Recent studies indicate a correlation between high expression of the POZ-ZF transcription factor Kaiso, and the aggressiveness of the triple negative breast cancer (TNBC) subtype. However, little is known about the biological roles of Kaiso in TNBC tumorigenesis and metastasis, which laid the foundation for this thesis. To elucidate Kaiso’s role in TNBC, we generated stable Kaiso depletion in two well-established TNBC cell lines – MDA-MB-231 and Hs578T – using RNA interference technology. Intriguingly, we observed that Kaiso depletion delayed the tumor onset of MDA-MB-231 but not Hs578T cells, and led to the reduced expression of the c-Myc oncoprotein in MDA-MB-231 but not Hs578T cells. We postulate that this reduction in c-Myc expression is partly responsible for the delayed tumor onset observed in MDA-MB-231 cells. Additionally, loss of Kaiso expression resulted in increased apoptosis of both MDA-MB-231 and Hs578T cells in vitro and in vivo, which was accompanied by reduced expression of the DNA repair protein BRCA1. Remarkably, bioinformatic analysis revealed that high Kaiso and BRCA1 mRNA expression correlates with the reduced survival rates of TNBC patients. Further characterization of the Kaiso-depleted cells revealed that loss of Kaiso expression strongly inhibited the metastatic abilities of MDA-MB-231 and Hs578T cells. Importantly, Kaiso depletion led to decreased TGFβ-receptor I and II (TGFβRI and II) expression that is essential for the activation of the TGFβ signaling cascade. Concomitantly, suppressing Kaiso led to reduced TGFβ signaling. As increased TGFβRI expression is independently associated with the poor prognostic outcome of breast tumors, and the TGFβ signaling pathway is highly involved in breast tumor metastasis, we hypothesize that Kaiso functions together with TGFβRI and the TGFβ signaling cascade to promote TNBC metastasis. An additional goal of this thesis was to investigate the role of Kaiso in the prevalence of TNBC in women of African ancestry (WAA) compared to Caucasian women – since increased Kaiso expression is implicated in the poor survival outcomes of breast cancer patients of African ancestry relative to their Caucasian counterparts. Using tissue microarray and immunohistochemical analyses, we revealed for the first time a high nuclear expression of Kaiso in TNBC tissues of WAA (Nigerian, Barbadian, African American) compared to TNBC tissues of Caucasian women. Collectively, these findings unveiled functional oncogenic roles for Kaiso in the tumorigenesis and metastasis of TNBC, and revealed a plausible link between high Kaiso expression, high African ancestry and the predisposition of young WAA to TNBC. / Thesis / Doctor of Philosophy (PhD)

We recently reported that intestinal-specific overexpression of the POZ-ZF transcription factor Kaiso produced two prominent phenotypes in 1-year old mice: Kaiso transgenic (KaisoTg) mice presented with chronic intestinal inflammation, and an increase in secretory cell types – a trait typical of Notch signalling inhibition. Despite these findings however, the factor(s) responsible for Kaiso-mediated inflammation and secretory cell increases had not been elucidated. The primary goal of this thesis was to begin filling in this knowledge gap, by shedding mechanistic insight on Kaiso’s role in governing these two prominent phenotypes. First, we elucidated Kaiso’s role in the Notch signalling pathway and found that Kaiso inhibited the expression of the Notch1 receptor, and its ligand Dll-1, but promoted the expression of the Jagged-1 ligand. We postulated that the Kaiso-mediated reduction in Dll-1 might be responsible for the increase in secretory cell types, whereas Kaiso-mediated regulation of Jagged-1, which is dispensable for cell fate decisions, may be implicated in colon cancer progression. Importantly, we also found that Kaiso’s effects on Notch pathway inhibition occurred prior to the onset of chronic intestinal inflammation. Our analyses of the chronic inflammatory phenotype in KaisoTg mice demonstrated that Kaiso overexpression drives pathogenic neutrophil-specific recruitment (as evidenced by increases in neutrophil-specific enzymatic activity, the formation of crypt abscesses, and augmented expression levels of the neutrophils-specific chemokine, MIP2); an increase in the pore-forming Claudin-2; reduction of the cell adhesion protein E-cadherin; and abnormal intestinal epithelial repair mechanisms. Together, these findings imply that the pathogenesis of Kaiso-mediated intestinal inflammation is a multi-factorial process. A secondary goal of this thesis was to initiate studies to elucidate how the Kaiso binding partner, Znf131, might play a role Kaiso-mediated transcriptional regulation. We found that Znf131 indirectly associated with several Kaiso target genes, including Cyclin D1 (CCND1). Importantly, Znf131 activated a minimal CCND1 promoter previously shown to be inhibited by Kaiso. Moreover, Kaiso overexpression attenuated Znf131-mediated transcriptional activation and Znf131 expression in intestinal cells. Together, these findings hint that Znf131 and Kaiso may exert opposing biological functions, which may have implications in Kaiso-mediated intestinal homeostasis and disease. / Thesis / Doctor of Philosophy (PhD)

