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Metastasis initiation is a complex process encompassing numerous steps. To identify molecular alterations associated with early and late stages of metastasis, we used high throughput screening techniques. Early events in metastasis were investigated by differential proteomic analysis of lymph node-negative and positive breast cancer samples. Two candidate biomarkers (DCN and HSP90B1) were discovered and further validated through tissue microarray analyses. To examine late events in metastasis formation, we prospectively evaluated genomic differences between disseminated tumour cells in bone marrow, and metastatic tumour cells obtained from computed tomography guided biopsies of bone metastases. Results indicate that specific subsets of genes are required for breast cancer cells to initiate bone metastases. Discovery of proteomic and genomic alterations specifically associated with metastases may yield biomarkers capable of stratifying patients into different risk categories. Proteins and genes identified in this work may form the foundation of a biomarker panel for metastatic risk assessment.

Background: Chylothorax occurs in ~4% of children undergoing cardiac surgery. Treatment requires transition to a medium chain triglyceride (MCT) based formula. Breast milk (EBM) is discontinued due to the presence of long chain triglycerides. Objective: To determine the effectiveness of a modified fat breast milk for the treatment of chylothorax. Methods: Infants with chylothorax were eligible. Treatment infants (n=8) received EBM that had been modified by removing the fat layer (centrifugation) from EBM and adding MCT and nutrients to provide 67 kcal/ml and 11 g/100 ml protein. Control infants (n=8) received MCT formula. Results: Volume of chest tube drainage was not different (p<0.40). Treatment infants experienced declines in mean weight (p<0.006), length (p<0.013) and head circumference (p<0.008) z-scores. Conclusion: Modified fat breast milk allowed for successful resolution of chylothorax. Strategies to address poor growth, however, need to be tested before clinical adoption of this novel treatment.

Metastasis initiation is a complex process encompassing numerous steps. To identify molecular alterations associated with early and late stages of metastasis, we used high throughput screening techniques. Early events in metastasis were investigated by differential proteomic analysis of lymph node-negative and positive breast cancer samples. Two candidate biomarkers (DCN and HSP90B1) were discovered and further validated through tissue microarray analyses. To examine late events in metastasis formation, we prospectively evaluated genomic differences between disseminated tumour cells in bone marrow, and metastatic tumour cells obtained from computed tomography guided biopsies of bone metastases. Results indicate that specific subsets of genes are required for breast cancer cells to initiate bone metastases. Discovery of proteomic and genomic alterations specifically associated with metastases may yield biomarkers capable of stratifying patients into different risk categories. Proteins and genes identified in this work may form the foundation of a biomarker panel for metastatic risk assessment.

Background: Chylothorax occurs in ~4% of children undergoing cardiac surgery. Treatment requires transition to a medium chain triglyceride (MCT) based formula. Breast milk (EBM) is discontinued due to the presence of long chain triglycerides. Objective: To determine the effectiveness of a modified fat breast milk for the treatment of chylothorax. Methods: Infants with chylothorax were eligible. Treatment infants (n=8) received EBM that had been modified by removing the fat layer (centrifugation) from EBM and adding MCT and nutrients to provide 67 kcal/ml and 11 g/100 ml protein. Control infants (n=8) received MCT formula. Results: Volume of chest tube drainage was not different (p<0.40). Treatment infants experienced declines in mean weight (p<0.006), length (p<0.013) and head circumference (p<0.008) z-scores. Conclusion: Modified fat breast milk allowed for successful resolution of chylothorax. Strategies to address poor growth, however, need to be tested before clinical adoption of this novel treatment.

