Breast cancer is the most common cancer and the leading cause of cancer-related death among Canadian women. Despite improvements in treatment and early detection, there is still a need to develop novel therapies for breast cancer management. Aberrant Notch signaling is tumorigenic and is associated with poor clinical outcomes in breast cancer, as well as in several other types of cancer. Activation of Notch signaling requires -secretase-mediated Notch receptor cleavage. Thus, strategies to inhibit Notch signaling, including -secretase inhibition, are being evaluated for potential anti-tumor effects. The strongest justification for targeting Notch in breast cancer, and more specifically for using -secretase inhibitors, came from two studies that reported that the -secretase inhibitor (GSI) Z-LLNle-CHO inhibited the growth of breast cancer cells both in vitro and in vivo without causing significant side effects. In Chapter 2, we compared the enzymatic activities and cytotoxicity of Z-LLNle-CHO to those of two other specific GSIs and three proteasome inhibitors and demonstrated that the cytotoxicity of Z-LLNle-CHO in breast cancer cells is mediated by proteasome inhibition, not by -secretase inhibition. In Chapter 3, we characterized the protein complexes formed in breast cancer cells by the intracellular domains (NICD) of the four Notch paralogs. We found that the assembly of NICD protein complexes is dose-dependent and availability of MAML proteins becomes the limiting factor for continuous formation of NICD/RBPj/MAML transactivation complex. This suggests that the formation of some non-canonical NICD complex might occur preferentially at high levels of NICD, conditions under which aberrant Notch signaling induces tumorigenesis in breast cancer. Consequently, these non-canonical interactions might be good targets to specifically block oncogenic, but not physiological, Notch signaling. In addition, we found that the relative affinities of individual NICD paralogs to several known NICD-interacting proteins were different. This may account for the paralog-specific activities of Notch that have been previously reported. Together, these results may be of value for the development of new reagents to block Notch signaling for therapeutic benefit in breast cancer treatment. / Experimental Oncology

Insulin-like growth factor-I (IGF-I) is a mitogenic polypeptide that induces proliferation and activation of kinase pathways in MCF-7 breast cancer cells. The role of estrogen receptor α (ERα) in mediating responses induced by IGF-I was investigated in cells transfected with small inhibitory RNA for ERα (iERα) or cotreated with the pure antiestrogen ICI 182780. The results showed that IGF-I-dependent phosphorylation of Akt and MAPK, induction of G1–S-phase progression and enhanced expression of cyclin D1 and cyclin E were dependent on ERα. Moreover, these IGF-I-induced responses were also inhibited by the antiestrogen ICI 182780, suggesting that the effects of ICI 182780 as an inhibitor of IGF-I induced responses in breast cancer cells are primarily related to downregulation of ERα. Chemoprotective phytochemicals exhibit multiple activities and interact with several cellular receptors, including the aryl hydrocarbon receptor (AhR). We investigated the AhR agonist/antagonist activities of the following flavonoids: chrysin, phloretin, kaempferol, galangin, naringenin, genistein, quercetin, myricetin, luteolin, baicalein, daidzein, apigenin, and diosmin, in MCF-7 breast cancer cells, HepG2 human liver cells and mouse Hepa-1 cells. The dietary phytochemicals exhibited substantial cell context–dependent AhR agonist as well as antagonist activities, and these are factors that must be considered in risk assessment of overall exposures to AhR agonists. Halogenated aromatic hydrocarbons (HAHs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7,8- pentachlorodibenzo-p-dioxin (PeCDD), 3,3’,4,4’,5-pentachlorobiphenyl (PCBP), 2,3,7,8- tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) bind and activate the aryl hydrocarbon receptor (AhR). It has been assumed that these compounds only differ in their potencies. The SAhRM-like activity of the 5 HAHs was investigated by determining ligand structure dependent differences in their induction of CYP1A1 and interactions of the AhR with a series of coactivators in a mammalian two-hybrid assay in three AhR-responsive cell lines, including mouse Hepa-1, Human HEK293 and human Panc1 cells. There were multiple structure-dependent differences in activation of luciferase activity in these cell lines transfected with VP-AhR and six different GAL4-coactivator chimeras and a GAL4-response element-luciferase promoter construct. The results show that HAHs selectively interact with coactivators and these interactions are dependent on cell-context, and even among HAHs, these compounds exhibit selective receptor modulator activity.

