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Peroxisome proliferator-activated receptor (PPAR)γ plays a role in tumorigenesis. Previous studies with PPARγ(+/-) mice suggest PPARγ normally suppresses dimethylbenz[a]anthracene (DMBA)-induced breast, and other, tumor progression. Since many cell types associated with the mammary gland express PPARγ, each with unique signaling pathways, the present study aimed to define which tissues are required for PPARγ-dependent anti-tumor effects. Conditional adipocyte and endothelial cell-specific PPARγ knockout mice (PPARγ-A KO and PPARγ-E KO respectively) were used to evaluate whether PPARγ signaling normally acts to prevent DMBA-mediated breast tumour progression in a stromal cell-specific manner. Twelve week old PPARγ KO mice and their congenic wildtype (WT) controls were randomly assigned to one of two treatment groups. All mice were treated by gavage once/week for 6 weeks with 1 mg DMBA and maintained on a normal chow diet. At week 7, mice in each group were divided into those continuing normal chow, and those receiving a PPARγ ligand (ROSI, 4 mg/kg/day) supplemented diet for the duration of the 25 week study, and monitored weekly. Tumour and tissue samples were collected at necropsy, and portions of each were fixed and frozen for future analysis. In both PPARγ-A KOs and PPARγ-E KOs versus PPARγ-WT mice, malignant mammary tumor incidence was significantly higher and mammary tumor latency was decreased. DMBA+ROSI treatment reduced average mammary tumor volumes by 50%. Gene expression analyses of mammary glands by qRT-PCR and immunofluorescence indicated that untreated PPARγ-A KOs had significantly decreased BRCA1 expression in mammary stromal adipocytes. Compared to PPARγ-WT mice, serum leptin levels in PPARγ-A KOs were also significantly higher throughout the study. In the PPARγ-E KO mice, both treatment groups saw a significant increase in thymic tumour incidence, a finding not established before with the study of other stromal cell knockout mice. These studies provide the first direct in vivo evidence that PPARγ signalling in stromal adipocytes and endothelial cells attenuates DMBA-mediated breast tumourigenesis. This study supports a protective effect of activating PPAR gamma as a novel chemopreventive therapy for breast cancer. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2012-12-24 11:28:17.668

L'antigène de prolifération cellulaire Ki-67 est exprimé de manière constitutive dans les cellules de mammifères. Ki-67 est régulièrement utilisé en tant que marqueur de prolifération cellulaire pour classer les tumeurs. Cependant, malgré son utilisation fréquente en histopathologie, ses fonctions sont encore mal caractérisées. Mes travaux de thèse ont eu pour objectif d'améliorer la compréhension des fonctions biologiques de Ki-67 ainsi que d’étudier l’importance de son expression dans l’initiation et la progression des cancers. Nous avons montré que Ki-67 était dispensable à la prolifération cellulaire. Quant aux souris mutantes Ki-67, elles ne présentaient aucune anomalie de développement, étaient fertiles et vieillissaient normalement. Néanmoins, l’expression de Ki-67 s’est révélée être requise pour l’organisation de l'hétérochromatine dans les cellules prolifératives. En étudiant le contrôle de l'expression de Ki-67, nous avons pu mettre en évidence que les différents niveaux d’expression de Ki-67, souvent observés dans les lignées cellulaires transformées ou non, les tissus et les échantillons de tumeurs des patients, seraient expliqués par une régulation via la machinerie du cycle cellulaire.En utilisant nos souris mutantes Ki-67, nous avons également montré que l’absence de Ki-67 permettait de protéger les souris contre la carcinogenèse intestinale dans les deux différents modèles expérimentaux utilisés. De plus, l'analyse de la conséquence de l'ablation de Ki-67 dans la lignée tumorale murine, 4T1, a révélé que Ki-67 est requis pour le maintien des propriétés souches de ces cellules cancéreuses. En outre, la déplétion de Ki-67 a fortement affecté la croissance des tumeurs et la formation de métastases pulmonaires chez les souris. De façon similaire, l'absence de Ki-67 a fortement altéré le développement des xénogreffes de la lignée MDA-MB-231 dans des souris immuno-déficientes. De plus, le séquençage de l'ARN dans les cellules 4T1 a révélé l’existence d’altérations importantes au niveau transcriptomique, suite à la déplétion de Ki-67.L’ensemble de ces résultats suggère une implication spécifique de Ki-67 dans l'initiation et la progression tumorale et que Ki-67 serait une cible thérapeutique potentielle et intéressante dans le traitement du cancer. / The cell proliferation antigen Ki-67 is constitutively expressed in cycling mammalian cells and is widely used as a cell proliferation marker to grade tumours. Despite its use in cancer histo-pathology its functions are poorly understood. The aim of this project is to improve understanding of Ki-67 functions and its requirements in cancer initiation and progression. We found that Ki-67 is dispensable for cell proliferation and Ki-67 mutant mice did not exhibit any developmental abnormalities, and were fertile and aged well. Although Ki-67 was uncoupled from cell proliferation, Ki-67 was found to promote heterochromatin organization in proliferating cells. Studying Ki-67 expression control, we have found that cell cycle regulation accounts for Ki-67 variability levels in normal human cells, proliferating tissues in mice, human cancer cell lines and caner patients.Using our Ki-67 mutant mice, we found that Ki-67 depletion can protect mice from intestinal carcinogenesis in two different experimental models used. Moreover, analysis of the consequence of Ki-67 ablation in the mouse breast cancer cell line, 4T1 has revealed its requirements for the maintenance of the stem-like proprieties of these cancer cells. More importantly, Ki-67 depletion strongly affects 4T1 tumour growth and formation of lung metastases in vivo. Similarly, Ki-67 absence strongly impaired the development of the TNBC-derived MDA-MB-231 xenografts in vivo. Moreover, comparison of Ki-67 dependent alterations in gene expression in 4T1 cells by RNA sequencing revealed widespread transcriptome changes following Ki-67 depletion. Together, these results suggest a specific involvement of Ki-67 in cancer initiation and progression and may constitute a potential therapeutic target in cancer therapy.