Since the discovery of the p120ctn binding partner, Kaiso, a BTB/POZ transcription factor, several studies have implicated the protein in both development and tumourigenesis. Most information about Kaiso’s function in vertebrate development has been gleaned from studies in Xenopus laevis embryos where Kaiso negatively regulates the Wnt signalling pathway. Since the Wnt signalling pathway is crucial in intestinal development, intestinal-specific Kaiso overexpressing mice were generated and characterized to elucidate Kaiso’s role in a mammalian context. Kaiso transgenic (KaisoTg/+) mice were viable and fertile but developed gross histopathological changes in the small intestine. The KaisoTg/+ mice exhibited enlarged crypts accompanied by increased secretory cell differentiation reminiscent of inhibition of the Notch pathway. Indeed, the Notch effector protein, HES1, is decreased in KaisoTg/+ mice. Additionally, KaisoTg/+ mice display a neutrophil-specific intestinal inflammation reminiscent of the knockdown of p120ctn. Interestingly, the KaisoTg/+ mice display decreased p120ctn localization at the membranes and an increase in the neutrophil adhesion molecule, ICAM-1, both of which induce neutrophilia. Notably, the KaisoTg/+ mice developed multiple crypt abscesses over time due to massive neutrophil infiltration of the epithelial cell layers. This is the first study to examine the in vivo roles of Kaiso in a mammalian context and our findings suggest a regulatory role for Kaiso in the inflammatory and Notch signalling pathways. / Thesis / Candidate in Philosophy

In mammals, epigenetic modifications and trans-acting effectors coordinate gene expression during development and impose transcriptional memories that define specific cell lineages and cell-types. Methylation at CpG dinucleotides is an epigenetic mechanism through which transcriptional silencing is established and heritably maintained through development. Functionally, DNA methylation regulates key biological processes such as X-chromosome inactivation, transposon repression and genomic imprinting. However, the extent to which DNA methylation is the primary regulator of single-copy gene expression and the precise mechanism of methylation-dependent silencing remain undetermined. Here, I identify a novel set of germline-specific candidate genes putatively regulated by DNA methylation. Analysis of one candidate gene, Tex19, demonstrates that promoter CpG methylation is the primary and exclusive mechanism for regulating developmental silencing in somatic lineages. Genetic or pharmacological removal of CpG methylation triggers robust de-repression of Tex19 and loss of transcriptional memory. Moreover, Tex19 critically relies on de novo methylation, mediated by Dnmt3b, to impose silencing in differentiating ES cells and somatic cells in vivo from embryonic day (E)7.5. Reporter gene and ChIP analysis demonstrate that Tex19 is strongly activated by general transcription factors and is not marked by repressive histone modifications in somatic lineages, consistent with differential DNA methylation per se being the primary mechanism of regulating expression. Full transcriptional silencing of Tex19 is critically dependent on the methyl-binding protein (MBP) Kaiso, which is only recruited to methylated Tex19 promoter. The reliance on DNA methylation and Kaiso for silencing in somatic cells establishes an epigenetic memory responsible for maintaining expression in germline and pluripotent cell types through successive developmental cycles. This thesis represents the first causal report of lineagespecific promoter DNA methylation directing silencing of an in vivo gene through recruitment of an MBP.

