The middle T-antigen (MT) encoded by polyomavirus induces tumours by mimicking an activated cell surface receptor. A hydrophobic region close to the C-terminus locates MT to the plasma membrane where it initiates oncogenic signalling. HA or FLAG tags added to the C-terminus of MT could be detected on the outside of cells, demonstrating that MT is a transmembrane protein. Addition of a KDEL sequence retained MT in the endoplasmic reticulum, where it failed to transform cells as a consequence of its lack of binding to ShcA, PI3K, and PLC-γ1 despite the phosphorylation of the appropriate binding sites. Additional studies show that MT binding to PP2A is probably required in addition to the TMD for MT to exit the ER and migrate to the plasma membrane. MT on the cell surface is present in discrete clusters that contain phosphorylated ShcA and so represent active signalling complexes. New mutations in the hydrophobic region prevented MT clustering, and also inhibited cell transformation without altering the association of MT with its known binding proteins. Overall, these data, together with previous publications, illustrate that MT associates with signalling proteins at discrete subcellular membrane sites in its maturation pathway. MT binds to PP2A in the cytoplasm, to c-Src at the ER, and to ShcA, PI3K, and PLC-γ1 at subsequent locations en route to the plasma membrane. Formation of a large macromolecular complex in the plasma membrane is probably required for MT signalling. Similar cell surface complexes are observed with activated growth factor receptors, so it is possible that normal and oncogenic signalling from receptors is also dependent upon the assembly of large macromolecular complexes at the cell surface membrane. There was no obvious association of MT clusters with lipid rafts but there was colocalisation with the actin cytoskeleton.

The use of anthracyclines such as doxorubicin (DOX) has improved mortality and morbidity in cancer patients, yet associated risks of cardiomyopathy have limited their clinical application. DOX-associated cardiotoxicity typically progresses to heart failure (HF). The knowledge of the mechanisms underlying DOX-related cardiac dysfunction is currently limited, hampering the development of cardioprotective strategies. MicroRNAs (miRNAs) are gene expression regulators that play potent roles in both cardiovascular disease and cancer. We wished to investigate DOX-induced changes in cardiac miRNA expression and the potential alteration of cellular processes downstream. Myocardial miRNA profiling was performed after DOX injury, either via acute administration to cardiomyocytes in vitro or chronic exposure in vivo, and compared to miRNA profiles from infarcted hearts. We identified an overlapping down-regulation of several members of the miR-30 family. Subsequent experimental validation of a bioinformatically predicted subset of target genes allowed us to confirm four novel miR-30 targets: β1- and β2-adrenoceptors (β1AR, β2AR), Gi alpha 2 (Giα-2) and the pro-apoptotic gene BNIP3L, all of which are essential in cardiomyocytes. The implications of the β-adrenergic pathway in HF are extensively described. Importantly, we show a preferential βAR inhibition by miR-30, having a β-blocker like effect. Additionally, we demonstrate that high miR-30 levels are protective against DOX insults and correlate with lower reactive oxygen species generation in cardiac cultures. Upstream of these mechanisms, we describe the transcription factor GATA-6 to be involved in mediating DOX-induced miR-30 down-regulation. Finally, when assessing the implications of miR-30 in breast cancer, we observed an inverse correlation between miR-30 expression levels and breast cancer cell migration in vitro. Moreover, bioinformatic analyses revealed reduced miR-30 levels in breast cancer patients. Taken together, our findings encourage a potential translational use for miR-30 as an early biomarker as well as a therapeutic strategy, combining a cardioprotective action with pleiotropic anti-cancer effects.

Background: Non-response to neoadjuvant therapy is a significant challenge for clinicians managing solid cancers. This thesis aimed to determine whether Epithelial Mesenchymal Transition (EMT) was associated with non-response to neoadjuvant therapy in patients with locally advanced rectal cancer. Methods: Representative tissue specimens from the tumour invasive front of consecutive patients undergoing resection of rectal cancer from 2009-2011 were used. Patients with marked regression to neoadjuvant therapy were classified as responders with the remainder as non-responders. Markers of EMT included: reduced immunohistochemical expression of membranous E-cadherin, increased nuclear beta-catenin expression and tumour budding. In-situ-hybridisation was used to assess the expression of microRNA-200c (mir200c), an upstream master-regulator of EMT. Real-time polymerase chain reaction was used to quantitate expression of the gene for E-cadherin. Results: From 103 patients undergoing resection of rectal cancer, 69 received neoadjuvant chemoradiotherapy; 65% of these were non-responders. Reduced mir200c expression was significantly associated with higher T grade. Reduced membranous E-cadherin, increased nuclear beta-catenin and tumour budding individually predicted the presence of extra-mural vascular invasion. Reduced E-cadherin, nucleic beta-catenin, reduced mir200c and tumour budding were all significantly associated with non-response to neoadjuvant therapy (all p<0.001). Reduced E-cadherin and mir200c expression were both associated with reduced cancer specific survival (log-rank p-value 0.036 and 0.009 respectively). E-cadherin gene expression was not related to radiotherapy response or tumour budding. Conclusion: Targeted biomarkers of EMT were associated with non-response to neoadjuvant therapy and reduced survival in advanced rectal cancer. EMT may provide a practical clinical biomarker and novel therapeutic target, to improve the proportion of patients who respond to neoadjuvant therapy.

Pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) is one of the most lethal tumour types world-wide. The majority of patients present late with locally advanced or metastatic disease. Therefore, despite advances in operative techniques, perioperative management and oncological treatments, the overall 5-year survival remains <5%. Determining tumoural factors that contribute towards its aggressive nature may help in identifying novel molecular biomarkers and/or therapeutic targets. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate target gene expression and are able to act as tumour suppressors or oncogenes. MiRNAs have been extensively profiled and implicated in the initiation and progression of PDAC. Furthermore, there is a possibility of translating miRNAs into clinically useful biomarkers. Here, I developed upon these initial observations and demonstrate that miRNAs can be used to differentiate low risk pancreatic benign cystic tumours (BCTs) from PDAC. We confirmed that these miRNAs regulate the expression of known PDAC oncogenes, and that miR-16, miR-126 and let-7d target BCL2, CRK and KRAS respectively. Next, in order to investigate the main contributors to tumourigenesis, an integrated molecular analysis (miRNA-mRNA) was performed in PDAC. By using a combination of network-based bioinformatics, miR-21, miR-23a and miR-27a were prioritised as important in PDAC progression. We demonstrated that the use of a combination of miRNA inhibitors (against miR-21, miR-23a and miR-27a) in a murine subcutaneous PDAC xenograft model was able to reduce tumour growth, better than oncomiR-21 inhibition alone. BTG2 and NEDD4L were found to be direct targets of the miRNA combination and were established as new candidate tumour suppressors in PDAC. The clinical relevance of this 3 miRNA signature was demonstrated, as high expressors of the combination have poor overall survival after surgical resection, independent of other clinicopathologic factors. Together, these studies identify specific miRNAs as important regulators of PDAC tumourigenesis and their possible use as biomarkers.

Aberrant choline metabolism is a characteristic shared by many human cancers. It is predominantly caused by elevated expression of choline kinase alpha, which catalyses the phosphorylation of choline to phosphocholine, an essential precursor of membrane lipids. In this thesis, a novel choline kinase inhibitor has been developed and its therapeutic potential evaluated. Furthermore the probe was used to elaborate choline kinase biology. A lead compound, ICL-CCIC-0019 (IC50 of 0.27 ± 0.06 μM), was identified through a focused library screen. ICL-CCIC-0019 was competitive with choline and non-competitive with ATP. In a selectivity screen of 131 human kinases, ICL-CCIC-0019 inhibited only 5 kinases more than 20% at a concentration of 10 μM (< 35% in all 131 kinases). ICL- CCIC-0019 potently inhibited cell growth in a panel of 60 cancer cell lines (NCI-60 screen) with a median GI50 of 1.12 μM (range: 0.00389-16.2 μM). Importantly, proliferation of normal cells was only minimally affected (MCF-10A, ST-T1b and CCD-18Co; GI50 30-120 μM). In HCT116 cells, ICL-CCIC-0019 potently inhibited the formation of phosphocholine (EC50 0.67 ± 0.28 μM), which consequently decreased the formation of phosphatidylcholine. The compound arrested cells in the G1 phase of the cell cycle, and induced endoplasmic reticulum stress and apoptosis. A single injection of ICL-CCIC-0019 at 10 mg/kg decreased tumour uptake of the choline kinase specific PET tracer [18F]fluoromethyl-[1,2-2H4]-choline at 24 hours (AUC0-60-23%). Treatment of HCT116 colon cancer cell xenograft bearing mice with 5 mg/kg ICL-CCIC-0019 i.p. resulted in strong tumour growth inhibition. Human breast cancer cell lines oncogenically transformed by HER2 exhibit increased levels of phosphocholine and are therefore more likely respond to CHKA inhibition. To identify such patients more readily, a novel, non-invasive, PET-imaging-based HER2- targeting diagnostic tool, [18F]GE-226, was developed. [18F]GE-226 (KD = 76 pM) uptake was 11 to 67-fold higher in 10 HER2 positive versus negative cell lines in vitro. Tumour uptake correlated with HER2 expression in 5 different tumour models (r2 = 0.78), and a fluorophore-labelled tracer analogue co-localised with HER2 expression. Tracer uptake was not influenced by short-term or continuous treatment with trastuzumab, but reflected HER2 degradation by short-term HSP90 inhibition. Taken together, these data further validate CHKA as a drug target and warrant the further development of ICL-CCIC-0019, potentially in the setting of HER2 positive cancers.

