Prostate cancer (PC) is the most common cancer and the second cause of cancer related death in men. Central to this, is the role of the androgen receptor (AR) which acts as a transcription factor, regulating the expression of genes required for normal prostate growth and cancer development. Consequently, the AR remains the primary target for therapeutic intervention. However, these treatments become ineffective, resulting in castrate resistant prostate cancer (CRPC) which generally retains AR expression. The AR interacts with several co-regulatory proteins which can perturb AR-targeted therapies in CRPC. Targeting these co-regulatory proteins to indirectly target the AR signalling cascade may prove beneficial. Recently, our group identified KMT5A as a potential regulator of AR through selective siRNA library screening. KMT5A is a lysine methyltransferase that mono-methylates histone 4 lysine 20 and non-histone proteins, including p53. Using a relevant in vitro CRPC model it was shown that KMT5A acquires AR co-activator activity which is in contrast to androgen sensitive models where KMT5A co-represses AR activity. This highlights the importance of studying KMT5A regulation. KMT5A protein levels are tightly regulated by multiple E3 ligases for cell cycle-dependent poly-ubiquitination-mediated degradation. KMT5A poly-ubiquitination by E3 ligases SCFβ-TRCF, CRL4Cdt2 and APCCdh1 promotes degradation in G1, S and late mitosis cell cycle phases, respectively. Moreover, the Skp2 E3 ligase has been suggested to play a role in KMT5A ubiquitination and degradation but direct supporting evidence is currently absent. Additionally, Skp2 is suggested to directly regulate the AR signaling pathway. It is also unknown whether KMT5A could be modified directly by ubiquitination without promoting its degradation. As such, we aimed to investigate KMT5A mono-ubiquitination and the role of Skp2 in regulating KMT5A as well as independently regulating the AR signaling cascade. Mono-ubiquitinated KMT5A was demonstrated in a panel of PC cell lines. Its existence was further confirmed by performing ubiquitination assays in COS7 cells. Furthermore, the KMT5A C-terminal SET domain was identified as the target for mono-ubiquitination. Moreover, mono-ubiquitinated KMT5A was highly enriched in S phase cells, coincident with extremely low levels of unmodified KMT5A. Mono-ubiquitinated KMT5A was exclusively cytoplasmic and its abundance was greatly enhanced by Skp2, but not associated with protein turnover. Together, this data suggests that cell cycle-dependent KMT5A mono-ubiquitination is an important mechanism to diminish nuclear, unmodified KMT5A levels to facilitate cell cycle progression. Thus, insight for the physiological significance of mono-ubiquitinated KMT5A ii may provide a novel therapeutic target to indirectly target the AR. Finally, Skp2 was not found to have a direct effect on AR signaling.

Alternative splicing is a co-transcriptional process which contributes to protein diversity in eukaryotes because it can generate different mRNAs arising from the same gene. Resulting proteins can play either redundant or counteracting roles, or they can lack functionality. Changes in the alternative splicing process have been associated to several diseases including breast cancer. The Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase (RTK) which is activated through hetero-dimerisation with other receptors including members of its own family (HERs). No ligand binds directly to the HER2 receptor; however, it is the favoured hetero-dimerisation partner amongst other HER family members. Following phosphorylation, HER2 can activate several signalling pathways, including RAS/MAPK (ERK1/2) and PI3/AKT, which promote cell proliferation and inhibit apoptosis. Therefore, HER2 is a breast cancer biomarker and its overexpression is commonly assessed in patients. HER2 is a target for therapies including the monoclonal antibody Trastuzumab (Herceptin®) and the tyrosine kinase inhibitor Lapatinib (Tykerb). Nevertheless, these therapies are not always efficacious and their efficiency decreases with the aggressiveness of the disease leading to drug resistance. Besides the HER2 (wild type) receptor, the HER2 gene can produce three functional alternative splicing variants. HER2Δ16, arising from skipping of exon 16, shows high oncogenicity and it is associated with drug resistance. Conversely, Herstatin and p100, arising from inclusion of intron 8 and intron 15 of HER2 respectively, have shown anti-oncogenic properties. In this study a speculative working model was developed; this partially explains regulation of the alternative splicing of Herstatin in the HER2 positive breast cancer cell line SKBR3. In this model the Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 promotes the inclusion of intron 8, whereas the SR protein SRSF1 prevents Herstatin formation. Moreover, using a HER2 minigene, RNA secondary structure and somatic mutations were shown not to affect the splicing of Herstatin. Also, two novel alternative splicing variants of HER2, arising from intron inclusion, were identified. Their protein expression was subsequently confirmed via an in vitro translation system using plasmid expression vectors. Furthermore, these novel variants were shown to enhance phosphorylation of ERK1/2 in the HER2 negative breast cancer cell line MCF-7. iii Finally, a CRISPR/Cas9 system was developed with the aim of promoting Herstatin expression in breast cancer cell lines by modifying the splicing of HER2. Expression of Cas9 protein, as well as its ability to cleave the DNA, was confirmed in the HER2 negative breast cancer cell lines MCF-7 and T47D. Here, modification of HER2 splicing and increased Herstatin expression was not confirmed, but this can be the subject of future research.

