Colorectal cancer is a major health problem, and many patients relapse despite apparently curative treatment. Local recurrence is an important factor, and is more common when cancer cells are present on the free serosal surface or circumferential resection margin. We hypothesised that detecting tumour cells in the peritoneal cavity during surgery (in peritoneal washings) or post-operatively (in drain fluids) would act as a marker for risk of local recurrence, and that the use of molecular biological techniques would allow for the sensitive detection of such cells. We collected samples of colorectal tumours, with washings from the peritoneal cavity at the start and end of surgery, and fluid from the surgical drain on the first and second postoperative day. Epithelial cells in the peritoneal samples were enriched using magnetic cell separation (MACS). Using mutation specific PCR or a Mismatch Ligation Assay we detected common mutations of K-ras, TP53, APe and BRAF in primary tumour samples, and when such a mutation was found, we studied the peritoneal samples from that patient for the same mutation. We detected 23 mutations in 22 (out of 46) tumours from 21 patients. In 16 patients, at least one of the peritoneal samples gave a positive result for the same mutant DNA. Using MACS increased the proportion of positive samples. Half of the patients with positive peritoneal samples had Dukes' stage A or B tumours. Follow up is not yet long enough to allow conclusions to be drawn on the significance of these results. We have described techniques allowing mutations to be characterised in half of colorectal tumours and have demonstrated the presence of cells with the same mutation in peritoneal samples from three-quarters of patients. Longer follow up will show whether our tests are too sensitive, or whether they provide useful information about likely local recurrence.

The genomes of higher eukaryotes are marked by distinct chromatin domains, which allow for the control of different gene expression states. It is thought that the boundaries of chromatin domains could be formed by DNA sequence elements called insulators. The paradigm HS4 insulator element is located at a boundary between the β-globin gene cluster and an adjacent condensed chromatin domain. Proteins that bind to the HS4 sequence recruit enzymes that mediate a number of histone modifications generally associated with chromatin accessibility. Inspired by yeast genetic studies, we hypothesised that H2B ubiquitination might be a key regulator of these ‘active’ marks. It was found that HS4 and another chromatin boundary at the neighbouring FOLR1 gene locus, HSA/HSB, are sites of H2B ubiquitination. The ubiquitination E3 ligase RNF20 was found to be necessary for global H2B ubiquitination and for methylation of H3K4, in a trans-histone modification pathway that is conserved from yeast to man. RNAi-mediated knockdown of RNF20 not only resulting in the depletion of H2B ubiquitination normally found at chromatin boundaries, but also disrupted their H3K4 methylation and acetylation at multiple histones. H2B ubiquitination is a master controller of the active chromatin state at the HS4 and HSA/HSB chromatin boundaries. Long term depletion of RNF20 expression leads to a compromise of the boundaries, allowing the spreading of heterochromatin into the FOLR1 and β-globin gene loci, resulting in gene silencing. This study also looked at the recruitment of factors that mediate the incorporation of the histone variant H2A.Z at chromatin boundary elements in vertebrates. It was found that insulator binding proteins control H2A.Z incorporation and acetylation.

The aims of this study were to develop novel in vitro models of Human Papillomavirus (HPV) -associated vulval and vaginal neoplasia and to use them to investigate the mechanism(s) of action of the nucleoside analogue, Cidofovir (CDV), for which a mechanism is unknown. Single cell clones were successfully isolated from heterogeneous vulval and vaginal parental populations. The clonal cell lines were characterised morphologically and in terms of cell proliferation; a range of growth rate and morphological variants were identified. Clonal lines were also characterised with respect to HPV integration state using Amplification of Papillomavirus Oncogene Transcripts (APOT) and Detection of Integrated Papillomavirus Sequences (DIPS). Cell lines were identified that represented naturally occurring episomal and integrated HPV infections. HPV gene expression analysis was also performed using quantitative real-time reverse transcription PCR (qRT-PCR) which displayed different expression profiles for each line. Patterns of HPV gene expression appeared to correlate with gene disruptions associated with integration. Characterised clonal lines were used to investigate the effects of CDV on cell viability, morphology and HPV gene expression. CDV (10 μM) reduced cell viability in all HPV-positive clonal lines and HPV negative HEK cells; viable cell counts showed that a more considerable response was detected in vulval lines compared to vaginal lines and that response in HEK lines was similar to vulval lines. Cell enlargement was observed in response to treatment in the clonal lines but not the HEK line. CDV treatment did not cause significant reduction in expression of HPV E6 or E7. mRNA sequencing confirmed HPV integration and gene expression profiles. Differentially Expressed Gene (DEG) analysis identified a large percentage of the top 20 most significant Gene Ontology (GO) categories to be involved in nucleotide synthesis and RNA polymerase activity. Gene Ontology Over Representation Analysis (GO-ORA) showed that 4 GO categories relating to cellular senescence were found in over 300 GO categories comprising the top 500 most significant gene transcripts.

