Neuroblastoma is the most common extracranial solid tumour in children, with a high mortality rate among patients with aggressive disease. In a previous study we showed that Clusterin (CLU) inhibits the transcription factor NF-ϰB in a neuroblastoma cell line by stabilizing the NF-ϰB inhibitors (IϰBs). Moreover, suppression of CLU could elicit NF-ϰB activation and increased the expression of markers for the epithelial-to-mesenchymal transition (EMT), a developmental process utilized by aggressive cancer cells for invasion, in a mouse neuroblastoma model. The expression of CLU is also negatively regulated by the proto-oncogene MYCN, which is associated with aggressive stages of neuroblastoma tumours. Thus, we hypothesised that CLU is a tumour suppressor gene, which negatively regulates NF-ϰB and metastasis. In this study, we investigated the role of the different isoforms of CLU in the regulation of signalling pathways. We also aimed at identifying the precise mechanisms by which CLU regulates NF-ϰB. The results show that intracellular, but not secreted CLU, inhibits NF-ϰB activity. Interestingly, extracellular CLU (secreted CLU) positively regulates AKT and the Phosphoinositide-3 Kinase (PI3K) pathway. Mass-spectrometry analysis and co-immunoprecipitation experiments demonstrated that a chaperone protein named Heat Shock Protein 60 (HSP60) is bound to the N-terminal region of intracellular CLU in neuroblastoma cells. Suppression of HSP60 by shRNA knock down experiments caused decreased neuroblastoma cell proliferation and increased cell death. Our results suggest that HSP60 exert its oncogenic activity by inhibiting the function of CLU and promoting NF-κB activity. In summary, in this report we demonstrate that a direct interaction between intracellular CLU and HSP60 could play an important role in the regulation of NF-κB activity and neuroblastoma development.

Human Papillomavirus positive (HPV+) head and neck squamous cell carcinoma (HNSCC) represents a distinct clinical and epidemiological entity compared with HPV negative (HPV-) HNSCC. In this thesis I conducted both an epigenomic and genomic analysis to test the possible involvement of epigenetic modulation by HPV in HNSCC and associated genetic changes. Using laser-capture microdissection of formalin-fixed paraffin-embedded (FFPE) HNSCCs, I generated both DNA methylation and genetic profiles of HPV+ and HPV— samples. I then used an independent clinical sample set and HPV+ and HPV- HNSCC cell lines for the validation of the obtained methylation data by two independent methods (Infinium 450k BeadArray and MeDIP-seq). Paired end sequencing of captured DNA, representing 3,230 exons in 182 genes often mutated in cancer was applied for mutation profiling. I validated the latter findings by Infinium copy number variation (CNV) profiling, Sequenom MassArray sequencing and immunohistochemistry. Significant differences in the methylation and genomic profiles between HPV+ and HPV- HNSCC were observed. Methylation analysis revealed a hypermethylation signature with involvement of Cadherins of Polycomb group target genes in HPV+ HNSCC samples. Integration with independent expression data showed strong negative correlation, especially for the Cadherin gene family members. Combinatorial ectopic expression of the two HPV oncogenes (E6 and E7) in an HPV— HNSCC cell line partially phenocopied the hypermethylation signature observed in HPV+ HNSCC tumours and established E6 as the main viral effector gene. Moreover, MeDIP-Seq data revealed methylation sites within integrated HPV genomes. These methylation sites were confirmed by bisulfite sequencing, both in HNSCC samples and HPV+ HNSCC cell lines. Validated genomic changes clustered HPV+ and HPV- oropharyngeal carcinomas into two distinct subgroups with TP53 mutations detected in 100% of HPV- cases. Abrogation of the G1/S checkpoint by CCND1 amplification and CDKN2A loss occurred in the majority of HPV- tumours, indicating that trials with CDK inhibitors in this disease subtype may be warranted. My data establish archival FFPE tissue to be highly suitable for these types of methylation and mutation analysis and suggest that HPV modulates the HNSCC epigenome through hypermethylation of Polycomb repressive complex 2 target genes such as Cadherins which are implicated in tumour progression and metastasis. Moreover, my findings reinforce the causal role of HPV in oropharyngeal cancer and indicate that therapeutic stratification according to somatic genomic changes, in addition to HPV status, could be the most appropriate future approach for these cancers.

