C-terminal Binding Protein (CtBP) is a transcriptional co-repressor that plays an important role in mammalian development and tumorigenesis. CtBP is known to interact with Ikaros, an important transcriptional regulator in the pituitary; however CtBP itself has not been examined in this gland. I examined the role of CtBP in pituitary cell growth and survival. Compared to control pituitary GH4 cells, CtBP1-deficient cells exhibit reduced proliferation and de-regulation of genes involved in cell cycle and growth factor signaling. CtBP1-deficient cells were more susceptible to hypoxia-induced apoptosis and showed a reduction in hypoxia-induced Ikaros expression. Interactions between CtBP and Ikaros isoforms were demonstrated in pituitary tumor cell lines. CtBP and Ikaros also bound a common region of the previously characterized Ikaros target, the LDL-R promoter. These results identify oncogenic properties of CtBP1 in the pituitary and set the groundwork for future studies into regulatory roles of CtBP and Ikaros in the pituitary.

C-terminal Binding Protein (CtBP) is a transcriptional co-repressor that plays an important role in mammalian development and tumorigenesis. CtBP is known to interact with Ikaros, an important transcriptional regulator in the pituitary; however CtBP itself has not been examined in this gland. I examined the role of CtBP in pituitary cell growth and survival. Compared to control pituitary GH4 cells, CtBP1-deficient cells exhibit reduced proliferation and de-regulation of genes involved in cell cycle and growth factor signaling. CtBP1-deficient cells were more susceptible to hypoxia-induced apoptosis and showed a reduction in hypoxia-induced Ikaros expression. Interactions between CtBP and Ikaros isoforms were demonstrated in pituitary tumor cell lines. CtBP and Ikaros also bound a common region of the previously characterized Ikaros target, the LDL-R promoter. These results identify oncogenic properties of CtBP1 in the pituitary and set the groundwork for future studies into regulatory roles of CtBP and Ikaros in the pituitary.

The oncogenic protein BCL-3, a member of the IκB family, was originally identified in a subset of human B-cell chronic lymphocytic leukemias that carry a translocation t(14,19), which results in BCL-3 overexpression. BCL-3 is also overexpressed in many solid tumors, such as in breast cancers and in cylindromas. This IκB protein activates or represses gene transcription through binding with the NF-κB proteins p50 and p52. Furthermore, BCL-3 is K63-linked polyubiquitinated, which leads to its translocation into the nucleus and to its target genes expression. BCL-3 is also K48-linked polyubiquitinated after GSK3 phosphorylation, which leads to its subsequent proteasomal degradation. However, the mechanisms underlying both its polyubiquitination and its ability to repress gene transcription remain poorly understood. In order to gain more insight into these BCL-3 functions, parallel screenings involving both yeast-two-hybrid experiments and biochemical purifications led to the identification of BCL-3-interacting partners. Those screenings identified CtBP as a molecule required for the ability of BCL-3 to repress gene transcription. CtBP is also required for the stability, for the oncogenic potential and for the ability of BCL-3 to inhibit UV-mediated cell apoptosis in keratinocytes. We also defined the E3 ligase TBLR1 as a key element involved in BCL-3 polyubiquitination and degradation through a GSK3-independent pathway and the proteasome subunit PSMB1 as a protein required for the GSK3-dependent and -independent proteasomal degradation of polyubiquitinated BCL-3. Importantly, all interactions require unique motifs within the amino-terminal domain of BCL-3. In conclusion, our data define multiple BCL-3-associated proteins that differentially and specifically regulate its function and stability and indicate that a better understanding of the mechanisms underlying the oncogenic properties of this IκB protein could be achieved through similar interactomic studies.

C-terminal binding proteins (CtBPs) are transcriptional co-repressors involved in developmental processes, and also implicated in a number of breast, ovarian, colon cancers, and resistance against cancer chemotherapy. CtBP is a validated novel potential anti-cancer target. In this project we sought to develop potent and selective small-molecule inhibitors of CtBP. Using a combination of classical medicinal chemistry and modern computational approaches, we designed a potent inhibitor HIPP (hydroxyimino-3-phenylpropanoic acid) that showed an IC50 of 0.24 μM against recombinant CtBP. Further elucidation of the structure-activity relationship (SAR) of HIPP led to the design of more potent inhibitors 3-Cl HIPP (CtBP IC50 = 0.17 μM) and 4-Cl HIPP (CtBP IC50 = 0.18 μM). These compounds also showed inhibition in HCT-116 colon cancer cells with GI50 values ~ 1-4 mM. The compounds showed no off-target toxicity against a closely related protein. This is a starting point for the development of CtBP inhibitors as anti-cancer therapeutics. The second part of this dissertation focuses on C–H activation chemistry. C–H activation is the most atom-economical method of introducing complexity into a molecule, even at late stages of drug/product development. We have used solid-supported palladium nanoparticle catalyst (Pd-MWCNT) to investigate the scope of C–H activation reactions it can catalyse. Pd-MWCNT was found to efficiently catalyse N-chelation directed C-H activation reactions – halogenations, oxygenations and arylations. The turn-over numbers for these reactions were significantly higher than that of the reported homogenous catalyst. The added advantages of reuse/recyclability of catalyst, low contamination of metal in the final product make this catalyst very attractive on an industrial scale. This work serves as a foundation for the further development of Pd-MWCNT catalyst in late-stage synthesis of drugs and/or diversification of products.

