclear protein Pinin

Abstract

Abstract Objectives: Ovarian cancer continues to be the leading cause of mortality from gynecological malignancy despite advancements in novel therapeutics. Our group recently demonstrated that C-terminal binding protein 2 (CtBP2), a transcriptional co-repressor that regulates gene expression via epigenetic mechanisms, is overexpressed in epithelial ovarian carcinoma. The target genes mediated by the CtBP2 complex remain largely unknown. We designed a study aimed at identifying the in vivo binding sites of the CtBP2 complex as well as examining the differential gene expression profiles between wild-type and CtBP2-knockdown cancer cell lines in an attempt to identify key pathways regulated by CtBP2. Methods: Gene expression profiling was performed on CtBP2 wild type and knockdown SKOV3 and MCAS cell lines. RNA was extracted, reverse transcribed to cDNA, labeled and hybridized to Affymetrix Gene 1.0 ST Microarray. Differentially expressed genes were identified using D-Chip and SAM statistical programs. The differentially expressed genes were integrated into the Ingenuity Protein Analysis system and key pathways were analyzed. Selected differentially expressed genes were validated using qRT-PCR. Expression of Pinin was examined using Immunohistochemistry (IHC) on 64 ovarian tissues and using Western blot analysis on 15 ovarian cell lines. The interaction between Pinin and CtBP2 was analyzed by co-immunoprecipitation assays and immunofluorescence microscopy. Results: Analyses of gene expression profiling data resulted in a 154-gene signature largely involved in DNA replication and estrogen receptor signaling and metabolism. Interestingly, the majority of differentially expressed genes are down-regulated in the knockdown cell lines, which is unexpected when CtBP2 is considered as a co-repressor. The nuclear protein Pinin was found to co-precipitate with CtBP2 and co-localized with CtBP2 in the nucleus. IHC and Western blot studies showed significant overexpression of Pinin in tumor specimens compared to benign specimens (P<0.001). Conclusions: Since the majority of differentially expressed genes are down-regulated in the knockdown cell lines, the co-repressor function of CtBP2 in ovarian cancers might be modified by co-regulators. The nuclear protein Pinin was found to interact closely with CtBP2. It is likely that Pinin binds to CtBP2 and regulates its gene expression programming function in ovarian cancer cells. Additional in-depth studies are underway to delineate the role of Pinin in gene expression regulation mediated by the CtBP2 complex and the effect this has on ovarian pathogenesis. Furthermore, Chromatin Immunoprecipitation and next-gen sequencing experiments are being carried out to identify the direct targets of the CtBP2 complex. 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 2036. doi:10.1158/1538-7445.AM2011-2036