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Wnt/β-catenin signalling plays a central role in the regulation of multicelluar organism development and in the maintenance of tissue homeostasis in adults. Dysregulation of Wnt signalling resulting in aberrant pathway activation is a key initiating step in the development of a diverse range of cancers, including colorectal cancer, and as such is an important target for therapeutic intervention. A novel Wnt pathway inhibitor, ‘MSC’, has been identified as blocking activated Wnt signalling, specifically through inhibiting the ability of CDK8 and CDK19 to activate nuclear β-catenin/TCF-dependent transcription. However, despite potently inhibiting Wnt-dependent transcription, the ability of MSC to reduce cellular viability was limited. This study aimed to determine genes that whose loss operated with MSC to reduce cell survival. A whole-genome RNAi chemical sensitisation screen identified 3 genes whose depletion in combination with MSC treatment conditionally reduced the viability of HCT116 cells in vitro. The outstanding hit of this screen was Histidyl Aminoacyl tRNA Synthetase (HARS). The identification of this enzyme as an MSC ‘interactor’ suggested links between Wnt signalling and the regulation of translation. BRAF and MED11 RNAi also conferred conditional sensitivity to MSC. Interestingly, MED11 is a component of the Mediator complex, a multiprotein transcription regulatory complex in which CDK8 functions to regulate β-catenin/TCF-dependent transcription, suggesting that mediator complex may be a key target of MSC action. A parallel overexpression screen was initiated to identify novel Wnt pathway activators, and subsequently used to map MSC resistance. Expression of the transcription factors GBX1 and HMGB2, determined to be novel regulators of TCF-dependent transcription, blocked MSC-mediated disruption of Wnt signalling. Overexpression of either gene in a clinical context might therefore be regarded as a contra-indication for MSC-class therapies. These studies have highlighted potential avenues for broadening the scope of MSC activity through the determination of survival and resistance mechanisms, thus the rational design of MSC-combination therapies could be of huge clinical benefit for the treatment of colorectal cancer.

The central aim of this study was to characterise the expression of the candidate tumour suppressor gene, LV/-J (lentivirus integration-I) and its products. The LVI-J gene (WDR76) was discovered as a target for disruption by proviral insertional mutagenesis in a case of pre-B cell lymphoma in a FIV infected cat {Beatty, 1998 5 lid; Beatty, 2002 7 lid}. As LV/-J is highly conserved, my work focused on the human and murine orthologues to take advantage of the superior resources available for these species. My work showed potentially important differences in expression of the human and mouse genes with respect to promoter use and length of 3' untranslated sequences. The murine gene is transcribed mainly from the distal PI promoter, which appears to be a bi-directional element shared with the adjacent MJapJ gene, while the human gene transcripts are derived exclusively from the proximal P2 promoter. Direct analysis by RT-PCR showed that the murine gene could also be expressed from the P2 promoter. These findings have significant implications for Lvi-l protein expression as the P2 transcripts are predicted to express larger proteins with a distinct N-terminal sequence. To characterise the LV/-J gene products, rabbit polyclonal antisera were raised to GST fusion proteins expressed in bacteria. Use of the murine anti-ml.vi-I antiserum in Western blotting identified a protein of the expected size (58 kDa) based on translation of the major mRNA species from the PI promoter. Immunofluorescence and confocal microscopy suggested that this protein is localised mainly in the cytoplasm. Although the function of LVI-I is unknown, its closest relative in the human genome is DDB2, a protein involved in repair of UV-induced DNA damage. Regulation of LVI-I expression was examined after UV irradiation, providing preliminary evidence of responses at transcriptional and post-transcriptional levels. Further leads were followed by analogy with the yeast orthologue of LVI-I, YDLI56W, but no evidence of a complex between LVI-I and MSH6 was found. In conclusion, while function of the LVI-I gene remains to be establishes, it provides the basis for future characterisation of this highly conserved and potentially important eukaryotic gene.

