Pathology - Theses

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    Investigating the role of hypoxia in tumour progression in breast cancer
    Chen, Anna ( 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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    Delineating the tumor suppressive role of Scribble in prostate cancer progression and metastasis
    Borsetti, Yvonne Christine ( 2013)
    Cell and tissue polarity are distinguished by the asymmetrical distribution of cytoplasmic and membrane components that allows the formation of structurally and functionally distinct domains within cells, and the organisation of multilayered tissues. This asymmetry is required for many cellular processes including migration, interaction with the microenvironment, diversification of cell shapes and development. To establish cellular polarity several polarity regulators are required, such as Scribble, whose loss results in deregulated cellular functions. Scribble is localised at the baso-lateral membrane, which is crucial for its normal functionality. Mislocalisation, which appears as diffuse expression within the cytoplasm, also results in disrupted cellular polarity and therefore impacts polarity regulated cellular functions. Scribble is widely accepted as an evolutionarily conserved tumor suppressor that is often deregulated in many human epithelial cancers, and is generally considered to contribute to tumor progression. Loss of cell and tissue polarity is a hallmark of epithelial cancers suggesting a crucial role for polarity regulators in suppressing tumor formation and progression(1). Interestingly, Scribble is also found to be overexpressed in many epithelial cancers, including prostate cancer, while mislocalization in prostate cancer rather than overexpression correlates with poor patient outcome in the clinic. Scribble has been shown to be a weak initiator of prostate neoplasia in mice owing to elevated Ras/MAPK signaling. Furthermore, Scribble depletion and K-Ras-oncogenic activation have been shown to cooperate in vivo, resulting in an increased incidence of invasive prostate carcinoma compared to single mutants, indicating that Scribble loss can contribute to tumor progression in the presence of an additional oncogenic mutation. Nonetheless, the molecular mechanisms underpinning tumour progression in the context of Scribble loss are not well understood. To directly assess the role of Scribble in prostate carcinogensis and metastasis, in vitro functional assessment of PC3 prostate cancer cells expressing constructs to knockdown (shSCRIB7), overexpress (hSCRIB) or mislocalize (SCRIB P305L) Scribble together with in vivo experimental models of metastasis have been performed. This work shows that Scribble is upregulated in PC-3 cells and that Scribble plays a role in coordinating directed cell migration and invasion, but does not mediate PC-3 cell cycle progression or proliferation in vitro. In addition, by employing the well characterized prostate cancer transgenic mouse model (PBCre+;Ptenfl/fl), in combination with Scribble depletion, it appears that loss of Scribble and Pten cooperate to facilitate invasion, associated with a reduction in the cell:cell adhesion molecule E-cadherin. Collectively, these findings establish that Scribble functions to coordinate prostate cancer cell migration and invasion and that deregulation of Scribble (either by over-expression, depletion or mislocalisation) causes aberrant migration and invasion. Further investigations into understanding how Scribble and the polarity network can regulate migration and invasion during prostate cancer on a molecular level may provide valuable insights into the mechanisms behind prostate cancer progression and prove to hold prognostic value in the clinic, as well as identify novel routes of therapeutic intervention.
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    The contribution of stromal caveolin-1 to breast cancer metastasis
    BURROWS, ALLAN ( 2012)
    In a previous study, our group has shown that the expression of caveolin-1 (CAV1) in normal tissue surrounding a primary breast cancer is a powerful prognostic indicator of subsequent metastatic disease. While reports linking expression of CAV1 within breast tumour cells to clinical outcome have not led to any clear conclusions, the finding of a strong positive correlation between loss of CAV1 expression in breast tumour stroma and poor prognosis by our group and others, is novel and exciting as it offers the potential of a reliable prognostic indicator of metastatic disease. These clinical observations have led us to propose the hypothesis that breast tumour cells decrease stromal CAV1 expression, and a reduction in stromal CAV1 expression leads to promotion of tumour growth and metastasis. The aims of this project were to model the progression in breast cancer following stromal CAV1 loss, and determine the underlying breast tumour-stroma paracrine metastasis. Tumours arising from weakly metastatic mammary cell lines 66cl4 and 4T1ch5, grow at a faster or similar rate respectively, when co-injected with CAV1 null mammary fibroblasts. In contrast, co-injection with CAV1 expressing mammary fibroblasts has a suppressive effect on tumour growth. Metastasis to lung was significantly higher in mice with resected primary tumours that arose from co-inoculation of 4T1ch5 cells and CAV1 null mammary fibroblasts, in addition to a significant increase in individual lung tumour nodule size. However no significant difference in immune infiltrate in these resected tumours was observed in preliminary flow cytometry analysis. No significant differences in primary tumour growth or metastasis were observed in human xenograft models. To mimic the phenotype observed in vivo, 3D co-culture assays were developed. Although these assays demonstrated a positive effect of fibroblasts on tumour cell invasion and proliferation, no stromal CAV1 specific effect was observed in response to fibroblast co-culture or conditioned medium. To further understand the consequences of the loss of stromal CAV1, profiling of CAV1 expressing and null mouse mammary fibroblasts was conducted using cytokine arrays and cDNA microarrays. A significant increase in Gas6 and a decrease in RANTES cytokine secretion were observed as a result of CAV1 loss, with no significant changes in their transcript levels. In summary, results from this project demonstrate that stromal CAV1 is an important prognostic factor in breast cancer progression. Based on these findings, a stromal targeted therapy to that restores or substitutes for CAV1 activity in stromal cells, or that targets CAV1 regulated cytokines such as Gas6, may be a viable therapy in the treatment of breast cancer metastasis.