Ovarian cancer has the highest mortality among malignancies of the female reproductive tract. Negative regulatory mechanisms within the tumour microenvironment inhibit anti-tumour T-cell function, leading to evasion from immune responses. The binding of PD-L1, expressed on antigen presenting cells to PD-1 on activated T cells, is thought to result in an important T-cell inhibitory mechanism and promote immune evasion. We have shown that increased PD-L1 expression on monocytes correlates with malignancy, advanced stage and surgical debulking status in ovarian cancer. We have also studied PD-L1 expression on T-cells in women with ovarian tumours and its relation with diagnosis. We have devised a sandwich ELISA to accurately detect the presence of soluble PD-L1 in the plasma and ascites of patients with ovarian cancer and have shown increased presence of soluble PD-L1 in women with ovarian cancer when compared with benign ovarian tumours and healthy female donors. We have found high expression of PD-L1 at mRNA and protein level in all serous ovarian cancer cell lines. We have investigated the role of PD-1/PD-L1 engagement in in vitro T-cell responses, using ovarian cancer cell lines. We have shown that PD-L1/PD-1 engagement in tumour and T-cell interaction is associated with reduced T cell proliferation. In an in vitro model of ovarian cancer cell line and stimulated T-cell co-culture we have shown that blocking PD-L1, PD-1 and key cytokines reverse T cell 'suppression' and increase proliferation. We have also demonstrated that IL-10 and TGF-β play an important role in regulation of PD-L1 expression on tumour associated monocytes. We have also shown that ascitic monocytes suppress the proliferation of both ascitic T-cells and healthy stimulated T-cells. This suppression by tumour associated monocytes can be completely reversed by combined blocking of PD-L1, PD-1 and IL-10 to the baseline proliferation of T-cells cultured. This research has shown that PD-L1 can be a potential biomarker and therapeutic target in ovarian cancer.

Introduction of bowel cancer screening programmes internationally has resulted in a significant shift in diagnosis towards early stage disease. In addition, the number of patients diagnosed with complex benign colorectal polyps is increasing. Advanced endoscopic techniques including endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) as well as transanal endoscopic microsurgery (TEMS) are being increasingly utilised for excision of early rectal neoplasia. An equivalent surgical technique is currently not available for colonic lesions, and endoscopic techniques are associated with a high risk of complications such as bleeding, perforation and recurrence than in the rectum. Hemicolectomy with en bloc mesenteric excision remains the gold standard treatment for patients with early, node negative colon cancer and large colonic polyps. Even when performed laparoscopically within an enhanced recovery protocol, problems such as death, anastomotic leakage and other complications occurring in up to 40% of patients make colectomy a morbid intervention. The introduction to this thesis reviews the literature on the development and staging of colorectal cancer as well as currently available endoscopic and surgical techniques. In order to improve our understanding of the morbidity associated with hemicolectomy for the treatment of benign colonic polyps, two studies examined short term outcomes after surgery. The results suggest similar 30-day outcomes to those after cancer resection. In addition, a two-part study was designed to assess bowel function and related quality of life in patients who underwent hemicolectomy for colonic neoplasia, a subject that is poorly documented in the literature. As an introduction to the laboratory work, a systematic review of endoscopic full thickness excision techniques is presented. The final three chapters of the thesis describe ex-vivo development and outcome data after a porcine survival study of a laparo-endoscopic excision technique for colonic lesions as a potential alternative to hemicolectomy.

This thesis investigates the clinical impact of a point-of-care diagnostic strategy for pulmonary tuberculosis (TB) in a setting at the heart of the TB and human immunodeficiency virus (HIV) epidemics in rural KwaZulu-Natal, South Africa. Although the identification and prompt treatment of active pulmonary TB disease remains the cornerstone of global TB control strategies, weak diagnostic systems contribute to substantial delays and default during the diagnostic process. As new diagnostic technologies are developed, evidence is needed around how best to deliver them within health systems in order to maximize their impact. The impact of positioning of a molecular diagnostic system (Xpert MTB/RIF) was investigated in a cluster randomised trial. Clusters (two-week time periods) were randomised to one of two strategies: centralised laboratory Xpert MTB/RIF testing or point-of-care Xpert MTB/RIF at the clinic. The trial enrolled 1297 adults with symptoms of pulmonary TB who were HIV infected and/or at high risk of drugresistant TB. There was some evidence that point-of-care placement shortened the time to initiation of treatment but there was no difference in the overall proportion of culture-positive pulmonary TB cases initiated on appropriate anti-TB treatment within 30 days. Overall mortality was lower than anticipated and, although it was higher with the point-of-care strategy, this effect was not maintained after adjusting for the presence of TB disease and CD4+ T-cell count. Further analysis suggested that the point-of-care strategy increased the proportion of valid Xpert results from the initial sputum specimen, increased the proportion of individuals receiving test results and allowed same-day treatment initiation for half of all culture-positive cases that tested positive with Xpert. The diagnostic performance of the Xpert MTB/RIF system was comparable under both strategies. However, delays in initiation of treatment for drug-resistant TB cases and for Xpertnegative/ culture-positive cases occurred similarly with both strategies, reducing the potential to detect a real impact on outcomes. Although not a primary focus of the study, the results highlighted deficiencies in the performance of sputum culture, which raise questions about its place as the gold standard diagnostic test. The development of simple, rapid diagnostics suitable for point-of-care use remains important for TB control in high burden settings. The findings will improve understanding of the key requirements for successful diagnostic strategies and the lessons learnt will help to inform future diagnostic clinical trials. Further research is needed to evaluate how different diagnostic strategies might impact on TB transmission in health care facilities and more broadly in the community.