Met has been found to be over-expressed in human breast cancer, correlating with disease progression and poor prognosis. Src and Stat3 have also been found to be over-expressed in many human cancers, including breast. Met, Src and Stat3 have all been proposed as potential therapeutic targets for basal-like breast cancer (BLBC), an aggressive subtype of breast cancer defined by expression of Cytokeratin 5/6 and epidermal growth factor receptor (EGFR). In addition, Src and Stat3 have been shown to act co-operatively to promote the transcription of HGF, while Stat3 can also increase the expression of Met mRNA. The goal of this study was to determine if Src and Stat3 affect the activity and expression of Met in human breast cancer. The study has also assessed the functional effect of Src, Stat3 and Met blockade on cultured breast cancer cells and a breast tumour xenograft model. Finally, a preliminary assessment was performed of Src, Stat3 and Met as biomarkers for distinct clinico-pathological parameters in a breast cancer cohort, and of the prognostic value of these markers in an online, publically available, breast cancer database. The results demonstrate a density- and Src-dependent increase in Met protein levels in cultured cells, through Stat3 (Src/Stat3-Met). Furthermore Src, Stat3 or Met blockade in tumour xenograft models were found to inhibit primary tumour growth. However treatment with Dasatinib (Src inhibitor) or Met knockdown had no effect on pulmonary metastasis, while Stat3 inhibition (CPA7) increased metastasis, indicating that Stat3 may have a protective function in metastatic breast cancer. Finally Src and the Stat3 target gene, Cyclin D1, were found to correlate with distinct clinico-pathological parameters in a human breast cancer cohort. My study has identified a Src/Stat3-regulated Met pathway in human invasive breast cancer. These findings further provide insight into the minimal effectiveness of Src and Met inhibitors as single agent therapeutics in breast cancer treatment. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2014-01-23 23:36:06.52

The C-terminal region of breast cancer susceptibility gene 1 (BRCA1) contains a pair of tandem BRCT repeats that are critical for the tumour suppressor function of BRCA1. BRCT repeats are present in a large of number of proteins that are implicated in the cellular response to DNA damage. A subset of tandem BRCT domains, including those of BRCA1, functions as phosphorecognition modules. Aside from BRCA1, the precise molecular mechanisms of the BRCT repeats of other proteins remain largely unknown. We determined the crystal structure of the tandem BRCT domain of human mediator of DNA checkpoint 1 (MDC1) at 1.45 Å resolution. Our structural and biochemical studies suggest that the tandem BRCT domain of MDC1 functions as the predominant histone variant, γH2AX phosphorecognition module and that the interaction is critically dependent on the free carboxylate group of the γH2AX C-terminal tail. We also determined the crystal structure of the tandem BRCT domain of human BARD1, the in vivo binding partner of BRCA1. Our structure uncovers a degenerate phosphopeptide binding pocket that lacks the key arginine critical for phosphopeptide interactions in other BRCT proteins. Our biochemical studies reveal that a flexible tether links ankyrin and BRCT domains in BARD1. Furthermore, the linker is required for the interactions between the CstF-50 WD-40 domain and BARD1, allowing the BARD1 C-terminus to convey DNA damage signals directly to RNA polymerase. Finally, using protease-based and phosphopeptide pull-down assays, we directly assessed the structural and functional effects of 117 single amino acid substitutions in the BRCA1 BRCT domain derived from breast cancer screening programs. None of the variants showing enhanced sensitivity to proteolytic digestion were found to be active in peptide binding, indicating that these missense mutations contribute to BRCA1 loss of function through protein destabilizing effects. A subset of structurally stable variants was defective in peptide binding activity, suggesting that these variants may disrupt the phosphopeptide binding pocket. Taken together, the results reveal that 32% of the variants show structural stability and peptide binding activity that were indistinguishable from those of wild type.

