FKBPL and its peptide derivative, AD-01, have already demonstrated well-established inhibitory effects on breast cancer growth and CD44 dependent anti-angiogenic activity. FKBPL stable overexpression or AD-O! treatment were highly effective at reducing the BCSC population measured by inhibiting mammosphere forming efficiency (MFE) in cell lines and primary breast cancer samples from both solid breast tumours and pleural effusions. Flow cytometry, to assess the ESA+/CD44+/CD24' subpopulation, validated these results. The ability of AD-01 to inhibit the selfrenewal capacity of BCSCs was confirmed across three generations of mammospheres, where mammospheres were completely eradicated by the third generation. Clonogenic assays suggested that AD-O! mediated BCSC differentiation, with a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones. In support of this, the stem cell markers, Nanog, Oct4 and Sox2 were significantly reduced following AD-01 treatment, whilst transfection of FKBPL-targeted siRNAs led to an increase in these markers and in mammosphere forming potential, highlighting the endogenous role of FKBPL in BCSC signalling. The clinical relevance of this was confirmed using a publically available microarray data set (GSE7390), where high FKBPL and low Nanog expression were independently associated with improved overall survival in breast cancer patients. When AD-01 was combined with other agents, we observed additive activity with the Notch inhibitor, DAPT and AD-Ol was also able to abrogate a chemo- and radiotherapy induced enrichment in BCSCs. Importantly, using 'gold standard' in vivo limiting dilution assays we demonstrated a delay in tumour initiation and reoccurrence in AD-O! treated xenografts. The anti-BCSC mechanism of action of the FKBPL endogenous protein and its peptide derivative, AD-01, involves the CD44 and Notch pathway. In summary, AD-Ol appears to have dual anti-angiogenic and anti-BCSC activity which will be advantageous as this agent enters clinical trial.

Acute myeloid leukaemia is a highly heterogeneous entity of disorders in haematopoietic progenitors, characterised by an arrest in differentiation and an outgrowth of myeloid blasts in the bone marrow. This uncontrollable proliferation of immature myeloid blasts leads to their escape from the marrow, causing severe leukocytosis. As AMl is considered a disease of the aging, many patients can't withstand the harsh standard chemotherapy and it comes with a bleak outlook. A novel approach to developing anti-cancer agents has arisen in the understanding of epigenetic regulation in cancer cells, such as chromatin remodelling. Acetylation of histones is a reversible process, whereby acetyl groups are transferred on the amino groups of specific lysine residues by a specific group of enzymes, histone acetyltransferases (HATs) and removed by histone deacetylase complexes (HDACs). These enzymes are recruited, as part of a regulatory system of controlling acetylation equilibrium within the cell . HDACs are involved in regulating a number of processes in the cell, such as cell proliferation, differentiation as well as apoptosis. Deregulation of the activity of these enzymes is associated with cancer; therefore it is important that the HAT and HDAC equilibrium is regained. This equilibrium can be improved through the inhibition of HDAC enzymes using HDAC inhibitors. Vorinostat is a HDAC inhibitor, clinically approved for the treatment of CTCl, and is in phase" clinical trials for AMl and a number of haematological malignancies. Studies have shown that some patients are non-responsive/resistant to Vorinostat; therefore a fuller characterisation of Vorinostat needs to be made so an adequate combination drug can be identified. The purpose of this study was to provide a comprehensive analysis of Vorinostat in AMl cell lines and identify potential synergistic therapies to be used in combination with Vorinostat to provide a better outlook in AMl. Through functional studies, the mode of Vorinostat induced cell death was characterised; mediated by an arrest in G2/M of the cell cycle, an increase in DNA damage and induction of reactive oxygen species. Furthermore, cell death appeared to be induced through the extrinsic pathway, evident through the activation of caspase B and down-regulation of the anti-apoptotic protein c-FLIP. In a collaborative study, the prognostic and therapeutic potential of c-FLIP was determined. Although primarily caspase-B driven, apoptosis was not exclusive to the extrinsic pathway. A gene signature was identified for Vorinostat in OCI-AMl3 cells; with functional group enrichment corroborating results generated characterising Vorinostat induced cell death. Microarray studies led to the identification of the Vorinostat induced up-regulation of HDAC1 expression; elucidated as being a determinant in Vorinostat resistance, in conjunction with an increase in c-FLIP. The global identification of genes associated with Vorinostat induced histone H3 lysine 9 (H3K9) acetylation as investigated using chromatin immunoprecipitation coupled with next generation sequencing (ChIPSEQ), made the overall observation that H3K9Ac was associated with transcriptional completion. Furthermore, the study identified the Vorinostat induced acetylation of the BET bromodomain protein BRD4, an epigenetic reader which facilitates in transcriptional activation. On further analysis, it was found to be up-regulated, providing rationale for BRD4 inhibition with the small molecule inhibitor JQ1 in combination with Vorinostat. Integrated analysis of data from gene expression studies and the ChiP-SEQ experiment provided evidence of enrichment of DNA damage response genes; eluding to the importance of DNA repair in relation to Vorinostat. Throughout this study, a number of potential therapeutic targets were identified which may be used in combination with Vorinostat, although these require further delineation. However it is hoped these novel treatment strategies may improve the clinical response and outlook in AML.

Despite adjuvant systemic therapies improving breast cancer survival rates, some patients are over- or under-treated. This is particularly problematic for the management of early stage ER+/LN- breast cancers. Identifying patients at a higher risk of relapse for whom additional adjuvant chemotherapy is necessary, remains a significant challenge. The primary focus of this thesis was to evaluate the prognostic potential of FKBPL within breast TMA cohorts, and as part of a meta-analysis. FKBPL was shown to be an independent prognostic marker, which is able to predict breast cancer specific survival in ER positive, node positive patients. I As many prognostic tests now focus on the inclusion of multiple markers, the prognostic ability of two FKBPL-associated proteins, phospho-ERα and p21, were also assessed, however both failed to correlate with survival. In an attempt to evaluate the prognostic potential of additional FKBPL-associated proteins, the relationship between FKBPL and an FKBPL-interacting protein, USP19, was characterised in breast cancer cell lines. After an interaction between the two proteins was confirmed, subsequent analysis identified a role for USP19 in the deubiquitination and stabilisation of FKBPL. USP19 was shown to positively regulate FKBPL protein expression in breast cancer cell lines, functionally suppressing breast cancer cell proliferation . . ALM201, an anti-angiogenic peptide based on FKBPLs active anti-angiogenic domain, is soon to enter Phase 1111 clinical trials in ovarian cancer patients. Therefore, the current study also aimed to evaluate FKBPL's prognostic potential within an ovarian cancer TMA cohort, with the potential to utilize the FKBPL biomarker as a companion diagnostic. Unfortunately, FKBPL was not prognostic in the ovarian cancer setting. In conclusion, FKBPL has the ability to further stratify ER+/LN- breast cancer patients, identifying patients who may benefit from additional adjuvant chemotherapy. Validating the prognostic potential of USP19, within the breast cancer setting might prove beneficial to further sensitise the marker.

Oropharyngeal cancers are increasing in incidence worldwide, and many of these tumours are caused by the human papillomavirus. This is a DNA virus that integrates into the host's genetic information, and can cause cancer through suppression of important tumour suppressors such as p53. TP53 is a gene that codes for p53, and in many cancers of the head and neck this is thought to be mutated. In NI, limited emigration provides an excellent context for research that depends on long-term follow up of patients. This study assesses molecular and demographic characteristics of patients and their tumours, in order to build a comprehensive of the emerging disease of oropharyngeal cancer. Methods Retrospective review of head and neck pathology reports was conducted for a twelve-year period, and all primary oropharyngeal squamous cell carcinomas were included. Detailed clinical and pathological information was gathered about patients and their tumours, and tissue microarrays were created for analysis of protein markers. In addition, DNA and RNA were extracted from a cohort of tumours, to study the tumour suppressor p53, and its family member p63. Different HPV testing methods were compared with one another, in order to ascertain the most reliable test for diagnosing presence of active virus in the oropharynx. Outcomes It was determined that testing for HPV is necessary in tumours arising in the oropharynx, and the best testing method for this appears to be detection of HPV DNA using chromatin in situ hybridisation. p53 mutations were found at a rate comparable with other studies in the head and neck, however the mutation profile appeared to be slightly different to that of other anatomical subsites. Studies of TP63 gene expression revealed that high levels of delta-Np63 are associated with disease recurrence and decreased survival, suggesting a role for this in tumour invasion and metastasis.

To date, very little is known about the aetiology of the plasma cell disorder multiple myeloma (MM) and its precursor condition monoclonal gammopathy of undetermined significance (MGUS). Chronic antigenic stimulation and, more recently, medications have been investigated as potential aetiological risk factors however findings from observational studies have been largely inconsistent. This thesis aimed to explore the impact of medical history and drug exposure on the risk of developing MGUS and MM. A systematic review of the literature revealed an elevated risk of MGUS/MM in association with prior autoimmune disease and in particular pernicious anaemia. The findings of this study were further supported by a population-based nested case-control study carried out within the UK Clinical Practice Research Oatalink (CPRO). Similarly an increased risk of MGUS and MM following exposure to common community-acquired infections was observed within studies carried out within the CPRO and the USA SEER-Medicare dataset. Autoimmune disease and infections diagnosed after MGUS were not associated with progression to MM or associated Iymphoproliferative disorders within the CPRO dataset. Oral statin and bisphosphonate use was investigated as a risk factor for the development of MGUS/MM and MGUS progression using the UK CPRO dataset. While there was evidence of a reduced risk of MGUS/MM in association with oral statin use, an increased risk of both MGUS and MM was observed among oral bisphosphonate users most likely as a result of detection bias and/or reverse causality. Post-diagnostic statin use was also associated with a reduced risk of MGUS progression to any Iymphoproliferative disorder but not MM. Overall, the studies conducted as part of this thesis support a role for chronic antigenic stimulation in the development of MGUS and MM, and suggest a potential role for statins as chemopreventive agents within the MGUS/MM setting. Further research is however warranted to confirm these findings.

Prostate carcinoma represents one of the leading causes of cancer death in men within the Western world, Several studies have hypothesised that chronic prostatic inflammation may predispose men to developing this malignancy. In 2005, the Gram-positive anaerobe, Propionibacterium acnes was identified as the predominant bacterium in cancerous prostate glands; with its pro-inflammatory nature illustrated following acute and chronic infection of such cells both in vitro and in vivo, The main objective of this study was therefore to further elucidate the potential role of p, acnes in the aetiology of prostate cancer. Firstly, the inflammatory response of normal prostate epithelial cells (RWPE-1s), following short-term exposure to a number of P. acnes phylogroups, was examined; with particular interest on the effects of infection on expression of pro-inflammatory molecules, such as PAR-2. Next, long-term infection studies Were performed to evaluate P. acnes persistence, and the mechanisms by which this bacterium stimulates chronic prostatic inflammation. Short-term P. acnes infection potently induced PAR-2 expression in RWPE-1s, as revealed by RT-PCR and Western! immunoblotling; with all strains also found to enhance secretion of inflammatory mediators, including IL-6 and IL-8. Distinct phylotypic differences were noted during these studies; thus underpinning reports of diversity among P. acnes phylotypes. Total viable counts demonstrated the ability of P. acnes to establish chronic RWPE-1 infection; thereby facilitating the induction of sustained prostatic inflammation, achieved through augmentation of PAR-2, and increased release of pro-inflammatory molecules, Moreover, qRT-PCR array analysis revealed P. acnes infection to upregulate many pro-angiogenic genes; a response reflected during in vitro tubule assays upon incubation of HMEC-1 s with conditioned media obtained from P. acnes-infected RWPE-1s. Taken together, the findings obtained these studies highlight prostatic P. acnes infection as a potential risk factor for prostate carcinogenesis, due to the strong inflammatory response that is generated upon exposure to such cells.

Radical radiotherapy, often in combination with hormone ablation, is a safe and effective treatment option for localised or locally-advanced prostate cancer. However, up to 30% of patients with locally advanced PCa will go on to develop biochemical failure, within 5 years, following initial radiotherapy. Improving radiotherapy response is clinically important since patients exhibiting biochemical failure develop castrate-resistant metastatic disease for which there is no curative therapy and median survival is 8-18 months. Loss of the tumour suppressor PTEN function is observed at a high incidence in advanced prostate cancer and is reported to be a prognostic factor for relapse following radiotherapy in locally advanced PCa. Unpublished studies within our group identified PTEN as synthetically lethal with ATM deficiency. We therefore aimed to test the efficacy of ATM inhibition to radiosensitise PTEN-deficient cells. PTEN-isogenic in vitro tumour and normal cell models as well as a Tet-inducible in vivo xenograft prostate model were used in this study. Response to clinically relevant dose's of ionising radiation and ATM kinase inhibitor KU-60019 treatment was assessed using clonogenic survival assays and tumour growth delay studies. The molecular mechanisms mediating selective sensitivity of PTEN-deficient cells to these agents were determined by western blotting, y-H2AX foci. Our results show the role of PTEN in cellular response to IR was both complex and context-dependent. Secondly, combination treatment with KU-6001 9 ATM kinase inhibitor and IR was shown to selectively target PTEN-deficient tumour cells with minimal toxicity to normal prostate RWPE-1 cells. Furthermore, combined treatment significantly inhibited PTEN-deficient tumour growth compared to PTEN-expressing counterparts, with minimal toxicity observed. This project shows A TM inhibition enhances radiation sensitivity of PTEN-deficient tumour cells. The mechanism of this effect appears to involve increased oxidative damage rather than the cells' capacities to repair DNA damage. These findings support the clinical evaluation of ATM inhibitors in combination with radiation in CRPC,

Basal-like breast cancers (BLBC) are an aggressive sub-type of breast cancer which associates with high rates of proliferation and metastasis. However, the molecular mechanisms underlying these tumours are still largely unknown as most BLBC are also triple-receptor negative, which is defined by the lack of expression of oestrogen (ER) and progesterone (PR) receptors, with normal HER2 status. Consequently, there are currently no targeted therapies available for these tumours, which display the worst prognosis of all breast cancer subtypes. TP53 mutations occur within 20% of all breast tumours, however, this rate is observed to increase dramatically to approximately 85% within the basal-like subtype. Moreover, TP53 missense mutations can lead to the production of mutant proteins which possess novel oncogenic capabilities, known as Gain-of-Function (GOF) p53 mutants. These GOF mutants may display wild-type protein conformation (contact mutants) or exhibit conformational alterations to the tertiary protein structure (structural mutants). We have shown that siRNA-mediated knockdown of endogenous mutant p53, particularly contact GOF mutants significantly reduces survival of BLBC cells. We also demonstrate that contact, but not structural, p53 mutant proteins can directly bind to the transcription factor Etsl, and are also involved in upregulation of its expression. We show that mutant p53 interaction with Etsl is a critical event for both survival of these cells and co-regulation of downstream target genes. We have identified several novel mutant p53-specific transcriptional targets, some of which play important roles in the proliferation and survival of BLBC cells. Additionally, we have putatively detected an alternatively-spliced p53 protein isoform following over-expression of a structural GOF p53 mutant in non-tumourigenic and tumour-derived cell lines.

