Surgery (Austin & Northern Health) - Theses

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    The Haemodynamic Effects of Mannitolcontaining Intravenous Paracetamol: A Pilot Program
    Chiam, Elizabeth Ann ( 2019)
    The haemodynamic effects of intravenous paracetamol: overview of clinical significance In households worldwide, paracetamol is an over-the-counter drug synonymous with its safety and efficacy for its antipyretic (fever relieving) and analgesic (pain relieving) properties. It is readily available to the public in both enteral (oral) and rectal formulations. In the hospital setting, with 100% bioavailability and ease of administration, the intravenous (IV) formulation of paracetamol is an attractive option for patients undergoing major surgery and the critically ill, where oral and rectal administration may be challenging. Studies have found IV paracetamol is often used in multimodal postoperative pain management and has been shown to improve pain, opioid consumption and overall patient satisfaction in the postoperative setting for both major and minor surgeries. As such, IV paracetamol is one of the most commonly ordered medications for surgery and critical care patients. However, there is a paucity of evidence-based research surrounding its safety profile in these patient subgroups. Emerging clinical data suggests it may have the propensity to produce hypotension in surgical patients and the critically ill. Intraoperative hypotensive events have been associated with morbidity and longer hospital stays. Additionally, maintenance of haemodynamic stability is a requirement for a patient to be discharged from intensive care. Despite the concern of such a significant, and potentially underreported side effect, the current corpus of science relating to the haemodynamic effects of IV paracetamol is limited. It is also worth noting an underappreciated excipient of IV paracetamol is mannitol. Mannitol is added to the IV paracetamol formulation as a stabilizing compound but is present in quantities close to 4% in the majority of IV paracetamol formulations available. Recent advances in invasive and non-invasive haemodynamic monitoring have allowed for the accurate measurement of variables that determine blood pressure, namely cardiac output and systemic vascular resistance. For this thesis, state-of-the-art haemodynamic monitoring will be used to quantify the haemodynamic impact of IV paracetamol by measuring the fundamental components of blood pressure. In order to understand the effects of IV paracetamol on blood pressure and the potential for mannitol to play a role in any haemodynamic alterations, this thesis will aim to investigate the haemodynamic effects of IV paracetamol by means of a comprehensive paracetamol program. This program will be specifically tailored to examine these effects in patient subgroups of varying baseline haemodynamic stability. This comprehensive paracetamol haemodynamic program involves the following: Stage 1 – Literature review: Comprehensive literature review to identify, evaluate and critically analyse the current knowledge base of the haemodynamic effects of IV paracetamol. Stage 2 – STUDY 1 Healthy volunteer study: To assess the haemodynamic effects of IV paracetamol in a healthy, normotensive population. Stage 2 – STUDY 2 Cardiac surgery patients (preoperative) study: To assess the haemodynamic effects of IV paracetamol in patients with pre-existing cardiac disease. Stage 2 – STUDY 3 Cardiac surgery patients (postoperative) study: To assess the haemodynamic effects of IV paracetamol in patients who have undergone major surgery and who are at greater risk of haemodynamic instability. Stage 2 – STUDY 4 Chronic liver disease patients study: To assess the haemodynamic effects of IV paracetamol in patients with pre-existing derangements of their systemic vascular system (similar to shock). It is hoped that this thesis will synthesize high quality results to add to the growing body of evidence that IV paracetamol may cause hypotension. Analyzing the haemodynamic effects in such different patient populations, may offer insight as to which individuals may be at greater risk of developing hypotension after the administration of IV paracetamol.