Recently, through reporter gene studies, the novel BTB/POZ protein, Kaiso, has been identified as a transcriptional repressor. The purpose of this study was to determine if Kaiso recruited the Histone Deacetylase Complex to mediate repression and if the previously identified Kaiso Binding Site (KBS; TCCTGCNA) is a physiological target regulated by Kaiso. The two objectives are complementary because an HDAC interaction identifies the mechanism of transcriptional regulation used by Kaiso and regulation of the KBS element identifies a novel, non-methylation dependent, physiological target under transcriptional regulation by Kaiso. Through coimmunoprecipitation and Western blot analyses, Kaiso does not interact with HDAC1, HDAC2 or mSIN3A. These results were surprising since all three of these proteins are common to a variety of repression complexes. mSIN3A is a common component of SIN3 mediated repression and HDAC1/HDAC2 are part of various repression complexes including SIN3, NuRD and CtBP. Although the remaining HDAC proteins were not assayed for an interaction, Kaiso transcriptional activity was demonstrated to be insensitive to the HDAC inhibiting drug, Trichostatin A (TSA). These results indicate either a non-HDAC mechanism of action or alternatively, transcriptional activation. Complementary to the observations of no Kaiso-HDAC interaction and TSA insensitivity was the findings that Kaiso activates transcription of the KBS cis-element in HCT116, HCA-7 and 293 cells, but not MOCK cells in reporter gene assays. Taken together, these results indicate that Kaiso is a dual functioning protein capable of both transcriptional activation and repression and that the mechanism of repression is not through the direct recruitment of HDAC proteins. / Thesis / Master of Science (MSc)

Multiple studies have implicated the POZ-ZF and methyl-DNA-binding transcription factor, Kaiso, in the regulation of genes and pathways that are important for development and tumorigenesis. In Xenopus embryos and mammalian cultured cells, Kaiso has been implicated as a negative regulator of the canonical Wnt signaling pathway. Paradoxically however, Kaiso depletion extends lifespan and delays polyp onset in the ApcMin/+ mouse model of intestinal tumorigenesis, where aberrant activation of Wnt signaling results in the development of neoplasias. These findings call into question Kaiso’s role as a negative regulator of canonical Wnt signaling and led us to hypothesize that Kaiso promotes intestinal tumorigenesis by a mechanism independent of its role in canonical Wnt signaling. To further delineate Kaiso’s role in intestinal tumorigenesis and to determine Kaiso’s role in regulating canonical Wnt signaling in the murine intestine, we generated a Kaiso transgenic mouse model expressing an intestine-specific murine Kaiso transgene. We then crossed our Kaiso transgenic mice with ApcMin/+ mice and analyzed the resultant progeny. Unexpectedly, Kaiso transgenic mice exhibited intestinal inflammation, increased expression of Wnt target genes and deregulated progenitor cell differentiation, although ectopic expression of Kaiso was not sufficient to drive tumorigenesis in the intestine. In agreement with previous studies, ectopic Kaiso expression in ApcMin/+ mice resulted in a significantly shortened lifespan and increased tumour burden. While we were unable to determine the precise mechanism by which Kaiso promotes intestinal tumour development, we found that Kaiso-induced inflammation is enhanced in the ApcMin/+ background and ectopic Kaiso expression further intensifies Wnt target gene expression in this model. Collectively, these studies have identified novel roles for Kaiso in regulating inflammation and cell-fate determination in the intestine. Furthermore, our findings suggest that Kaiso may contribute to intestinal tumorigenesis by promoting inflammation, which has been shown to be a predisposing factor for colorectal cancer development. Lastly, we have demonstrated distinct tissue and organism-specific roles for Kaiso in regulating canonical Wnt signaling. While, the aforementioned studies were the primary focus of this thesis, we also examined Kaiso’s role in DNA methylation-dependent repression of two tumour-associated genes, cyclinD1 and HIF1A. Our studies revealed that Kaiso binds and regulates the cyclinD1 locus via both sequence-specific and methylation-dependent DNA binding, suggesting that these alternate modes by which Kaiso binds to DNA may not be mutually exclusive. Furthermore, we identified a previously unexplored role for Kaiso in regulating the expression of the master regulator of hypoxia, HIF1A, which implicates Kaiso in modulating hypoxia-driven tumorigenic processes. / Thesis / Doctor of Philosophy (PhD)

