Metastatic prostate cancer cells display EphB receptor-mediated attraction when they contact stromal fibroblasts but EphA-driven repulsion and contact inhibition of locomotion (CIL) when they contact one another. The impact of these social interactions between cells during cancer cell invasion and the signalling mechanisms downstream of Eph receptors are unclear. Here, I show that EphA receptors drive prostate cancer cell dissemination in a 20 dispersal assay and a 3D cancer cell" spheroid assay by activating repulsive interactions and CIL between contacting prostate cancer cells. I show that EphA receptors interact with the exchange factor Vav2 to activate RhoA, and that both Vav2 and RhoA are required for prostate cancer cell-cell repulsion. Using pharmacological inhibitors I show actomyosin contractility is not a key driver of CIL. I find instead that microtubule dynamics are important for generating the front-rear polarity switch required during CIL, and that EphA2/EphA4, Vav2 and RhoA affect microtubule stability in prostate cancer cells. Furthermore, I find that in EphA2/EphA4, Vav2 or RhoA knockdown cells, contact repulsion can be restored by partial microtubule destabilisation. I propose that EphAVav2- RhoA-mediated repulsion between contacting cancer cells at the tumour edge could enhance their local metastatic invasion and dissemination from the primary tumour. Subsequently, EphB-mediated attractive migration and failure of CIL, between prostate cancer cells contacting ephrin-B2· expressing fibroblasts, could facilitate cancer cell invasion through the surrounding stroma. Stimulation of prostate cancer cells with ephrin-B2lFc leads to filopodia formation and activation of Cdc42. I show that Cdc42-silenced PC-3 cells have significantly impaired migration towards surface coated ephrin-B2 compared with control siRNA-treated cells. Furthermore, Cdc42 is required for attractive migration and defective CIL during collisions b~tween advanced prostate cancer cells and ephrin-B2-expressing fibroblasts. Using organotypic 3D gel invasion assays, I show that ephrin-B2 expressing fibroblasts enhance prostate cancer cell invasion. These data suggest that EphB-Cdc42-mediated attractive interactions with fibroblasts and defective CIL might facilitate prostate cancer cell invasion through the surrounding stroma.

Prostate cancer is the most commonly diagnosed cancer in British men and the second most common cause of cancer mortality, exceeded only by lung cancer. Oxidative stress is known to play a role in cancer and cardiovascular disease, implying a connection between diets low in antioxidants and chronic disease development. Apparently involved in chronic illnesses such as cancer and atherosclerosis, transcription factor Nuclear Factor kappaB (NF-κB) has attracted widespread interest. NF-κB is activated during the inflammatory response. Being a redox sensitive transcription factor, it is also affected by oxidative stress. The factor is ubiquitously expressed in the cytoplasm of all cells, where it regulates expression of genes that control immune system. growth and inflammation.

Reactive oxygen species (ROS) are mechanistically implicated in the development and progression of prostate cancer. Intracellular ROS may cause oxidative DNA damage, resulting in the formation of adducts, such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxo-dG) and the cyclic pyrimidopurinone N-1, N-2 malondialdehyde-2'deoxyguanosine (MidG). These adducts have been associated with carcinogenesis, genomic instability and clonal evolution. It has previously been shown in vitro that testosterone and dihydrotestosterone (DHT) increase ROS levels in the androgensensitive, human prostate cancer cell line, LNCaP.

Prostate cancer (PCa) is the second most common cancer in Western men; with over 30,000 new cases are diagnosed in the UK each year. Primarily treatment involves targeting the androgen-signalling pathway by blocking production of testicular androgens and/or inhibiting AR function. Current treatments depend on the age and health of the patient. These treatments range from a watch and wait strategy to androgen ablation therapy, by chemical or surgical castration, with the addition of using anti-androgens to block the AR function. The drawbacks of these treatments include loss of bone mineral density, urinary dysfunction, bowel dysfunction, erectile dysfunction, loss off libido and fertility.

Androgen ablation therapy is frequently used in locally advanced androgen-dependent prostate cancer, however the cancer often returns with a more aggressive, androgen- independent phenotype. The causes of this are poorly understood, however androgen withdrawal is known to induce hypoxia in androgen-sensitive tissue; this is important as hypoxia is known to drive malignant progression. Tumour oxygenation was measured in LNCaP tumours grown in SCID mice treated with a single dose of three anti-androgens; within 24h all had reduced tumour oxygenation with the greatest reduction caused by bicalutamide (10 mg/kg). LNCaP-Luc tumour-bearing mice were then treated daily with a clinically relevant bicalutamide dose (2mg/kg). Oxygenation showed a profound drop to almost zero by day 7 persisting until day 14 when it started to increase up to day 28. This was confirmed ex vivo using the hypoxic marker G lut-I. LNCaP-Luc tumour fragments implanted in a dorsal skin fold chamber and treated with daily bicalutamide showed a marked loss of small vessels at days 7 and 14, revascularisation was complete within the next week suggesting that vascular changes caused the oxygenation changes. Quantitative PCR showed changes in VEGF, IL8, Runx 2 and androgen receptor expression consistent with the oxygen changes and progression to a more malignant phenotype. A control and treated tumour were excised on day 28 and re-established in vitro; cells from treated tumours were shown to have a more malignant phenotype than vehicle-treated controls. Growth delay studies compared daily bicalutamide and its combination with AQ4N which targets hypoxic cells. Bicalutamide increased tumour growth after 16 days and lung metastasis by day 28; AQ4N (single dose, day 7) blocked these effects. AQ4N alone provided the greatest reduction in metastasis. This study shows that bicalutarnide-induced hypoxia selects for cells that show malignant progression; targeting the pre-existing hypoxic cells may therefore be of greater clinical benefit.

In addition to the more traditional roles associated with Vitamin D, such as the formation and maintenance of healthy bones, regulation of calcium-phosphate homeostasis and parathyroid hormone regulation, Vitamin D is involved in many other processes in the body including regulation of immune function, neurological function and control of cellular proliferation. Many studies have shown the chemoprotective and therapeutic effects of Vitamin D in the treatment of many ailments including cancer, with many studies investigating as a potential treatment option. Vitamin D therapy has limited success in prostate cancer with tumours becoming resistant to treatment and developing a more aggressive phenotype. Intracrine signalling has been put forward as a theory as to how tumours continue to grow in patients who have undergone androgen ablation therapy; this hypothesis suggests that androgens are synthesized de novo within the tumour microenvironment from adrenal precursors. Cytochrome P450 enzymes are involved in the metabolism of endogenous and exogenous compounds, including steroid hormines, to prevent toxic accumulation. Previous studies in this laboratory have shown that the major hepatic CYP, CYP3A4, can be regulated by the Vitamin D Receptor (VDR) in epithelial cells of the colon when it is liganded to toxic bile acids which in turn facilitates their excretion. This thesis demonstrates a novel finding whereby the VDR directly modulates expression of CYP3A4 in the well known prostate cancer cell line LNCaP. CYP3A4 expression is found to be up-regulated in the presence of the most biologically active form of the vitamin (1,25D) in a LNCaP cells, with a subsequent increase in enzymatic activity and metabolism of testosterone as evidenced by HPLC MSIMS analysis. Interestingly mutation analysis uncovered the finding that the more novel vitamin D response element, ER-6, has important roles to play in the VDR mediated transcriptional response. Additionally VDR was also found to interact with the receptor interacting protein of 140kDa (RIP140) in a ligand dependent manner resulting in dose dependent repression of VDR transcriptional activation while gene silencing of RIP140 resulted in a significant increase in CYP3A4 expression. Considering these results together, CYP3A4 and RIP140 may provide new therapeutic targets in the treatment of prostate cancer.

Surgically induced unilateral cryptorchidism in juvenile rats was used as a method of inducing a known pre-malignant state for testicular tissue. The cell-cell contacts in the testis were then quantified using the newly developed second order stereological technique of one-stop stereology. The stereological analysis showed significant differences in the cell contacts between basal lamina - Sertoli cells, Sertoli cells - spermatogonia, spermatogonia - spermatocytes, and spermatocytes - spermatids. In addition, the effect of bisphenol A (BP A) on the development of the rat testis was investigated. BP A is known to have estrogenic properties but is used in a wide variety of products including baby bottles and dental sealants. Incomplete polymerisation of the BPA monomer can result in leaching of BP A from these products. Female pregnant rats were given a daily dose of either 2 ng/g bisphenol A (BPA) or 20 ng/g BP A. The testes of the male offspring were then examined at the age of 8 weeks. Stereological analysis of the testes exposed in utero revealed significant differences in the cell-cell contacts between basal lamina - Sertoli cells, spermatogonia - spermatocytes, and spermatocytes - spermatids. Furthermore, abnormal cells were observed in the stereological analysis only in the samples exposed to 2 ng/g BPA. The subtle levels of tissue dysgenesis revealed by one stop stereology, which cannot be detected on qualitative slide review, indicate its power as a tool for highlighting the main areas disruption of cell-cell interactions in a tissue. This will make it a powerful tool for investigating the Tissue Organisational Field Theory (TO FT), as an alternative to the Somatic Mutation Theory (SMT) of carcinogenesis, by helping to steer investigators to the sensitive changes within a tissue.

The utilisation of Androgen Deprivation Therapy (ADT) in prostate cancer patients is associated with a number of adverse side effects including: changes in body composition; an increase in fat mass and a decrease in muscle mass, increased fatigue and a reduced Quality of Life (QoL). Existing literature suggests that physical activity plays a key role in alleviating some of the associated side effects of ADT; however studies in this area are based on supervised facility-based exercise programs without dietary modification. The aim of this thesis is to present the rationale and methodology of a Randomised Controlled Trial (RCT) to test the efficacy of a 6 month combined dietary and walking intervention on alleviating the Health-Related QoL (HRQoL) issues associated with ADT treatment. Ninety-four men were recruited and randomised to the intervention arm or a standard care control arm. The trial had a significant impact on reducing the adverse body composition changes as well as improving the functional capacity of intervention patients compared to control patients. A systematic review was also completed to determine if expression of the lipogenic enzyme Fatty Acid Synthase (FAS) differs in prostate cancer tissue compared with normal prostate tissue. and if F AS expression in cancer tissue has a role in prostate cancer progression. The number of studies in the area was limited, however 6 of the 7 studies included in the review reported elevated F AS expression in prostate cancer tissue when compared to normal tissue. Additionally. using data from the Prospective Epidemiological Study of Myocardial Infarction (PRIME) study. the association between body composition measurements (body mass index, height, waist circumference and waist-hip ratio). physical activity. smoking status and prostate cancer risk was explored using Cox Proportional Hazard analysis. None of the included' lifestyle behaviours or body composition measurements had a statistically significant effect on prostate cancer risk .

Prostate cancer is the second most common diagnosed cancer and the third most common cause of death related to cancer among men in developed countries. Several epidemiological studies, prospective cohort studies and animal tumour models state an inverse relationship between selenium status and cancer incidence. Se- methylselenocysteine (SeMSC), present in garlic, onions, leeks and broccoli, has been shown to be the most effective anti-carcinogenic selenium form in animal models. The aim of the work presented in this thesis was to investigate the influence of selenium compounds (Se-methylselenocysteine and selenomethionine) on prostate cancer progression and metastasis using various human cell lines (LNCaP, OU145 and PC3). Standard 20 gel and SILAC (stable isotope labelling with amino acids in cell culture) proteomics were used, in combination with mass spectrometry, to identify selenium- responsive proteins. IMPOH2, GPI, EZR and RGS10 were validated by western blot, while POIA3 and 00X5 showed a selenium response under serum depleted conditions. Some proteins require more scrutinizing (galectin-1, XRCC5, TAGLN2, 00X5 and FLT) as conflicting results were obtained during validation. Preliminary analysis using 20 gel proteomics revealed galectin-1 to be selenium-responsive in PNT1A cells, although this could not be confirmed by Western blot or an in-house ELlSA. Previously, it has been shown that SeMSC decreased the expression of collagen I and increased that of collagen IV and collagen VI. A LNCaP 3D gel suspension model was developed to allow further investigation of extracellular matrix components using fluorescence microscopy. In addition, the effect of selenium exposure on the migration and invasion of PC3 cells was investigated using a transwell kinetic assay and revealed a dose response increase, especially under low baseline selenium concentrations. In order to optimize future selenium in vitro projects the dynamics of several selenium biomarkers were investigated using different conditions, enabling better comparison between cell lines and/or selenium compounds.

Green tea catechins have been recognized as a potential chemopreventive agent for prostate. Prostatic intraepithelial neoplasia (PIN) lesion is widely accepted as the pre-invasive stage of adenocarcinoma and a predictive marker of prostate cancer development. This study confirmed that novel catechin-derivatives showed a greater inhibitory effect on proliferation in PIN cells than in PCa cells. In addition, two minor existing catechins, (-)-epigallocatechin (EGC) and (-)- epicatechin (EC) showed a synergistic inhibitory effect on PIN cell growth, invasion, migration and progression of PIN to a mesenchymal-like invasive phenotype by inhibition of Epithelial Mesenchymal Transition (EMT)-associated transcriptional factors such as Twist, Snail, Slug and Zeb-l. In this study, a carcinoma-associated fibroblast (CAF)-like human bone marrow- derived mesenchymal stem cells (hMSCs) was established via exposing hMSCs to human prostate tumour-conditioned medium (TCM). TCM-exposed hMSCs exhibited the ability to promote tumour cell growth of prostate and colon cancer and PIN cell invasion. Green tea catechin treatment inhibited PIN cell invasion by reversal of EMT. A chemo-resistant (CR) prostate cell line (CR-PC3M) was established and exhibited a greater resistance to several chemotherapeutic drugs such as 5- fluorouacil, Taxotere and Doxorubicin, and expressed a high level of a drug resistance-associated transporter ABCG2 and the cancer stem cell (CSC) marker