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    Characteristics, management and outcomes of acute liver failure in australasian intensive care units
    Warrillow, Stephen Joseph ( 2019)
    Introduction Acute liver failure (ALF) is a form of rapidly progressive liver injury. It induces encephalopathy, cerebral oedema, deranged haemostasis, haemodynamic compromise, renal failure, and metabolic abnormalities. Emergency liver transplantation (ELT) is sometimes necessary for survival. Unfortunately, little is known about ALF in Australia and New Zealand (ANZ). Aims To develop a comprehensive description of ALF in ANZ. Methods Data for ALF patients were obtained from the ANZ Intensive Care Society Centre for Outcomes and Resource Evaluation Adult Patient Database and the ANZ Liver Transplant Registry. The Australasian Management of Acute Liver Failure Investigator group was formed and contributed data from all six liver transplant ICUs in ANZ. Analyses of these data were used to develop a broad and representative understanding of ALF across ANZ. The approach to key management challenges was evaluated and outcomes were described. Results ALF is rare but increasing in incidence across ANZ. Illness severity and mortality are higher than the general ICU population and most cases due to paracetamol overdose. Deranged measures of haemostasis are universal, with hypofibrinogenaemia and thrombocytopaenia more strongly associated with bleeding risk than the international normalised ratio. Blood products are often administered but without clear correlation with bleeding complications. Continuous renal replacement therapy is provided in most cases, and often in the absence of classical renal failure indications but with effective correction of hyperammonemia. Hypertonic saline is often administered to induce hypernatraemia, fever is uncommon, and ventilatory strategies achieve a low normal PaCO2 in most patients. Despite low ELT utilisation compared to other regions, outcomes are similar. Conclusions These studies form the most comprehensive evaluation of ALF across ANZ to date. They identify key aetiologies, patterns of illness, current management, utility of prognostic criteria and clinical outcomes. These findings can be used to inform the evolution of practice in this field.
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    Protection of the kidney against Ischaemia-Reperfusion injury using zinc
    O'Kane, Dermot Bernard ( 2019)
    Acute kidney injury (AKI) continues to be a major cause of morbidity and mortality worldwide. Septic shock, hypovolaemia, and renal ischaemia related to major surgeries are the primary contributors to AKI in hospitalised patients. AKI is associated with a four-fold increase in mortality in hospitalised patients, and a two-fold increase in the likelihood of discharge to a short- or long-term care facility. As a result, the estimated healthcare costs associated with AKI in hospitalised patients in the US alone exceeds US$10billion per year. Studies have also demonstrated that AKI resulting from ischaemia-reperfusion (IR) is a causative determinant in the development, and progression, of chronic kidney disease (CKD). Despite major medical advances to the current day, short of supportive measures there is still no definitive therapeutic option available to prevent AKI in these settings. Preconditioning (PC) against renal IR injury has been heralded as a promising solution to abrogate this major healthcare problem, and an extensive volume of research has amassed in this area. Preconditioning is a phenomenon whereby an innate tissue adaptation occurs in response to a sublethal stimulus, which leads to protection of an organ or tissue against a subsequent insult. PC was first discovered in the context of ischaemic PC (IPC), where brief sublethal periods of ischaemia led to protection against a subsequent more sustained period of ischaemia in the canine heart. Since the discovery of the IPC phenomenon in 1986, tissue protection against ischaemia by means of IPC has been demonstrated by a number of methods in a variety of tissues, including the heart, brain, liver, kidney, and striated smooth muscle. The promise of these findings has also prompted research into the use of alternative methods of tissue PC, and studies have since investigated the use of pharmaceutical agents to promote these tissue adaptations by pharmacological preconditioning (PPC). The race for a pharmaceutical agent capable of eliciting protective adaptations against tissue ischaemia has involved many classes of pharmaceutical compounds, endogenous proteins, and trace elements. Zinc (Zn) is a metal that is essential to many biological functions, including cell growth and survival. The omnipresence of Zn in cellular interactions, and its importance in so many biological processes has led to the investigation of augmenting Zn homeostasis as a means of protection against tissue ischaemia. This is the primary topic of this thesis. The promise of enabling organ protection against IR injury has major clinical implications, spanning many areas of medicine. However, despite the extensive volume of clinical and basic science research in this area, there is still currently no effective method of PC that will protect the human kidney against IR injury. The aims of this thesis are to investigate if parenteral Zn can be used as a therapeutic strategy to protect the kidney against IR injury. The body of research on the topic of tissue PC has highlighted some potential targets for PC agents, however to date a definitive mechanism has not been elucidated, and therefore a further aim of this thesis is to investigate the potential mechanisms through which Zn effects renal tubular and glomerular epithelial cell survival in the setting of renal IR.