Cancer results from the sequential accumulation of heterogeneous mutations and epigenetic changes, leading to “onco-selection” of cells with survival/growth advantages in specific microenvironments. Chemotherapy and radiotherapy act by damaging DNA; uncoupling DNA damage from cell death can cause treatment resistance. Deregulation of the DNA damage response (DDR) can result from aberrant expression of DDR-related genes, due to chromosomal aberrations. Solid tumours often have multiple, recurrent chromosomal aberrations, suggesting coordinated somatic evolution of cancer genomes via co-selection of interdependent chromosomal aberrations. This thesis investigates the hypothesis that “onco-selection” acts on unstable cancer genomes by selecting for multiple changes together. A comprehensive DDR signalling network was constructed. The cytogenetic location of its genes was analysed. The non-random genomic organisation of DDR genes partly coincided with regulation by shared transcription factors, notably oncogenic SNAIL. SNAIL expression was investigated in relation to hypoxia and apoptosis in xenografts of four human colorectal cancer cell lines. SW1222 and LS174T had weak and strong SNAIL expression, respectively, so could be used to investigate the role of SNAIL in treatment resistance through regulation of DDR genes. Bayesian Networks (BNs) were constructed to model probabilistic dependencies between the proposed co-selected, DDR-related chromosomal aberrations in breast, colorectal, lung, ovarian and prostate cancer. The significance of the models was assessed using newly-developed software. Breast cancer involved both co-selection of aberrations on different chromosomes and loss/gain of adjacent regions, whereas loss/gain of adjacent chromosomal regions predominated in the other cancers. A therapeutic combination of DDR targets (RELA, DVL1, E2Fs, CDC45L and MAP2K1/2) was proposed by mapping the predicted dependencies in the breast cancer BN model onto the DDR network, and analysing gene function and network topology. DDR network motif analysis further supported the importance of targeting RELA in combination therapies. This thesis provides insight into cancer resistance to DNA damaging therapies, and may help to predict new personalized combinations of targets to overcome treatment resistance.

This thesis investigates the expression of dopaminergic constituents within human B lymphocytes and selected malignancies thereof while addressing the impact of chemically modifying a structural analogue of dopamine - 3,4-methylenedioxymethamphetamine (MDMA) - on its anti-neoplastic potential. With regards the former, it was concluded that B cells are 'dopaminoceptive' in character rather than dopaminergic. Thus while transcripts for the D2 receptor (D2R) and dopamine transporter (DAT) were expressed, mRNA for dopamine-synthesising tyrosine hydroxylase was undetectable amongst B cells studied. Upon B-cell activation, the D2R and DAT transcripts were translated into readily detectable protein. There was evidence for different D2R isoforms being expressed which, in model B-cell lines, were shown to be localised intracellularly. The recently identified trace amine-associated receptor 1 (Taarl) associated with dopaminergic signalling in the central nervous system was also found expressed at the protein level in human B cells. B-cell Taarl was functional: selective agonists triggering apoptosis in susceptible lines at the low micromolar level. MDMA has been reported to promote apoptosis in susceptible malignant B-cell lines such as those derived from Burkitt's lymphoma. Unfortunately, concentrations required to elicit killing were at least lOO-fold higher than those that could be reached safely in vivo leading to the study herein on analogues of the parental compound iteratively modified at the a-carbon andlor nitrogen site. Modifications resulting in increased potency were indeed found and attempts to unravel mechanisms-of-action of the more potent analogues were begun. The dopaminoceptive system of B cells and their pathologies may therefore offer novel therapeutic opportunities.