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    Integrin β3 as a therapeutic target for breast cancer metastasis to bone
    Carter, Rachel Zoe ( 2011)
    Breast cancer is the most common cancer in women, with patients currently having high survival rates if the disease is confined to the breast. However, these survival rates drop drastically if the cancer has metastasised to distant sites. Previous studies have shown both tumour and stromal integrin β3 are involved in breast cancer metastasis, yet questions remain regarding its specific role in the process. In particular, whether integrin β3 is essential for metastasis and if so, to which organ and at which stage of the metastatic cascade it is required. Furthermore, the relative contribution of stromal and tumour integrin β3 in tumour growth and metastasis needs to be clarified. To address these questions, the transplantable 4T1 model of breast cancer with spontaneous metastasis was utilised. RNA interference was used to suppress tumour integrin β3 expression in the highly metastatic 4T1.2 and 4T1BM2 cells, and investigate whether tumour integrin β3 is essential for metastasis. Additionally, the contribution of stromal cell populations expressing integrin β3 was investigated in integrin β3 knockout mice. Lastly, the therapeutic potential of targeting integrin β3 was investigated using the disintegrin DisBa-01. Downregulation of tumour integrin β3 expression induced a coordinated decrease in the surface level of the integrin αv subunit. Functional assays revealed integrin αvβ3 dependent decreases in adhesion, migration and MMP9 secretion. In vivo, downregulation of integrin αvβ3 had no affect on primary tumour growth, but significantly reduced spontaneous metastasis to bone, lung and other soft tissues, indicating integrin β3 is required for metastasis to multiple sites. Surprisingly, unlike studies in other tumour types, integrin β3 suppression in 4T1.2 cells did not impact on experimental breast cancer metastasis to lung or bone, suggesting it is acting at an early stage of metastasis. Unlike reports in other models, primary tumour growth was not affected by the loss of stromal integrin β3, indicating orthotopic growth of breast tumours is not dependent on the expression of integrin β3 in stromal cell populations. However, metastasis to bone, but not lung, was reduced in integrin β3 null mice. These observations allow the reconciliation of previous studies that reported conflicting conclusions with regard to the role of stromal integrin β3 in tumour growth and metastasis. In vitro treatment of 4T1BM2 cells with DisBa-01 achieved similar results to down-regulation of the protein by shRNA, suppressing integrin αvβ3 dependent adhesion, proliferation, migration and MMP9 secretion. Unfortunately, these promising results did not translate to effects on tumour growth and metastasis in vivo, and neither experimental nor spontaneous metastasis were suppressed by DisBa-01 treatment under the conditions tested. However, the results obtained provide valuable information with regards to future protocol design. This study demonstrates that tumour expressed integrin β3 is essential for spontaneous breast cancer metastasis to multiple organs, and provides evidence the protein acts at an early stage in the process. It also supports the role of stromal integrin β3 in metastasis to bone, but not to lung. Taken together, these data suggest both tumour and stromal integrin β3 are potential therapeutic targets, with tumour integrin β3 contributing to the process to a greater degree.
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    Contribution of tumour derived cysteine cathepsin B to breast cancer metastasis
    Withana, Nimali P. ( 2012)
    The major cause of mortality associated with breast cancer is the development of distant meatastases to sites such as lung and bone. Approximately 70% of patients who die from breast cancer have evidence of metastases in the skeleton. The consequences of bone metastasis are always devastating. The clinical outcomes of severe pain, pathologic fractures, spinal cord and nerve compression, leading to hypercalcemia and acid/base imbalance severely diminish the quality of life. Once tumour cells home to bone, curative therapy is no longer possible in most patients and only palliative therapy is available. This emphasises the importance of understanding the mechanisms of primary tumour cell invasion and spread to bone, to be able to identify molecular drivers of bone metastasis as new therapeutic targets. Proteases are known to contribute to tumour cell invasion and angiogenesis and are commonly associated with metastasis. Recent studies in our laboratory support a role for cysteine cathepsin proteases in bone metastasis. Using our unique 4T1.2 syngeneic model of spontaneous bone metastasis, we identified the endogenous cysteine cathepsin inhibitor stefin A as a metastasis suppressor. Tumour cell expression of stefin A significantly reduced bone metastasis in the murine model. The fact that stefin A is a potent metastasis suppressor indicates that its targets, the cysteine cathepsins, have essential roles in distant metastasis. Of the cysteine cathepsins, cathepsin B was co-expressed with stefin A in primary tumours and metastases, suggesting that the mechanism of metastasis suppression by stefin A was through inhibition of cathepsin B. Cathepsin B is highly upregulated in a wide variety of cancers, including breast and prostate cancer and is linked to enhanced tumourigenesis. In this study, using the 4T1.2 spontaneous bone metastasis model, we evaluated the function of cathepsin B in bone metastasis and the potential of selectively targeting this protease as a novel therapeutic. Cathepsin B was abundant in 4T1.2 mammary tumours and matched spine metastases, mimicking that of the human disease. We have demonstrated a critical function for tumour-derived cathepsin B in bone disease. Stable knockdown of cathepsin B in tumour cells significantly reduced collagen I degradation in vitro and bone metastasis in vivo. Additionally, use of a highly selective cathepsin B inhibitor CA-074 significantly reduced metastasis to lung and bone, a reduction that was not observed when using a broad spectrum cysteine cathepsin inhibitor. This study reveals the pro-metastatic role of cathepsin B in distant metastasis and the therapeutic benefit of its selective inhibition in a murine model of breast cancer.