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    Development of a cell-free DNA methodology to assess organ rejection after liver transplantation
    Goh, Su Kah ( 2019)
    Background: Liver transplantation has revolutionised the prognosis of patients with fulminant liver failure, chronic liver disease, and liver cancer. Although liver transplantation is safe, organ rejection is a common complication after such a procedure. The gold-standard for diagnosing organ rejection after liver transplantation is a tissue biopsy. Liver biopsies are invasive. There is thus an unmet clinical need for accurate blood tests to diagnose the episodes of organ rejection after liver transplantation. Donor-specific cell-free DNA (dscfDNA) is an emerging biomarker of organ rejection. Measuring dscfDNA using current methodologies such as next generation sequencing can be both complex and expensive. Novel tests that overcome these limitations would favour adoption of such methodologies for the quantification of dscfDNA and implementation for the surveillance of organ rejection after transplantation. Objectives: The first objective of this thesis was to develop a cell-free DNA based assay for the accurate quantification of dscfDNA that could overcome some of the limitations of existing methodologies. The second objective of this thesis was to deploy this assay to monitor episodes of organ rejection in a prospective cohort of recipients. Main findings: A probe-free droplet digital PCR-based methodology was developed. The methodology overcame some of the common limitations that were observed in next generation sequencing-based and other PCR-based methodologies. The newly developed approach was accurate, economical, and rapid which facilitated the rapid turnaround of results as well as enabled early clinical decision-making (Chapters 3 and 4). The application of this approach to measure dscfDNA was shown to be feasible for the monitoring of dscfDNA in a prospective cohort of forty recipients after liver transplantation (Chapter 5). The levels of dscfDNA were reflective of organ health. Furthermore, a calculated threshold of 898 copies of dscfDNA per mL of recipient plasma identified majority of the recipients with biopsy-proven acute rejection requiring treatment. The diagnostic performance of dscfDNA, in this cohort, was superior compared to routine liver function tests in identifying organ rejection. Conclusion: This thesis presented the application of a novel cfDNA methodology to measure dscfDNA in a prospective cohort of recipients after liver transplantation. The results demonstrated the promising utility of dscfDNA as a marker of organ rejection after liver transplantation. These pertinent findings warrant further validation with a view towards clinical implementation.
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    Targeting p21-activated kinases in the treatment of pancreatic cancer
    Wang, Kai ( 2019)
    Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies worldwide, with a very poor prognosis and a 5-year survival rate less than 9%. This dismal outcome is largely due to lack of early diagnosis, quick disease progression, high rate of post-surgery recurrence and resistance to conventional therapies. Oncogenic Kras mutation is a well-defined hallmark of pancreatic cancer. It is presented in over 95% cases and leads to constitutively active form of Kras protein. Kras still remains as an undruggable target due to absence of a well-defined drug-binding domain. With the aim to fight against Ras-driven cancers, high priority has been given to the novel therapeutic strategies targeting Ras-dependent signalling. P21-activated kinases (PAKs) are a family of serine/threonine kinases that are important down-stream effectors of multiple small GTPases including Ras, Rac1 and Cdc42. Based on the difference in the structure and sequence, all the six members of PAK family are divided into two groups: group I (PAK1-3) and group II (PAK4-6). PAK1 and PAK4 are the most widely studied members and have been reported to be up-regulated in PDA. PAK1 is situated at the convergence of multiple signalling pathways that are associated with cell proliferation, survival/apoptosis, migration/invasion and cytoskeletal regulation. Immunotherapy is now emerging as a promising treatment in the era of personalised anti-cancer therapeutics. However, it can only bring limited clinical benefits for PDA patients, which is largely attributed to the immunosuppressive tumour microenvironment (TME). The role of PAK1 has not been fully elucidated in pancreatic cancer, especially its involvement in re-programming TME. The work presented in this thesis investigated the role of PAK1 in tumour biology and therapeutic regimens, with a focus on its linkage to stroma modulation and anti-tumour immune response. Firstly, the anti-tumour effect of a potent PAK inhibitor (PF-3758309) was determined on a panel of clinical patient-derived PDA cell lines (TKCC 2.1, TKCC15, TKCC18, TKCC22, TKCC23, TKCC26). PF-3758309 treatment inhibited cell proliferation and sensitized PDA cells to different chemotherapies (fluorouracil, gemcitabine and nab-paclitaxel) with a synergistic effect, which was associated with reduction in PAK1 and PAK4 activity and down-regulation of HIF-1α, α-SMA and palladin in vitro. Combination of PF-3758309 and gemcitabine maximally suppressed tumour growth in vivo and had a comparable or even greater therapeutic efficacy compared to combination of gemcitabine and nab-paclitaxel. As mentioned above, the expression of α-SMA was observed in PDA cells. All-trans retinoid