Introduction: Over ten years ago the Saskatchewan Breast Cancer Network (SBCN) was created to work towards better meeting the needs of breast cancer patients and survivors in the province. With funding from the Cancer Program of the Public Health Agency of Canada in June 2009, the SBCN implemented the A Thinking, Learning and Caring Collaboration: Promoting a Healthy Lifestyle program in various communities throughout Saskatchewan.<p> Purpose: The purpose of this study was to evaluate the program implemented in 2009/2010 to: (1) determine to what extent the program was implemented as proposed; and (2) assess to what extent the program met its own goals.<p> Methods: Using a constructivist ethnographic mixed methodology, the data collected included self-report surveys, observational data, and semi-structured interviews, which were conducted with seven members of the Advisory Committee and five breast cancer survivors representing urban, rural, senior, young and Aboriginal demographics. Descriptive analysis was used to tabulate the surveys' results and qualitative analysis followed the structure of the program logic model.<p> Results: The results of this evaluation reveal that the program was implemented as proposed despite unforeseen challenges, which necessitated slight alterations in the timing of events. The program was successful in meeting many of its goals; however, the sustainability of these impacts are unlikely due to the disintegration of the formal Network following the completion of the program.<p> Conclusions: Future research should focus on the role of current medical service providers and grassroots organizations in meeting the needs of breast cancer survivors, beyond treatment for physical symptoms. Evidence surrounding the motivations to adopt healthy choices will facilitate effective program delivery for the diverse group of Canadian breast cancer survivors.

The aryl hydrocarbon receptor (AHR) and estrogen receptor alpha (ERα) are ligand-activated transcription factors. Reciprocal crosstalk between these two receptor systems has been previously established but the exact molecular mechanisms of their interactions remain incompletely understood. Using chromatin immunoprecipitation followed by DNA microarrays (ChIP-chip), I assessed the role of ERα in AHR signalling after dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) treatment in the T-47D human breast cancer cell line. I determined that ERα is recruited to a subset of AHR target genes suggesting that it is a gene-specific modulator of AHR activity. Transcription factor binding site analysis of our data set also revealed that forkhead motifs were over-represented, implying that they may be important in AHR signalling. To address this, I focused on the regulation of cyclin G2 (CCNG2) to determine the importance of FOXA1 (forkhead box A1) in AHR signalling. CCNG2 is a negative regulator of cell cycle and known to be repressed by ERα. Using ChIP, Co-IP, CCNG2 reporter gene constructs and RNA interference targeting FOXA1, I demonstrated that FOXA1 was important for the AHR-mediated and TCDD-dependent induction of CCNG2. Another finding from the ChIP-chip study was that AHR was recruited to estrogen target genes. To determine the importance of this I used zinc-finger nuclease mediated knockout of AHR and studied ERα signalling as well as the role of AHR in the cell cycle using breast cancer cell lines. Focusing on the regulatory regions of trefoil factor 1 (TFF-1) and gene upregulated in breast cancer 1 (GREB1) I determined that AHR had an inhibitory effect. Cell cycle analysis indicated that AHR facilitated cell cycle progression with cells accumulating in both the G¬1 and G2/M phases in the absence of AHR. My novel findings demonstrated the complexity of AHR-ERα crosstalk, its importance in the cell cycle, and the need for further study.

