Ovarian cancers are frequently diagnosed at advance stage with a high mortality rate. Investigating prospective biomarkers are crucial to understand the molecular defects behind ovarian cancer. WPM encodes the protein Microcephalin involved in the DNA damage response and cell cycle pathways. MCPH1 is also known, as BRIT1 (BRCT-repeat inhibitor of hTERT expression). MCPH1 is a potential tumour suppressor gene since defects in these pathways may cause carcinogenesis. Microcephalin expression was identified by immunofluorescence in primary EOC cultures derived from epithelial ovarian cancer (EOC) patients. Patients with weak nuclear Microcephalin presented with high grade tumours. Cytoplasmic Microcephalin increased with tumour grade (p = 0.04). Patients with weak nuclear Microcephalin had a reduced survival rate (p = 0.081). MCPH1 might be a tumour suppressor gene and aberrant localization of Microcephalin involve in tumour development. Microcephalin was evaluated in EOC tissue by immunohistochemistry. Low Microcephalin expression was identified in high grade (p < 0.0001) and advance stage EOC (p = 0.0438). Therefore, Microcephalin is a potential prognostic biomarker. However, immunofluorescence and florescence activated cell sorting indicated that there was no correlation between Microcephalin expression and mitotic defects or aneuploidy. The association between MCPH1 and telomerase activity and alternative splicing of the catalytic subunit (hTERT) was examined. Four different hTERT splice variants were assayed by qRT-PCR. A negative correlation was identified between MCPH1 and the WT hTERT (p = 0.03). Strong positive associations with the hTERT sand a/13-deletion hTERT were reported (p = 0.03, p = 0.04 respectively). This supports the regulatory effect of MCPH1 on telomerase activity. The association between MCPH1 and a-deletion hTERT was confirmed in ovarian cancer cell lines using a hTERT a-deletion plasmid construct. To further elucidate the function of MCPH1, gene expression profiling was performed. Thirty-two pathways were affected by MCPH1 siRNA knockdown including mRNA processing. We proposed that MCPH1 might have a novel role in the mRNA processing pathway.

Aims: Identify the mean prevalence of CR-PTSD, factors related to trauma, and the development and maintenance of PTSD, in cancer survivors. Background: Systematic reviews reveal that CR-PTSD is uncommon, and it is unclear a) what makes this experience traumatic, and b) what factors are implicated in the development, and maintenance of PTSD in this population. Methods: A mixed-methods sequential explanatory approach was used. Phase 1 consisted of three studies: a) random-effects meta-analysis of PTSD prevalence statistics and moderating factors in cancer survivors (k=25, n=4189); b) a cross-sectional analysis of PTSD and contributing factors in a PTSD Clinic for cancer survivors (n=60); and c) a prospective analysis of the role of emotion schemas and processing styles and how they predict adaptation to stress in a sample of students (n=24). Phase 2 was conducted to find follow-up explanations for Phase 1 results. Study 4 (Phase 2) consisted of two clinical case studies from the PTSD Clinic – one with adjustment disorder, and the other with severe chronic CR-PTSD. Results: Study 1 revealed that PTSD prevalence in breast cancer survivors was 5.8% (95% CI=3.3-10%), and that there were no significant study-level moderators that predicted differences in prevalence. Similar results were found for Study 2, although when adjusted for age, those with CR-PTSD suffered from more impoverished emotional experiences than those without CR-PTSD. These differences were rendered non-significant when depression symptoms were added as a covariate. Study 3 revealed that increases in anxiety during a stressor were best predicted by emotion schemas related to the lack of comprehensibility of emotions. Findings from Study 4 suggested that aspects of the cancer experience was very traumatic for both patients, but that the course/development of disorder was influenced by the social-cognitive processes involving the interaction of the patient’s emotion schemas and coping strategies, with the quality of their support system. Conclusions: Cancer can be traumatic under certain conditions, and PTSD is uncommon in cancer survivors, but clinical samples of cancer survivors with and without PTSD suggest that CR-PTSD is characterised by severe problems experiencing, linking, and labelling emotions. Preliminary evidence from case studies reveal that the combination of a) an appraisal of the cancer as traumatic, b) an invalidating social network, and c) emotionally avoidant coping styles throughout the cancer treatment, may predispose traumatised cancer survivors to PTSD.

