Surgery (Austin & Northern Health) - Theses
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ItemRole of p21-activated kinases in pancreatic cancer( 2016)Pancreatic cancer remains one of the most lethal of all solid tumours with an overall 5-year survival rate of 7%. Management has not improved significantly over the last thirty years and based on current trends, is expected to become the second leading cause of cancer-related mortality by 2030. Treatment options are limited and gemcitabine-based chemotherapy remains the standard of care as a single agent. Furthermore, the presence of the dense stroma, characteristic of pancreatic cancer, contributes to therapeutic resistance and poor therapeutic response. Thus, a better understanding of the underlying genetic and molecular mechanisms is urgently required to find targeted and effective therapies. There is growing evidence that p21-activated kinases (PAKs) are involved in pancreatic carcinogenesis. The PAK family consist of six isoforms, two of which, PAK1 and PAK4, are upregulated and/or hyper-activated in pancreatic cancer. PAK1 can mediate many different cellular processes including the regulation of cytoskeletal dynamics and cell adhesion, the evasion of apoptosis, the promotion of cell survival, proliferation, migration and invasion, the fibrosis that constitutes the stroma, and the interplay between cancer cells and the stroma. PAK1’s role has not been fully elucidated in pancreatic cancer and has not been evaluated as a target for therapeutic intervention. The work presented in this thesis investigates the role of PAK1 in pancreatic cancer and the effect of PAK1 inhibitors, alone and in combination with gemcitabine, on pancreatic cancer growth, metastasis, stroma, and survival. First, we investigated the effect of glaucarubinone, a known inhibitor that reduces the activity of PAK1 and PAK4, on pancreatic cancer growth, migration and murine survival. Using 4 human and 2 murine pancreatic cancer cell lines, PAK1 and PAK4 was expressed in all pancreatic cancer cell lines tested and proliferation and migration/invasion inhibited by treatment of glaucarubinone with reduction in PAK1 and PAK4 activity in vitro. Synergistic inhibition was observed when combined with gemcitabine with decrease in pancreatic cancer proliferation in vitro, decrease in pancreatic cancer growth in human xenograft tumours in vivo, and increase in murine survival in an orthotopic immunocompetent model in vivo. This was one of the first studies that showed clinical benefit of targeting and reducing PAK1 in pancreatic cancer. Using more direct methods of reducing PAK1 activity, shRNA knockdown systems, and a PAK1 selective inhibitor, FRAX597, were utilised. shRNA knockdown of PAK1 resulted in a reduction in pancreatic cancer cell proliferation and survival and sensitised cells to gemcitabine in vitro. PAK1 was also found to be key regulator of signalling pathways such as PI3K and HIF1α. FRAX597 treatment decreased pancreatic cancer cell proliferation and migration/invasion and synergised with gemcitabine to decrease cell proliferation in vitro. FRAX597, combined with gemcitabine, reduced pancreatic tumour volume and increased murine survival in preclinical orthotopic immunocompetent murine models in vivo. Although, further clinical validation is required, it illustrates the clinical potential of a PAK1 inhibitor, FRAX597, combined with gemcitabine to improve pancreatic cancer patient outcomes. PAK1’s role was investigated in pancreatic stellate cells (PSCs), which are primarily responsible for the fibrosis that constitutes the pancreatic cancer stroma. This was the first study to show the presence of PAK1 activity in isolated human PSCs. The treatment of the selective PAK1 inhibitor, FRAX597, on PSCs resulted in a reduction in their activation, proliferation, and increase in apoptosis in vitro. PAK1 knockout mice tumours had decreased expression and activity of PAK1, associated with increased murine survival, showing the effect of depleting host PAK1 in an orthotopic immunocompetent murine model in vivo. These results implicate PAK1 as a regulator of PSC activation, proliferation and apoptosis and targeting stromal PAK1 could increase therapeutic response and survival of patients with pancreatic cancer. Together, these results illustrate the importance of PAK1 signalling in pancreatic cancer and the possible therapeutic benefit of targeting PAK1 with gemcitabine on pancreatic cancer growth and the stroma to increase the survival of pancreatic cancer patients.