Retroviruses possess several biological features that differentiate them from all other infectious agents. The obligatory integration step of the retrovirus genome into the host genome has allowed these viruses to associate, modulate and alter the biology of the cell with a variety of unique mechanisms. Integration of retroviruses into the germ line of the host results in the formation of vertically transmitted “endogenous” retroviruses (ERVs). It is now becoming apparent that ERVs have often been selected as they provided evolutionary advantages to the host. Sheep Betaretroviruses provide a unique biological system to study the complex interaction between retroviruses and their hosts. Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a naturally occurring lung cancer of sheep. The JSRV Env glycoprotein is a dominant oncoprotein and its expression is sufficient to induce cell transformation in vitro and in vivo. Thus, OPA is a unique large animal model for the study of lung carcinogenesis. The sheep genome harbours at least 27 copies of ERVs highly related to JSRV (enJSRVs). Studies on enJSRVs have provided evidence supporting the idea that ERVs, exogenous retroviruses and the host have coevolved through a dynamic process throughout evolution. enJSRVs play a critical role in conceptus development and placental morphogenesis, and can block JSRV replication in vitro at both early and late stages of the replication cycle. The work presented here focuses on the study of the exogenous and endogenous JSRV Envs and their role in cell transformation and trophoblast differentiation respectively. We were able to show that: I) the JSRV Env transforms epithelial cells in vitro independently from its cellular receptor; II) both the exogenous and endogenous JSRV Envs interact with the receptor tyrosine kinase RON and that the cytoplasmic tail of the Env is the major determinant modulating the biological effects of the Env-RON interaction; III) the molecular chaperone Hsp90 regulates JSRV Env induced cell transformation, in part by downregulating Akt; and IV) OPA is a useful large animal model for the evaluation of new anti-cancer therapeutic agents. Moreover, we characterized the transforming properties, receptor usage and fusogenic activity of enJSRVs Envs to gain insight into their role in placental morphogenesis. The studies described in this thesis contributed to the understanding of JSRV induced cell transformation and the biology of enJSRVs.

Osteosarcoma (OS) is the most common bone cancer in dogs. It is biologically aggressive and <20% survive >2 years with standard therapy. Hence, new approaches must be considered. TP53 is altered in ~50% of human and canine cancers, including OS, making it a candidate for targeted suicide gene therapy strategies. Canine OS is considered to be a good model for human OS. The aims of this study were to:  examine the site incidence of canine OS retrieved from Glasgow University Veterinary School (GUVS) histology database;  perform TP53 mutational analysis in canine OS cases diagnosed at GUVS;  investigate delivery of exogenous wild-type canine TP53 into D17, CMT3, CMT7 and CMT8 canine OS cell lines;  design and construct vectors for a TP53-targeted suicide gene strategy, which can selectively target canine OS cells containing accumulated TP53, and initially analyse using Dual-Luciferase® reporter assays (DLR);  perform suicide gene/prodrug assays using nitroreductase (NTR) in combination with CB1954 or nitrofurazone (NFZ) in several canine cell lines;  replace luciferase with NTR in vectors for TP53-targeted suicide gene strategy, and with CB1954, determine if survival of CMT7 cells possessing accumulated TP53 are reduced, in comparison to D17 cells, containing wild-type TP53. OS were most commonly found in appendicular areas, followed by axial and extraskeletal sites; this agrees with published findings. No TP53 mutations were found in 7 biopsies removed from 4 dogs, 5 were OS, due to analysis of a small sample number, but still fits within published data. TP53 expression did not have a significant negative effect on canine OS cell growth. Contrasting results have been shown in canine and human OS cells. Luciferase expression levels following transfection with designed constructs were higher in CMT7 cells, than in D17 cells. Similar results were shown in NTR/CB1954 assays as reductions in cell survival only occurred in CMT7 cells but not in D17 cells. NFZ was not suitable as a prodrug for NTR in canine cells as there were no differences in cell survival with cells not expressing NTR. Hence, the TP53-targeted suicide gene therapy strategy appears to selectively reduce survival of canine OS cells possessing accumulated TP53, warranting further investigation as a treatment modality for OS, in both dogs and humans.

‘Living with Cancer in Old Age’ is an exploration of older people’s experiences of living with cancer, using qualitative research methods. A qualitative systematic review of international literature found that the experience of living with cancer in old age is characterised by ambiguity. There are sources of suffering, imposed by cancer itself, by treatments for cancer and by co-morbid disease. At the same time older people have access to sources of comfort and strength, both internal (attitudes of mental fortitude) and external (strong relationships with family, friends, communities and health professionals) which mitigate the worst effects of suffering. This literature study synthesised and interpreted findings from 11 studies covering a heterogeneous population of people aged 55-90+, representing a wide range of cancers at different stages of progress and treatment, across four countries (Israel, Canada, Sweden, USA) and using a range of qualitative methods. A subsequent empirical study using narrative methods focused on a more homogenous population of older people aged 74-87, all resident in the same geographical region (NW England), with one of the four most common cancers (breast, colon, prostate and lung) at different stages of progress and treatment, but treated at the same cancer centre. In this study a biographical/narrative method of interviewing was used, in which 20 participants (13 men and 7 women) were invited to tell the ‘story’ of their life both before and after cancer. Interpretation of life history data reported by participants in this study suggests that the overriding features of life with cancer for people in their 70s and 80s are hope and hardiness, together forming a kind of resilience which appears to be psychologically protective and which fosters a determination to continue living positively, even at an advanced stage of illness. Whereas this ‘fourth age’ has been presented by sociologists as a life stage of inevitable decline, findings from the two studies conducted in this doctoral study indicate a quality of continuing robustness in the lives of some older people which runs counter to common assumptions about their vulnerability and frailty.