<p> Downregulation of E-cadherin is a frequent event in epithelial cancers and it correlates with weakened cell-cell adhesion and the induction of an epithelial-to-mesenchymal transition (EMT). It is postulated that E-cadherin downregulation liberates the catenin p120 and allows p120's translocation to the nucleus where it interacts with and functionally regulates the novel BTB/POZ transcription factor, Kaiso. Kaiso mediates transcriptional repression of various tumourigenesis-associated genes via methylated CpG dinucleotides or a sequence-specific Kaiso binding site (KBS). The Kaiso/p120 interaction has been detected in E-cadherin expressing cells of various origins, but is seldom detected in N-cadherin expressing cells or cells that have undergone EMT. We hypothesize that p120 and Kaiso play a role in EMT by modulating the expression of EMT-associated genes. We demonstrated that TGF-β-induced EMT occurs in a dose- and time-dependent manner in NMuMG cells but not in FHL-124 cells. In both cells lines, the Kaiso/p120 interaction occurred irrelevant of EMT induction by TGF-β. In NMuMG cells, the expression of p120 increased with EMT induction, while the expression of Kaiso remained unchanged. Finally, misexpression of Kaiso and p120 in mammary epithelial cells affected TGF-β-mediated EMT induction by delaying the upregulation of the positive mesenchymal markers, N-cadherin and α-SMA.</p> / Thesis / Master of Science (MSc)

Kaiso is a novel member of the BTB/POZ (Broad complex, Tramtrak, Bric a brac,/Pox virus and zinc finger) zinc finger family of transcriptional regulators that have many roles in development and tumorigenesis. Kaiso was first identified as a binding partner for p 120ctn, an Armadillo catenin with roles in cell adhesion and stabilization of cadherins at the cell membrane. Kaiso is both an activator and repressor of gene transcription and interacts with two distinct types of DNA sequence; a consensus Kaiso binding site (KBS) TCCTGCNA and methylated CpG dinucleotide pairs (i.e. CpGCpG). Thus far p120's nuclear role is to inhibit Kaiso-mediated regulation of its target genes. Some of the Kaiso target genes identified to date include, matrilysin, rapsyn, and MTA2. The Kaiso homologue in Xenopus laevis (frog) has also been shown to regulate the cell cycle regulator CCND 1. Sequence analysis of the human CCND1 promoter revealed several potential Kaiso binding elements including both KBS and methylatable CpGs. My research demonstrated that Kaiso binds to the CCND1 promoter in vitro and in vivo to both KBS-specific and CpG-specific regions. Furthermore, I determined that Kaiso may act as either a repressor or activator of the human CCND 1 gene depending on the cellular environment. Altogether these data support my hypothesis that Kaiso is a regulator of the CCND1 gene. Future studies looking at the significance of KBS versus CpG-binding on Kaiso's mechanism of regulation are required to determine the significance of this regulation. Furthermore, studies examining the cell cycle-dependent changes in Kaiso levels may reveal how alterations in Kaiso expression affect Kaiso target genes including CCND1. / Thesis / Master of Science (MSc)