Immunotherapy is a valuable approach to target tumour cells by stimulating the body’s adaptive immune system. To achieve this objective, it is important to identify and study antigens that are distinctively expressed in cancer and can be used effectively to initiate or enhance immune responses. T21 (Testis clone 21) was identified as a member of cancer/testis antigen (CTAs) family of proteins using a modified SEREX technique and can be considered a promising prostate-associated tumour antigen, shown to elicit a humoral immune response in prostate cancer patients. T21 has also been shown to be over-expressed in malignant glands of the prostate compared to benign glands and stroma at the mRNA level. Since T21 shares significant similarity with the Centrosomal Protein, CEP290, which has been implicated in several cilia associated syndromic disorders such as Joubert syndrome, it was necessary to determine similarities and differences in the expression and functionality of these two molecules to facilitate further studies on the role of T21 in prostate cancer tumourigenesis. As with the majority of identified cancer/testis antigens, the role of T21 in cancer remains undetermined. Therefore, investigations were initiated to understand the potential function of T21 in cancer cells. Next Generation Sequencing (NGS) data have been previously obtained following T21 knockdown/silencing in PC3 cells and the expression profiling of this data indicated that T21 function was related to several pathways involved in tumourigenesis. Genes that were either up or down regulated in the presence of T21 were validated by qRT-PCR and the results provided evidence that T21 may share functional activity these various genes and be implicated as a key driver of important signaling pathways. The up or down regulation of molecules associated with key signaling pathways was established from the NGS data and validated by qRT-PCR. The effect of T21 silencing on the associated MAPK pathways was further investigated by using proteome profiler arrays. The results indicated the potential role that T21 may play in cancer cell growth regulation and suggested that T21 is potentially a central player in the cancer process. Finally, the data presented here strongly supports the hypothesis of T21 having a significant role in the biology of malignant cells. This, together with its potential use in immunotherapy, that warrants further investigation.

Phosphatidylinositol 3-kinase (PtdIns3-K) signalling is a crucial survival pathway in multiple malignancies, and an exciting target for drug development. The pathway is subject to multiple regulatory mechanisms including ubiquitylation, a reversible process that can influence protein stability, localisation and activity. A family of approximately 80 deubiquitylating enzymes (DUBs) are responsible for the cleavage of ubiquitin and ubiquitin chains from protein substrates. The DUBs are attractive drug targets and have increasingly been implicated in cellular processes germane to malignancy, including the PtdIns3-K pathway. The aim of this study was to systematically identify DUBs involved in regulating the pathway, with a particular view to identifying potential novel drug targets. The project employed a DUB siRNA library to perform a series of RNAi screens using two complementary approaches. In the first approach, the effects of DUB depletion on the protein level of nine PtdIns3-K pathway components (p110α, p110β, p110γ, p85α, p170, PDK1, PTEN, Akt, and mTOR) were assayed by immunoblotting. DUBs whose depletion either increased or decreased the protein level of these components were subject to further validation. Of particular interest were five DUBs (PRPF8, TNFAIP3, USP32, USP34 and OTUD1) whose depletion decreased the level of PDK1. In addition, depletion of three closely related members of the Josephin family of DUBs (ATXN3, ATXN3L and JOSD1) resulted in an increase in the protein level of the tumour suppressor PTEN. In view of the potential clinical utility of upregulating PTEN, the latter DUBs were prioritised for further investigation. Initial investigation indicates that all three DUBs alter PTEN level at a transcriptional level. Moreover, the effects of ATXN3 appear to be independent of its known function in modulation of histone acetylation status. The second approach utilised a U2OS cell line stably transfected with EGFP tagged FOXO3, in which PtdIns3-K dependent FOXO3 translocation could be assessed by live-cell imaging. This enabled the design of a functional screen in which the effects of DUB depletion on downstream PtdIns3-K signalling were assessed. Among the DUBs identified in this screen was USP45, depletion of which enhanced nuclear translocation of FOXO3-EGFP in response to PtdIns3-K inhibition. Several DUBs regulating FOXO3-EGFP abundance were also identified in this screen, including USP1 and USPL1 whose depletion respectively decreased and increased FOXO3-EGFP levels. The screen additionally identified three DUBs (USP8, OTUD4 and DUB4) whose depletion was synthetically lethal with PI-103 treatment. In summary, several DUB modulators of the PtdIns3-K pathway were identified that, subject to further mechanistic and functional studies, may increase the options available for targeting this vital pathway in malignancy.

