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Progression to metastatic disease is the leading cause of deaths resulting from breast cancer. Understanding the mechanisms underlying a cell's ability to move away from its site of origin and populate a distant site is important for the future development of therapies. The interactions between a tumor cell and the microenvironment can modulate a cell's ability to invade through tissues and access distant organs. In this study we present evidence indicating the differential modulation of invasive and proliferative phenotypes by hyaluronan present in the cellular microenvironment.We establish the role of CD44, the primary receptor for hyaluronan, in breast cancer progression and metastasis through the use of transgenic mouse models of breast cancer. While no differences were seen in the onset of primary breast tumors, mice expressing CD44 had a reduced rate of pulmonary metastasis compared to mice that lacked CD44. This establishes an anti-invasive role for CD44 in breast tumor progression. We also identify a decreased population of alveolar macrophages in CD44 negative mice that could affect metastatic breast cancer cell colonization of the lungs.We then focused our study in vitro, where we assessed the invasive properties of breast cancer cells as they move through three dimensional (3D) matrices containing or lacking hyaluronan. We show that in 3D type I collagen gels, breast cancer cells invade more readily in the absence of hyaluronan compared to when hyaluronan (HA) is embedded within the gel. HA mediated inhibition of invasion is dependent on CD44 binding as demonstrated through the use of a CD44 functional blocking antibody.We also show that HA promotes differential phenotypes of breast cancer cell. HA promotes filopodia formation and invasion when soluble in the cell microenvironment. Alternatively, matrix-embedded HA inhibits invasion and promotes migration through the formation of lamellipodia. The differential HA invasive and proliferative phenotypes are mediated by differential activation of ERK or γPAK. Activation of γPAK is mediated by CD44 while ERK activation by HA occurs by CD44 independent mechanisms.We also demonstrate an inhibition of MMP9 mediated invasion by HA when embedded within a type IV collagen matrix, but not a type I collagen matrix. This differential activity indicates that it is not only the immobilization of HA in a matrix that determines its activity, but also the context in which it is present within the matrix.These data underscore the importance of studying matrix components in an environment that closely resembles in vivo conditions. HA is a prime example as it has the capability of both promoting and inhibiting invasion depending on how it is presented to a cell. Differential HA activity also underlies the importance of understanding extracelluar matrix degradation and the release of matrix components as these can adversely affect disease progression.

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.

Gene expression profiles have identified five major molecular breast cancer subtypes (Luminal A, Luminal B, Basal-like, HER2+/estrogen receptor− , and Normal Breast-like) that show significant differences in survival. The cost and complexity of gene expression technology has impeded its clinical implementation. By comparison, immunohistochemistry is an economical technique applicable to the standard formalin-fixed, paraffin-embedded material commonly used in hospital labs, and has the advantage of simultaneously interpretation with histomorphology. In this thesis, I hypothesize that a surrogate panel of immunohistochemical biomarkers can be developed to discriminate the breast cancer biological subtypes. The main study cohort consists of over 4000 primary invasive breast tumors, assembled into tissue microarrays. These patients were referred to the British Columbia Cancer Agency between 1986-1992 and have staging, pathology, treatment and follow-up information. In summary, our results demonstrate that (1) the rabbit monoclonal antibody, SP1, is an improved standard for immunohistochemiscal estrogen receptor assessment in breast cancer; (2) the transcription factor, GATA-3, is almost exclusively expressed among estrogen receptor positive tumors but does not seem to predict for tamoxifen response among estrogen receptor positive patients; (3) the proliferation marker, Ki-67, together with HER2 can segregate Luminal A from Luminal B subtypes, which carry distinct risks for breast cancer relapse and death; and (4) the inclusion of the basal markers EGFR and ck5/6 to “triple negative” breast cancers provides a more specific definition of basal-like breast cancer that better predicts patient survival. These results consistently demonstrate that an immunopanel of six biomarkers (estrogen receptor, progesterone receptor, HER2, Ki-67, epidermal growth factor receptor and cytokeratin 5/6) can be readily applied to standard pathology specimens to subtype breast cancer samples based on their underlying molecular biology. These findings have been considered sufficient to justify application of this panel onto NCIC (MA5, MA12) and CALGB (9341 and 9741) clinical trials specimens. This followup work which is underway and will determine if the six marker immunopanel can guide decisions about which patients need aggressive systemic drug treatment, and thereby ensure patients get the most effective, individualized adjuvant systemic therapy for their breast tumor.

Breast cancer is one of the leading causes of death of women in North America. Signal Transducer and Activator of Transcription (Stat)3 and Stat5 are transcription factors involved in normal breast development, and are hyperactivated in 30-50% of breast cancers. Stat5 is required for breast epithelial cell differentiation and is over-expressed in breast cancers with a more differentiated phenotype. Constitutive over-expression of Stat3 in breast cancer, on the other hand, can drive expression of genes involved in survival, migration and angiogenesis. Our group has previously elucidated a novel activation mechanism of Stat3 by cadherin engagement in densely growing cells. We hypothesize that Stat5 and Stat3 are regulators of the balance between differentiation and transformation of breast epithelial cells: Stat3 activation is modulated by a cadherin/Rac1 pathway, and this may be necessary for differentiation. Stat5 on the other hand may play a role in differentiation independent of cell density. Therefore, Stat3 and Stat5 may turn out to be independent prognostic markers for breast cancer. We show here, through pharmacological inhibition experiments, that Stat3 is required for differentiation of HC11 breast epithelial cells, measured by β-casein expression. On the other hand, we also show that constitutively active Rac, a molecule downstream of cadherin in the Stat3 activation pathway, blocks the differentiation of breast epithelial cells. Stat5 is upregulated by hydrocortisone, insulin and prolactin, but is unaffected by density. Stat5 is also required for differentiation; moreover we show that expression of activated Stat5 in HC11 breast epithelial cells promotes a more differentiated phenotype. As an initial approach to biomarker development, we have optimized a quantitative analysis method to assess protein expression profiles of clinically relevant robust biomarkers using a 63 tumour breast cancer cohort. Automated quantitative analysis of protein expression in human breast cancer specimens, including target genes of Stat3 and Stat5, cyclin D1 and p21, are statistically similar to manual scoring, and correlate with clinico-pathological parameters. These data provide a vital link between benchwork and clinical studies, and could lead to possible future predictive biomarkers. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2013-12-24 12:00:40.389

Flaxseed oil (FO), rich in α-linolenic acid, has been shown to inhibit breast cancer growth. One suggested mechanism is through modulation of HER2 expression and signalling. This study determined the effect of FO on the growth of established HER2-overexpressing breast tumours (BT-474) and its interaction with two doses of a primary anti-HER2 therapy, trastuzumab (TRAS), in athymic mice. FO alone had no effect on tumour size, cell proliferation and apoptosis. TRAS (2.5 and 5 mg/kg) reduced tumour size and cell proliferation but had no effect on apoptosis. TRAS (2.5 mg/kg) combined with FO reduced tumour size and cell proliferation and increased apoptosis compared to TRAS (2.5 mg/kg) alone and was just as effective as 5 mg/kg TRAS. TRAS (5 mg/kg) resulted in almost complete tumour regression with or without FO. In conclusion, FO has no effect on BT-474 tumour growth but can enhance the effectiveness of low dose TRAS.

Breast cancer is the most commonly diagnosed cancer, and the second leading cause of cancer-related deaths, among women world-wide. Improved understanding of breast tumourigenesis may facilitate the development of more effective therapies. Peroxisome proliferator-activated receptor (PPAR)γ is a transcription factor that regulates the genes involved in insulin sensitivity and adipogenesis. In vitro and in vivo studies also suggest that PPARγ suppresses breast tumour progression; however, the mechanisms remain to be clarified. In the current study, I investigated the mammary epithelial cell-specific role of PPARγ in 7,12-dimethylbenz[a]anthracene (DMBA)-mediated breast tumourigenesis. Mammary epithelial cell-specific PPARγ knockout (PPARγ-MG KO) mice and their congenic, wild-type controls (PPARγ-WT) were treated with either DMBA alone or in combination with a PPARγ ligand (rosiglitazone)-supplemented diet, and followed for tumour formation. DMBA-mediated mammary tumour multiplicity decreased 4.5-fold among PPARγ-WT, but only 1.2-fold in PPARγ-MG KO mice upon co-treatment with rosiglitazone. Similarly, compared to respective DMBA alone groups, mammary tumour volumes were decreased, and onset was delayed, more among DMBA + Rosiglitazone treated PPARγ-WT versus PPARγ-MG KO mice. To assess whether DMBA could alter cell growth, in vitro studies using two human breast cancer cell lines were performed. Human MCF-7 and MDA-MB-231 cells were treated with DMBA, rosiglitazone or both, and assessed for changes in proliferation, apoptosis and target gene expression. DMBA exerted minimal effects on proliferation; whereas, treatments induced apoptosis in MCF-7, and necrosis in MDA-MB-231, cells. The expression of MCF-7 PPARγ1 protein increased with all treatments, while MDA-MB-231 PPARγ2 protein and BRCA1 mRNA expression increased following rosiglitazone or co-treatment. This work advances our understanding of the mammary epithelial cell-specific role of PPARγ signaling in DMBA-mediated breast tumourigenesis, and supports a role for PPARγ activation in the suppression of breast tumour progression. These findings may assist with the development of more effective anti-breast cancer agents. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-09-04 11:45:16.472