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An assay of minichromosome maintenance (mcm) protein 5 in bile was developed to determine whether it was a more sensitive marker of pancreatic malignancy than routine biliary brush cytology. The potential for immunotherapy and photodynamic therapy (PDT) for hepatobiliary cancer was addressed. Methods: 1) Prospective study of mcm protein expression in bile and tissue sampled from biliary strictures; 2) Longitudinal and cross-sectional studies of T cell responses in patients with primary liver cancer treated with transarterial embolisation or PDT, 3) Preclinical studies of interstitial verteporfin PDT and a phase II clinical study of endoscopically delivered meso-tretrahydroxyphenylchlorin (mTHPC) PDT in patients with malignant biliary strictures. Results and Conclusions: Mcm proteins were expressed in all tissue layers in malignant biliary strictures and confined to the basal proliferative compartment in benign tissue. Mcm 5 in bile sediment, detected by an immunofluorometric test, was significantly more sensitive than routine brush cytology for the diagnosis of malignancy in biliary strictures. Tumour (alphafetoprotein)-specific CD4+ T cells were significantly expanded in-vivo in hepatocellular carcinoma patients during and after embolisation. Novel AFP-derived epitopes were identified which could be targeted in an AFP based vaccine for hepatocellular carcinoma. The unmasking of AFP-specific CD4+ T cells was significantly associated with greater tumour necrosis and an improved clinical outcome, which may provide a rational for combining immunotherapy with embolisation in HCC patients. In patients with malignant biliary obstruction, mTHPC PDT caused efficient tumour necrosis but there was a significant risk of vascular complications. Verteporfin PDT was safe in the normal hamster pancreas and had a similar efficacy to previous photosensitisers, with the advantages of a shorter drug-light interval and drug elimination time. Phase 1 studies of verteporfin PDT in patients with locally advanced pancreatic cancer are planned.

Despite advances, the impact on long-term survival for individuals with epithelial ovarian carcinorna (EOC) remains modest. To date, efforts to find novel treatment strategies have focused on complex upstream oncogenic signalling pathways. However exploiting the molecules which govern these redundant pathways in the clinical setting has proved difficult. An alternative approach is to focus on the cell cycle machinery, which acts as an integration point for information transduced through upstream pathways. The DNA replication licensing factors and Aurora kinases are important cell cycle regulatory proteins which are showing promise as novel biomarkers and therapeutic targets in a broad range of tumour types. However there is a paucity of information regarding the role of these G1/S and G2/M regulatory molecules in EOC. Here I show that there is aberrant regulation of the DNA replication licensing system and Aurora kinases in EOC. Moreover, dysregulation of these molecules is associated with aggressive, genomically unstable tumour phenotypes, which in turn translates into important prognostic information with regards to patient survival. Furthermore, I have shown that multi-parameter expression analysis of these proteins allows assessment of cell cycle kinetics in individual pathological specimens, parameters linked to the biological behaviour of these tumours. This form of analysis could be utilised as a predictive test for therapeutic agents targeting the cell cycle machinery or upstream growth signal transduction pathways that accelerate cell cycle progression. Notably, my work identifies the Cdc7 and Aurora A kinases as promising predictive biomarkers and molecular targets in this complex tumour type, an intriguing finding in view of the nature of these molecules and their amenability to small molecule inhibition. Thus, finally, I have utilised in vitro RNA interference studies to provide early proof-of-principle validation that Cdc7 kinase represents a novel mechanism-based therapeutic target in this heterogeneous malignancy. Collectively, these studies highlight the prognostic, predictive and therapeutic utility of the DNA replication licensing system and Aurora kinases in EOC.

This thesis describes investigations into mechanisms responsible for cancer cell motility in vivo. Chapters 1 and 2 provide a review of current literature in this field and also describe the techniques used to generate the following the results. Chapter 3 describes a candidate-based approach to investigate whether ROCK1 might be regulated by phosphorylation. Mutagenesis of ROCK1 was carried out at 3 chosen sites (T233 T380 T398) in the activation loop and the hydrophobic domain and the phenotypes of the mutants were analysed. Chapter 4 describes a parallel approach finding phosphorylation sites in ROCK1 by mass spectrometry. From these results T518 was chosen for further investigation and its possible function is investigated. Chapter 5 describes an siRNA screen designed to identify novel regulators of the cortical acto-myosin cytoskeleton. The read-out for this was based on the disruption of rounded blebbing morphology of A375 cells cultured on 3D gel matrices. The rounded morphology is similar to that observed in amoeboid cancer cell motility in vivo, therefore we hypothesised that genes required for contracted, rounded morphology might also be required for motility. Results identified PDK1 amongst other genes as a potential regulator of contractile forces in A375 cells and the role of PDK1 was investigated further. It was found that PDK1 was required both in vitro and in vivo for amoeboid cell motility. Chapters 6,7 and 8 detail the investigations into the mechanism of how PDK1 regulates the cytoskeleton and amoeboid cell motility. It was shown that PDK1 was responsible for the localisation of ROCK1 but not ROCK2 at the plasma membrane. This regulation was achieved by the direct binding of ROCK1 to PDK1. It was further found that PDK1 was able to compete with and prevent RhoE, a negative regulator of ROCK1, from binding. Chapter 9 investigates the relationship between cell morphology, motility and pigment production. It was found that it was possible to image melanin containing vesicles using multiphoton excitation, and using this technique, the motile behaviour of pigmented melanoma cells was observed in vivo. It was found that motile invasive cells tended to contain less melanin than non-motile cells suggesting that they were less well differentiated. This chapter details investigations into what differences in signalling could be responsible for a switch to a de-differentiated, more invasive/metastatic phenotype. The final chapter discusses the findings contained within this thesis and the possible implications.

An increased concentration of insulin-like growth factor-1 (IGF-1) is recognized to be an independent risk factor for pre-menopausal breast cancer. Tamoxifen is thought to initially reduce IGF-1 concentrations and increase levels of the IGF binding proteins 1 and 3. Isoflavones are oestrogen-like plant compounds, which may, because of their structural similarities to tamoxifen, also alter IGF status. This thesis first compares IGF-1, IGF binding protein 1(BP-1) and IGF binding protein 3 (BP-3) levels in breast cancer patients (n=14) compared with control subjects (n=23) and then assesses the effect of tamoxifen on IGF status after 9, 18 and 27 months of treatment. Concentrations of IGF-1, BP-1 and BP-3 at baseline did not differ between cases and controls. However on tamoxifen treatment, BP-1 and BP-3 were both significantly increased after 18 and 27 months while the IGF-1/BP-3 ratio was significantly decreased after 9 and 18 months. A feasibility study was then conducted to compare the effects of acute (single 80 mg load) versus chronic (80 mg/day for 7 days) administration of isoflavone- containing soy and linseed cereal bars on IGF-1, IGFBP-1 and IGFBP-3 in healthy female volunteers (n=10). Assays were established for the analyses of serum IGF-1, BP1 and BP3 concentrations and GCMS analysis of isoflavones in urine. Concentrations of IGF-1 were unchanged following the single 80 mg load. However, IGF-1 and BP-3 concentrations were significantly elevated after a week of supplementation with the soy and linseed bar. A larger study was then carried out to assess the effect of one-month isoflavone supplementation (80 mg/d) in tablet form, on IGF status in healthy pre- (n=16) and post-menopausal (n=7) women. This was a randomized, placebo-controlled crossover study with a minimum two-month washout period. Hormonal, antioxidant and lipid altering effects of the supplement were also examined, as was background diet. For pre-menopausal subjects, while there was an effect of time on IGF-1 (p=0.005), BP-1 (p=0.004), and BP-3 (p<0.001) confirming that IGF profile is influenced by menstrual cycle, this did not differ between placebo and isoflavone supplement. When the change in IGF-1 over the whole supplementation period was compared between the supplement and placebo phases, there was a non-significant reduction in change in IGF-1 (p=0.06) on isoflavone supplement compared to placebo. However, this may have been due to the non-significantly higher baseline concentrations of IGF-1 during the supplement phase. In post-menopausal subjects, there was no effect of isoflavone supplementation in comparison with placebo on IGF-1, BP-1 or BP-3. Finally, experiments were done in vitro to study the effect of isoflavones on DNA synthesis and proliferation in ERa positive MCF-7 and ERa negative MDA-MB 231 human breast cancer cells. In MCF-7 cells, low concentrations of isoflavones (0.1uM-10uM) stimulated DNA synthesis while high concentrations of genistein and equol were able to inhibit DNA synthesis with IC50 values of 93uM and 55uM respectively compared with 1.17uM tamoxifen. In MDA-MB231 cells the Isoflavones did not stimulate DNA synthesis at any concentrations but significantly inhibited DNA synthesis with IC50 values of 114uM, 14.1 uM and 14.55uM for daidzein, genistein and equol respectively compared with 1.35uM tamoxifen. This work suggests that isoflavone supplementation may alter IGF profile in pre menopausal women, and could suggest a role for these dietary compounds in breast cancer prevention. This should be further investigated in long term intervention studies with isolated isoflavone supplements.