The three human Ras proto-oncogenes encode for four highly homologous protein isoforms, H-, K(A)-, K(B)- and N-Ras, that function as molecular switches and timers to transduce signals from activated cell surface receptors to modulate signalling pathways responsible for cell proliferation, growth, survival and apoptosis. Oncogenic mutations of Ras occur in ~16% of all human cancer cases and most often affect codons 12, 13 and 61, each resulting in constitutive activation of the protein. Currently, it is uncertain how many Ras molecules are present per cell and which isoform is most abundant, essential information for the emerging field of systems biology, which utilises mathematical modelling to understand the behaviour of complex signalling pathways in a holistic approach. In the first part of this thesis, a selected reaction monitoring (SRM)-based Ras quantification technique is established for the measurement of each major isoform, along with the generation of isotope-labelled Ras protein standards that support quantification using the protein standard absolute quantification (PSAQ) strategy. Combining targeted SRM-based proteomics with the PSAQ strategy enabled the most accurate measurement of cellular Ras isoform abundance to date, as detailed in the second part of this thesis. The target cell lines in the present thesis were a panel of isogenic SW48 colorectal cells, which express a variety of heterozygous Ras mutations following the exchange of a wild-type allele for a mutant Ras sequence through targeted homologous recombination. Over 250,000 Ras molecules per wild-type SW48 cell were quantified, with K(B)-Ras the major Ras isoform. ~114 Ras molecules are predicted to be bound per μm2 of plasma membrane, with the cellular molarity of Ras at 253 mM. Intriguingly, the presence of certain K-Ras mutations induced significant changes in cellular Ras abundance, which may be attributable to the specific transforming potential of each mutant protein. The presented data also suggests that, in addition to their structural and biochemical variations, Ras mutant proteins may exert their different biological effects through differential protein expression. Many cancer types demonstrate a preference for a single Ras isoform to be mutated and present a codon-specific mutation signature. In colorectal cancer, K-Ras is the most frequently mutated isoform, with over 75% of mutations accounted for by G12D, G12V and G13D in colon tumours with a K-Ras mutation. Clinical data suggests that patients harbouring codon 12 or 13 K-Ras mutations have different overall survival rates and responsiveness to treatment. Isogenic SW48 cells harbouring the aforementioned K-Ras mutations were subject to mass spectrometry-based proteome and phosphoproteome quantitative analysis, to investigate the effects of different amino acid substitution (G12D vs. G12V) or codon mutation (G12D vs. G13D) on oncogenic Ras signalling. The presented data in the third part of this thesis provide the first quantitative proteomic and phosphoproteomic profile of signatures associated with specific K-Ras mutant proteins expressed at endogenous levels. Each K-Ras mutation induced a distinct signalling output, with the most variability observed between codon 12 mutants and G13D. This indicates that the position of a Ras point mutation may have more impact on signalling than an amino acid substitution. Several proteins relevant to colorectal carcinogenesis were found to specifically up-regulated by codon 12 or 13 mutations, with one notable example being the recently described colon cancer stem cell marker double cortin-like kinase 1, which was sensitive only to codon 12 K-Ras mutations. Together, the data presented in this thesis provides a novel insight into the behaviour of a range of Ras mutations in a colorectal cancer setting.