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ItemThe role of hypoxia inducible factor 1 alpha (HIF1α) in prostate cancer( 2016)Prostate cancer (PC) is one of the most prevalent cancers in men. Although many PCs are indolent, a significant proportion will metastasize and develop resistance to therapy. Contemporary screening tests lack the finesse to accurately differentiate aggressive PCs from indolent tumours, potentially leading to over-diagnosis and over-treatment. While cellular hypoxia often plays an integral role in carcinogenesis and tumour progression, this connection has been difficult to demonstrate in PC. However, a downstream marker of hypoxia, Hypoxia inducible factor 1α (HIF1α), which is a transcription factor that protects cells against noxious stimuli, is frequently over expressed in PC. Therefore, the role of HIF1α in PC was investigated in this thesis. The Castrate resistant PC (CRPC)-like human PC cell lines PC3 and DU145 were found to over-express HIF1α protein compared to an androgen-sensitive cell line LNCaP under normoxic conditions. Using HIF1α 5’UTR-luciferase constructs in PC3 cells, further experiments revealed that increased translation of HIF1α mRNA regulated by a 70bp GC-rich, secondary structure in the 5’UTR of the HIF1α promoter may be responsible for normoxic HIF1α overexpression. Cell proliferation assays revealed that PC3 cells over-expressing HIF1α were more resistant to destruction by cytotoxic agents (H2O2 and 5-fluorouracil) than androgen-dependent LNCaP cells. Reduction of HIF1α expression in PC3 cells using RNA interference decreased both the resistance towards cytotoxic agents and cell migration. Conversely, in the androgen-dependent LNCaP cells overexpression of HIF1α increased the resistance to cytotoxic agents. One hundred prostate tumours were then immune-stained for HIF1α and outcomes measured. On multivariate analysis HIF1α was an independent risk factor for progression to metastatic PC (Hazard ratio (HR) 9.8, p = 0.017) and development of CRPC (HR 10.0, p = 0.021) in patients on androgen-deprivation therapy (ADT). Notably the tumours that did not express HIF1α did not metastasise or develop CRPC. Next, the effects of non-specific HIF1α inhibitors (digoxin, metformin and angiotensin-2 receptor blockers) were investigated in ninety-eight patients who had continuous ADT as first line therapy and developed CRPC. The median CRPC-free survival was longer in men using HIF1α inhibitors compared to those not on inhibitors (6.7 yrs vs. 2.7yrs, p=0.01) and there was a 71% reduction in the risk of developing CRPC (p=0.02) and an 81% reduction in the risk of developing metastases (p=0.02) after adjustment for Gleason score, age and PSA. Finally, the effects of metformin were investigated in 2055 men treated for PC with external beam radiotherapy. Surprisingly, metformin did not result in any improvement in time to biochemical failure, time to metastases or overall survival in men undergoing radiotherapy, but there was an 1.5 fold increase in PC-specific deaths (p<0.05) in men on metformin who received ADT when adjusted for cancer risk and co-morbidities. In conclusion, the results presented in this thesis indicate that HIF1α is a promising marker in PC, which may be used for early identification of cancers that potentially will progress to metastases and develop resistance to ADT. HIF1α is likely to contribute to metastasis and chemo-resistance of CRPC, targeted reduction of HIF1α may improve outcomes of aggressive PC.
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ItemGastrin-mediated adaptive responses to hypoxia in colorectal cancer( 2014)Over the past two decades the potential biological activities exerted by gastrin precursors on colorectal tumourigenesis have gradually widened to include mitogenesis, apoptosis resistance, stimulation of angiogenesis and promotion of cell migration and invasion. However, the molecular mechanisms underlying this plethora of biological effects are unclear. Furthermore, the interplay between gastrin precursors and the colorectal tumour microenvironment has been a relatively neglected area of gastrin research. This thesis investigates these two important areas of gastrin biology and is the first study to report that hypoxia-inducible gastrin gene expression in colorectal cancer cells mediates resistance against hypoxia-inducible cell death in vitro and in vivo and may contribute to the development of distant metastatic disease.