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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.

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.

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.

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.

The problem investigated Four studies investigated the phenomenon of cancer-related fatigue (CRF) in women who had received adjuvant treatment for early-stage breast cancer, with a view to reducing the diagnostic uncertainty surrounding the syndrome and thus facilitating progress in both clinical management and aetiological research. Procedures and results A cross-sectional study of 109 women compared a ???cancer-specific??? self-report questionnaire (FACT-F) (canvassing fatigue symptoms) and a more generic questionnaire (SPHERE) (identifying depression and fatigue). Thirty-seven percent of women reported fatigue. Overall in 20%, fatigue was associated with psychological distress. Seventeen percent of women had fatigue but no depression. A qualitative study utilised focus groups to identify and compare the distinctive features of CRF with those of women with chronic fatigue syndrome (CFS). A similar set of symptoms was found in both groups, including overwhelming fatigue, un-refreshing sleep and subjective concentration problems. However, women with CFS also reported myalgia and arthralgia. Using the Structured Clinical Interview for Neurasthenia- SCIN, the third study compared the symptoms of three groups of women with fatigue: those with CRF, CFS or major depression. The detailed ???interviewer guide??? provided explicit directions for evaluating and classifying symptoms. This study confirmed the core symptom of ???profound fatigue unrelieved by rest???, and additional features that distinguished between the clinical diagnoses. The fourth study compared features of the evolution of clinically-identified fatigue syndromes in women from two prospective cohort studies; women with post-cancer fatigue (PCF) and women with post-infective fatigue syndrome (PIFS). Major conclusions A syndrome of PCF, present at least six months following adjuvant treatment and unexplained by medical or psychiatric disorder was investigated. The characteristics of PCF and those of CFS are very similar, with the fatigue state having indistinguishable descriptors. Longitudinal evaluation of the symptom complexes of PCF and PIFS suggests divergent pathways may be relevant. Co-morbid features like sleep disturbance; physical deconditioning and mood disturbance may be implicated as factors in the evolution and prolongation of PCF. These studies provide a basis for a more uniform and rigorous classification system - a necessary first step towards advancing the field both in investigating aetiology and new intervention strategies.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / A major clinical dilemma in the management of patients with early stage breast cancer is determining which patients are at risk of subsequent recurrence so that these patients may be offered adjuvant therapies. Currently, the major prognostic factors used in determining prognosis including nodal status and tumour size fail to identify up to 30% of patients classified as having low risk disease who subsequently recur with breast cancer. Therefore, there is a great need for new methodologies that can sensitively and specifically identify disseminated disease. During this study, the major aims were to use immunobead-based and methylation-based methods for the detection of occult disseminated disease in peripheral blood samples obtained from breast cancer patients. Firstly, an RT-PCR assay was developed that identified a panel of gene specific markers that could be used for sensitive and specific detection of any circulating epithelial cells with minimal risk of false positive results from contaminating haematopoietic compartment cells. These gene markers were used to assess breast cancer patients using immunobead enrichment prior to RT-PCR (IB RT-PCR) and resulted in the detection of circulating epithelial cells in 21157 peripheral blood samples. Importantly, some of these patients had been deemed lymph node negative and at low risk of relapse. Furthermore, it was decided to screen for gene promoter region methylation in freely circulating DNA in the plasma fraction, and in DNA from the nuclei of epithelial cells captured using the immunomagnetic enrichment method. The analysis of gene loci methylation of patient samples was conducted concurrently with the analysis of the frequency of gene loci methylation in mononuclear cells from normal individuals. Surprisingly, a high proportion of normal individuals were methylated at high frequency at gene loci that have previously been reported to be tumour specific. This finding had implications for the concurrent study using methylation as a marker of occult disseminated disease in breast cancer patients. The analysis of blood samples using the methodology presented in this thesis for the early detection of breast cancer dissemination, has the potential to be developed to a point where it can be introduced into the clinical setting. Once fully developed and validated, this minimally invasive methodology, that causes little discomfort to the patient, could help to improve breast cancer staging, and provide important prognostic information ensuring the identification of those early stage breast cancer patients with the greatest risk of relapse and in most need of adjuvant therapies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1097066 / Thesis (Ph.D.) -- University of Adelaide, Dept. of Medicine, 2003

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / A major clinical dilemma in the management of patients with early stage breast cancer is determining which patients are at risk of subsequent recurrence so that these patients may be offered adjuvant therapies. Currently, the major prognostic factors used in determining prognosis including nodal status and tumour size fail to identify up to 30% of patients classified as having low risk disease who subsequently recur with breast cancer. Therefore, there is a great need for new methodologies that can sensitively and specifically identify disseminated disease. During this study, the major aims were to use immunobead-based and methylation-based methods for the detection of occult disseminated disease in peripheral blood samples obtained from breast cancer patients. Firstly, an RT-PCR assay was developed that identified a panel of gene specific markers that could be used for sensitive and specific detection of any circulating epithelial cells with minimal risk of false positive results from contaminating haematopoietic compartment cells. These gene markers were used to assess breast cancer patients using immunobead enrichment prior to RT-PCR (IB RT-PCR) and resulted in the detection of circulating epithelial cells in 21157 peripheral blood samples. Importantly, some of these patients had been deemed lymph node negative and at low risk of relapse. Furthermore, it was decided to screen for gene promoter region methylation in freely circulating DNA in the plasma fraction, and in DNA from the nuclei of epithelial cells captured using the immunomagnetic enrichment method. The analysis of gene loci methylation of patient samples was conducted concurrently with the analysis of the frequency of gene loci methylation in mononuclear cells from normal individuals. Surprisingly, a high proportion of normal individuals were methylated at high frequency at gene loci that have previously been reported to be tumour specific. This finding had implications for the concurrent study using methylation as a marker of occult disseminated disease in breast cancer patients. The analysis of blood samples using the methodology presented in this thesis for the early detection of breast cancer dissemination, has the potential to be developed to a point where it can be introduced into the clinical setting. Once fully developed and validated, this minimally invasive methodology, that causes little discomfort to the patient, could help to improve breast cancer staging, and provide important prognostic information ensuring the identification of those early stage breast cancer patients with the greatest risk of relapse and in most need of adjuvant therapies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1097066 / Thesis (Ph.D.) -- University of Adelaide, Dept. of Medicine, 2003