Public health concerns associated with high prevalence of foodborne salmonellosis, the emergence of antibiotic-resistant organisms and the identification of poultry meat and products as one of the most common sources of Salmonella support the need for new pathogen control strategies in the poultry industry. Scientific research has focused on the use of bacteriophages as therapeutic agents for humans and animals; however, limited studies have been conducted on bacteriophage application on food safety, especially on poultry. Therefore, the objective of this study was to evaluate the phage density and exposure time required to reduce Salmonella load on experimentally inoculated chicken meat. In Experiment 1, serovars of Salmonella were tested for antimicrobial susceptibility and rifampicin-resistant isolates were generated. Cocktails of the serovars Enteritidis, Kentucky and Typhimurium (EKT), and Hadar and Heidelberg (HH), were inoculated on chicken breast samples to a target of 104 CFU/g. A mixture of three lytic bacteriophages, active against multiple Salmonella serovars was applied to chicken samples. A total of 84 samples (25 +/- 2 g) per each cocktail were distributed among a negative control, Salmonella-inoculated positive control, Salmonella-inoculated samples treated with the phage mixture at differing titers (105, 106, 107, 108, and 109 PFU/ml) with two identical samples at 0, 15, 30, 60, 120, 360 min at 4 degrees C. Experiment 2 evaluated nalidixic acid-resistant Salmonella Typhimurium among negative control, Salmonella-inoculated control (positive control), Salmonella with two phage titers (105 and 109 PFU/ml) at 0, 30, 60 and 120 min at 25 degrees C and 4 degrees C. Results showed differences in means for Salmonella cocktail EKT ranged from 0.1 to 0.7 log10 CFU/g with 0.7 log10 for 108 PFU/ml, 30 min, 4 degrees C. For Salmonella cocktail HH, reductions ranged from 0.1 to 0.4 log10 CFU/g with 0.4 log10 on samples treated with 108 PFU/ml, 120 min, 4 degrees C. For the Experiment 2, a higher phage concentration (109 PFU/ml) at 120 min post-inoculation storage at 25 degrees C was required to yield a 0.9 log10 difference in means. These findings showed that higher concentrations of bacteriophage were more effective controlling Salmonella than lower ones at both temperatures. In addition, temperature, time and bacterial attachment may influence phage efficacy.

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.

Breast cancer is the most common malignancy affecting women in developed countries. The BRCA1 and BRCA2 germline mutations predispose carriers to breast and ovarian cancers and account for approximately 10% of all breast cancer cases. Diets rich in micronutrients, such as carotenoids, folate, vitamin C and E are associated with reduced breast cancer risk. / Folate functions in one-carbon metabolism, and is an important factor in DNA-synthesis, DNA repair as well as DNA methylation. Moderate folate deficiency induces global DNA hypomethylation, gene promoter CpG hypermethylation and excessive uracil incorporation into DNA, causing genome instability and successively increasing cancer risk. Genome instabilities such as chromosome 17 mal-segregation and Her2 amplification are frequently observed in human breast tumours. / Methionine, another key factor in one-carbon metabolism, is the sole methy-donor for DNA methylation. Many human tumours are methionine-dependent, a phenotype characterised by the inability of cells to grow when methionine is replaced by its precursor homocysteine, causing elevated homocysteine levels, global DNA hypomethylation when methionine is restricted. In addition, genetic polymorphisms may affect enzyme activity and modulate cancer risk. / This thesis describes a study on the impact of selected nutrients, growth hormones and in vivo genome stability on breast cancer risk in BRCA1 or BRCA2 germline mutation carriers. Peripheral blood lymphocyes of BRCA germline mutation carriers and healthy non-carrier controls were studied for the impact of folic acid deficiency on genome damage and the methionine-dependence phenotype (MDP; in combination with common polymorphisms in one-carbon metabolism) on breast cancer risk, respectively. Plasma IGF-1 and IGFBP-3 were determined and chromosome 17 aneuploidy and Her2 amplification were assessed in mononucleated lymphocytes to establish the association of these markers on breast cancer risk in BRCA germline mutation carriers, independently or in combination with plasma folate, vitamin B12, homocysteine, selenium and common gene variants in the one-carbon metabolism, DNA repair genes or glutathionine S-transferase. / Results indicated that folic acid deficiency was a much more important factor affecting chromosome instability than carrying a BRCA1 or a BRCA2 germline mutation. In addition, MDP was associated with development of breast cancer in BRCA1 germline mutation carriers and appeared to be affected by common polymorphisms in methyltetrahydrofolate reductase. The methionine synthase (MTR) A2756G polymorphisms was associated with elevated cell growth when methionine is present in excess and was the only polymorphism studied that was associated with breast cancer risk in BRCA1 and BRCA2 germline mutation carriers. In addition, chromosome 17 aneuploidy, Her2 amplification and plasma IGF-1 and IGFBP-3 did not directly affect breast cancer risk; however, these biomarkers were significantly correlated with each other and MTR A2756G, suggesting a common mechanism for their inter-relationship. Plasma folate, vitamin B12, homocysteine and selenium of BRCA1 and BRCA2 germline mutation carriers were not associated with breast cancer risk and did not differ from non-carrier relatives. Further research with larger study populations are required to confirm these findings. / Thesis (PhDPharmacy)--University of South Australia, 2006.