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

In ER+ breast cancer initial responses to antihormones are variable, complete responses are rare and resistance is eventually acquired by many patients. It is important to model these events to discover predictive markers of antihormone outcome and so targeted strategies can be developed to maximise antihormone effectiveness. To date, most studies have employed the MCF-7 cell line which fails to represent the variability of ER+ disease. Focusing on Faslodex, the thesis objective was to use 4 cell lines in vitro encompassing ER+/HER2- (MCF-7/T47D) and ER+/HER2+ (BT474/MDA-MB-361) disease to (i) characterise the magnitude of initial antihormone response, (ii) monitor the onset of resistance by prolonged treatment and (iii) detail gene expression changes during Faslodex treatment. All models were initially growth-inhibited by Faslodex, with superior responses in HER2- lines. Microarray analysis revealed gene cohorts affected by Faslodex treatment differed between HER2+ and HER2- models. While MCF-7, BT474 and MDA-MB-361 cells acquired Faslodex resistance, this failed to develop in the T47D line, providing a model of complete-response. A filtering process identified genes involved in the varying Faslodex responses and clinical relevance was determined using the NEWEST Faslodex clinical trial dataset. Of interest was the Faslodex-induction of CXCR4, as a potential mediator of acquired resistance, while suppression of the RET signalling pathway related to improved initial response in the ER+/HER2- setting. Importantly up-regulation of DCN by Faslodex was associated with improved Faslodex response in T47D cells and also with proliferation (Ki67) fall in the NEWEST clinical trial. shRNA knockdown of DCN reduced the sensitivity of T47D cells to Faslodex and enabled development of resistance. This thesis has successfully identified novel elements of Faslodex response and resistance and further work is now required to clarify the importance of these mediators and to determine if DCN could prove a useful clinical biomarker of Faslodex response.

The anti-viral acyclic nucleoside monophosphate compound Cidofovir has shown efficacy in treatment of Human Papillomavirus (HPV) associated genital intraepithelial neoplasia; however, the mechanism of action of Cidofovir in this setting has not been determined. This investigation focused on modifying nucleoside analogue compounds to increase their efficacy in HPV positive cell models of disease, in addition to determining the molecular mechanism of action of Cidofovir in premalignant HPV associated intraepithelial neoplasia. ProTide modification increases the efficacy of nucleoside analogue compounds by increasing their cellular permeability. Cidofovir was not amenable to ProTide manipulation; however, ProTide derivatives of its sister compounds, Adefovir and Tenofovir, were synthesized. Parent Adefovir and Tenofovir and a range of their respective ProTide modified daughter compounds were examined for inhibition of cell growth and effect on cell size and morphology in HPV positive and negative transformed cell lines. The most effective compounds were further examined for dose response in normal HPV negative untransformed Human Epidermal Keratinocytes (HEKs) and naturally HPV immortalized short term (NHIST) cell lines cloned from vulval and vaginal intraepithelial neoplasia biopsies. ProTide analogues displayed striking increased efficacy in comparison to their parent compounds; however, they did not show specificity to transformed or HPV positive cell lines. Cidofovir did not show specificity to HPV positive cells when examined for growth inhibitory effect in HPV positive and negative cell models. A variety of molecular processes were examined to determine the mechanism by which Cidofovir inhibits cell growth in validated NHIST cell lines and HEK cells. At the concentrations investigated, Cidofovir did not cause apoptosis in HPV positive or negative cells and its growth inhibitory effect appeared likely to be associated with cell cycle arrest or senescence. The effects of radiation on the molecular response induced by Cidofovir were also evaluated as previous studies suggested Cidofovir can function as a radiosensitizer. Cidofovir combined with gamma radiation did not result in apoptosis but was associated with an augmented molecular response in NHIST cell lines. On the contrary, Cidofovir combined with gamma radiation caused a major apoptotic response in HPV negative HEKs, suggesting such a combination could result in disadvantageous effects on healthy tissue if it were used in vivo.