Disseminated breast cancer cells have been found in the perivascular niche of lung, brain and bone marrow. Research has suggested that thrombospondin 1 (TSP1), an antiangiogenic protein secreted by endothelial cells, is involved in cancer dormancy. My research aims to clarify the role of TSP1 in cancer dormancy. On the other hand, breast tumours overexpress indoleamine 2,3-dioxygenase (IDO1) which degrades intracellular tryptophan, a key amino acid of TSP1. I aim to investigate whether cancer cells induce endothelial IDO1 expression and therefore limit TSP1 synthesis. I hypothesize that the decrease of TSP1 might enable cancer cell proliferation and angiogenesis. To evaluate whether endothelial cells can induce cancer dormancy, MDA-MB-231 cells were cultured on the top of an endothelial monolayer or treated with endothelial conditioned medium. Ki67, p21 and cell cycle analysis showed that endothelial cells induce cell cycle arrest in MDA-MB-231 cells but not senescence. ki67 was also decreased when MDA-MB-231 cells were cultured with TSP1. MDA-MB-231 revealed to be more resistant to docetaxel, a breast cancer drug, when pre-cultured with TSP1. Conditioned medium experiments showed that MDA-MB-231 cells are capable of inducing endothelial IDO1 expression and it also increased tryptophan degradation, which was prevented by siRNA IDO1 knockdown. Interestingly, endothelial TSP1 secretion was revealed to be decreased under low tryptophan concentration. Immunohistochemistry of breast cancer tissue showed that there was a negative correlation between vascular IDO1 and stromal TSP1. IFNγ, a potent inducer of IDO1, showed to be able to induce endothelial IDO1 and a decrease in endothelial TSP1. Taken together, the data presented here suggests that endothelial cells induce breast cancer dormancy and drug resistance via TSP1. My research also suggests that an IFNγ/IDO1 pathway might decrease TSP1 synthesis leading to cancer cell proliferation and angiogenesis.

Despite recent advances in the treatment and detection of breast cancer it still remains the third most common cause of death from cancer in the UK as a result of its final metastatic stage, which is currently incurable. Numerous targets have been identified in an attempt to prevent and treat this highly aggressive form of cancer with limited success, however recent work has highlighted B-cell lymphoma 3 (Bcl-3) as a promising therapeutic target. Bcl-3 is a mediator of the well characterised NF-kB signalling pathway and both have been implicated with promoting tumour growth and progression. The role of Bcl-3 in mediating tumour proliferation and apoptosis has been extensively researched; however more recent work has also implicated Bcl-3 with an important role in regulating metastasis. Interestingly, suppression of Bcl-3 expression has been shown to inhibit breast cancer metastasis, a result which has been replicated through the use of small-molecule inhibitors designed to disrupt Bcl-3 binding to both p50 and p52. Despite this little is known, other than a reduction in cell motility, on how Bcl-3 is mediating its effects on metastasis, therefore the aims of this project were to elucidate the mechanisms through which Bcl-3 is regulating metastasis. In this work we have shown how Bcl-3 can regulate a variety of single-cell and collective migratory phenotypes through inhibiting Rac1 and Cdc42 GTPase activity. We have also shown for the first time Bcl-3 to be upregulated during EMT, which appears to help regulate the expression of a number of EMT-inducing genes. Interestingly, we have also shown that prolonged inhibition of Bcl-3 results in a loss of cell viability through either senescence or apoptosis which appears to be dependent on cells expressing wildtype or mutant p53 respectively. Finally we have identified a novel set of small molecule Bcl-3 inhibitors that are capable of mimicking Bcl-3 suppression to regulate human breast cancer cell lines as well as both prostate and colorectal cell lines, offering a new therapeutic option for the treatment of breast and other human cancer types.

Chronic lymphocytic leukaemia (CLL) is a mono-clonal B-cell malignancy characterised by heterogeneous clinical course and response to treatment. Recent studies with whole genome or whole exome sequencing have identified novel recurrent genomic lesions, and associated them with adverse clinical outcome of this disease. Owing to their limited sensitivity, the true incidence of these mutations, subclonal complexity and evolution and their roles in disease progression and treatment resistance are still not quite clear. To gain insight into these issues, I developed a highly sensitive (with an average coverage depth being 2250x and the lowest limit of detection 1%) and robust next generation sequencing (NGS) test using HaloPlex and Ion Torrent PGM to target exons of 15 recurrently mutated genes including TP53, ATM, SF3B1, PCLO, NOTCH1, LRP1B, SAMHD1, FBXW7, BIRC3, HISTIH1E, XPO1, CHD2, MYD88, POT1 and ZFPM2 in CLL. In the initial study using this technique, samples from a cohort of 32 cases with progressive and/or therapy resistant CLL before (n = 10) or after chemotherapy (n = 22) were screened. 87.5% of the patients were found to carry at least one somatic non-synonymous mutation in the first 12 targeted genes (VAF: 2-98%). The most commonly mutated genes were SF3B1, ATM, TP53 and PCLO identified in 11, 10, 9 and 8 cases, respectively. Mutations in TP53 and its upstream regulator ATM appeared to be dominant over other concurrent gene mutations compared to other genes (P = 0.011). Combining NGS and global SNP array analyses revealed a significant association between genomic aberrations in the ATM-p53 pathway and genomic instability. Moreover, prior exposure to DNA-damaging chemotherapy was associated with the bigger numbers of mutation events and mutated genes, although the increases were borderline significant. These results suggest that ATM-p53 pathway defects contribute to the acquisition of additional genomic aberrations and that treatment with DNA-damaging chemotherapy may play a role in the induction or selection of mutations. In the subsequent longitudinal study of 33 additional samples of 23 mutated cases from the same cohort, I showed the existence of different mutational processes operative in CLL including mutations related to AID, ageing and other factors. I confirmed the significant contribution of AID-related mutations to CLL clonal evolution, implying on-going activity of this enzyme in off-target genes. In addition, I demonstrated the predominance of a linear path of clonal evolution in this cohort. Thus, 89.3% of the mutated clones/subclones identified at advanced disease stages were detectable at time of diagnosis or prior to treatment. Hence, the early detection of these mutations may potentially serve as predictive biomarkers to inform on therapeutic decisions. I also documented convergent clonal evolution with priori-selection of clones carrying deleterious mutations in 2 target genes including ATM and TP53. This observation suggests that not all the mutations in the same gene play an equal role in disease progression and/or treatment resistance. Analysis of mutation doubling time revealed that driver mutations, including those in TP53, BIRC3, NOTCH1 and SF3B1, were significantly correlated with faster evolution as indicated by the shorter doubling time of variant allele frequency. Importantly, I found that increased subclonal sizes were strongly associated with shorter treatment-free survival in patients with driver mutations. Taken together, the results from this thesis have provided strong evidence emphasising the importance and usefulness of applying the ultra-sensitive NGS test in the early detection and subsequent monitoring of these recurrent somatic mutations in CLL. A translational study based on findings in this thesis is now ongoing, with an aim to convert this test into a regional clinical service.

