Active-Site Tryptophan, the Target of Antineoplastic C-Terminal Binding Protein Inhibitors, Mediates Inhibitor Disruption of CtBP Oligomerization and Transcription Coregulatory Activities

Abstract

C-terminal binding proteins (CtBP1/2) are oncogenic transcriptional coregulators and dehydrogenases often overexpressed in multiple solid tumors, including breast, colon, and ovarian cancer, and associated with poor survival. CtBPs act by repressing expression of genes responsible for apoptosis (e.g., PUMA, BIK) and metastasis-associated epithelial-mesenchymal transition (e.g., CDH1), and by activating expression of genes that promote migratory and invasive properties of cancer cells (e.g., TIAM1) and genes responsible for enhanced drug resistance (e.g., MDR1). CtBP's transcriptional functions are also critically dependent on oligomerization and nucleation of transcriptional complexes. Recently, we have developed a family of CtBP dehydrogenase inhibitors, based on the parent 2-hydroxyimino-3-phenylpropanoic acid (HIPP), that specifically disrupt cancer cell viability, abrogate CtBP's transcriptional function, and block polyp formation in a mouse model of intestinal polyposis that depends on CtBP's oncogenic functions. Crystallographic analysis revealed that HIPP interacts with CtBP1/2 at a conserved active site tryptophan (W318/324; CtBP1/2) that is unique among eukaryotic D2-dehydrogenases. To better understand the mechanism of action of HIPP-class inhibitors, we investigated the contribution of W324 to CtBP2's biochemical and physiologic activities utilizing mutational analysis. Indeed, W324 was necessary for CtBP2 self-association, as shown by analytical ultracentrifugation and in vivo cross-linking. Additionally, W324 supported CtBP's association with the transcriptional corepressor CoREST, and was critical for CtBP2 induction of cell motility. Notably, the HIPP derivative 4-chloro-HIPP biochemically and biologically phenocopied mutational inactivation of CtBP2 W324. Our data support further optimization of W318/W324-interacting CtBP dehydrogenase inhibitors that are emerging as a novel class of cancer cell-specific therapeutic.

NADH/NAD+ binding and linked tetrameric assembly of the oncogenic transcription factors CtBP1 and CtBP2

“…Similarly, the best-fit molecular masses of CtBP2 in the presence of NAD + or CtBP1 with either nucleotide also correspond to tetramer. These results agree with our previous light scattering analysis of CtBP1 and CtBP2 [31] and indicate that the SV feature near 6 S populated in the presence of nucleotide is associated with the tetramer rather than dimer, as has been previously suggested [36]. We then assign the 4.1 S feature to dimer.…”

“…Here, we investigate the NAD(H)-linked assembly of CtBP through analytical ultracentrifugation and isothermal titration calorimetry (ITC). Despite earlier interpretations of sedimentation velocity (SV) data as deriving from a monomer to dimer assembly [36], we definitively show using both SV and sedimentation equilibrium (SE) that the NAD(H) bound forms of both CtBP1 and CtBP2 are predominantly tetrameric in solution at micromolar concentrations. This tetrameric assembly is fairly stableour SV experiments show that the tetramer to dimer dissociation constants for CtBP1 and CtBP2 in the presence of saturating NADH or NAD + are around 100 nM.…”

“…Previous SV experiments on truncated CtBP2 , in which the first 30 residues and the critical last 91 residues are missing, identified a predominant species with a sedimentation coefficient of 6.0-6.7 S [36]. This species was assumed to be dimeric.…”

CtBP determines ovarian cancer cell fate through repression of death receptors

“…Pharmacologic reduction of NADH depolymerizes CtBP, resulting in induction of certain normally repressed target genes 54,55 . Since CtBP dimers can be disrupted not only by reduction in NADH level, but also by small molecule CtBP dehydrogenase inhibitors 56 , a promising approach for treatment of HGSOC and other CtBP dependent cancers that is currently under investigation may be combining small molecule CtBP inhibition with strategies that reduce cellular NADH level 57 .…”

CtBP2 interacts with TGIF to promote the progression of esophageal squamous cell cancer through the Wnt/β‑catenin pathway

“…Furthermore, it may function as a transcriptional co-repressor, negatively regulating certain tumor suppressors, thereby promoting the occurrence and development of tumors (17,18). Recent studies have revealed that the CtBP family plays a crucial role in the occurrence and development of breast, colon, ovarian and prostate cancer (19)(20)(21).…”