The increasing prevalence of breast cancer (BC) in different parts of the world, particularly in the UK, highlights the importance of research into the aetiology and pathology of the disease. BC is the most common malignancy affecting women worldwide. Aquaporins (AQPs) are membrane protein channels that regulate cellular water flow. Recently, studies have demonstrated that expression of AQP3 is up-regulated in cancerous breast tissue. The present study examines the role of AQP3 in BC cell biology. Examination of clinical cases of BC showed higher AQP3 gene and protein expression in cancer tissues compared to healthy border tissues. In distinct clinicopathological groups however there were no differences observed with regards to AQP3 expression, suggesting that AQP3 expression may not be a predictor of lymph node infiltration or tumour grade. shRNA technology was used to knockdown gene expression of AQP3 in the invasive MDA-MB-231 BC cellular model. Cellular proliferation, migration, invasion, adhesion and response to the 5- fluorouracil (5-FU) based chemotherapy treatment were investigated in parental and knockdown cell line. AQP3 knockdown cells showed reduction in cellular proliferation, migration, invasion and increase in cell sensitivity to 5-FU compared with wild type (WT) or scrambled control (SC) cells. The effects of AQP3 knockdown on cellular glycolytic ability and ATP cellular content were quantified. Indirect glucose uptake was also measured by quantifying reconditioned media. AQP3 knockdown cells showed significantly lower levels of glucose uptake as compared to WT or SC. However there was no difference in the glycolytic ability and ATP content of the cells suggesting AQP3 has no role in cancer cell energetics. These data collectively suggest AQP3 expression is associated with the BC disease clinically and plays a role in multiple important aspects of BC pathophysiology, thus AQP3 represents a novel target for therapeutic intervention.

Tissue transglutaminase (TG2) has been suggested to be a key player in the progression and metastasis of chemoresistant breast cancer. One of the foremost survival signalling pathways implicated in causing drug resistance in breast cancer is the constitutive activation of NFκB (Nuclear Factor -kappa B) induced by TG2. This study provides a mechanism by which TG2 constitutively activates NFκB which in turn confers chemoresistance to breast cancer cells against doxorubicin. Breast cancer cell lines with varying expression levels of TG2 as well as TG2 null breast cancer cells transfected with TG2 were used as the major cell models for this study. This study made use of cell permeable and impermeable TG2 inhibitors, specific TG2 and Rel A/ p65 targeting siRNA, TG2 functional blocking antibodies, IKK inhibitors and a specific targeting peptide against Rel A/p65 to investigate the pathway of activation involved in the constitutive activation of NFκB by TG2 leading to drug resistance. Crucial to the activation of Rel A/p65 and drug resistance in the breast cancer cells is the interaction between the complex of IκBα and Rel A/p65 with TG2 which results in the dimerization of Rel A/p65 and polymerization of IκBα. The association of TG2 with the IκBα-NFκB complex was determined to be independent of IKKα/β function. The polymerized IκBα is degraded in the cytoplasm by the μ-calpain pathway which allows the cross linked Rel A/ p65 dimers to translocate into the nucleus. Using R283 and ZDON (cell permeable TG2 activity inhibitors) and specific TG2 targeting siRNA, the Rel A/ p65 dimer formation could be inhibited. Co-immunoprecipitation studies confirmed that the phosphorylation of the Rel A/p65 dimers at the Ser536 residue by IKKε took place in the cell nucleus. Importantly, this study also investigated the transcriptional regulation of the TGM2 gene by the pSer536 Rel A/ p65 dimer and the importance of this TG2- NFκB feedback loop in conferring drug resistance to breast cancer cells. This data provides evidence that TG2 could be a key therapeutic target in the treatment of chemoresistant breast cancer.