The paralogous C-terminal binding proteins (CtBP) 1 and 2 are evolutionarily conserved transcriptional coregulators that target and disrupt the expression of several genes essential for multiple cellular processes critical to regulating tumor formation. CtBP’s ability to govern the transcription of genes necessary for apoptosis, tumor suppression, invasion/migration and EMT gives rise to its oncogenic activities. Both isoforms of CtBP are found to be overexpressed in cancers including colorectal, pancreatic, ovarian, and breast, with higher levels correlating to lower overall median survival. Although multiple lines of evidence suggest CtBP plays a role in tumorigenesis, it has never been formally characterized as an oncogene. For this reason, the goal of this dissertation was to design a set of experiments to determine the transforming ability of CtBP2 in vitro using both murine and human fibroblast and in vivo using the Apcmin/+ mouse model of cancer. Specifically, we demonstrate that overexpression of CtBP2 alone can drive transformation of NIH3T3 cells leading to loss of contact inhibition, increased x invasion/migration, and anchorage independent growth. In addition, CtBP2 was found to cooperate with the large T-antigen (LT) component of the simian virus 40 (SV40) to lead to transformation of murine embryonic fibroblasts (MEFs) and with both LT and small T-antigen (ST) to induce migration/invasion and anchorage-independent growth in BJ human foreskin fibroblasts. To confirm the role of Ctbp2 in a mouse tumor model with Ctbp overexpression, we bred Apcmin/+ mice to Ctbp2 heterozygous (Ctbp2+/-) mice, which otherwise live normal lifespans. CtBP is a known target of the APC tumor suppressor and is thus stabilized in APC mutated human colon cancers and is found in high levels in Apcmin/+ polyps. Remarkably, removing an allele of Ctbp2 doubled the median survival of Apcmin/+ mice (P <0.001) and reduced polyp formation to near undetectable levels. These data suggest the importance of CtBP2 in driving cellular transformation and identify it as a potential target for prevention or therapy in APC mutant backgrounds.

<p>In this study, adenovirus E1A has been used to target and analyse the transcriptional function of the cellular C-terminal Binding Protein (CtBP).</p><p>Transcription is a complex biochemical process that represents a major regulatory step in gene expression. Formation of condensed chromatin by histone deacetylation and inhibition of efficient assembly of the transcription machinery are hallmarks of transcriptional repression. During a virus infection, an extensive modulation of the host cell gene expression in favour of viral gene expression can be observed. For example, the transcription regulatory E1A protein from adenovirus has been proven to be a valuable research-tool in characterising cellular proteins controlling eukaryotic gene expression.</p><p>Expression of a CtBP-binding peptide, encoded by the second exon of E1A, de-repressed transcription from a broad range of promoters, suggesting that CtBP functioned as a repressor of transcription. Artificial promoter recruitment of CtBP, by using different Gal4-fusion proteins, confirmed that CtBP functioned as a repressor. Repression of transcription by Gal4E1A-recruited CtBP was efficiently prevented by a CtBP binding competent E1A peptide, indicating that E1A relieved CtBP mediated repression by displacing CtBP from the promoter. Furthermore, Gal4CtBP repressed both basal and activated transcription in a distance dependent manner, suggesting that CtBP might repress transcription by interfering with the assembly of the basal transcription machinery. Interestingly, CtBP was found to interact with histone deacetylase-1 (HDAC-1) both <i>in vivo</i> and <i>in vitro</i> and endogenous CtBP could also recruit histone deacetylase activity. This might indicate that histone deacetylation was involved in CtBP mediated repression. However, Gal4CtBP mediated repression was insensitive to inhibition of histone deacetylase activity, suggesting an alternative function of HDAC-binding in CtBP mediated repression.</p><p>In conclusion, this work demonstrates that CtBP can act as a general repressor of activated and basal transcription. Furthermore, although CtBP was shown to recruit histone deacetylase activity the relevance of this binding remains unclear.</p>

