Prostate cancer has high cancer incidence in males and is the second highest cause of male cancer related mortality. Currently the main stay therapy for localised and metastatic disease is maximum androgen blockade (MAB). This aims to inhibit androgen production or action, thereby reducing stimulation of the androgen receptor (AR). This in turn prevents the activation of androgen-regulated genes, which normally result in on-going growth and survival. Inhibition of testicular androgen production may be achieved surgically (bilateral orchidectomy) or chemically, using gonadotropin-releasing hormone (GnRH) agonists. The latter induces castrate levels of testosterone by down-regulating pituitary GnRH receptors (and therefore gonadotropin hormone production) through constant stimulation. The action of androgen may be blocked at a peripheral level using anti androgens, which inhibit ligand binding to AR and subsequent activation. Although this approach has initial response rates of over 80% the majority of men relapse with castrate resistant prostate cancer (CRPC) and this is the cause of significant morbidity and mortality. To overcome this and to improve patients treatment options the mechanisms of castrate resistance need to be addressed. The PI3K/Akt cascade regulates several cellular processes such as proliferation and apoptosis. Akt activation results in phosphorylation of multiple substrates and has been implicated in prostate carcinogenesis and castration resistance. Research has suggested that Akt interacts with signallingcascades implemented in carcinogenesis, in particular the NFkB cascade and AR signalling. The current study investigated the hypothesis that the expression and activation of PI3K/Akt cascade influences the progression to castrate resistant disease using clinical prostate cancer tumours. Fluorescent insitu hybridisation and Immunohistochemistry revealed that PTEN deletion was a common event in castrate resistant prostate cancer and low PTEN protein expression was significantly associated with a poor outcome. PTEN negatively regulates PI3K signalling. Consequently increased levels of PI3K and activated Akt (pAkt ser 308 and pAkt ser 473) were significantly associated with a shorter time to biochemical relapse and shorter disease specific survival. Inhibition of PI3K resulted in a significant reduction in cellular proliferation and Akt phosphorylation. The downstream affects of Akt activation were also investigated. Akt has been reported to directly activate the NFkB signallingcascade both directly and indirectly but no correlations between Akt and NFkB were observed in the current study. Using an immunohistochemical approach NFkB, IкBα and MMP-9 expression were observed to be significantly associated with shorter time to death from relapse and disease specific death. MMP-9 and IкBα expression were also significantly associated with metastases at relapse. Using paired hormone naive and castrate resistant LNCaP cells lines allowed the functional consequences of NFkB inhibition to be investigated. Reduced NFkB activation significantly inhibited cellular proliferation and induced apoptosis in both cell lines. Having shown a significant link between expression and activation of the PI3K cascade and progression to castrate resistant disease, the interaction between Akt and the AR was investigated in both clinical prostate tumours and cell lines. The phosphorylation of AR at the Akt consensus site serine 213 (pARser213) was significantly associated with disease progression. Patients with high expression pARser213 had a significantly shorter time to death from relapse and disease specific survival. Additionally 42% of patients displayed an increase in pARser213 expression, these patients also had a significantly shorter time to death from relapse and disease specific survival. Inhibition of PI3K resulted in a reduction of pARser213 expression in both cell lines and using siRNA knockdown to target PI3K p85 regulatory subunit reduced pARser213 expression. This research highlights the impact of both the PI3K/Akt and NFkB signallingcascades on prostate cancer progression and development of castrate resistant disease. In particular this study highlights the impact of Akt phosphorylation in castrate resistant prostate cancer patients. Therefore phosphorylation of AR at serine 213 may serve as a diagnostic tool to predict patient outcome in response to maximum androgen blockade and inhibition of AR 213 phosphorylation via the Akt cascade may be an effective therapeutic avenue to investigate for treatment of prostate cancer.

