Pathology - Theses
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ItemInvestigating the role of hypoxia in tumour progression in breast cancer( 2015)Metastasis is a major cause of morbidity and mortality in breast cancer patients. The molecular processes and mediators that underpin this process have yet to be completely delineated. Hypoxia, the state of reduced oxygen conditions, occurs frequently in solid tumours and is a factor of poor prognosis for patient outcome. The upregulation of HIF-1α, the main mediator of the hypoxic response pathway, has been implicated in several different facets of tumour progression, including tumour growth, angiogenesis, therapy resistance and metastasis. Hypoxia has been shown to induce Epithelial-to-Mesenchymal Transition (EMT), a highly conserved developmental program that facilitates tumour cell dissemination. It is thought that EMT is co-opted by epithelial tumour cells in order to acquire a degree of plasticity, allowing them to undergo a number of genetic, biochemical and morphological changes to adopt a mesenchymal phenotype. This results in the loss of polarity, and the gain of migratory and invasive capabilities. EMT is regulated by a core cassette of transcription factors, including Snail, Slug, Twist, Zeb1 and Zeb2. Zeb1 is the most proximal transcription factor, however, how hypoxia modulates Zeb1 expression is not known. This study demonstrates that Siah, a family of E3 ubiquitin ligases and a master regulator of HIF-1α protein expression, binds to and targets Zeb1 for proteasomal degradation. Loss of Siah2 is sufficient to cause spontaneous EMT in tumour cells derived from the PyMT murine model of breast cancer. On the other hand, EMT induction led to the decrease in Siah protein expression. This work is the first to describe a post-translational mechanism of regulation of Zeb1 and further defines the relationship between hypoxia and EMT. There are, in fact, two forms of hypoxia in a growing tumour, chronic hypoxia and intermittent hypoxia. Chronic hypoxia describes the long-term limitations on oxygen diffusion caused by abnormal tumour vasculature. While intermittent hypoxia refers to the fluctuations of oxygen tension in a tumour, caused by the aberrant and temporary closing and reopening of tumour-supplying blood vessels. The consequences of these two different types of hypoxia in breast cancer have not yet been well characterised. Using the orthotopic, syngeneic PyMT murine model of breast cancer, it was found that intermittent hypoxia-treated cells gave rise to a greater number of larger lung metastases in vivo. This was facilitated by an enhanced ability for anchorage-independent growth, increased clonogenicity, the induction of a pro-tumourigenic gene expression and secretory profile, and the increase in tumour-initiating capacity through the gain of cancer stem cell properties. RNA sequencing of hypoxia-treated cells found distinct gene expression patterns between treatment groups. While, pathway analysis revealed a marked enrichment of immune-related pathways and a downregulation of DNA replication and cell cycle pathways, by both chronic and intermittent hypoxia. Interestingly, chronic hypoxia also upregulated extracellular matrix degradation pathways, in spite of the lack of an overt EMT in cells. These results unveil novel mechanisms and pathways involved in hypoxia-mediated metastasis while highlighting the extensive effects of hypoxia signalling in cancer. Taken together, this work demonstrates the complexity of hypoxia signalling in tumour progression. Not only does it endow tumour cells with an aggressive, tumour-initiating phenotype, but it also contributes to the priming of the tumour microenvironment to be pro-inflammatory and immunosuppressive and ultimately, tumour-promoting.
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ItemBiomarkers in ductal carcinoma in situ( 2016)Ductal carcinoma in situ (DCIS), a non-invasive form of breast cancer and a non-obligate precursor of invasive carcinoma of the breast, displays heterogeneous behaviour. Most DCIS are adequately managed by local surgical excision alone, but in 20-30% of cases, disease recurrence occurs after local surgical excision either as DCIS or invasive carcinoma. Accurate identification of these two clinical outcome groups at the time of diagnosis is desirable to allow appropriate treatment allocation. In this thesis, genomic and epigenetic alterations in DCIS epithelium, including copy number aberrations, somatic mutations, and DNA methylation were investigated as markers of DCIS biology and outcome. In addition, the expression and significance of LRH-1, a nuclear receptor which acts as a transcription factor, was investigated in both invasive carcinoma and DCIS. Copy number analysis of DCIS of known clinical outcome identified amplification of 20q13 to be associated with disease recurrence, but this was unable to be validated on an independent cohort. Targeted next generation sequencing of a panel of breast cancer-relevant genes revealed that the mutational profile of DCIS was similar to that reported for invasive carcinomas, with the most frequently mutated genes being GATA3, PIK3CA, and TP53. A high prevalence of GATA3 mutations in DCIS was observed and TP53-mutant DCIS was associated with high stromal tumour infiltrating lymphocytes. Mutations of RUNX1 were a novel finding, not previously reported in DCIS. Promoter methylation of a candidate gene panel, consisting predominantly of known tumour suppressor genes, was associated with adverse tumour features in DCIS. Methylation load varied among DCIS cases, suggesting that methylation differs in importance in the tumorigenesis of DCIS, and that assessment of methylation may be useful as a biological classifier of DCIS. Finally, LRH-1 mRNA expression patterns in breast cancers was similar to that reported for breast cancer cell lines and distinct LRH-1 immunohistochemical staining patterns were associated with tumour phenotype in both invasive breast carcinoma and DCIS. The results of this thesis demonstrate that copy number alterations, somatic mutations, DNA methylation, and LRH-1 expression are indicative of DCIS phenotype and hence biology. These markers showed promise as prognostic biomarkers, although validation of their utility was hampered by the small number of pure DCIS cases with both adequate genomic material and long-term clinical outcome data. Nonetheless, the findings of this thesis indicate that assessment of these biomarkers can be performed in routine diagnostic tissue material and provide several avenues for future research.