In this study, adenovirus E1A has been used to target and analyse the transcriptional function of the cellular C-terminal Binding Protein (CtBP). Transcription is a complex biochemical process that represents a major regulatory step in gene expression. Formation of condensed chromatin by histone deacetylation and inhibition of efficient assembly of the transcription machinery are hallmarks of transcriptional repression. During a virus infection, an extensive modulation of the host cell gene expression in favour of viral gene expression can be observed. For example, the transcription regulatory E1A protein from adenovirus has been proven to be a valuable research-tool in characterising cellular proteins controlling eukaryotic gene expression. Expression of a CtBP-binding peptide, encoded by the second exon of E1A, de-repressed transcription from a broad range of promoters, suggesting that CtBP functioned as a repressor of transcription. Artificial promoter recruitment of CtBP, by using different Gal4-fusion proteins, confirmed that CtBP functioned as a repressor. Repression of transcription by Gal4E1A-recruited CtBP was efficiently prevented by a CtBP binding competent E1A peptide, indicating that E1A relieved CtBP mediated repression by displacing CtBP from the promoter. Furthermore, Gal4CtBP repressed both basal and activated transcription in a distance dependent manner, suggesting that CtBP might repress transcription by interfering with the assembly of the basal transcription machinery. Interestingly, CtBP was found to interact with histone deacetylase-1 (HDAC-1) both in vivo and in vitro and endogenous CtBP could also recruit histone deacetylase activity. This might indicate that histone deacetylation was involved in CtBP mediated repression. However, Gal4CtBP mediated repression was insensitive to inhibition of histone deacetylase activity, suggesting an alternative function of HDAC-binding in CtBP mediated repression. In conclusion, this work demonstrates that CtBP can act as a general repressor of activated and basal transcription. Furthermore, although CtBP was shown to recruit histone deacetylase activity the relevance of this binding remains unclear.

No description available.

Cancer involves the dysregulated proliferation and growth of cells throughout the body. C-terminal binding proteins (CtBP) 1 and 2 are transcriptional co-regulators upregulated in several cancers, including breast, colorectal, and ovarian tumors. CtBPs drive oncogenic properties, including migration, invasion, proliferation, and survival, in part through repression of tumor suppressor genes. CtBPs encode an intrinsic dehydrogenase activity, utilizing intracellular NADH concentrations and the substrate 4-methylthio-2-oxobutyric acid (MTOB), to regulate the recruitment of transcriptional regulatory complexes. High levels of MTOB inhibit CtBP dehydrogenase function and induce cytotoxicity among cancer cells in a CtBP-dependent manner. While encouraging, a good therapeutic would utilize >100-fold lower concentrations. Therefore, we endeavored to design better CtBP-specific therapeutics. The best of these drugs, 3-Cl and 4-Cl HIPP, exhibit nanomolar enzymatic inhibition and micromolar cytotoxicity and showed that CtBP enzymatic function is subject to allosteric interactions. Additionally, the function of the substrate-binding domain has yet to be examined in context of CtBP’s oncogenic activity. To this end, we created several point mutations in the CtBP substrate-binding pocket and determined key residues for CtBP’s enzymatic activity. We found that a conserved tryptophan in the catalytic domain is imperative for function and unique to CtBPs among dehydrogenases. Knowledge of this and other residues allows the directed synthesis of drugs with increased potency and higher CtBP specificity. Early work interrogated the importance of these residues in cell migration. Taken together, this work addresses the utility of the CtBP substrate-binding domain as a target for cancer therapeutics.

<p>Adenovirus E1A is needed to activate early viral genes and induce cell cycle progression to optimise the conditions for viral replication. This is mostly achieved through interactions between the first exon of E1A and cellular transcriptional regulatory proteins. The carboxy terminus of E1A binds the cellular corepressor of transcription C-terminal Binding Protein (CtBP), resulting in derepression of CtBP target genes. </p><p>Inducible stable U2OS cell lines were established, expressing wild type E1A (E1Awt) and a mutant unable to bind CtBP (E1A∆CID). Low inducible levels and loss of protein expression after prolonged induction together with induction of apoptosis were consistent with the fact that wild type E1A is a cytotoxic protein and correlated with the ability of CtBP to repress proapoptotic genes. E1A∆CID did not induce apoptosis and could be expressed at high levels for prolonged time periods. Moreover, the binding of CtBP contributed to E1A-induced activation of viral E1B and E4 genes, through possible targeting of Sp1 and ATF transcription factors.</p><p>In a micorarray study on mRNA levels in E1A-expressing cells, several genes consistent with the tumour suppressive and apoptotic properties of E1Awt were identified as differentially expressed. Furthermore, the differences between the two cell lines correlated with the presence of binding sites for CtBP-interacting transcription factors in the promoters of regulated genes, enabling the possible identification of new CtBP target genes. </p><p>Finally, a molecular characterisation of the CtBP mechanism of repression revealed that positioning proximal to the basal promoter element was required for efficient repression, suggesting that CtBP interferes with the basal transcriptional machinery. Two separate domains were identified in CtBP, conferring transcriptional repression and activation when expressed alone, achieved through their interaction with HDACs and HATs, respectively. However, together they cooperate to ensure maximal repression through recruitment of histone deacetylase and inhibition of histone acetyl transferase activity.</p><p>Together, these data shows important modulation of E1A activities by the binding of CtBP and suggests the involvement of acetylation/deacetylation complexes for the regulation of E1A function.</p>