To determine whether recurrent genomic imbalances are a feature of HL, CD30-positive HRS cells were laser microdissected from 20 cHL cases and 4 HL-derived cell lines and subjected to analyses by CGH. In primary tumours, the most frequently involved chromosomal gains were 17q (70%), 2p (40%), 12q (40%), 17p (40%), 22q (35%), 9p (30%), 14q (30%), 16p (30%), with minimal overlapping regions at 17q21, 2p23-13, 12q24, 17p13, 22q13, 9p24-33, 14q32, 16p13.3 and 16p11.2. The most frequent losses involved 13q (35%), 6q (30%), 11q (25%) and 4q (25%), with corresponding minimal overlapping regions at 13q21, 6q22, 11q22 and 4q32. Statistical analysis revealed significantly more gains of 2p and 14q in the older adult cases; loss of 13q was associated with a poor outcome. The results suggest that there is a set of recurrent chromosomal abnormalities associated with cHL and provide further evidence that cHL is genetically distinct from nodular lymphocyte predominance Hodgkin lymphoma (NLPHL). Combined immunophenotype and interphase cytogenetic (FICTION) studies were used as techniques for follow-up studies. High expression of both STAT3 and STAT5a has been described in cHL and their genes are located in 17q21.2. Constitutive activation of NF-?B has been found to be a feature of the HRS cells in cHL and has been shown to facilitate escape from apoptosis. The c-rel gene encodes for a subunit of NF-?B and is located on chromosome 2p16. REL amplification has been shown in some cHL cases. Non-functional inhibitor proteins of NF-?B, such as I?Ba, have been described as an alternative mechanism for the aberrant activation of NF-?B. As part of the follow-up studies, IKBa gene mutation status and loss of heterozygosity (LOH) in the HRS cells were determined by sequence analysis and SNP assays. FICTION confirmed that gains of 2p involved the REL gene but gains on 17q were not due to amplification of STAT3/5a. Frequent IKBa mutations were detected but many are not considered to be of functional importance. REL gain, EBV status, IKBa mutation or LOH were not mutually exclusive mechanisms in the pathogenesis of cHL.

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that is upregulated in a variety of human cancers. While there is evidence that FAK is implicated in a wide range of crucial cellular processes that are perturbed in malignancy including proliferation, cell cycle, adhesion and invasion, there is limited information regarding the role of FAK in radiation survival. We aimed to evaluate whether FAK is a novel radiosensitising target by studying clonogenicity in wt p53 FAK +/+ versus FAK -/- squamous cell carcinoma (SCC) cell lines generated in this laboratory. Surprisingly, the absence of FAK was associated with increased radioresistance. In this particular context, FAK indirectly inhibits p53 mediated transcriptional regulation of p21 in response to ionising radiation. Why FAK should repress the pro-survival function of p53 is unclear, but this data indicates that inhibition of FAK in combination with radiation may not always be advantageous in the clinical setting and contributes to an increasing body of literature highlighting a close interaction between FAK and p53.

Background: Colorectal cancer is the third most common cancer in the UK for both men and women. Survival rates are improving and more than half of those diagnosed will survive for more than five years. The configuration of care for colorectal cancer patients is likely to shift in line with survivorship strategies which favour self care, a personalised approach, a holistic perspective, and a reduction in specialist care. Aim: The overall aim of the study is to explore the physical, social and psychological impact of colorectal cancer at diagnosis and one year post diagnosis, with a particular focus on the impact of health care professionals on patients’ experience. Methods: In-depth interviews with 24 participants with a new diagnosis of colorectal cancer, and then follow-up interviews with 19 of the 24 twelve months later. Findings: This physical illness can largely be understood as an experience of uncertainty. Uncertainty as a reaction to colorectal cancer is associated with lower quality of life and the findings here demonstrate that uncertainty proves profoundly unsettling (in spite of efforts to interpret the experience positively). Current health care provision does not reflect this yet there is clear scope for mitigating the experience of uncertainty. Conclusion: Primary care is well placed to play a central role in the care of colorectal cancer patients and has the capacity to mitigate aspects of uncertainty that characterise the experience.

It was shown that LS310 is 2.3 times more resistant to cisplatin and shows a 50% reduction in MLH1 expression when compared to LS274 (p < 0.001). It was demonstrated that the hMLH1 promoter region of LS310 exhibited methylation whereas the LS274 promoter region did not. Neither of these lines exhibited methylation of the p16, MINT 25 or DAPK loci suggesting that de novo methylation was not responsible for the methylation specific PCR results. Further work demonstrated that treatment of the LS310 cell line with the demethylating agent decitabine increased its cisplatin sensitivity as well as increasing the level of MLH1 expression of the cell line. No such changes were demonstrated in the LS274 cell line after treatment with decitabine. In summary, this research project was limited by the availability of samples. However it has demonstrated that collaborative multidisciplinary prospective planned translational research can be done and emphasises the need for a translational component to be an integral part of future lung cancer studies.