Doctor of Philosophy(PhD) / Melanotransferrin or melanoma tumour antigen p97 (MTf) is a transferrin homologue that is found predominantly bound to the cell membrane via a glycosylphosphatidylinositol anchor. The molecule is a member of the transferrin super-family that binds iron through a single high affinity iron(III)-binding site. Melanotransferrin was originally identified at high levels in melanoma cells and other tumours, but at lower levels in normal tissues. Since its discovery, the function of MTf has remained intriguing, particularly regarding its role in cancer cell iron transport. In fact, considering the crucial role of iron in many metabolic pathways e.g., DNA and haem synthesis, it is important to understand the function of melanotransferrin in the transport of this vital nutrient. Melanotransferrin has also been implicated in diverse physiological processes, such as plasminogen activation, angiogenesis, cell migration and eosinophil differentiation. Despite these previous findings, the exact biological and molecular function(s) of MTf remain elusive. Therefore, it was important to investigate the function of this molecule in order to clarify its role in biology. To define the roles of MTf, six models were developed during this investigation. These included: the first MTf knockout (MTf -/-) mouse; down-regulation of MTf expression by post-transcriptional gene silencing (PTGS) in SK-Mel-28 and SK-Mel-2 melanoma cells; hyper-expression of MTf expression in SK-N-MC neuroepithelioma cells and LMTK- fibroblasts cells; and a MTf transgenic mouse (MTf Tg) with MTf hyperexpression. The MTf -/- mouse was generated through targeted disruption of the MTf gene. These animals were viable, fertile and developed normally, with no morphological or histological abnormalities. Assessment of Fe indices, tissue Fe levels, haematology and serum chemistry parameters demonstrated no differences between MTf -/- and wild-type (MTf +/+) littermates, suggesting MTf was not essential for Fe metabolism. However, microarray analysis showed differential expression of molecules involved in proliferation such as myocyte enhancer factor 2a (Mef2a), transcription factor 4 (Tcf4), glutaminase (Gls) and apolipoprotein d (Apod) in MTf -/- mice compared with MTf +/+ littermates. Considering the role of MTf in melanoma cells, PTGS was used to down-regulate MTf mRNA and protein levels by >90% and >80%, respectively. This resulted in inhibition of cellular proliferation and migration. As found in MTf -/- mice, melanoma cells with suppressed MTf expression demonstrated up-regulation of MEF2A and TCF4 in comparison with parental cells. Furthermore, injection of melanoma cells with decreased MTf expression into nude mice resulted in a marked reduction of tumour initiation and growth. This strongly suggested a role for MTf in proliferation and tumourigenesis. To further understand the function of MTf, a whole-genome microarray analysis was utilised to examine the gene expression profile of five models of modulated MTf expression. These included two stably transfected MTf hyper-expression models (i.e., SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type with downregulated MTf expression (i.e., SK-Mel-28 melanoma). These findings were then compared with alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the microarray data were also assessed in another model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and upregulation, three genes were identified as commonly modulated by MTf. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and Tcf4 were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and proliferation/survival, respectively. Hence, this study identifies novel molecular targets directly or indirectly regulated by MTf and the potential pathways involved in its function, including modulation of proliferation. To further understand the function of MTf, transgenic mice bearing the MTf gene under the control of the human ubiquitin-c promoter were generated and characterised. In MTf Tg mice, MTf mRNA and protein levels were hyper-expressed in a variety of tissues compared with control mice. Similar to the MTf -/- mice, these animals exhibited no gross morphological, histological, nor Fe status changes when compared with wild-type littermates. The MTf Tg mice were also born in accordance with classical Mendelian ratios. However, haematological data suggested that hyper-expression of MTf leads to a mild, but significant decrease in erythrocyte count. In conclusion, the investigations described within this thesis clearly demonstrate no essential role for MTf in Fe metabolism both in vitro and in vivo. In addition, this study generates novel in vitro and in vivo models for further investigating MTf function. Significantly, the work presented has identified novel role(s) for MTf in cell proliferation, migration and melanoma tumourigenesis.