Prostate cancer metastasis is a complex process, involving multiple pathways in its orchestration. Malignant cells are influenced by different growth factors from the extracellular environment which promote or inhibit cell movement and metastasis. HGF has been implicated in progression and metastasis of prostate cancer. A cell interacts with the environment through surface molecules like integrins. These interactions are further translated in to different responses through various intracellular machineries. Furthermore organization of the actin cytoskeleton is vital for many cellular functions. WAVEs are member of WASP family of proteins, which have important role in regulation of actin dynamics through regulation of actin related protein (ARP 2/3). The role of individual members of WASP family has been investigated in development and progression of different cancers. We documented the expression of different WAVE family members in various prostate cancer cell lines. Expression of WAVE-3 was effectively knocked down with the use of hammer head ribozymes. Loss of WAVE-3 expression resulted in reduced cell movement and invasion in the PC-3 cell line. These cells failed to show any significant increase in cellular movement and invasive potential following treatment with HGF. Further experiments to investigate the underlying mechanism of this phenotypic change revealed that optimum levels of phosphorylated paxillin play an important role in this change. Our study also indicates that reduced potential of invasive capability following WAVE-3 knock down, may be related to reduced availability of MMP-2 in the cellular environment.

Telomeres are specialised structures that that cap the ends of chromosomes; and prevent the natural end of a chromosome from being recognised as a double stranded DNA break. Telomeres erode with ongoing cell division ultimately leading the induction of replicative senescence that provides a proliferative lifespan barrier. In the absence of a functional DNA damage response, telomeres are prone to end-fusions, creating dicentric chromosomes that can initiate breakage fusion–bridge cycles. Evidence from studies in well-defined mouse models of cancer, and also human tumours, have shown that telomere dysfunction may be a key event in the progression of disease via the increasing genomic instabilities that arise from telomere fusion events. The key aim of this thesis was to test the hypothesis that telomere dysfunction occurs during the progression of breast cancer and that this can drive the large-scale genomic rearrangement frequently observed in this disease. Technology development was attempted in the hope to allow single-molecule telomere fusion detection of more complex mutational structures, with limited success but possible future potential. Telomere length analysis was carried out using high resolution single molecule PCR strategies (STELA) in a panel of DNA samples derived from invasive ductal carcinoma. Telomere length data analysed alongside clinical data received for all samples was used for the potential utility of telomere length as a potential prognostic indicator in breast cancer. A key finding of this work was to demonstrate the use STELA combined with statistical tests to show that short telomere length is highly significant in terms of prognosis in breast cancer. Telomere length stratification could thus potentially be used as a method of defining new breast cancer subtypes in terms of severity.

In recent years, substantial evidence has been generated demonstrating the importance of the immune system in preventing and controlling the growth of many cancers, including colorectal adenocarcinomas. In particular, populations of tumour-specific effector T cells appear to play a crucial role in restricting the generation and expansion of transformed neoplastic cells. However, the fact that tumours continue to grow in the presence of a seemingly intact immune system suggests that these responses are often inadequate. CD4+Foxp3+ T regulatory cells (Tregs) have been shown to play a key role in modulating the immune system by keeping immune responses to self-antigens in check, thereby preventing autoimmunity. These cells also appear to be employed by tumours to protect against recognition and eradication, and have been demonstrated to impinge upon the anti-tumour immune response in humans. Furthermore, it appears that the tumour microenvironment facilitates the development of highly immunosuppressive T cells, which may also allow subsequent tumour progression. In colorectal cancer, the relationship between Tregs and tumour progression is less clear – despite their well-documented ability to impinge on anti-tumour immune responses, increased tumour infiltrates have also been associated with prolonged survival. In this thesis, the phenotype and function of CD4+ T cells derived from PBMC, colon and tumour samples were analysed for suppressive markers by FACS, and anti-tumour responses by IFN-γ ELISpot. CRC patients with more advanced tumours responded to fewer epitopes and generated a significantly weaker epitopespecific T cell response to the oncofoetal antigen, 5T4 than healthy donors. Human depletion experiments both in vitro and in vivo indicated suppression by Foxp3+ regulatory CD4+ T cells. These cells were found in abundance amongst tumourinfiltrating lymphocytes; however, another equally prominent population of IL-10 and TGF-β-producing CD4+Foxp3- T cells were found to be >50-fold more suppressive. Thus, a major caveat to cancer immunotherapy is the suppressive tumour microenvironment, which contributes to the selective decline of measurable antitumour CD4+ T cell responses as tumours progress. These responses were enhanced in metastatic CRC patients by depleting regulatory T cells. It is hoped such findings will augment our understanding of how anti-tumour CD4+ T cells are activated and conversely regulated, with the intention of designing better treatment for patients with colorectal cancer.