Insulin resistance associated with obesity has been suggested to contribute to an increased risk and poor prognosis for breast cancer. In this study, a HER2/Neu transgenic mouse model of breast cancer was used to assess how obesity-induced insulin resistance and hyperinsulinemia can influence the development and progression of breast cancer. We investigated the effect of a high-fat diet and found a tumor-promoting effect in the absence of overt insulin resistance. In contrast, a high-fat combined with fructose diet induced significant hyperinsulinemia but no tumor promoting or growth effect was observed. Treatment with the anti-diabetic, insulin-lowering agent metformin led to a delay in tumor onset in mice on control diet, but this effect was abrogated by the high-fat fructose diet. These data indicate that the effects and potential interactions of insulin, nutrition and drugs on breast cancer development and progression are complex and require further study.

The aryl hydrocarbon receptor (AHR) and estrogen receptor alpha (ERα) are ligand-activated transcription factors. Reciprocal crosstalk between these two receptor systems has been previously established but the exact molecular mechanisms of their interactions remain incompletely understood. Using chromatin immunoprecipitation followed by DNA microarrays (ChIP-chip), I assessed the role of ERα in AHR signalling after dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) treatment in the T-47D human breast cancer cell line. I determined that ERα is recruited to a subset of AHR target genes suggesting that it is a gene-specific modulator of AHR activity. Transcription factor binding site analysis of our data set also revealed that forkhead motifs were over-represented, implying that they may be important in AHR signalling. To address this, I focused on the regulation of cyclin G2 (CCNG2) to determine the importance of FOXA1 (forkhead box A1) in AHR signalling. CCNG2 is a negative regulator of cell cycle and known to be repressed by ERα. Using ChIP, Co-IP, CCNG2 reporter gene constructs and RNA interference targeting FOXA1, I demonstrated that FOXA1 was important for the AHR-mediated and TCDD-dependent induction of CCNG2. Another finding from the ChIP-chip study was that AHR was recruited to estrogen target genes. To determine the importance of this I used zinc-finger nuclease mediated knockout of AHR and studied ERα signalling as well as the role of AHR in the cell cycle using breast cancer cell lines. Focusing on the regulatory regions of trefoil factor 1 (TFF-1) and gene upregulated in breast cancer 1 (GREB1) I determined that AHR had an inhibitory effect. Cell cycle analysis indicated that AHR facilitated cell cycle progression with cells accumulating in both the G¬1 and G2/M phases in the absence of AHR. My novel findings demonstrated the complexity of AHR-ERα crosstalk, its importance in the cell cycle, and the need for further study.

Insulin resistance associated with obesity has been suggested to contribute to an increased risk and poor prognosis for breast cancer. In this study, a HER2/Neu transgenic mouse model of breast cancer was used to assess how obesity-induced insulin resistance and hyperinsulinemia can influence the development and progression of breast cancer. We investigated the effect of a high-fat diet and found a tumor-promoting effect in the absence of overt insulin resistance. In contrast, a high-fat combined with fructose diet induced significant hyperinsulinemia but no tumor promoting or growth effect was observed. Treatment with the anti-diabetic, insulin-lowering agent metformin led to a delay in tumor onset in mice on control diet, but this effect was abrogated by the high-fat fructose diet. These data indicate that the effects and potential interactions of insulin, nutrition and drugs on breast cancer development and progression are complex and require further study.