Lung cancer is the leading cause of cancer-related death and is usually diagnosed at advanced stage leading to poor patient survival. Therefore there is a pressing need for early detection of disease. DNA methylation is an early event in carcinogenesis and a limited number of diagnostic markers have been developed for clinical use. This thesis seeks to address whether the development and application of novel DNA methylation assays can diagnose lung cancer at early stage. Previously identified DNA methylation biomarkers, along with novel targets identified by methylation microarray, were developed in multiplex assay format. Twelve markers were used to screen 417 bronchoalveolar lavage specimens from Liverpool Lung Project (LLP) subjects divided into training and validation sets. The optimal biomarker panel (CDKN2A, RARB and TERT) demonstrated improved clinical sensitivity and specificity (Sensitivity/Specificity: 85.7%/93.8%, AUC: 0.91) compared to previous studies. The optimal methylation algorithm detected more than 60% of stage T1 tumours and 93 cytologically occult lung cancer cases. Eight methylated DNA assays were optimised for use with the newly developed Droplet DigitalTM PCR (ddPCR) platform and a targeted pre-amplification technique, MethPlex enrichment, was developed. I established a comprehensive analytical framework to compare performance of methylation-specific ddPCR and quantitative methylation-specific PCR directly and in combination with MethPlex enrichment. ddPCR demonstrated greater precision and linearity, lower limit of detection (WT1 MethPlex ddPCR LOD95 = 1.86 GE), and discriminated twofold differences in methylated DNA input. MethPlex ddPCR detected DNA methylation more frequently in lung cancer patient plasma than in controls in a retrospective case-control study. Technical methylation controls were consistently and precisely detected at inputs as low as 3 methylated copies. Discriminatory efficiency of marker combinations was inadequate, presumably due to limitations in DNA extraction methodology. DNA methylation biomarker diagnostic performance in bronchoalveolar lavage merits further validation in a prospective trial. MethPlex ddPCR analysis showed great promise, demonstrating highly sensitive DNA methylation detection in technical assessment. It is expected that appropriate DNA extraction procedures and higher cfDNA yields will lead to much improved clinical discriminatory efficiency.

The impact of randomized controlled trials is frequently diminished by disparate outcome reporting, precluding the comparison of results between trials or synthesis of data in meta-analyses. This is particularly problematic in lower incidence conditions such as oropharyngeal squamous cell carcinoma (OPSCC), where the need to synthesise data from competing trials is greater. Minimum outcome reporting standards, known as Core Outcome Sets (COS) have been shown to increase the consistency of outcome reporting between trials of comparable interventions, thus facilitating the comparison or synthesis of trial data. The objective of the work in this thesis was to identify outcomes of importance to patients, carers and healthcare professionals and define a COS for OPSCC. The methods used comprised a systematic review of OPSCC RCTs to identify the outcomes reported and establish whether there was outcomes heterogeneity as suggested by other studies; semi-structured qualitative interviews with patients and carers to establish their outcomes of importance; a Delphi Study of patients, carers and healthcare professionals, to reach consensus on the outcomes that should be included in a COS for OPSCC. The systematic review described in chapter two identified significant heterogeneity in outcome reporting; 58 outcomes were reported in 43 RCTs, only three outcomes were measured in more than 50% of studies, and only 41% of outcomes were measured in more than one study. The qualitative study identified 136 outcomes. Survival and late adverse effects of treatment are of greatest priority to patients and carers. The Delphi study successfully reached consensus on eight outcomes for inclusion in the COS. There is substantial heterogeneity in the outcomes measured in contemporary RCTs in OPSCC. Yet, there is strong consensus between stakeholder groups in the outcomes of importance. Implementation of the COS will increase the consistency of outcome reporting thus facilitating the comparison of data from competing trials and synthesis of data in meta-analyses. Further consideration must be given to ways in which the uptake of COS can be maximised to have the highest impact. The COS is applicable to trials of interventions used in current clinical practice, however the advent of new treatment strategies may require that this is reviewed and adapted.

In clinical breast cancer, endocrine therapy and cytotoxic chemotherapy are the mainstay of treatment. Progesterone receptor (PR) analogues can be radiolabelled by Positron Emission Tomography (PET) to assess response to endocrine therapy while molecular imaging of apoptosis biomarkers can be utilised to monitor early response to chemotherapy. To develop a pre-clinical PR PET imaging agent, LA036 (a PR analogue), based on non-steroidal tanaproget core, was selected due to high affinity to PR with a relative binding affinity of 140%, compared to progesterone. Cell uptake studies in various PR cell lines and blocking studies with progesterone demonstrated specificity of [18F]LA036 for PR. PET imaging of T47D xenografts with [18F]LA036 revealed uptake in the tumour: AUC0-60(%ID/ml)(area under the curve from 0-60min) 498.79±29.7%, muscle: (AUC0-60 423.62±26.3). The tracer was unstable in vivo, with high uptake due to metabolism noted in various organs with deflourination observed in bone. [18F]ICMT-11, an isatin analogue is a novel PET radiotracer for detection of apoptosis. Treatment of a breast cancer xenograft MDA-MB-231 with combination chemotherapy resulted in increased uptake of [18F]ICMT-11 in tumour within 24 hours of treatment and sustained up to 96 hours. The corresponding NUV60(%ID/ml) of time-points was 0.203 (Control), 0.789 (24h), 0.832 (48h), 0.903 (72h) and 0.757 (96h). PET based voxel intensity histograms revealed an increase in voxel intensity maintained between 24 and 96h post-chemotherapy. TUNEL and caspase immunofluorescence of tumours correlated with PET data of increased apoptosis with chemotherapy while Ki67 index correlated with reduced proliferation of the tumour. In clinical breast cancer, [18F]ICMT-11 was utilised to study the optimum time point of apoptosis following chemotherapy. Patients were imaged at baseline and 24-48h post chemotherapy, exhibited an increase in standardised uptake value (SUV) in the lymph nodes; SUVav 0.39±0.02 (pre), SUVav 0.45±0.03 (post), and SUVmax 0.87± 0.02(pre),1.22±0.12 (post). In patients imaged at baseline and 2 weeks post chemotherapy, tumour to breast ratio (TBR) and tumour to muscle ratio (TMR) were increased. Immunohistochemistry with caspase and TUNEL labelling from breast biopsies was related to apoptosis observed in the PET data. Endocrine therapy and chemotherapy in breast cancer treatment can therefore be monitored by utilisation of molecular imaging using PET.