The Apc gene encodes the Adenomatous polyposis coli tumour suppressor protein, the germ line mutation of which characterizes Familial Adenomatous Polyposis (FAP), an autosomal syndrome characterized by multiple colorectal lesions. Inactivation of the Apc gene is recognized as a key early event in the development of colorectal cancers and leads to the deregulation of the Wnt pathway and the activation of TCF/LEF target genes. This project focuses on the proto-oncogene c-Myc as it is a key Wnt target gene which is activated following loss of Apc in vivo. This upregulation is noteworthy as c-Myc is implicated in stem cell survival, proliferation, apoptosis and tumourigenesis. Previous studies have shown c-Myc dependency for both apoptosis and proliferation following activation of the Wnt pathway, however little is known about the role c-Myc plays in inducing apoptosis following DNA damage in vivo. To study this I have conditionally deleted c-Myc from the intestinal epithelium and examined the response of intestinal enterocytes following DNA damage. Remarkably, following DNA damage, c-Myc deficient enterocytes were unable to upregulate p53 and induce apoptosis, which was mechanistically due to an upregulation of MDM2. Taken together, results from this study showed for the first time in vivo, a key role for c-Myc in inducing apoptosis following DNA damage through control of p53. Previous studies from this lab have shown that within the intestinal epithelium, c-Myc is absolutely required for the hyper-proliferative phenotype that is observed following loss of Apc. Therefore one of the key aims of this thesis is to look downstream of c-Myc in order to delineate how c-Myc induces and controls this proliferation. Given that one of the key postulated functions of c-Myc is the transcriptional repression of p21, this thesis examines this hypothesis by investigating the significance of the upregulation of p21 following c-Myc deletion in Apc deficient intestinal enterocytes. To do this, I have generated triple knockout (TKO) intestines by intercrossing p21 knockout mice to mice where we can conditionally delete both Apc and c-Myc within the murine intestinal epithelium. Surprisingly, the levels of proliferation were the same between double knockout Apc Myc and TKO intestines, which had markedly less proliferation than Apc deficient intestines. However, unlike double knockout enterocytes, TKO intestinal enterocytes no longer moved up the crypt-villus axis and failed to generate villus. To examine which of these phenomena were key to tumourigenesis (differentiation or proliferation), we investigated whether TKO intestines could form intestinal adenomas and found that even in the absence of p21, c-Myc deficient cells were unable to form tumours. Taken together we have identified a novel role for p21 in driving differentiation following Apc and Myc deletion. This is consistent with the expression of p21 in the normal crypt at the crypt villus junction. Remarkably this function of p21 is independent of its key role as a cell cycle inhibitor. Moreover, this study also examined the importance of the upregulation of the Cyclin D/CDK4 complexes following Apc loss and their role in c-Myc dependent proliferation. Results from these studies showed that Cyclin D2 is required for efficient proliferation immediately following loss of Apc as well as for tumourigeneis in the Apc Min/+ mouse. Taken together, results from these studies showed that the upregulation of Cyclin D2 and CDK4 are c-Myc dependent and that the upregulation of these complexes are key for Wnt driven proliferation and tumourigenesis. Lastly, in this study I have examined whether Apc loss within the intestinal epithelium, where it is a bona fide tumour suppressor gene, can provoke senescence, and compared this to the ability of Apc gene deletion to trigger senescence in the renal epithelium, where it is not mutated in human cancer. This study showed that deletion of Apc within the renal epithelium invoked a p21 dependent senescence response, and Apc deficient renal epithelial cells were cleared and very rarely initiated tumourigenesis. However, combined Apc and p21 gene deletion rapidly initiated tumourigenesis, with all mice developing renal carcinoma by 2 months of age. In contrast to Apc deficient intestinal epithelium, this process was unaffected by loss of c-Myc. However within the intestinal epithelium, deletion of Apc did not invoke senescence, but lead to a highly proliferative, p21 independent response. Combined Apc and p21 gene loss had no impact on either the short term phenotypes of Apc loss or upon tumourigenesis. Taken together these results show for the first time that Apc loss in vivo can invoke a senescence program but in a context dependent fashion. This implies escape from senescence is not a crucial pathway in colorectal cancers that are initiated by Apc loss, and goes to explain why renal carcinoma is not observed in FAP patients who are germline heterozygous for APC. Therefore the aims for this thesis are: • To investigate the role of c-Myc in inducing apoptosis within the intestinal crypt, and whether this is p21 dependent? • To investigate the role of p21 in causing senescence of Apc deficient cells, and whether this is c-Myc dependent? • To determine the functional importance of repression of p21 by c-Myc in Apc deficient cells. • To determine the significance of Cyclin D2 upregulation within Apc deficient cells.

Rho-associated kinase 1 (ROCK1) is a serine/threonine kinase important for the regulation of the cellular cytoskeleton through the induction of actin stress fibres and acto-myosin contractility. The cleavage and subsequent activation of ROCK1 by caspase 3 during apoptosis is believed to cause many morphological phenomena associated with programmed cell death such as dynamic membrane blebbing. I now formally prove the necessity of ROCK1 cleavage for apoptotic blebbing by knocking-in a caspase cleavage resistant mutant of ROCK1 in a genetically modified model. In addition, animals homozygous for non-cleavable ROCK1 demonstrate a phenotype consistent with auto-immune disease suggesting that apoptotic blebbing is important to mediate rapid efferocytosis, which is a rapid phagocytic clearance of the cellular corpse, and thus maintain self-tolerance. Furthermore, apoptotic blebbing is important for the clearance of apoptotic cells and I demonstrate a novel mechanism for ROCK to mediate the release of factors participating in macrophage migration to dying cells. ROCK induced apoptotic blebs and bodies lose membrane integrity prior to secondary necrosis and leak intracellular material. Using quantitative mass spectrometry I identified numerous proteins that were previously unrecognized to be released during apoptosis. The release of protein was found to be impaired following ROCK antagonism with Y27632 which underscores the importance of ROCK activity in apoptotic protein release. One of these proteins, gelsolin, was released following caspase cleavage and encourages macrophage motility towards apoptotic cells. Finally, I now demonstrate that the three nonsynonymous somatic mutations in the ROCK1 gene identified in the Cancer Genome Project lead to elevated kinase activity and drive actin cytoskeleton rearrangements that promote increased motility and decreased adhesion, characteristics of cancer progression. Mapping of the kinase-interacting regions of the carboxy-terminus combined with structural modeling provides insight into how these mutations likely affect the regulation of ROCK1. Consistent with the frequency of ROCK1 mutations in human cancer, these results support the conclusion that there is selective pressure for the ROCK1 gene to acquire ‘driver’ mutations that result in kinase activation.