Changes in DNA methylation patterns are a hallmark of both cancer and ageing, and may underlie susceptibility to developing age-related diseases such as cancer. To uncover the impact that such variation has on health and ageing, we assessed DNA methylation patterns in the peripheral blood leukocytes (PBL) of 480 participants in the Newcastle 85+ study to determine the levels, and degree of inter-individual variation, of DNA methylation in the promoter region of a panel of genes by Pyrosequencing. We found considerable inter-individual variation in promoter CpG methylation in several genes and a remarkable similarity to leukaemic patterns of aberrant methylation. This included specific methylation of the same sets of genes, strong correlations between methylation of the genes (in a pattern reminiscent of the CpG island methylator phenotype observed in cancer) and the presence of densely methylated alleles in highly methylated individuals, identical to patterns observed in cancer cells. This suggests that ageing and cancer related methylation may be closely linked. Further analysis of PBL DNA methylation levels in Newcastle 85+ study participants with a previous history of cancer (n=113) versus cancer-free individuals (n=113) found significantly higher methylation in those with a cancer history (10.71% vs. 10.21%, p=0.04). Further, a separate group of 72 individuals diagnosed with cancer within the 3 year duration of the study had similarly increased methylation levels (10.96% vs. 10.36%, p=0.03), suggesting that pre-existing methylation in normal cells may increase risk of cancer and may be evident prior to clinically detectable disease. In addition, individuals with increased DNA methylation were more likely to be categorised as frail (as defined by Fried) than those with lower DNA methylation measures, indicating that disrupted methylation patterns are associated with detrimental effects on healthy ageing. Subsequently, a GWAS analysis found that SNPs in two genes, DSCAM and DSCAML1, appeared to be associated with determining DNA methylation levels in the Newcastle 85+ study participants.

Breast cancer is a complex set of diseases with different biological and clinical characteristics. An important contribution to this diversity is provided by germ-line genetic variations. The HER2-positive breast cancers have been extensively studied with particular regard to their biology and targeted treatments. However, the influence of pharmacogenetic (PG) factors on these aspects remains largely unexplored. This research focused on the possible effects of common single nucleotide polymorphisms (SNPs) on specific aspects of HER2-positive disease. Initially we analysed two coding SNPs in the HER2 gene (Ile655Val and Ala1170Pro) in breast cancer patients and evaluated their potential association with HER2 expression in tumour samples. The proline variant of the Ala1170Pro SNP was associated (odds ratio = 1.7, p = 0.01) with HER2 over-expression/amplification in over 360 breast cancer patients. In contrast, Ile655Val was not associated with HER2 over-expression/amplification. Bioinformatics tools predict that Ala1170Pro might affect the structure or function of the HER2 protein. The same variants were explored in the context of DNA extracted from the patients’ primary tumours in 241 patients. We hypothesized that the proline allele of Ala1170Pro could undergo allele-specific amplification during the development of HER2-positive tumours. This hypothesis, however, was not confirmed. Although the association of the proline allele of Ala1170Pro with HER2 positivity is intriguing, the role of the two SNPs in HER2 over-expression/amplification remains to be elucidated. Trastuzumab has radically changed the treatment of HER2-positive breast cancer. However, resistance to treatment and toxicity can limit its effectiveness. The second objective of this project was the analysis of PG, biomarker and pharmacokinetic (PK) parameters in trastuzumab-treated patients. Fc Gamma Receptors (FcgRs) are key proteins in the trastuzumab-induced Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and two coding SNPs in these genes (FCGR2A His131Arg and FCGR3A Phe158Val) were analysed. The measurement of trastuzumab in plasma was made possible by the development of a novel cell-based ELISA. Only 28 patients with advanced disease treated with trastuzumab were recruited. However, we observed a possible association of the valine allele of the FCGR3A Phe158Val SNP with a longer time to progression (p = 0.03). Cardiac toxicity was assessed in a group of 139 patients treated with adjuvant trastuzumab. Although a role of germ-line genetic variants could not be demonstrated, the analysis highlighted the challenges and limitations encountered in the conduct of an observational pharmacogenetic study. This project leaves a legacy archive composed of germ-line DNA samples, tumour DNA samples, plasma samples and tumour FFPE blocks from over 360 breast cancer patients. These samples and data are available for the exploration of further potential factors which might influence the biology of the disease and/or its response to treatment.