The characterization of Kaiso transgenic mice, a potential model for the IBD-to-CAC transition. / Colitis-associated cancer (CAC) is a poorly characterized subgroup of colorectal cancers (CRC) that afflicts ~20% patients suffering from inflammatory bowel disease (IBD). The limited understanding of CAC stems from the lack of suitable mammalian model systems, as well as a general gap in knowledge regarding the molecular mechanisms of this disease. Currently, colitis is modelled by the use of the detergent dextran sodium sulfate (DSS) to induce inflammation in the intestines of mice. Studies have shown that increased expression of the transcription factor Kaiso causes intestinal inflammation and early-stage tumorigenesis in mice, even without additional intestinal insult. This inflammatory progression mimics the beginnings of CAC in humans, and we postulate that with a “second-hit” caused by a carcinogen such as azoxymethane (AOM), the mice will cross the threshold from inflammation to carcinogenesis. Wildtype (WT), KaisoTg mice, and APCmin/+/KaisoTg crossed mice were exposed to various combinations of the pro-inflammatory detergent DSS, the carcinogen AOM and the general anti-inflammatory, aspirin. Intestinal tissues were collected for gross morphological assessment, polyp quantification and Immunohistochemistry (IHC) analysis, in order to determine the relative expression level and localization of pro-inflammatory and tumorigenic proteins. We hypothesized that exposure to DSS or AOM will exacerbate Kaiso-mediated intestinal inflammation and lead to colitis-associated cancer (i.e. polyp formation), while aspirin will rescue the APCmin/+/KaisoTg accelerated tumour forming phenotype. KaisoTg treated with AOM or DSS exhibited an impeded weight gain phenotype, extensive intestinal hyperplasia and altered gene expression. IHC analysis revealed that two key adhesion proteins, p120ctn and E-Cadherin exhibit aberrant expression and localization in KaisoTg, independent of treatment. Additionally, it was observed that AOM treatment and Kaiso overexpression work synergistically to produce an ectopic expression profile for the proliferation marker, Ki67. Together these finding suggest a role for Kaiso in intestinal inflammation, cancer initiation via altered proliferation, and the destabilization of adherens junctions, leading to a compromised intestinal epithelial barrier. / Thesis / Master of Science (MSc)

<p>The POZ-ZF transcription factor ZBTB4 was initially identified due to its sequence homology to the dual-specificity DNA-binding transcription factor Kaiso. Subsequent characterization of ZBTB4 revealed that it is also a dual-specificity DNA-binding protein; it recognizes a specific oligonucleotide sequence C^T/_CGCCATC, coined the <strong>Z</strong>BTB<strong>4</strong> <strong>B</strong>inding <strong>S</strong>equence (Z4BS) as well as methylated CpG-dinucleotides. Interestingly, ZBTB4 also binds to the highly similar consensus <strong>K</strong>aiso <strong>B</strong>inding <strong>S</strong>ite (KBS) <em>in vitro</em>.</p> <p>ZBTB4 is misexpressed in cancer, and follows a stage-specific pattern of expression in breast carcinoma tissues; low ZBTB4 levels are found in late stages while high ZBTB4 expression is detected in early stages of disease progression. Ongoing studies have begun to elucidate the molecular interactions that mediate ZBTB4’s apparent tumour suppressor role in tumourigenesis, however no study has investigated the nature of ZBTB4’s ability to interact with both the Z4BS and the KBS <em>in vivo</em>, and how this may expand ZBTB4’s repertoire of potential target genes.</p> <p>Recently Kaiso has been characterized as a transcriptional repressor of the cell cycle regulatory gene <em>cyclin D1</em>, and thus we used <em>cyclin D1 </em>as a model to investigate the nature of ZBTB4’s interaction with the KBS <em>in vivo</em>. The <em>cyclin D1</em> minimal promoter contains two partial Z4BS at the same location as the KBS sites and we found that ZBTB4 binds to the +69 Z4BS/KBS site, but not to the -1067 site. Because the +69 Z4BS/KBS is immediately flanked by a CpG dinucleotide, this interaction may be a methylation-dependent interaction. To determine the consequence of this interaction, we conducted minimal promoter luciferase assays, and observed that ZBTB4 mediates an activation of the -1748-CD1 minimal promoter activity.</p> / Master of Science (MSc)