Metastasis, the leading cause of mortality in cancer patients, is the dissemination of cancer cells from the primary tumour and spread to the distant sites of the body. This is a complex process during which tumour cells need to overcome several natural barriers to gain entry into the bloodstream and thus allow formation of secondary tumour in distant location. In the last two decades, much effort has been focused on showing that tumour cells use specialized actin-based membrane protrusions termed invadopodia to perform matrix degradation. Invadopodia gain their protrusive capacity combining the mechanical force of actin polymerization with the chemical activity of matrix degradation. As such, invadopodia are F-actin- rich structures enriched in integrins, tyrosine kinases signalling machinery, soluble and membrane proteases, including matrix metalloproteases (MM Ps) , and actin-associated proteins. How all these components are specifically recruited to the ECM degradation sites has not been fully clarified yet. An emerging model describes invadopodia as dynamic cellular platforms where the signalling, membrane trafficking and cytoske~eton remodelling converge upstream of ECM degradation - at spatially confined cholesterol-rich membrane compartments. Despite the field of invadopodia biogenesis and function is still a very recent, it is witnessing an increasing interest and an increasing number of molecular players have been identified in the last two decades. Although the existence of the invadopodia-like structures in vivo settings still needs to be determined, invadopodia represent powerful experimental paradigm to study the tight integration between the signalling, the membrane trafficking and cytoskeleton remodelling upstream of ECM degradation, the rate-limiting step in cell invasion, and might provide better understanding of cancer cell invasion and metastasis. In order to investigate the cholesterol-rich lipid raft feature of invadopodia, I followed three lines. First, I showed that inhibition of cholesterol formation at penultimate step of its biosynthesis, and subsequent accumulation of desmosterol, blocks formation and function of invadopodia; .thus demonstrating that the central role of cholesterol is connected to its presence in functional lipid rafts. In the second approach, I found that the SFKs inhibitory kinase Csk is a negative regulator of invadopodia-mediated ECM degradation and its role depends on the localization in the cholesterol-rich lipid rafts. Finally, I demonstrated that free cholesterol-dependent ARF6-associated recycling pathway might be involved in the trafficking to invadopodia, while the ARF6-pathway constituents are localized at ECM degradation sites and ARF6-specific cargo CD147 is recycled to invadopodia. Taken together, my findings provide novel insight towards the elucidation of invadopodia as specialized cholesterol-dependent membrane domains where signal transduction and membrane trafficking events might be temporally and spatially confined.

Colorectal cancer (CRC) remains one of the most common malignancies in the world. Development of resistance to conventional therapies is frequently observed in advanced stages of the disease and frequently involves MAPK signaling. Recent studies identified p38a. MAPK as a mediator of resistance to irinotecan and 5-fluorouracil in CRC. Our genetic and pharmacologic studies revealed that p38a is required to maintain CRC cell metabolism, as its inhibition leads to Fox03A activation, autophagy, cell death, and tumor growth reduction both in vitro and in preclinical mouse models. Furthermore, our data show that p38 is activated in response to cisplatin treatment and mediates chemoresistance in HT29-xenografted tumors. MEKl inhibition has been found to reduce CRC cell proliferation in vitro and to decrease tumor growth in xenograft models and we recently reported that MEK-ERKl/2 inhibition is followed by over-activation of the p38 MAPK pathway. We found that p38a and MEK combined inhibition specifically induces apoptosis by enabling TR. Aa signaling propagation through t-Bid and caspase 3, and fosters cell death in CRC cells. Sorafenib is reported to inhibit nine protein kinases, including BRAF, VEGF and the DGF-out conformation state ofp38a. Our results show that the SB202190 (type I p38a inhibitor) and Sorafenib (type II p38a inhibitor) synergize at the molecular and biqlogical level enhancing tumor growth inhibition and induction of apoptosis in CRC both in vitro and in vivo. The combined use of SB202190 and PD0325901 (MEKl inhibitor), Sorafenib or Cisplatin significantly reduced tumor growth by inducing a higher degree of apoptosis compared to each single treatment. Our data validate in vivo the combined inhibition of the p38a. and ERK pathways or their association with chemotherapy as a promising approach to treat CRC. Moreover, they suggest that the phosphorylation status of p38 MAPK may be a marker of resistance in CRC.