Childhood acute lymphoblastic leukaemia (ALL) does not possess a propagating cell hierarchy, at least as defined by B-cell precursor immunophenotype. Indeed, many, or even all, leukaemic blasts may have the potential to propagate the disease. This unusual characteristic mirrors the substantial capacity for clonal expansion demonstrated by fully differentiated normal lymphoid cells. This Fellowship aimed to investigate the genetic programmes underlying the propagation of acute lymphoblastic leukaemia. An initial candidate approach confirmed the expression of PIWIL2, a gene critical to the maintenance of germline stem cells, in both cell line and primary ALL. Knockdown of PIWIL2 resulted in reduced cellular proliferation and significant prolongation of doubling time in two ALL cell lines, SEM (MLL/AF4) and 697 (E2A/PBX1). Unexpectedly, PIWIL2 was also found to be expressed in peripheral lymphoid cells from healthy donors, but not terminally differentiated cells of myeloid origin, suggesting that PIWIL2 may have a previously unidentified function in both normal and malignant lymphoid cells. A second project has developed an in vitro genome-wide RNAi screen to identify candidate genes involved in the clonal propagation of ALL. This project has assessed a serial re-plating assay using feeder cell co-culture to provide a surrogate niche environment. Initial results have demonstrated the feasibility of such an approach. The benefit of using a co-culture re-plating assay, as compared to a standard suspension culture approach, remains under investigation. ii Finally, this Fellowship developed a protocol for the lentiviral transduction of patient-derived leukaemic blasts and cloned and validated a novel lentiviral vector capable of in vitro analysis, in vivo disease monitoring and RNAi. With these, it will now be possible to validate candidate leukaemic propagation genes in vivo, using primary leukaemic material. The results of these studies will provide candidates for the development of novel therapeutic agents for children with ALL.

Colorectal cancer typically arises through the sequential accumulation of mutations in different genes. Mutations in the adenomatous polyposis coli (APC) gene are thought to be the initiating step in this sequence of events and are found in the majority of early colorectal tumours. Investigation of these lesions has revealed that selection of 'optimal' combinations of mutations at the APC locus is in place, but the roles of such selected combinations have never been clarified. In the work presented in this thesis, I have demonstrated that similar constraints on APC mutations are active in tumours from attenuated FAP (AFAP) patients and that given the sub-optimal location of the germline APC mutation in these patients, additional somatic mutations are often required, especially in patients with germline mutations in the alternatively spliced region of exon 9. I have also shown that APC promoter hypermethylation does not appear to play a fundamental role in the selection of optimal APC genotypes. I have shown that the optimum combinations of mutations at APC are those that allow retention of some APC activity with respect to β-catenin degradation and that this has effects on the resulting activation of the Wnt signalling pathway. Optimal combinations of APC mutations result in intermediate nuclear β-catenin levels, which surprisingly highly activate a selection of Wnt targets. In an attempt to identify novel Wnt targets that are important for tumorigenesis and could serve as therapeutic targets, I have validated results from a cross-species comparison that identified a set of genes showing consistent differential expression between early tumour samples carrying APC mutations and normal tissue. In addition, I have investigated the biological function of one such-identified molecule, the serum/glucocorticoid-regulated kinase (SGK1), and I have revealed its potential role as a key regulator of intestinal cell differentiation and apoptosis.

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