Breast cancer is characterized in part by chronic inflammation and tissue remodelling in the mammary gland. Adipocyte enhancer binding protein 1 (AEBP1), a pro-inflammatory protein, is up-regulated in breast cancer and enhances cytokine secretion in the mammary tumour microenvironment. AEBP1 over-expression in cultured macrophages resulted in increased enzymatic activity of MMP-9, a matrix metalloproteinase implicated in processing cytokines and stimulating tumour cell growth and mobility. MMP-9 activates the cytokine tumour necrosis factor-alpha (TNF?), and is required for the transformation of epithelial cells by the cytokine interleukin 6 (IL6). Treatment of epithelial cells with TNF? and IL6, both of which promote tumourigenesis, induced AEBP1 expression. Chromatin immunoprecipitation results suggested AEBP1 induction is directly mediated by pro-inflammatory transcription factors NF-?B and STAT3, downstream effectors of TNF? and IL6, respectively. AEBP1 induction may enhance inflammation, thereby contributing to cell proliferation and survival.

Doctor of Philosophy(PhD) / Melanotransferrin or melanoma tumour antigen p97 (MTf) is a transferrin homologue that is found predominantly bound to the cell membrane via a glycosylphosphatidylinositol anchor. The molecule is a member of the transferrin super-family that binds iron through a single high affinity iron(III)-binding site. Melanotransferrin was originally identified at high levels in melanoma cells and other tumours, but at lower levels in normal tissues. Since its discovery, the function of MTf has remained intriguing, particularly regarding its role in cancer cell iron transport. In fact, considering the crucial role of iron in many metabolic pathways e.g., DNA and haem synthesis, it is important to understand the function of melanotransferrin in the transport of this vital nutrient. Melanotransferrin has also been implicated in diverse physiological processes, such as plasminogen activation, angiogenesis, cell migration and eosinophil differentiation. Despite these previous findings, the exact biological and molecular function(s) of MTf remain elusive. Therefore, it was important to investigate the function of this molecule in order to clarify its role in biology. To define the roles of MTf, six models were developed during this investigation. These included: the first MTf knockout (MTf -/-) mouse; down-regulation of MTf expression by post-transcriptional gene silencing (PTGS) in SK-Mel-28 and SK-Mel-2 melanoma cells; hyper-expression of MTf expression in SK-N-MC neuroepithelioma cells and LMTK- fibroblasts cells; and a MTf transgenic mouse (MTf Tg) with MTf hyperexpression. The MTf -/- mouse was generated through targeted disruption of the MTf gene. These animals were viable, fertile and developed normally, with no morphological or histological abnormalities. Assessment of Fe indices, tissue Fe levels, haematology and serum chemistry parameters demonstrated no differences between MTf -/- and wild-type (MTf +/+) littermates, suggesting MTf was not essential for Fe metabolism. However, microarray analysis showed differential expression of molecules involved in proliferation such as myocyte enhancer factor 2a (Mef2a), transcription factor 4 (Tcf4), glutaminase (Gls) and apolipoprotein d (Apod) in MTf -/- mice compared with MTf +/+ littermates. Considering the role of MTf in melanoma cells, PTGS was used to down-regulate MTf mRNA and protein levels by >90% and >80%, respectively. This resulted in inhibition of cellular proliferation and migration. As found in MTf -/- mice, melanoma cells with suppressed MTf expression demonstrated up-regulation of MEF2A and TCF4 in comparison with parental cells. Furthermore, injection of melanoma cells with decreased MTf expression into nude mice resulted in a marked reduction of tumour initiation and growth. This strongly suggested a role for MTf in proliferation and tumourigenesis. To further understand the function of MTf, a whole-genome microarray analysis was utilised to examine the gene expression profile of five models of modulated MTf expression. These included two stably transfected MTf hyper-expression models (i.e., SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type with downregulated MTf expression (i.e., SK-Mel-28 melanoma). These findings were then compared with alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the microarray data were also assessed in another model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and upregulation, three genes were identified as commonly modulated by MTf. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and Tcf4 were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and proliferation/survival, respectively. Hence, this study identifies novel molecular targets directly or indirectly regulated by MTf and the potential pathways involved in its function, including modulation of proliferation. To further understand the function of MTf, transgenic mice bearing the MTf gene under the control of the human ubiquitin-c promoter were generated and characterised. In MTf Tg mice, MTf mRNA and protein levels were hyper-expressed in a variety of tissues compared with control mice. Similar to the MTf -/- mice, these animals exhibited no gross morphological, histological, nor Fe status changes when compared with wild-type littermates. The MTf Tg mice were also born in accordance with classical Mendelian ratios. However, haematological data suggested that hyper-expression of MTf leads to a mild, but significant decrease in erythrocyte count. In conclusion, the investigations described within this thesis clearly demonstrate no essential role for MTf in Fe metabolism both in vitro and in vivo. In addition, this study generates novel in vitro and in vivo models for further investigating MTf function. Significantly, the work presented has identified novel role(s) for MTf in cell proliferation, migration and melanoma tumourigenesis.

As the primary antagonist to the tumorigenic PI3K/AKT pathway, PTEN is classified as a tumor suppressor. The inactivation of PTEN through genetic or post-translational modifications is a critical step in the tumorigenesis of many breast cancers (BCs). SIPL1 is a novel protein which was identified as a PTEN negative regulator. To further explore SIPL1-mediated PTEN inactivation, we analyzed 17 datasets covering 3484 BC cases and 228 normal individuals. SIPL1 gene amplification and increased mRNA expression correlates with the progression and poor prognosis of ER and/or PR positive tumors. Furthermore, examination of a BC tissue microarray containing 224 tumor cases revealed elevated SIPL1 protein expression in ER+ and PR+ tumors and was associated with greater AKT activation. Additionally, ectopic expression of SIPL1 in CHO-K1 cells resulted in increased AKT activation and cell proliferation, and cytoskeleton reorganization alongside with PTEN downregulation. SIPL1 contributes to the linear polyubiquitination of NEMO, suggesting a role for SIPL1 in PTEN ubiquitination. Indeed, it was SIPL1, not the SIPL1-∆UBL (a PTEN-binding defective mutant) which robustly induced PTEN polyubiquitination in a lysine (K) 63-dependent but K48-independent manner. While K48-linked polyubiquitin chains direct protein degradation, K63-linked chains regulate a variety of protein functions. SIPL1 binds polyubiquitinated PTEN with significantly higher affinity than non-ubiquitinated PTEN. A SIPL1 mutant, SIPL1-TFLV, is unable to cause PTEN ubiquitination but is capable of PTEN association. Collectively, our results reveal that SIPL1 interacts with PTEN with a low affinity, which results in PTEN polyubiquitination, and that the modification may stabilize the association between SIPL1 and PTEN. We propose a model where SIPL1 mediates the K63-linked ubiquitination of PTEN inactivating it. The downregulation of PTEN, when paired with the tumor-promoting effects of ER and/or PR, stimulates breast tumorigenesis. SIPL1 is an important BC marker and future research should focus on its potential as a therapeutic target. / Thesis / Doctor of Philosophy (PhD)

<p> Kaiso is a unique member of the BTB/POZ (Broad complex, Tramtrak, Bric à brac,/Pox virus and zinc finger) zinc finger family of transcription factors with established roles in development and tumourigenesis. Kaiso was originally identified as a novel binding partner of the Armadillo catenin p120^ctn, a cytosolic co-factor and regulator of the cell-cell adhesion molecule and tumor suppressor E-cadherin. In addition to their roles in cell adhesion, the multifunctional Armadillo catenins also regulate gene expression, thus providing at least two mechanisms for their contribution to tumourigenesis. The discovery of a novel interaction between p120^ctn and the transcription factor Kaiso was therefore consistent with gene regulatory roles for Armadillo catenins. Interestingly, Kaiso represses transcription via a sequence-specific DNA binding site (TCCTGCnA) as well as through methylated CpG di-nucleotides, and one role of nuclear p120^ctn is to inhibit Kaiso DNA-binding and transcriptional repression. We recently identified sequence-specific Kaiso binding sites in a subset of Wnt/β-catenin/TCF tumour-associated target genes, and here we present data characterizing cyclin D1 as a putative Kaiso target gene.