Pathology - Theses
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ItemThe contribution of stromal caveolin-1 to breast cancer metastasis( 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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ItemIntegrin β3 as a therapeutic target for breast cancer metastasis to bone( 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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ItemContribution of tumour derived cysteine cathepsin B to breast cancer metastasis( 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.