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The term cancer comprises a family of genetic diseases that share a fundamental biological commonality, the abnormal proliferation of cells. Targeted cancer therapies are strategies intended to exploit particular cancer features, for the delivery of tailored treatments that spare injury to normal tissues. Comparative functional profiling through RNA interference (RNAi) screens, offers a powerful framework for the identification of both, novel appropriate targets for therapy and, in the case of existent targeting agents, markers of susceptibility to drug resistance. In this thesis, RNAi technology, which renders gene suppression and provides an indirect model for the pharmaceutical inhibition of a target protein, was used to systematically survey the effect of targeting unbiased collections of individual genes, on the modulation of: (i) survival of cells deficient in PTEN a gene frequently mutated in cancer, and (ii) breast cancer response to the breast cancer drug tamoxifen. The high prevalence of PTEN tumour suppressor deregulation in cancer gives it considerable therapeutic pertinence. Until fairly recently, tumour suppressor loss-offunction was considered an undruggable cancer specific aberration, but with the unravelling of synthetically sick/lethal associations, targeting cancers that harbour absent or dysfunctional tumour suppressors is now possible. In the first part of this thesis, two complementary approaches were used with the purpose of identifying targets that sensitise PTEN deficient tumour cells. Both strategies shared the use of isogenic cell models to recapitulate endogenous PTEN loss-of-function, Firstly, a hypothesis-driven approach, demonstrated that DNA repair defects exhibited in the context of PTEN deficiency sensitise to treatment with PARP inhibitors. Secondly, a screening approach focussed on a siRNA kinome set, revealed vulnerability of PTEN deficient cells to the targeting of mitotic regulators such as TTK and PLK4, and led to the identification of a novel potential PTEN synthetic partner, NLK, targeting of which could potentially be useful in tackling a range of tumourigenic conditions characterised by PTEN deficiency. 4 In the second half of this thesis, attention was directed at investigating a pioneering targeted therapy, tamoxifen, which inhibits estrogen receptor (ER) signalling. Despite being the most common and effective treatment for breast cancer types reliant on estrogen stimulation, the effectiveness of this endocrine treatment is, nevertheless, limited by the frequent development of resistance, and the mechanisms attributed to this are not yet fully deciphered. A genome-wide RNAi screen was conducted to uncover novel determinants of tamoxifen sensitivity, and a compendium of molecules validated as possible modulators of response to tamoxifen. Inspection of retrospective clinical data showed that for some of these genes, their expression levels in patients with ER+ disease treated with tamoxifen correlated with relapse outcome, suggesting that monitoring of these genes could aid prediction of patient response to antiestrogens. Similar analyses indicated the suitability of targeting certain candidate genes to prevent or combat cancer recurrence in hormonal therapy. Several previously unexplored determinants of tamoxifen response were identified.

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Autophagy is an evolutionarily conserved catabolic process activated by various cellular stresses. In response to stress, cells degrade their cytoplasmic contents, macromolecules and organelles to generate amino acids, free fatty acids and ATP, providing building blocks for critical biosynthetic pathways. Thus, autophagy promotes the survival of cells by generating a nutrient pool and removing damaged cellular components. Consequently, deregulated autophagy has been implicated in many human pathologies, including cancer. The role of Unc51 -Like Kinase 1 and 2 (ULK1 and ULK2) in regulating cancer cell autophagy in response to nutrient deprivation was investigated. ULK1 and ULK2 silencing promoted cell type specific morphologic and molecular alterations in cancer cells from multiple origins that lead to inhibition of cell growth, cellular senescence and apoptotic cell death. At the molecular level, these affects were concomitant with impaired autophagy activity. Over-expression of ULK1 led to the identification of a novel phosphorylation event on Beclin1, a component of the hVps34 complex, essential for autophagosome formation. ULK1 phosphorylated Beclin1 at Ser30 in cells over-expressing active ULK1, or following nutrient deprivation. Further analysis revealed that ULK1 -dependent Ser30 phosphorylation of Beclin1 impairs the of ability of Beclin1/hVps34 to form complexes with UVRAG and Atg14L. Finally, the identification of a novel small molecule inhibitor of ULK1 (iULK1) is described that inhibits autophagosome formation. The iULK1 also exhibited semi-selective inhibition of receptor tyrosine kinase signalling and inhibited mTORC1 signalling in intact cells. Combined inhibition of ULK1 and mTORC1 signalling induced cell death in transformed cells and to a lesser extent in immortalised counterparts. Taken together, the data presented herein describe novel aspects of ULK biology, new mechanisms of ULK1 -dependent autophagy regulation and the validation of a small molecule inhibitor of the autophagy pathway that shows potential as a new class of cancer therapeutic.