acid (ATRA), a well-known compound that induced quiescence of pancreatic stellate cell (PSC) by decreasing the expression of α-SMA, was utilized to investigate its anti-tumour effect and association with PAK protein and α-SMA in PDA cells. Inhibitory effect of ATRA on PDA cell growth and migration and its synergism with gemcitabine was observed in both wildtype and gemcitabine-resistant PDA cell lines. Expression of PAK1, PAK2, PAK4 and α-SMA was down-regulated by ATRA. Inhibition of PAK1 by shRNA knockdown or PF-3758309 sensitized PDA cells to ATRA. This was the first study to demonstrate the role of PAK proteins in ATRA treatment. The role of PAK1 in tumour immune response was evaluated using an orthotopic mouse model of pancreatic cancer. Inhibition of PAK1 by PF-3758309 or genetic knockout up-regulated intra-tumoural infiltration of CD3+ lymphocytes and splenic CD3+ or CD8+ lymphocytes. Combination of PF-3758309 and gemcitabine synergistically inhibited PDA cell growth in vitro and in vivo. This study not only confirmed the anti-tumour effect of PF-3758309 and its synergism with gemcitabine, but also revealed the potential role of PAK1 in anti-tumour immunity. Finally, the underlying mechanisms of PAK1 in regulating anti-tumour immunity was investigated. Immunohistochemistry was performed on human tissue microarray and KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) mice samples. PAK1 was identified to be a negative prognostic marker and positively correlated with α-SMA expression. Depletion of PAK1 decreased PSC activity, reduced PSC-stimulated PDA cell proliferation and migration and increased intra-tumoural infiltration of CD4+ or CD8+ lymphocytes. Inhibition of PAK1 decreased both intrinsic and PSC-stimulated PD-L1 expression in PDA cells, which could enhance lymphocyte-induced PDA cell death. This was the first study to demonstrate the important role of PAK1 in regulating PSC activity and PD-L1 expression in PDA. In a summary, these studies revealed the importance of PAK signalling in PDA development, the therapeutic value of PAK inhibitors and its synergism with gemcitabine in PDA treatment. Most importantly, PAK1 is emerging as a potential target to enhance anti-tumour immunity and to facilitate the development of novel immunotherapies.
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    18F-fluorodeoxyglucose positron emission tomography as a biomarker for colorectal cancer liver metastases
    Lau, Lawrence F. ( 2017)
    Background: Colorectal cancer is the second most common cause of cancer-related death in Australia. The majority of patients with colorectal cancer develop liver metastases but only those amenable for surgical resection have a possibility of long term survival. Recent advances in achieving macroscopic resectability of colorectal liver metastases needs to be balanced urgently, by an ability to assess systemic micrometastatic disease. Tumour staging by 18F-fluorodeoxyglucose positron emission tomography (PET) is a non-invasive tool already in routine use. Aim: To explore metabolic characteristics assessed by PET as biomarkers for colorectal cancer liver metastases. Methods / Results: Four studies were performed, each addressing separate aspects regarding the utility of tumour metabolic assessment. The first three studies were performed on retrospective cohorts while the fourth study was a prospective study. The studies and main novel findings are summarized below: 1) The Prognostic Impact of Tumour Metabolism an a Single PET Scan after Preoperative Chemotherapy Various parameters that characterize and quantify tumour metabolism were assessed for their prognostic ability. These parameters were compared to clinical and pathological features as well as previously verified prognostic scoring systems. The metabolic parameters corresponding to metabolic tumour burden were found to be most prognostic on a single PET scan following preoperative chemotherapy. 2) The Prognostic Impact of Tumour Metabolic Response to Preoperative Chemotherapy The prognostic ability of metabolic response to preoperative chemotherapy was assessed using the serial assessment of various metabolic parameters. In comparison, tumour size shrinkage on computed tomography and pathological response, the current gold standards of chemotherapy response evaluation, were assessed. Metabolic response to preoperative chemotherapy was shown to be the best prognostic indicator. 3) Metabolic Response Correlated to Biological Mechanisms The biological mechanisms underlying the prognostic impact of metabolic response was explored. Immunohistochemical analysis of six tumour biomarkers showed an inverse correlation between metabolic response and the expression of Ki-67, a marker of cellular proliferation; and a direct correlation between metabolic response and the expression of p16, a tumour suppressor. 4) Early Metabolic Response Assessment The use of early tumour metabolic response after only the first cycle of preoperative chemotherapy was assessed for the ability to predict eventual metabolic response. Early tumour metabolic response after one cycle of chemotherapy did not predict eventual metabolic response or clinical outcome. Conclusion: This thesis showed tumour metabolism to be a powerful prognostic indicator for patients with colorectal cancer liver metastases. In particular, it reveals the burden of disease as well as the sensitivity of the metastases to systemic chemotherapy. PET assessment of tumour metabolic response to chemotherapy should be routinely performed, particularly in patients undergoing complex liver surgery.
