Abstract
Abstract C-terminal Binding Proteins (CtBP) 1 and 2 constitute a family of oncogenic transcriptional co-regulators overexpressed in tumor tissues that are associated with worse prognostic outcome and aggressive tumor characteristics in multiple cancer types. Specifically, CtBP has been found to repress expression of genes responsible for apoptosis and EMT (eg. BIK and CDH1) and promote expression of genes that partake in the migration of cancer cells and those that are responsible for enhanced drug resistance (eg. TIAM1 and MDR1). CtBP2 is also critically required for colon cancer stem cell self-renewal. CtBP is unique among transcription co-regulators in harboring a conserved D-isomer specific 2-hydroxyacid dehydrogenase (D2DH) domain, which reduces an alpha-keto acid substrate to an alpha-hydroxy acid in the presence of NADH. The presence of NADH also facilities oligomerization of CtBP, leading to assembly of higher order complexes of CtBP with both DNA binding transcription factors and histone modifying enzymes that then leads to modulation target genes. We have identified hydroxyimino-3-phenylpropanoic acid and its 4-chloro derivative (HIPP; 4-Cl-HIPP) as potent substrate competitive inhibitors of the CtBP dehydrogenase (IC50’s=240nM, 180 nM), which also disrupt CtBP oligomerization, promoter localization, and transcriptional regulation. Co-crystallization of HIPP and CtBP1/2 indicated a strong π-π interaction between the HIPP phenyl ring and the indolyl ring in tryptophan W318/324 of CtBP1/2, indicating that this tryptophan is critical to CtBP interaction with HIPP inhibitors, dehydrogenase function, and quaternary structure. Of note W318/324, though conserved in CtBP1/2, is unique among D2DH, suggesting that a better understanding of W318/324 role and function in CtBP structure and function is critical to optimizing design of inhibitors that targeting this evolutionary unique residue among dehydrogenases. To further elucidate the mechanism of action in catalysis and oligomerization, as well as functional importance for transcription of CtBP2 W324, we analyzed enzyme kinetics, oligomerization, transcriptional co-regulatory activity and cell migration in a series of CtBP2 W324 mutants overexpressed in breast and colon cancer cell lines with concomitant knockdown (siRNA) or knockout (CRISPR/Cas9) of CtBP2. Our data demonstrates that mutation of W324 abrogated dehydrogenase activity, oligomerization, transactivation of the validated CtBP target gene TIAM1 and induction of migration. In summary, our findings suggest that the W324 residue is critical for CtBP2’s function and its unique conservation in CtBP1/2 vs. other dehydrogenases will allow the development of high specificity CtBP W318/324 inhibitors to limit potential toxicity due to off target inhibition of related metabolic dehydrogenases. Citation Format: Martin M. Dcona, Benjamin L. Morris, Priyadarshan K. Damle, Zaid Nawaz, Francisco Zarate Perez, Michael J. Dennis, Sahib J. Singh, William E. Royer, Keith C. Ellis, Steven R. Grossman. Tryptophan 318/324, the target of C-terminal binding protein (CtBP) inhibitors, plays a critical role in CtBP enzymatic activity, oligomerization and transcriptional coregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3204. doi:10.1158/1538-7445.AM2017-3204
Published Version
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