Abstract 2199: Evaluation of critical residues in the C-terminal binding protein (CtBP) dehydrogenase domain contributing to substrate binding, catalysis, and oncogenic activity

Abstract

Abstract C-terminal binding proteins (CtBP) 1 and 2 are transcriptional coregulators that are upregulated in several cancers, including a majority of studied breast, colorectal, and ovarian tumor samples. CtBPs drive many cellular oncogenic properties, including migration, invasion, proliferation, and survival, in part through repression of tumor suppressor genes, such as E-cadherin, PTEN, BRCA1, and p16. CtBPs encode an intrinsic dehydrogenase activity regulated by both intracellular NADH concentration and the putative substrate 4-methylthio-2-oxobutyric acid (MTOB). NADH binding induces CtBP dimerization, which regulates the recruitment of transcriptional regulatory complexes. High levels of MTOB appear to inhibit CtBP dehydrogenase function and induce cytotoxicity among cancer cells in a CtBP-dependent manner. However, the function of the substrate-binding domain has yet to be examined with regard to CtBP's oncogenic activity. To this end, we have created several point mutations in the substrate-binding pocket of CtBP and have determined key residues for CtBP's enzymatic activity using an in vitro dehydrogenase assay. We have found that a conserved tryptophan in the catalytic domain, and specifically its aromatic moiety, is imperative for enzyme activity. This tryptophan is unique to CtBP family proteins and distinguishes the CtBP substrate-binding domain from that of all other families of dehydrogenases. Additionally, we found arginine and histidine residues lining the substrate pocket that are necessary for CtBP dehydrogenase function. Knowledge of these residues allows the directed synthesis of drugs with increased potency and higher CtBP specificity. Moreover, to investigate the importance of the catalytic domain on CtBP's oncogenic potential, we have transfected human cancer cell lines with several CtBP substrate-binding domain mutants and observed the effects on cellular processes, including growth, survival, migration, and recruitment of key components of the transcriptional repressor complex. From this work we have investigated the utility of CtBP, and specifically the CtBP substrate-binding domain, as a target for cancer therapeutics. We also provide preliminary insights into the function of this domain in cellular models of cancer. Citation Format: Benjamin L. Morris, Priyadarshan Damle, Zaid Nawaz, Steven R. Grossman. Evaluation of critical residues in the C-terminal binding protein (CtBP) dehydrogenase domain contributing to substrate binding, catalysis, and oncogenic activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2199. doi:10.1158/1538-7445.AM2015-2199