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    Role of p21-activated kinases in pancreatic cancer
    Yeo, Dannel ( 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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    The role of hypoxia inducible factor 1 alpha (HIF1α) in prostate cancer
    Ranasinghe, Weranja Kalana Bodhisiri ( 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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    Gastrin-mediated adaptive responses to hypoxia in colorectal cancer
    Westwood, David Alexander ( 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.
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    Interaction between p21-activated kinase 1 and beta-catenin
    Liu, He ( 2012)
    Colorectal cancer (CRC) was the second most frequently occurring cancer and the second leading cause of cancer death in Australia in 2007 (AIHW2010). Hyper-activation of the Wnt/β-catenin signaling pathway is a hallmark of colorectal cancer. The Wnt signaling pathway plays a critical role in embryonic development and homeostasitic maintenance in mature tissues, particularly in regeneration of intestinal epithelium (Lynch and Lynch 2005). In studies of human colon cancer over the last two decades, mutations have been identified in genes coding for Wnt/β-catenin pathway components, such as axin, adenomatous polyposis coli (APC) and β-catenin, which are known to contribute to tumor progression. Tumor genetic studies have revealed that mutations in these members of the Wnt/β-catenin pathway occur in approximately 90% of colorectal cancers (Bienz and Clevers, 2000; Cottrell et al., 1992; Morin et al., 1997; Polakis, 2000; Powell et al., 1992; Vogelstein and Kinzler, 2004). Under normoxic conditions (having a normal atmospheric oxygen concentration of 20~21%), the transcription factor 4 (TCF4) stably binds to β-catenin in the nuclei of colon carcinoma cells and is constitutively activated. This activation stimulates cell migration and proliferation, and contributes to the development of colorectal tumors (Munemitsu et al., 1995). Under hypoxic conditions β-catenin interacts with the heterodimeric transcription factor hypoxia inducible factor-1α (HIF-1α), enhances HIF-1-mediated transcription, and further promotes cell survival and adaptation to hypoxia (Kaidi et al., 2007). In a mouse model carrying a mutation in the APC gene, the gastrin gene has been identified as a downstream target of the β-catenin/TCF4 signaling pathway (Koh et al., 2000). Similarly the expression of a constitutively active β-catenin causes a threefold increase in gastrin promoter activity (Koh et al., 2000). In previous studies from this laboratory, p21-activated kinase 1 (PAK1) was found to interact with β-catenin and to be required for the regulation of the β-catenin signaling pathway by gastrins (He et al., 2008). PAK1 kinase activity has been implicated in various cellular processes such as gene regulation, cytoskeletal reorganization, cell growth, motility, and morphogenesis (Kumar et al., 2006). PAK1 also functions as a key node in various signaling pathways leading to cell growth, migration and survival. PAK1 has oncogenic functions in a broad range of cancers including CRC and its hyper-activation has been well documented in breast cancer (Dummler et al., 2009; Kumar et al., 2006). PAK1 expression has also been reported to increase in the progression of colorectal carcinomas to metastasis (Carter et al., 2004). However, the specific role of PAK1 in β-catenin signaling and the mechanism by which PAK1 interacts with β-catenin in CRC have not been investigated in detail. The studies in Chapter 3 demonstrate that PAK1 is required for maximal expression of β-catenin and its downstream targets and is important for Wnt signaling pathways in CRC, that β-catenin/TCF4 transcriptional activity is also significantly reduced in PAK1 knockdown cells, and that knocking down PAK1 decreases cell