Breast cancer has a high propensity to metastasize to bone. While the genetic and epigenetic changes associated with metastatic breast cancer progression are being identified, the changes that drive metastatic progression are poorly understood. Proteases, and in particular matrix metalloproteinases (MMPs), have been shown to play a pivotal role in certain aspects of tumor metastasis by modifying the affected microenvironment. Bone matrix-depositing mouse MC3T3 osteoblasts were co-cultured with metastatic human MDA-MB-23 1 (MDA23 1) cells or the bone-homing MDA-MB 231-1 833/TR (1 833/TR) variant in an effort to identify novel, osteoclast-independent, changes to the tumor/bone microenvironment. Co-culture-induced changes in the complete “protease and inhibitor” expression profile in the osteoblasts and the tumor cells were then determined using targeted murine and human specific microarray chips (CLIP-CHIP TM ). This analysis revealed an increase in the RNA expression of collagenase-3 (MMP 13) in the co-cultured osteoblasts that was confirmed by qPCR. Further, Western blotting indicated increased MIvIP13 protein secretion into the bone matrixltumor microenvironment by the co-cultured MC3T3 cells. The elevation in osteoblast-produced MMP13 was observed when the co- cultured tumor cells were in direct contact or separated by filters. Additionally, the elevation was also induced by conditioned medium derived from separate MDA23 1 or 1 833/TR cultures, which indicates that a soluble factor produced by the tumor cells is capable of inducing MMP 13. One soluble factor that appears to be produced by 1 833iTR cultures is oncostatin M. Oncostatin M is an interleukin-6 family cytokine that is known to upregulate MMP13 synthesis and secretion during chondrogenesis. Genome-wide Affymetrix® analysis revealed, and qPCR analysis confirmed, that oncostatin M receptor-specific subunit RNA was also significantly upregulated in co-cultured osteoblasts. Therefore, breast tumor cells may be capable of initiating protein degradative changes in the bone microenvironment that are independent of the much studied osteolytic degradation initiated by osteoclast activation.

Introduction: Over ten years ago the Saskatchewan Breast Cancer Network (SBCN) was created to work towards better meeting the needs of breast cancer patients and survivors in the province. With funding from the Cancer Program of the Public Health Agency of Canada in June 2009, the SBCN implemented the A Thinking, Learning and Caring Collaboration: Promoting a Healthy Lifestyle program in various communities throughout Saskatchewan.<p> Purpose: The purpose of this study was to evaluate the program implemented in 2009/2010 to: (1) determine to what extent the program was implemented as proposed; and (2) assess to what extent the program met its own goals.<p> Methods: Using a constructivist ethnographic mixed methodology, the data collected included self-report surveys, observational data, and semi-structured interviews, which were conducted with seven members of the Advisory Committee and five breast cancer survivors representing urban, rural, senior, young and Aboriginal demographics. Descriptive analysis was used to tabulate the surveys' results and qualitative analysis followed the structure of the program logic model.<p> Results: The results of this evaluation reveal that the program was implemented as proposed despite unforeseen challenges, which necessitated slight alterations in the timing of events. The program was successful in meeting many of its goals; however, the sustainability of these impacts are unlikely due to the disintegration of the formal Network following the completion of the program.<p> Conclusions: Future research should focus on the role of current medical service providers and grassroots organizations in meeting the needs of breast cancer survivors, beyond treatment for physical symptoms. Evidence surrounding the motivations to adopt healthy choices will facilitate effective program delivery for the diverse group of Canadian breast cancer survivors.

VCAM-1 (CD106) is a transmembrane glycoprotein and involved in many pathological inflammatory processes. VCAM-1 plays an important role in leukocyte adhesion, leukocyte transendothelial migration and cell activation by binding to integrin VLA-1 (£\4£]1). In our preliminary data, we observed 2-3 fold increase in the expression of VCAM-1 in the side population of ovarian cancer, which exhibits stem cell-like properties in ovarian cancer. In addition, we have also found VCAM-1 is upregulated in many breast cancer epithelial cells and directly correlated with breast tumor progression; however, its mechanism of action in tumor biology remains unknown. Here, we describe the establishment and use of breast cancer cell line model systems to dissect the functional roles of VCAM-1 activation in the manifestation of malignant phenotype of human breast cancer. We show that VCAM-1 overexpression in the NMuMG breast epithelial cells increase cell motility rates and chemoresistance to doxorubicin and cisplatin in vitro, conversely, in an established metastatic breast cancer cell line, MDAMB231, we find that knockdown endogenous VCAM-1 expression by small interfering RNA reduced the migration rate . Furthermore, we also demonstrated that knockdown endogenous VCAM-1 expression in MDAMB231 cells reduced the tumor formation in SCID xenograft mouse model. In conclusion, our findings are consistent with the hypothesis that overexpression of VCAM-1 facilitates breast cancer progression by enhancing the malignant properties of breast cancer cells and suggests that targeting of VCAM-1 induced pathways are attractive strategies for therapeutic intervention.