Background There is growing interest in preoperative chemotherapy for patients with colorectal liver metastases (CRLM) but personalising treatments to maximise response and minimise toxicity remains a challenge. Transcatheter hepatic therapy with irinotecan-eluting beads (DEBIRI) allows targeted delivery of irinotecan direct to CRLM. However, the safety and efficacy of DEBIRI in a preoperative setting has not yet been defined. In addition, very little is understood about why response to DEBIRI varies between patients. Aims This thesis had 2 key aims: (1) To assess the safety and efficacy of neoadjuvant DEBIRI (2) To investigate inter-patient variations in treatment response. Methods Patients with resectable CRLM received a single treatment with DEBIRI 1 month prior to surgery (maximal dose 200mg). The primary end-point of the study was R0 tumour resectability. Hepatic parenchyma and CRLM were sampled at the time of resection. Hepatic expression of key metabolising enzymes was assessed using mass spectrometry based proteomics. Hepatic irinotecan metabolism was characterised and correlated with tumour response. Results DEBIRI was successfully administered in 40 patients. 1 patient (3%) developed post-DEBIRI pancreatitis. All 40 proceeded to surgery, with 38 undergoing resection. 30 day operative mortality was 5%, morbidity 27.5% (Clavien-Dindo 1-4). 63 discreet lesions were targeted, with 74% R0 resection rate. Histopathological examination found no residual tumour in 17% of lesions, <50% residual tumour in 59% and >50% tumour in 24%. 91% of treated lesions demonstrated stable disease by RECIST, with 9% demonstrating disease progression. RECIST was a poor predictor of pathological response or long-term outcome. At a median follow up of 293 days, fourteen patients (36%) had disease recurrence. On multivariate analysis, only tumour KRAS status was predictive of long-term outcome (p=0.02). There was a strong correlation between hepatic CES-2 expression and irinotecan activation (p < 0.001). Patients with a UGT1A1*28 6/7 SNP showed no difference in drug metabolism or pathological response. Hepatic CES-2 mediated activation of irinotecan clearly correlated with tumour replacement by fibrosis (p = 0.01). Conclusions This study demonstrates the safety and efficacy of neoadjuvant DEBIRI for CRLM, with impressive pathological response rates. Systemic exposure to irinotecan was low. The ability of hepatic tissue to activate irinotecan into SN-38 clearly correlated with tumour replacement by fibrotic tissue, suggesting that hepatic CES-2 activation is the key step in the effectiveness of DEBIRI. These preliminary data provide a pharmacological rationale for whole lobe embolisation and suggests a potential predictive biomarker of treatment efficacy for future validation.

MDM2 is a proto-oncogene well known for its role as a negative regulator of the p53 tumour suppressor. It has also been demonstrated that co-upregulation of MDM2 and wild-type p53 is linked with reduced disease specific survival in renal cell carcinoma (RCC). Moreover, MDM2 expression has been shown to promote cell motility and invasiveness in several cancer cell types including RCC cells in a p53- and RING-finger- independent manner, suggesting a role for protein-protein interactions in mediating this effect. The work presented in this thesis has aimed to study the role of MDM2 interaction with other proteins, in particular with members of the NME (Non-metastatic protein) family of metastasis suppressing genes, in order to decipher the mechanisms of action by which MDM2 may contribute to a more aggressive phenotype in cancer cells. To accomplish this we have used a range of methodologies including investigating the consequences of MDM2 expression using exon array analysis in an attempt to identify changes in gene expression induced by wild type MDM2 (Clone 9) and an MDM2 RINGmutant (RFM9) expressed stably in clonal derivatives of H1299 cells. In parallel studies in our research group, an interaction was identified between MDM2 and NME2. Since NME2 is a member of a family of genes that has been implicated in metastasis suppression, and since metastatic spread is a primary determinant of patient survival, we also focused on an analysis of this interaction. Thus the primary aim of this work became an examination of the mechanistic details and consequences of MDM2-NME interaction in cancer cells. The two most highly expressed forms of NME genes in most cell types are the highly related NME1 and NME2 genes which both encode nucleoside diphosphate kinase (NDPK) activity, essential for maintaining cellular pools of nucleoside triphosphates. They are also metastasis suppressor genes with an ability to suppress cellular motility. Since MDM2 possesses an E3 ubiquitin ligase activity, we have investigated whether MDM2 has an ability to modify NME proteins by ubiquitinating them and also whether MDM2 modulates the NDPK activity of NME proteins and/or their motility suppressing activity. Our results suggest that MDM2 may abrogate motility suppressive effects of NME2 through down-regulation of NME2 (and also of NME1) protein levels with potential consequences for suppression of NDPK activity. More interestingly, our studies have identified NM23-LV, which is derived from a specific read-through transcription and alternative splicing event of the adjacent NME1 and NME2 genes, as an MDM2-interacting protein. In addition, we have identified NM23-LV as a novel substrate for MDM2-mediated ubiquitination that occurs in an MDM2 E3 ligase activity-dependent manner. Ubiquitination of NM23-LV by MDM2 seems to be highly specific, since neither NME1 nor NME2 are substrates for MDM2-mediated ubiquitination. Furthermore, our results have also shown that NM23-LV can substantially promote MDM2 stabilisation and enhance MDM2-dependent p53 inactivation, adding another layer of complexity to the interactions between MDM2 and NME family members. Functional studies of NM23-LV have suggested that NM23-LV plays a role in promoting cell motility, which contrasts with the canonical role of NME1 and NME2 as metastasis suppressors. Taken together, studies presented in this thesis have identified a novel relationship between MDM2 and NM23-LV, which could provide valuable insights into the underlying mechanisms leading to increased cell motility promoted by MDM2 and suggests that new studies examining how MDM2-NME interactions may regulate cell motility and cancer metastasis are needed. Given the role of MDM2 in promoting more aggressive RCCs, insights gained from these studies may ultimately identify opportunities for therapeutic interventions for RCC, as well as for other human cancers.