proliferation, migration, HIF-1α expression and cell survival. The mechanism by which PAK1 interacts with β-catenin was further investigated by studying the signaling networks of both proteins in Chapter 4. Cellular β-catenin expression is regulated at the protein level through phosphorylation by glycogen synthase kinase 3-beta (GSK-3β). In the cytoplasm, β-catenin forms a complex with APC, axin, GSK-3β and casein kinase 1 (CK1) (Giles et al., 2003; Kikuchi et al., 2006), and GSK-3β then induces serine-threonine phosphorylation at the amino-terminal of β-catenin, and the phosphorylated β-catenin binds to βTrCP, an E3 ubiquitin ligase that promotes the degradation of β-catenin (Wu et al., 2003). Without phosphorylation by GSK-3β, the stabilized β-catenin accumulates and is translocated to the nucleus, where it interacts with transcription factors of the TCF/LEF-1 family (mainly TCF4), leading to the increased expression of genes which stimulate cell proliferation and migration, and contribute to the development of tumors (He et al., 1998; Shtutman et al., 1999; Tetsu and McCormick, 1999). According to the results of the studies of Chapter 4, there is no change in either expression of GSK-3β protein or in kinase activity of GSK-3β as measured by phosphorylation at Serine 9 in PAK1 knockdown cells. These results indicate that PAK1 may not regulate β-catenin through GSK-3β signaling in CRC cell lines. Other effectors of β-catenin activity have also been studied in Chapter 4. The integrin-linked kinase (ILK) has been reported to be involved in β-catenin/TCF4 signaling through multiple mechanisms (Novak et al., 1998; Tan et al., 2001). The Inhibitor of β-catenin and TCF4 (ICAT) is reported to inhibit β-catenin nuclear signaling by competing with TCF4 for binding with β-catenin (Tago et al., 2000). ICAT is also located downstream of ILK in progastrin-mediated signaling in CRC tumors (Pannequin et al., 2007). Interestingly, PAK1 has been shown to be responsible for phosphorylation-dependent translocation and gene regulation of ILK (Acconcia et al., 2007). The studies of Chapter 4 demonstrate that PAK1 is required for ILK activity, that knocking down PAK1 increases ICAT expression and that PAK1 regulates β-catenin through the ILK/ICAT signaling pathway. After the investigation of the PAK1 and β-catenin interaction in CRC cells in vitro, the role of PAK1 in β-catenin signaling is further explored in vivo using animal models in Chapter 5. In this study, the in vivo role of the PAK1 protein and its interaction with β-catenin was examined using Severe Combined Immunodeficiency (SCID) mice and genetically modified APCΔ14/+ mice, and siRNAs were used as a treatment to inhibit expression of the proteins of interest. The growth of CRC cell lines as xenografts in SCID mice was studied and tumor histology in the genetically modified CRC mouse model APCΔ14/+ mice was analysed. In xenograft studies with human CRC cells in Chapter 5, PAK1 knockdown suppressed tumor growth by inhibition of proliferation and stimulation of apoptosis. In addition, PAK1 siRNA treatment delayed the growth of wildtype human CRC cells at an early stage of tumor development and contributed to tumor necrosis at later stages. In studies with APCΔ14/+ mice, expression of both PAK1 and β-catenin protein was reduced in tumors from APCΔ14/+ mice treated with PAK1 siRNA compared with mice treated with control siRNA. PAK1 siRNA treatment decreased tumor numbers significantly and slowed the bodyweight drop caused by tumor development. This thesis provides detailed information on the Wnt/β-catenin and PAK1 signaling pathways, contributes to understanding of the mechanism of human CRC development, and provides a novel direction for effective CRC treatment. The results presented here indicate that PAK1 could be a suitable target for CRC therapy. Future studies focusing on PAK1 as a drug development target may have promising outcomes in clinical trials as well as in cancer therapy.