The development of colorectal cancer (CRC) occurs sequentially through the accumulation of genetic changes, or mutations, resulting in unrestrained cellular proliferation and survival. The genetic changes leading to CRC are well defined, with aberrant activation of the Wnt signalling pathway and loss of the tumour suppressor p53 playing key roles. This project aimed to investigate the role of the orphan nuclear receptor liver receptor homologue-1 (LRH-1) in the development of CRC. Previous work in this laboratory identified LRH-1 as an important mediator of the estrogen response in breast cancer cells. LRH-1 has also been implicated in the development of CRC, where it promotes cell cycle progression and synergises with β-catenin. Here I provide evidence for novel mutations in the DNA binding domain of LRH-1 in CRC. I found that CRC-associated LRH-1 mutations affect its ability to bind LRH-1 response elements. To better define the mechanisms of LRH-1 action in CRC, gene expression microarray analyses were performed in two CRC cell lines following siRNA-mediated LRH-1 knockdown. Several previously undescribed candidate LRH-1-regulated genes were identified and validated. While I found no evidence for crosstalk between LRH-1 and the Wnt pathway in the CRC cell lines examined, pathway analyses coupled to gene expression profiling suggested a role for LRH-1 in p53 signalling. LRH-1 silencing was shown to be associated with growth inhibition and up-regulation of the cell cycle inhibitor p21 in CRC cells. This occurs in a p53-dependent manner and was not observed in cell lines where p53 is mutated or deleted. I further demonstrated LRH-1-mediated modulation of p53 activity at the p21 promoter. Collectively, this work demonstrates a novel role for LRH-1 in the suppression of p53 signalling in CRC tumours that retain wild-type p53, and identifies LRH-1 as an important prospective target for treatment of these tumours.

The lymphatic system plays a critical role in normal physiology and is associated with pathologies from lymphoedema to cancer metastasis. A primary role of the lymphatic system is to transport lymph containing pathogens and immune cells from tissues to lymph nodes (LNs) where humoral and cellular adaptive immune response is initiated. Despite the importance of fluid and proteins transport to specific regions of the LN in proper immune response, little is known about fluid distribution and its modulation under different pathologic conditions such as inflammation. Four studies in this thesis set out to improve our understanding of how lymph transport in the LN modulates its function. The first study established a computational model of fluid flow in the LN demonstrating its important role in fluid exchange with blood vessels, and determined medulla hydraulic conductivity as the key parameter for controlling hydraulic resistance of the LN. In the second study, the experimentally measured LN resistance showed an increase after inflammation, which was associated with medulla hydraulic conductivity. The third study demonstrated an application of this model in providing insight into the role of lymph transport in formation of interfollicular chemokine gradients in the LN that are crucial for antigen presenting cell entry to LN paracortex. In the fourth study, the effect of shear stress that is present in the sinuses of the LN was examined on the calcium dynamics of the lymphatic endothelium. Overall, this research revealed that lymph flow both modulates (e.g. chemokine gradient formation and calcium signalling) and is modulated by (e.g. hydraulic resistance change with inflammation) LN function. The lymph flow plays a critical role in fluid balance and immune response and has a great potential as a therapeutic target for modulating immune response.