Colorectal cancer remains one of the most common cancers in the United Kingdom with around 40,000 new cases being diagnosed each year. Around 25% of patients will have liver metastases at the time of presentation, with up to 50% developing metastases at some point in their life. Advances in surgical technique and developments in chemotherapy have increased the number of patients with advanced disease for whom potentially curative treatment is possible. The use of chemotherapy in a neo-adjuvant setting has improved the outcome for patients with liver metastases who have initially irresectable or borderline disease. Chemotherapy-induced hepatotoxicity affects up to 78% of patients receiving standard chemotherapy for colorectal cancer and can lead to increased morbidity and mortality. Current gold standard serum-based biomarkers of drug-induced hepatotoxicity have their limitations and there remains a need for more sensitive and specific novel biomarkers to detect early hepatotoxicity. The use of serum-based microRNAs, in particular microRNA-122 (miR-122), a hepatocyte-specific molecule has been proposed as a possible biomarker for chemotherapy-2induced hepatotoxicity. The work described in this thesis assessed the characteristics of serum miR-122 in a healthy human population. It also assessed serum levels of miR-122 in different diseases including primary liver cancer. In order to assess the role of serum miR-122 in chemotherapy-induced hepatotoxicity, a pilot study was carried out in patients receiving chemotherapy for advanced colorectal cancer. In a healthy human population (n=129) serum levels of miR-122 were measured to investigate the degree of variation and define a normal reference range that could be used to assess changes found in patients with hepatic injury and disease. In addition to miR-122, two endogenous (U6snRNA/let27d) and one exogenous controls (c.lin24) were measured. Inter-individual variation was low for miR-122 (CV% 5.21) and also for all three controls (CV% <4%). There was no circadian variation in serum miR-122 (ANOVA p=0.1254). Analysis of intra-patient variation over three consecutive days was similarly low (p=0.66). In a human population with underlying chronic liver disease (n=90) and primary liver cancer (n=104), serum miR-122 was significantly raised in the both cohorts (p=<0.001) but no difference was seen between the chronic disease and cancer cohorts (p=0.338). Patients with an underlying inflammatory condition had significantly raised serum miR-122 (p=<0.001) compared to those with underlying cirrhotic or fibrotic change (p=0.372). ROC analysis supported this finding (AUC 0.79 vs 0.54). In a pilot study of serum miR-122 during neo-adjuvant chemotherapy for colorectal cancer liver metastases, 11 patients were recruited. Serial blood sampling during chemotherapy treatment revealed a non-significant rise in miR-122 (p=0.14). Clinically insignificant levels of liver toxicity were seen in the ten patients who completed the treatment and had surgery. In those with histology changes known to be associated with chemotherapy, there was a significant rise in serum ALT (p=0.0082 and 0.0085) while the miR-122 did not rise significantly (p=0.053). This work confirmed the low variation in serum miR-122 that is a requirement for a novel biomarker. Furthermore, it confirmed the detectable increase of serum miR-122 in patients with liver disease, particularly those with an inflammatory pathophysiology. Finally, in a human model of chemotherapy-related hepatotoxicity, the role of miR-122 remains unclear at present, but the non-significant changes in level related to clinically insignificant liver perturbation compared to the significant changes in ALT suggest that, although more work is required, it may be a valuable biomarker with potential in this field.

Renal cell carcinoma (RCC) is a notoriously difficult cancer to treat. RCC has an unpredictable course, is radio- and chemo-resistant and survival in response to treatment with current therapies for the metastatic disease is generally very poor. This underscores the necessity for novel therapeutics for this condition. The tumour suppressor p53 is rarely mutated in RCC and up-regulation of p53 and its negative regulator, MDM2, has recently been shown to be an independent prognostic indicator for patients with poor outcome. The function of p53 in RCC cells in culture appears to be partially intact and is regulated by MDM2. Rescue of p53 from the inhibitory effects of MDM2 using inhibitors of the p53-MDM2 interaction results in cell cycle arrest or senescence, not apoptosis, three cell fates that p53 can induce depending on the tissue or tumour type. Because of the potentially deleterious effects of senescent cells on the tissue microenvironment, it was decided that apoptosis would be the preferable cell fate for the therapeutic reactivation of p53 in RCC cells. To that end, the MDM2 inhibitor Nutlin-3 was combined with the Bcl-2 family inhibitor ABT-737. This combination of drugs synergistically inhibited the proliferation of RCC cells harbouring wild type p53 but not in cells harbouring mutant p53. This synergistic inhibition of proliferation was shown to be p53-dependent and was confirmed to be inducing apoptosis, but only in RCC cells that had high levels of p53 protein. The synergistic inhibition of proliferation was not recapitulated when an inhibitor of MDM2 ubiquitin ligase activity was combined with ABT-737. The combination of Nutlin-3 and ABT-737 induced pro-apoptotic Puma and Noxa in RCC cells and the apoptosis was shown to be substantially Bax-dependent. This study suggests that the combination of Nutlin-3 and ABT-737 should be considered as a candidate for pre-clinical development in the treatment of RCC.

The over-expression of C-FABP plays an important role in promoting tumorigenicity of prostate cancer. It has been hypothesized that the overexpressed C-FABP may transport an excessive amount of intracellular fatty acids into cancer cells to activate their nuclear receptor PPARγ to trigger a chain of molecular events that may lead to a facilitated malignant progression of the cancer cells. To investigate whether PPARγ involved in activities exerted by C-FABP in prostate cancer, their expression status was assessed in prostate cell lines and tissues. Results showed that their expression levels in malignant cell lines and tissues (cytoplasm and nucleus) were significantly higher than those expressed in benign cells and in BPH and it appeared that their expression levels were increased as the increasing malignancies of the cell lines and tissues. The increased expression of both C-FABP and PPARγ were significantly correlated to a reduced survival time. Suppression of PPARγ in highly malignant prostate cancer cells produced a significant reduction in growth rate (up to 53%), invasiveness (up to 89%) and anchorage-independent growth (up to 94%) in vitro. Suppression of PPARγ in PC3-M cells could significantly reduce the sizes of tumours formed in nude mice by 99%. Tumour incidence was reduced to 10% and the latent period was significantly increased by 3.5 fold. The results in this study also showed that C-FABP promoted VEGF expression and angiogenesis by PPARγ (through the stimulation of the fatty acids transported by C-FABP). When PPARγ was blocked with its antagonists, cells did not respond to stimulation signal produced by fatty acids, even when high level of fatty acids was available. Further study showed that the activated PPARγ regulated VEGF expression through acting with the PPREs in the promoter region of VEGF in prostate cancer. Although androgen can modulate VEGF expression through Sp1/Sp3 binding site on VEGF promoter in androgen-dependent prostate cancer cells, this route was disappeared and gradually replcaced by the C-FABP-PPARγ route as the cells gradually lost their androgen dependency. The results of this study suggested that C-FABP, together with fatty acids, PPARγ and VEGF should be considered as key factors in the fatty acids-initiated signalling pathway that promoted the malignant progression. Therefore, the C-FABP-PPARγ-VEGF axis may be a novel therapeutic target for prostatic cancer.