CLL is a malignant disease of B-cells characterised by a heterogeneous clinical outcome. Some patients require an early treatment and have low survival time, while others never need treatment. Many prognostic markers have been established and used to help predict the clinical course of CLL. Despite advances in understanding the biology of CLL, the molecular differences underlying the variable clinical outcome of CLL are not yet fully understood. The hypothesis of this study was that the heterogeneous outcome of CLL could be driven, in part, by the aberrant expression of proteins in the two forms of CLL. Therefore, this study aimed to identify these proteins using proteomics approaches. In an attempt to achieve this goal, four steps were performed. Firstly, a cellular fractionation method was developed to extract cellular proteins into two different fractions (NP40 fraction for cytosolic protein enrichment and SDS fraction for nuclear protein enrichment). Secondly, extracted proteins were subjected to qualitative proteomics analysis using 2D nano-LC and MALDI TOF-TOF mass spectrometry in order to identify CLL proteins. Integrating the identified proteins (n=900) with previously published transcriptome of CLL cells and normal B-cells highlighted 20 proteins with preferential expression in CLL cells - some of which were linked to human cancer. Thirdly, iTRAQ technology coupled with 2D nano-LC and MALDI TOFTOF mass spectrometry was used to measure the relative expression of proteins in different CLL samples. This workflow identified 15 altered proteins in the two forms of CLL and detected 14 proteins with variable expression. Finally, six proteins were selected for investigation in an additional CLL cohort. Of these proteins thyroid hormone receptor-associated protein 3 (TR150), T-cell leukaemia/lymphoma protein 1A (TCL-1) and S100A8 showed association with poor prognosis CLL and early requirement for treatment. Additionally, myosin-9 exhibited reduced expression in poor prognosis CLL samples.! ! Overall, this study identified proteins with potential importance in CLL prognosis and pathology. These proteins merit investigation in a larger CLL cohort to further confirm their relevance to CLL. In addition, this study showed the usefulness of combining cellular fractionation with proteomics and transcriptomics to identify proteins with potential role in CLL.

Birt–Hogg–Dubé (BHD) syndrome is a rare inherited autosomal dominant disorder first described by Birt, Hogg and Dube in 1977 (Birt et al,. 1977). BHD affects approximately 100 families worldwide. Approximately one third of diagnosed BHD patients also develop renal cell carcinoma (RCC) (Schmidt et al., 2005). BHD arises as a result of loss of function of the Folliculin protein expressed from the BHD gene, suggesting than Folliculin serves as a tumour suppressor (Vocke et al., 2005). Although it is considered that FLCN represses cell growth, the role that FLCN plays in cancer progression and/or initiation is currently unresolved. It has been observed that tumours taken from BHD patients have reduced levels of mitochondria (Yang et al., 2008). Previous studies have also suggested that aberrant levels of mTOR activity are observed in Folliculin-deficient cell lines (Baba et al., 2006; Baba et al., 2008). Aberrant levels of mTORC1 activity have been associated with increased levels of Hypoxia inducible factor transcriptional activity, decreased autophagic activity (Land and Tee, 2007; Hands et al., 2009). There are several genes within the mTOR pathways which act as Tumour Suppressors. Mutations within these genes result in inherited genetic disorders, including Tuberous, Sclerosis complex (TSC). Loss of function of TSC1/TSC2 results in TSC which is clinically similar to BHD (Gomez et al., 1999). Arbarrant levels of mitochondrial biogeneisis and HIF activity have been observed in cells deficient in TSC1 ad TSC2 (Land and Tee, 2007; Chen et al., 2008). Increased levels of mTORC1 activity have also been shown to result in decreased levels of autophagic activity in TSC2 deficient cells (Parkhitko et al., 2011). In order to characterise the tumour suppression function of Folliculin, the effects of loss of function of Folliculin on mitochondrial biogenesis and mitochondrial function, hypoxia inducible factor (HIF) transcriptional activity, and autophagic activity were investigated using multiple cell lines. The results from this study suggest that loss of function of FLCN results in increased production of ROS species. This leads to compromised mitochondrial membrane potential and decreased ATP production as a result of increased expression of uncoupling proteins in order to try and reduce the increased levels of ROS. FLCN is also phosphorylated at multiple residues by both mTORC1 and AMPK and activity of both mTORC1 and AMPK is increased in response to the depleted ATP levels as a result of increased UCP production in response to the increased ROS production observed upon loss of functional FLCN. This results in increased levels of mitochondrial biogenesis and increased levels of glycolytic activity via increased activation of HIFα proteins in order to compensate for this energy deficit. Furthermore, it appears that both mTORC1 and AMPK could drive HIF transcription and mitochondrial biogenesis through modulation of FLCN phosphorylation. ULK-mediated autophagy also appears to be upregulated upon loss of functional FLCN, possibly as a result of the increased levels of AMPK activation in order to provide a protection for these cells. This may be via the catabolising the dysfunctional mitochondria observed in cells upon loss of BHD, a process which may be exploited as a potential therapeutic target for BHD patients. Further investigations into these cellular processes may also provide clues to potential therapeutic targets for treatment of BHD patients.