Loss of the tumour suppressor PTEN is a common feature of prostate cancer (PCa) and has recently been identified as a prognostic factor for patient relapse following radiotherapy. Published studies in our laboratory have identified CXCL8 signalling as a mediator of PTEN-depleted disease progression and therapeutic resistance. Therefore, experiments were designed to determine whether ionising radiation (IR) could selectively induce CXCL8 signalling in PTEN-deficient cells. Furthermore, we aimed to determine whether therapeutic targeting of the CXCL8 pathway could enhance PCa cell radiosensitivity. The results in this thesis show how exposure to IR increased gene and protein expression of CXCL8. In addition, inhibiting CXCL8 signalling with receptor-targeted siRNA or peptides increased PTEN-depleted cell sensitivity to IR. In vivo, treatment of PTEN-deficient xenografts with IR and a CXCR1/2-targeted pepducin (x1/2pal-i3) resulted in significant tumour growth delay. Subsequent analysis of tumour material confirmed that this was mediated by modulation of IR-induced anti-apoptotic proteins. Similar to CXCL8 signalling, macrophage infiltration has been associated with enhanced disease progression and poor therapeutic outcomes in prostate cancer patients. Unpublished data in our . laboratory has shown that loss of PTEN can predict for macrophage infiltration in prostate patient samples. Experiments were therefore designed to determine the impact of IR on macrophage-mediated paracrine signalling. Using the THP-1 cell line to model the macrophage component, co-culture systems demonstrated that the presence of microenvironment cells can enhance prostate cancer cell radioresistance. Furthermore, IR was shown to induce secretion of the cytokine TNF-α and this was sufficient to initiate NFKB-mediated upregulation of anti-apoptotic protein expression. Inhibition of cellular inhibitor of apoptosis protein-1 (clAP-1) by Smac-mimetics overcame TNF-α pro-survival effects and reduced cell viability by 50%. In addition, pre-treatment with Smac-mimetics sensitised DU145 cells to ionising radiation following macrophage co-culture.

FKBPL is a secreted anti-angiogenic protein, with a therapeutic peptide, ALM201, based on the active domain of FKBPL shortly entering phase I clinical trials. The aim of this thesis was to characterise the effects of FKBPL modulation on physiological and pathological angiogenesis, to identify FKBPL-associated pathways and to determine how FKBPL transcription, translation and secretion is mediated. To study the effects of FKBPL modulation on angiogenesis and tumour growth, a novel amphipathic peptide, RALA, was used to deliver FKBPL siRNA and FKBPL cDNA in vitro and in vivo, to ZR-75-1 xenografts; increased FKBPL was I associated with delayed tumour growth, prolonged survival and decreased microvessel density (MVD), whilst decreased I expression resulted in increased MVD and stemness. The physiological impact of endogenous FKBPL was established by development of a Fkbpl+/- mouse; Fkbpl-/- mice I were embryonically lethal prior to E8.5, suggesting a critical role for FKBPL in embryonic development. However, whilst Fkbpl+/- embryos showed some vascular irregularities, the mice developed normally. In murine angiogenesis models including the aortic ring, sponge, and tumour growth assays, Fkbpl+/- mice exhibited significantly increased sprouting, enhanced vessel recruitment and faster tumour growth, respectively, compared to their wild -type littermates, supporting the anti-angiogenic function of FKBPL. Furthermore, Fkbpl+/ mice were more prone to obesity and were less able to regulate glucose levels; interestingly, ALM201 was able to normalise this phenotype. SIRT1, a key gene involved in ; obesity and diabetes was positively regulated by FKBPL, both in vitro and in vivo, going some way to explaining these effects. Furthermore, manipulation of the SIRT pathway also potentiated the anti-tumour activity of FKBPL. The regulation of FKBPL by pro-angiogenic stimuli, and its secretory pathway was,also investigated. FKBPL was secreted via the Golgi body to a greater extent in human microvascular endothelial cells compared to tumour cells, in keeping with its anti-angiogenic role. Protein and mRNA expression was unaffected by hypoxia and other angiogenic cytokines, VEGF, bFGF or IL8; whilst hypoxia inhibited its secretion in a normal endothelial cell line, but not in a cancer cell line. In conclusion, this indicates that FKBPL is a potent secreted anti-angiogenic protein, and is essential for normal 1 physiological development. As well as being an anti-angiogenic drug with potential for use in cancer treatment, ALM201 also has the potential to reduce weight gain and to normalise blood glucose in patients deficient in FKBPL, opening up further opportunities for future study.

FLIP is an anti-apoptotic protein over expressed in multiple types of cancer, including non-small cell lung cancer and associated with drug resistance and poor prognosis. Histone deacetylase inhibitors (HDACi) are identified as a class of drug which down-regulate FLIP expression in NSCLC and induce apoptosis in a FLiP-, caspase-8- and death receptor-dependent manner. Rational combinations of HDACI with the chemotherapeutic cisplatin or TRAIL are result in synergistic cell death in non-small cell lung cancer cell line models but not in normal lung fibroblast cells. More selective HDACi elicited equal or greater responses as pan-HDACi. FLIP down-regulation in response to HDACi occurs via the ubiquitin-proteasome system (UPS). Fundamental biology of FLIP's ubiquitination is examined, including identifying FLIP's ubiquitination at the death inducing signalling complex (DISC). FLIP's ubiquitination is mediated by Iysines 192 and 195 as well as phosphorylation of serene 193. The conjugation of a ubiquitin moiety to a substrate is mediated by E3 ligases, and the removal of ubiquitin is dependent on deubiquitinating enzymes. Two screens were performed to identify novel E3 ligases and DUBs which regulate FLIP's stability and ubiquitination. From these we identified potential candidates. These were validated and appear to be bona fide regulators of FLIP's ubiquitination. Better understanding of the fundamental biology of FLIP has the potential to open new avenues for therapeutic intervention for cancer treatment.