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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ItemThe role of hypoxic signalling in the primary tumour microenvironment and metastasis in breast cancer( 2013)Intratumoural hypoxia is a poor prognostic factor associated with reduced disease-free survival in many cancer types, including breast cancer. Under hypoxic conditions, tumour cells signal via the hypoxic response pathway and stabilisation of the HIF-1 transcription factor to increase the expression of genes that promote survival, proliferation, angiogenesis, invasion and metastatic spread. The factors secreted from hypoxic tumour cells help to recruit various stromal and bone marrow-derived cell (BMDC) lineages that together create a growth-supporting primary tumour microenvironment. The aims of this thesis are twofold; to investigate novel treatments targeting HIF-1 in the primary tumour microenvironment, and to investigate the effects of hypoxic signalling on pre-metastatic niche formation in breast cancer. Antioxidants have been identified as potential anti-cancer drugs that reduce tumour growth and metastasis in a HIF-1-dependent manner. N-acetylcysteine (NAC) is one such antioxidant that has a promising but somewhat controversial role in cancer therapy. This study utilised three orthotopic, syngeneic murine models of breast cancer, the PyMT, EO771 and 4T1.2 models, to investigate the ability of NAC to regulate the hypoxic response in breast tumour cells, and prevent tumour growth and metastasis. NAC demonstrated an ability to prevent HIF-1 stabilisation and signalling via the hypoxic response pathway in breast tumour cells in vitro. When administered in vivo however, NAC treatment did not inhibit primary tumour growth or metastasis. Furthermore, NAC treatment promoted metastases formation in an experimental metastasis model. This work brings into question the validity of NAC as an anti-tumourigenic agent in breast cancer, and highlights the need to further investigate its properties in vivo in different cancer models. Recently, it has been demonstrated that a primary tumour promotes metastasis through the formation of supportive microenvironments, termed pre-metastatic niches, in secondary organs predisposed to metastases. Cytokines, chemokines and growth factors secreted from tumour cells promote the mobilisation and recruitment of BMDCs to pre-metastatic organs, where they interact with the extracellular matrix and other stromal components to create tumour-promoting niches. While hypoxic tumour cells themselves have an increased ability to metastasize, they also secrete a variety of factors independently identified in pre-metastatic niches. This study investigated the combinatorial effect of factors secreted from hypoxic breast tumour cells on pre-metastatic niche formation in distant organs. Using immune competent eGFP bone marrow chimeric mice and different syngeneic mammary cancer models, hypoxic factors were shown to create an immunosuppressed environment in pre-metastatic organs. A subset of granulocytic CD11b+/Ly6Cmed/Ly6G+ myeloid-derived suppressor cells with increased CCR2 expression and STAT3 signalling were identified in the pre-metastatic niche. As were CD3-/NK1.1+ Natural Killer cells with reduced maturity, cytotoxicity, and expression of the activating receptor NKG2D. Together, factors secreted from hypoxic breast tumour cells promote metastasis by creating immunosuppressed pre-metastatic niches in distant organs. This work identifies novel components of the pre-metastatic niche that provide potential avenues for therapeutic intervention in metastatic disease.