</p> <p> Kaiso binds the cyclin D1 promoter in vitro and in vivo, and artificial promoter assays revealed that Kaiso overexpression results in the repression of a cyclin D1 promoter luciferase reporter. Since cyclin D1 is highly amplified in ~50% of human breast tumours, and a cancer profiling array demonstrated that Kaiso is misexpressed in ~40% of human breast tumours, we hypothesized that Kaiso represses and regulates cyclin D1 expression to inhibit breast tumourigenesis. In fact, examination of Kaiso expression in human breast cell lines demonstrated that cyclin D1 mRNA levels were upregulated in Kaiso-depleted cells. My studies further revealed that methylation-dependent Kaiso-DNA binding may contribute to Kaiso's transcriptional repression of the cyclin D1 promoter. We also determined that Kaiso inhibits, while p120^ctn activates, β-catenin-mediated activation of the cyclin D1 promoter. These findings further support a role for Kaiso and p120^ctn in breast tumourigenesis via their modulation of the canonical Wnt signaling pathway which is highly implicated in human tumourigenesis. Together these findings support our hypothesis that Kaiso regulates cyclin D1 expression. However, further studies are required to elucidate the mechanism employed by Kaiso to elicit cyclin D1 repression and to examine how this activity may contribute to breast tumourigenesis.</p> / Thesis / Master of Science (MSc)

The Signal Transducer and Activator of Transcription-3 (Stat3) is a transcription factor that is required for transformation by a number of oncogenes, while a constitutively active form of Stat3 alone is sufficient to induce neoplastic transformation. It was previously demonstrated that cell to cell adhesion causes a dramatic increase in the activity of Stat3 in both normal and tumour cells. This hinted for the first time at the possibility that the role of Stat3 may differ upon cellular confluence. To examine such a mechanism, it is important to evaluate the effect of Stat3 downregulation at different time-points relative to confluence. To examine this, two different approaches for Stat3 downregulation were used: (1) the introduction of high levels of peptidomimetics analogs, which block the Stat3-SH2 domain by using a technique of in situ electroporation. (2) Treatment with two platinum compounds that inhibit Stat3 binding to activated receptors and DNA. The results demonstrated that Stat3 downregulation in vSrc or TAg transformed mouse fibroblast cells or in breast carcinoma lines, induced apoptosis which was more pronounced post-confluence at the time of its peak activity. In contrast, in sparsely growing normal mouse fibroblasts, Stat3 inhibition induced merely a growth retardation. However, in densely growing normal fibroblasts, Stat3 inhibition induced apoptosis. At least in part, apoptosis induced by Stat3 inhibition was mediated by p53, as shown by the resistance to cell death by Stat3 downregulation in colon carcinoma cells, HCT116, where the p53 gene is ablated. Overall, our observations point to the possibility that constitutive activation of Stat3 may lead to tumourigenesis by downregulating wt-53 in cancers that do not have p53 mutations. As a result, targeting Stat3 in cancers with wt-p53 may be a promising therapeutic approach for restoring p53 function, thereby inducing p53-mediated apoptosis. Next, we examined the effect of constitutively activated Stat3 as an oncogene. Stat3C expression in rat F111 fibroblasts induced anchorage independence, but to a lower degree compared to other oncogenes, such as vSrc. Surprisingly Stat3C expression increased gap junction intercellular communication, despite the fact that other oncogenes such as vSrc or vRas effectively block gap junctions. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2009-04-26 01:09:21.654

<p>Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the <i>MEN1</i> and <i>VHL</i> tumour suppressor genes, is still elusive. The protein product of the <i>MEN1</i> gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling.</p><p>Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous <i>Men1</i> mice. For comparison, normal and <i>MEN1</i> non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between <i>Men1</i> expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in <i>Men1</i> mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and <i>Men1</i> mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of <i>Men1</i> and wt mice were observed.</p><p>In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression.</p><p>Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.</p>