The aim of this study was to investigate the role of the adipokine visfatin as a possible molecular mediator between obesity and prostate cancer. Visfatin, an adipokine that is elevated in obesity and has many proposed roles and has been linked to a variety of cancers. No data pertaining to the role of visfatin in prostate cancer existed and this was an area that this study looked to address. It is suggested that obesity is a significant risk factor for prostate cancer; in particular, aggressive disease and adipokines have been investigated as a link for this hypothesis. This study presents novel data demonstrating the expression of visfatin in the LNCaP and PC3 cell lines as well as in benign and cancerous prostate tissue at both mRNA and protein level. Furthermore visfatin is shown to have functional roles in autoregulation and promoting increased cell proliferation in PC3 cells and also showed further effects with respect to cell migration across a wound. These data gave promise to develop the study further and evaluate potential mechanisms of action including common second messenger systems such as MAPK and also other oncologically multifunctional molecules in the forms of MMP-2/-9. We then demonstrated that visfatin up-regulated MAPK phosphorylation and MMP mRNA/protein expression and more importantly MMP-2/-9 zymographic activity. This provided possible mechanisms by which visfatin may mediate a role for obesity driven aggressive prostate cancer. The study then looked to evaluate NMN (the byproduct of visfatin catalysed biosynthetic activity), as well as the visfatin inhibitor FK866 which is being evaluated as chemotherapeutic agent. Unsurprisingly NMN and FK866 had opposing actions on proliferation and FK866 was naturally proapoptotic. NMN was able to rescue the effect of FK866 on PC3 cell apoptosis. Prior studies have shown that NMN did not affect oncogenes however NMN was found to significantly reduce BAX mRNA expression in PC3 cells. The findings are consistent with other studies linking visfatin with cancer states. These novel data indicate roles for visfatin in prostate cancer and possible mechanisms linking obesity and prostate cancer.

TMEFF2 (transmembrane protein with EGF-like and two follistatin motifs 2) is a transmembrane protein expressed in brain and prostate and over-expressed in prostate cancer (Uchida et al. 1999; Horie et al. 2000). The role of TMEFF2 in prostate cancer is controversial. Several data indicate that TMEFF2 has cancer-promoting activity (Glynne- Jones et al. 2001; Ali and Knäuper 2007), while others suggest that TMEFF2 inhibits progression of cancer (Gery et al. 2002; Gery and Koeffler 2003). TMEFF2 is cleaved by membrane-anchored proteases, including disintegrin and metalloproteases (ADAMs) and -secretase (Ali and Knäuper 2007), but the biological meaning of TMEFF2 shedding is not known. It was hypothesized that the opposing findings describing the role of TMEFF2 in prostate cancer result from proteolytic processing of TMEFF2 by different proteases which are co-expressed with TMEFF2 in prostate cancer cells, such as the type II transmembrane serine proteases (TTSPs), prostasin and ADAMs. To support this hypothesis co-expression of TMEFF2 and serine proteases was analyzed in prostate cancer cell lines and clinical samples. The shedding of TMEFF2 by ADAMs and serine proteases was investigated using HEK293 cells expressing AP/V5 TMEFF2 or shedding resistant AP/V5 303-320TMEFF2 mutant (Ali and Knäuper 2007). The data obtained from AP activity assay and Western blot analysis of cell lysates showed that TMEFF2 is cleaved by serine proteases (matriptase and hepsin) and ADAMs (ADAM9, ADAM12). Moreover, serine proteases and ADAMs cleave TMEFF2 in different positions, generating several soluble TMEFF2 fragments. To establish the biological role of TMEFF2 processing, N-terminal TMEFF2 fragments predicted to be generated by TTSPs and ADAMs were expressed in E. coli and mammalian cells. Preliminary experiments using HEK293 and PNT2-C2 cells indicated that soluble TMEFF2 does not signal through ErbB receptors and suggested several signaling pathways that might be regulated by TMEFF2. The fate of TMEFF2 C-terminus following ectodomain shedding was examined by confocal microscopy and Western blotting, indicating that TMEFF2 cytoplasmic domain is likely degraded following the release of TMEFF2-ECD