The mammary gland consists of an organized network of epithelial ducts and lobules. This histoarchitecture can be recapitulated in vitro by culturing mammary epithelial cells as 3D spheroids embedded in a reconstituted basement membrane. I first used this assay to characterize the role of cell-cell and cell-ECM adhesion in the formation and polarization of the apical junction complexes in normal mammary epithelial cells. Cell-cell adhesion alone was sufficient to initiate polarized junction assembly. However, the addition of exogenous ECM generated a spatial polarity signal dependent on laminin-1 and α6 and β1 integrins. This caused clusters of mammary epithelial cells to re-localize the junctional complexes to the center of the spheroid prior to lumen formation. In ductal breast carcinoma, a critical hallmark is the loss of normal polarized tissue architecture without the induction of an epithelial-to-mesenchymal transformation (EMT). Thus, misregulation of molecules that function as polarity determinants may contribute to ductal tumor progression. Podocalyxin is an anti-adhesive glycoprotein that may be involved, as it is important in epithelial morphogenesis, and its overexpression in clinical breast tumors is associated with poor outcome. Despite this, overexpression of podocalyxin in normal mammary epithelial cells did not disrupt 3D morphogenesis or apicobasal polarity. However, its overexpression in non-metastatic breast tumor cells did perturb the architecture and growth of tumor spheroids in vitro and it facilitated subcutaneous tumor growth in vivo without causing an EMT. Mechanistically, podocalyxin localized to and expanded non-adhesive membrane domains and induced microvillus formation that was dependent on its extracellular domain and Rho GTPase-regulated actin polymerization. Podocalyxin also recruited its intracellular binding partners NHERF-1 and ezrin via its cytoplasmic tail. Strikingly, the formation of this protein complex was not required for microvillus formation. Additionally, podocalyxin delayed cell-cell aggregation and decreased the initial adhesion, spreading and strength of attachment of tumor cells to fibronectin where it restricted β1 integrin localization to the basal/attached domain. These alterations in adhesion possibly contributed to podocalyxin's ability to increase growth factor-dependent tumor cell migration. Altogether, these data indicate that podocalyxin overexpression may facilitate a ductal tumor-like progression that involves EMT-independent alterations in tissue architecture.

Cationic antimicrobial peptides (CAPs) are small peptides that constitute an important defence against microbial pathogens. Certain CAPs also possess anticancer properties. NRC-03 and NRC-07 are pleurocidins derived from winter and yellowtail flounder, respectively. The purpose of this investigation was to study the anticancer properties of NRC-03 and NRC-07. NRC-03 and NRC-07 killed breast cancer cells, including P-glycoprotein-overexpressing cells, in a time-dependent manner that peaked at 4 h. NRC-03 and NRC-07 lysed breast cancer cells by a mechanism that involved cell binding, mitochondrial destabilization, nuclear localization, and significant membrane damage. Interestingly, NRC-07, but not NRC-03, caused DNA fragmentation. NRC-03 and NRC-07 killed normal human epithelial cells, but did not kill endothelial cells or fibroblasts, or lyse human erythrocytes. NRC-03, and to a lesser extent NRC-07, had chemo-sensitizing properties, suggesting promise for their inclusion in combinational treatment regimens. Importantly, intratumoural injections of NRC-03 or NRC-07 inhibited tumour growth in a mouse model of breast cancer. Fetal bovine serum dose-dependently reduced cell killing by NRC-03. NRC-03 was degraded in human and mouse serum, which limited its potency. NRC-03- and NRC-07-induced cytotoxicity correlated with expression of several different negatively-charged molecules, rationalizing the generation of [D]-NRC-03, which carries the same positive charge as NRC-03, and was more potent but less selective for cancer cells than NRC-03. [D]-NRC-03 was also cytolytic and exhibited in vivo anticancer properties. To further test the clinical potential of NRC-03- and NRC-07-resistant cells were generated. NRC-03 and NRC-07 bound to resistant cells to a lesser extent than parental cells and were phenotypically distinct. Importantly, NRC-03- and NRC-07-resistant cells were killed by chemotherapeutic drugs, as well as [D]-NRC-03. These studies demonstrate that NRC-03, NRC-07, and [D]-NRC-03 are cytolytic peptides that kill breast cancer cells in vitro and in vivo. While more potent than NRC-03, [D]-NRC-03 requires further modification to minimize its toxicity toward normal cells. Although cancer cells may become resistant to NRC-03 and NRC-07 over time, resistant cells are still killed by other cytotoxic drugs, thereby reinforcing the value of adding these peptides to combinational regimens for the treatment of breast cancer.

Most human breast tumors have mutations in the growth factor/phosphatidylinositol 3’ kinase (PI3K) pathway. These can occur in genes coding for receptors, adaptor proteins, catalytic and regulatory subunits of PI3K, downstream kinases, or antagonistic tumor suppressors. While each genetic change results in elevated PI3K signaling, and all major breast cancer subtypes show pathway activation, the specific mutations involved in any one tumor may play an important role in defining tumor subtype, prognosis and sensitivity to therapy. Here, I describe mouse models of PI3K-induced breast cancer. First I generated mice that express Pik3ca cDNA under control of the ROSA26 promoter, in a Cre-dependent and therefore tissue specific way. I have generated four strains of knock-in mice: R26-Pik3cawt, R26-Pik3caE545K, R26-Pik3caH1047R, and R26-Pik3caE545K-H1047R, which can be induced to express wild type, helical domain, kinase domain and double mutant forms of mouse p110α, respectively. Mice expressing mutant Pi3kca develop mammary tumors, but the phenotypic spectrum for each mutation is unique. Indeed, many E545K mammary tumors are ii vascularized, whereas H1047R tumors are not. Using these models, I have compared downstream signaling properties of E545K and H1047R. The potential for improved breast cancer treatment lies in combination therapies that target more than one oncogenic pathway. To develop such treatments, we need good mouse models, and an understanding of the oncogenic network. To this end, my Pik3caH1047R model was mated to p53 and PTEN knockout mice, and to mice with active Notch1 signaling. In each case, genetic cooperation was observed and characterized. Oncogenic PI3K cooperated with p53-loss and active Notch1 to decrease survival and alter tumor phenotype in distinct ways. Loss of PTEN cooperated with oncogenic PI3K to alter tumor type, decrease average age at end point, and increase the number of tumors per mouse. Overall, I have shown that Pik3caE545K and Pik3caH1047R are sufficient to induce mammary tumors, and that tumor characteristics differ with these mutations, and with cooperating genetic changes.

Breast cancer is the leading cause of cancer death among women worldwide and polyphenols are under investigation as an alternative to conventional treatment approaches of breast cancer. The anti-inflammatory and anti-proliferative activities of polyphenols have been demonstrated in many studies, yet cellular targets and the underlying cellular mechanisms remain unclear. The overall goal of this study was to investigate the anti-inflammatory and cytotoxic properties of polyphenol compounds extracted from the mango variety Keitt in MCF-12A breast non-cancer and MDA-MB231 breast cancer cells by assessing the modulation of signaling pathways involved in inflammation and carcinogenesis. Mango polyphenols were identified by HPLC-MS analysis. The generation of reactive oxygen species was performed using fluorescence intensity in the DCFH-DA assay. Gene expression was analyzed by qRT-PCR, and protein expression was conducted by Western Blotting and Multiplex Bead assay analysis. Bioactive compounds identified in the mango pulp by HPLC-MS included a great variety of polyphenols such as gallic acid, galloyl glucosides with different degree of polymerization and other polyphenols. The anti-inflammatory activities of mango polyphenols were evaluated in MCF-12A non cancer breast fibroblasts. An inflammatory microenvironment for MCF-12A breast cells was induced with tumor necrosis factor alpha (TNF-α). The generation of reactive oxygen species was suppressed significantly compared to cells induced with TNF-α, where there was no significant difference between the concentrations of mango polyphenol extract. Results showed a significant down-regulation of mRNA and protein expression of inflammatory genes involved in the PI3K/AKT pathway and related downstream targets such as NF-κB and mTOR involved in biological processes including cell growth, proliferation and survival. Moreover, mango polyphenols had a significant impact on the miRNA-126-PI3K/AKT axis which plays an important role in inflammation and carcinogenesis, suggesting a potential anti-inflammatory underlying mechanism. The cytotoxic effects of mango polyphenols were investigated in MDA-MB231 breast cancer cells. Mango polyphenols decreased the production of reactive oxygen species; however no significant differences were found between the tested concentrations of mango polyphenols. The gene expression of proapoptotic factors involved in the intrinsic mitochondrial pathway such as cytochrome C and caspase-3 were significantly regulated after mango polyphenol treatment. In addition, the suppression of the PI3K/AKT/mTOR pathway and downstream effectors such as HIF-1α and VEGF as well as the disruption of the miRNA-21-PTEN/AKT axis were identified as potential underlying mechanism of the cytotoxic properties of mango polyphenols. Overall, findings from this study show that mango polyphenols counteract inflammatory and cancerous cell signaling processes; therefore the potential of mango polyphenols in the prevention of breast-cancer focusing on the PI3K/AKT/mTOR-axis should be further investigated.

Decreased expression of the tumor suppressor gene BRCA1 (breast cancer 1, early onset) is frequently observed in sporadic breast tumors, with the decrease not attributed to mutations or hypermethylation of the promoter. A critical regulator of the BRCA1 promoter is the GA-Binding Protein (GABP), a heterotetramer of GABPα and GABPβ. Previous analysis of the GABPβ promoter revealed a regulatory multi-protein complex containing Nuclear Respiratory Factor 1 (NRF-1), which was aberrant in SK-BR-3 cells, resulting in decreased GABPβ and BRCA1 expression. To identify the unknown co-regulators of the NRF-1-containing complex at the GABPβ promoter, an immobilized-template assay containing the complex binding site was utilized. Despite optimization of binding and elution conditions through variations in salt content, Mg2+ concentration and pH, non-specific DNA-binding proteins were present in the column eluate. Further experimentation is therefore required to distinguish between non-specific proteins and complex co-activators. As this complex containing NRF-1 is also able to modulate BRCA1, a regulator of luminal progenitor differentiation, a defect in NRF-1 or complex co-activators could contribute to the abnormal morphogenesis and differentiation of BRCA1-deficient tumors. We therefore investigated the role of NRF-1 in mammary cell morphogenesis and its link to the GABP/BRCA1 pathway. As both GABPβ and NRF-1 are known regulators of nuclear-encoded mitochondrial proteins, this association also provides a link between mitochondrial metabolism and breast differentiation, both of which are frequently disrupted in breast cancer. An inducible lentiviral system was used to generate NRF-1 knockdown cell lines to examine its effect on morphogenesis. Growth in 3D culture resulted in abnormal cell structures having impaired cell polarization and lumen formation. In monolayer culture, prolonged NRF-1 knockdown did not result in decreased BRCA1 or GABP expression. However, these cells did display notable mitochondrial dysfunction, accompanied by the downregulation of several NRF-1 target genes involved in mitochondrial biogenesis including Tfam and cytochrome c. These results suggest a role for NRF-1 in mediating mammary morphogenesis through maintenance of functional mitochondria. Further investigation into the role of the NRF-1-containing complex at the GABPβ promoter during differentiation might also provide insight into the altered cell metabolism and differentiation observed in cancer cells. / Thesis (Master, Biochemistry) -- Queen's University, 2012-09-06 14:39:04.852

No description available.

Most human breast tumors have mutations in the growth factor/phosphatidylinositol 3’ kinase (PI3K) pathway. These can occur in genes coding for receptors, adaptor proteins, catalytic and regulatory subunits of PI3K, downstream kinases, or antagonistic tumor suppressors. While each genetic change results in elevated PI3K signaling, and all major breast cancer subtypes show pathway activation, the specific mutations involved in any one tumor may play an important role in defining tumor subtype, prognosis and sensitivity to therapy. Here, I describe mouse models of PI3K-induced breast cancer. First I generated mice that express Pik3ca cDNA under control of the ROSA26 promoter, in a Cre-dependent and therefore tissue specific way. I have generated four strains of knock-in mice: R26-Pik3cawt, R26-Pik3caE545K, R26-Pik3caH1047R, and R26-Pik3caE545K-H1047R, which can be induced to express wild type, helical domain, kinase domain and double mutant forms of mouse p110α, respectively. Mice expressing mutant Pi3kca develop mammary tumors, but the phenotypic spectrum for each mutation is unique. Indeed, many E545K mammary tumors are ii vascularized, whereas H1047R tumors are not. Using these models, I have compared downstream signaling properties of E545K and H1047R. The potential for improved breast cancer treatment lies in combination therapies that target more than one oncogenic pathway. To develop such treatments, we need good mouse models, and an understanding of the oncogenic network. To this end, my Pik3caH1047R model was mated to p53 and PTEN knockout mice, and to mice with active Notch1 signaling. In each case, genetic cooperation was observed and characterized. Oncogenic PI3K cooperated with p53-loss and active Notch1 to decrease survival and alter tumor phenotype in distinct ways. Loss of PTEN cooperated with oncogenic PI3K to alter tumor type, decrease average age at end point, and increase the number of tumors per mouse. Overall, I have shown that Pik3caE545K and Pik3caH1047R are sufficient to induce mammary tumors, and that tumor characteristics differ with these mutations, and with cooperating genetic changes.

Evidence exists that epidermal growth factor (EGF) and alpha transforming growth factor (αTGF) are important in breast cancer. An inverse relationship between epidermal growth factor receptor (EGF-R) and oestrogen receptor (ER) has been reported by some, (1) but not all workers (2). The aim in this thesis was to develop assays to measure, levels of EGF, and determine EGF-R status in human breast tumours. These results were then correlated with each other, with ER and node status and histological grade (Bloom & Richardson). An additional aim in this thesis was to develop a source of αTGF in conditioned median (CM) from a transformed cell line. After extraction and purification, the αTGF was intended for use as an immunogen to produce a polyclonal antiserum which could be used in either an RIA or ELISA. EGF was measured by a radioimmunoassay (RIA) utilising a rabbit antimouse EGF antiserum. This assay (sensitivity 0.1ng/ml) was demonstrated to have no cross reactivity with αTGF. The EGF-R assay was similar to that described by Sainsbury. (1) In a series of 88 human breast tumours 47 (53.4%) were found to contain extractable EGF. Forty eight (54.5%) were EGF-R positive and 39 (44.3%) were ER positive. A direct relationship between EGF and ER+ ve status was found (p < 0.01). Significantly higher levels of EGF were extracted from ER+ ve tumours (p = 0.049) compared with that from ER-ve tumours. However no relationship between EGF-R and EGF or ER status was found, or between EGF levels and histological grade or node status. A suitable cell line which produced αTGF, was obtained and culture conditions optimised. Alpha-TGF was assayed by a radioreceptor assay which utilised a cell line rich in EGF-R (A431). Extraction of αTGF was based on the principles of molecular grading by gel filtration (Sephadex G50), and ion exchange (Sephadex CM C25). By this process the αTGF was purified and separated it from any EGF present. By this method 20μg of αTGF was produced from 61t of CM. 1) Sainsbury JRC, Farndon JR, Serbet GV, Harris AL. Epidermal-growth-factor-receptors and oestrogen receptors in human breast cancer. Lancet 1985; <i>1</i>: 364-368. 2) Fitzpatrick SL, Brightwell J, Wattliff JL, Barrows GH, Schultz GS. Epidermal growth factor binding by breast tumour biopsies and relationship to oestrogen receptor and progestin receptor levels. Cancer Res 1984; <i>44</i>: 3448-3453.

No description available.

Breast cancer is a major health issue, affecting annually approximately 1.4 million women worldwide. It is a highly heterogeneous disease with the different subtypes having distinct clinical outcomes and different sensitivity to various treatment modalities. The focus of the present dissertation was the identification of novel proteomic prognostic markers for patients with early stage breast cancer. Three different approaches to identify potential prognostic markers were undertaken. First, we hypothesized that since different breast cancer subtypes have distinct clinical outcomes, breast cancer subtype-specific proteins may retain prognostic potential. Second, given the central role of estrogen signaling in breast epithelial cell biology, we hypothesized that estrogen-regulated proteins may be useful in predicting patient outcome. Finally, we hypothesized that genes related to survival based on meta-analyses of publicly available breast cancer tissue microarray data, may also demonstrate prognostic potential at the proteome level. As such, a variety of mass spectrometry-based approaches and biological samples were utilized for the discovery of these potential prognostic protein markers resulting in twenty-four candidates. Upon the identification of candidate biomarkers, a mass spectrometry-based assay for the simultaneous quantification of these proteins in breast cancer tissue samples was established. The developed assay was used for measuring the relative expression levels of the potential biomarkers in a cohort of 96 breast cancer tissue samples from untreated patients with early stage breast cancer. This exercise uncovered two proteins that showed the potential to discriminate between ER-positive patients at high and low risk of disease recurrence, namely KPNA2 and CDK1. In conclusion, the present dissertation describes the development of a preclinical exploratory study, from the discovery to the preliminary verification of potential prognostic biomarkers for breast cancer patients.