In this study a CD31-expressing co-culture system was used to establish whether differential CD31/CD38 signalling may contribute to the poor prognosis associated with CD38 expression in CLL. Using western blot analysis, a PKB phospho-substrate antibody was used in combination with phospho-specific antibodies to identify ribosomal protein S6 and GSK3β as key signalling molecules that were augmented following short-term CD31-expressing co-culture. CD31-expressing co-culture did not alter the phosphorylation of STAT6. However, the addition of IL-4 to the cultures was a potent mediator of this signalling pathway. This highlights the specificity of signalling molecules to different external stimuli. Multi-colour flow cytometry was employed to quantify the expression of cell surface activation markers as well as intracellular phospho-proteins. The CD31-expressing co-culture led to a significant up regulation of the activation markers CD38, CD49d and CD69. Selective pharmacological inhibition of the phosphorylation of S6, STAT6 and ERK resulted in the down regulation of activation markers. Furthermore, the inhibition of p-STAT6 and p-ERK resulted in increased levels of apoptosis, which indicates that these signalling pathways are directly involved in CLL cell survival. Multi-colour flow cytometry was also used to quantitate the levels of phospho proteins, p-S6 and p-ERK. Similar to the results observed by antibody detection following western blotting, basal and inducible levels of p-S6 and p-ERK were elevated in primary CLL cells expressing high levels of CD38. Taken together, the work carried out in this project highlights the importance of using co-culture systems to stimulate CLL cells in vitro in order to mimic some of the key stimuli encountered in vivo. The dissection of the signalling pathways activated as a result of CD31/CD38 interactions provides a rational for the poor prognosis associated with elevated CD38 expression in this disease and identified candidate therapeutic targets that might particularly benefit this group of patients.

In this thesis, we aimed to identify genetic factors that influence the risk of colorectal cancer (CRC). We also sought alleles that contribute to the likelihood of extreme adverse reactions to treatment. We validated five previously identified low penetrance variants using our training phase cohort, consisting of 2,186 advanced CRC (aCRC) from the COIN and COIN-B trials and 2,176 geographically matched controls. Using this cohort we also identified a variant in RAD1 that was significantly associated with risk (X2=13.51, P=2x10-4). However, we failed to replicate these findings in an aCRC validation cohort consisting of 1,053 cases and 1,397 geographically matched controls (X2=2.76, P=0.1), potentially as a result of a lack of power due to insufficient sample numbers. We identified ten patients from the COIN trial with severe peripheral neuropathy associated with oxaliplatin (PNAO) treatment. Through exome resequencing we identified a novel stop gain variant (Ser613X) in the nucleotide excision repair gene (NER), ERCC4. Following analysis of 54 additional patients from the COIN trial with PNAO, we identified three rare nonsynonymous variants (Pro379Ser, Arg576Thr and Glu875Gly) that were predicted to interfere with protein function. Consistent with the rare variant hypothesis of common disease, two of these variants were seen to collectively contribute to the risk of the phenotype (7/63 [11.11%] of patients with PNAO compared to 86/1763 [4.88%] of patients without PNAO; X2=4.89, P=0.03). Using the fission yeast, Schizosaccharomyces pombe, we sought to elucidate functional effects of these variants in ERCC4 by creating a model system. Using cre recombinase mediated cassette exchange, we introduced the variants of interest into the ERCC4 homolog, rad16. Following treatment with a range of DNA damaging agents, we observed an increased sensitivity following introduction of the novel stop gain, indicating a defect in the NER pathway. Additionally, there was a clear pattern of oxaliplatin-specific sensitivity of strains with the introduced rare nonsynonymous variants, suggesting a defect of XPF in other repair processes associated with interstrand crosslinks.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Electrical and Electronic Engineering / Master / Master of Philosophy

published_or_final_version / abstract / Pharmacology / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy