ABSTRACT Objective The use of conformal radiation therapy in the treatment of carcinoma of the prostate has allowed for dose escalation and improved local control. The dose to the rectum is an important consideration in determining complication rates. This study aims to evaluate the effect of a Foleys rectal catheter balloon on the dose volume histograms to the rectum and to assess the effect of the balloon catheter on prostate gland immobilization during treatment of intermediate risk cancer of the prostate. Design and methods Ten patients with intermediate risk prostate cancer, each acting as his own control, were recruited in the study; eight patients had complete data for analysis. CT scans were done at intervals during treatment, with and without a rectal balloon filled with 30 ml of contrast. 3 pairs of CT scans for each patient were performed and were available for analysis. All patients were treated with 6-field conformal radiotherapy up to 66 Gy followed by a boost of 12 Gy in 3 fractions to the prostate using a rectal balloon and a 3- field plan. Dose volume histograms were calculated for the boost plan with and without the rectal balloon. Movements of the prostate in the superior-inferior and the anteriorposterior directions were measured with and without the balloon for each treatment. There was a slight reduction in the dose received by 1% and 2 % of the rectal volume with the balloon (55% and 52% respectively), compared to without a balloon (57% and 54.3% respectively) (p> 0.05). Results There was a non significant increase in the dose received by 50% of the rectum (p>0.05) with the use of the rectal balloon due to the rectum being pushed towards the symphysis pubis by the balloon. With the use of rectal balloon, the superior / inferior displacement of the prostate was reduced (p=0.04) and a displacement of more than 5 mm was observed in one out of eight patients. The anterior / posterior displacement of the prostate was decreased with the rectal balloon with a mean of 4 mm compared to 5 mm with no rectal balloon. This was not statistically significantly (p>0.05). However, displacement of more than 5 mm was observed in 2 patients with the rectal balloon. No grade 3 acute rectal toxicity was recorded in the 8 patients. Conclusion There was no significant change in the percentage dose received by the rectum with the use of the rectal balloon in this study. The study showed however that the rectal balloon significantly reduced prostate movement during treatment.

Prostate cancer is the most common male-specific form of cancer in the U.K. Current treatments for the aggressive disease by androgen-deprivation therapy gives a rapid initial response, but the disease ultimately progresses into an androgen-independent state for which there are no effective treatments. α-Methylacyl-CoA racemase (AMACR, P504S) is an enzyme which is involved in metabolism of branched-chain fatty acids and the pharmacological activation of some NSAID drugs, such as Ibuprofen and most other ‘profens’. AMACR is over-expressed in prostate cancer and some other cancers, including colon and breast cancers. Reduction of AMACR protein levels inhibits proliferation of prostate cancer cells and restores the requirement for androgens for growth. Although the exact role of AMACR in prostate cancer progression is currently unknown, several other experiments show that AMACR is functionally important for prostate cancer proliferation, validating it as a drug target. There is no convenient high-throughput assay for AMACR and as a result only a few inhibitors have been reported to date. This thesis reports a study on whether other reactions can be catalysed by AMACR. 2-Methyl-3-enoyl-CoA esters are good substrates of AMACR but do not undergo double bond migration, while 2-methyl-2-enoyl-CoA esters are not converted to products. Acyl-CoA esters that contain a fluorine atom at carbon-3 undergo a fluoride elimination reaction to give 2-methyl-2-enoyl-CoA esters. This elimination reaction was investigated for use in the development of a high-throughput assay. A fluorescent binding assay, which can be adapted for the screening of large libraries of compounds, was developed and several known and novel inhibitors were tested. Finally, metabolism of mandelic acid was investigated. It was shown that chiral inversion of mandelic acid in humans proceeds via a different pathway to Ibuprofen and related drugs, in contrast with previous reports.

Prostate cancer (PCa) is a unique and controversial disease. This is at least due to the high prevalence of latent disease, increasing amounts of which is being diagnosed, most of which is indolent and not lead to death, and for which treatment carries significant risks. An increasing concern in medical sociology is how various social structures and actors contribute to the diagnosis and experience of conditions. For PCa, these include print media as an information source for men with prostate cancer (MWPCa), and PCa organisations (PCaOrgs) which have recently emerged in the UK. Yet, there is a distinct lack of UK studies of print media representation of PCa, of PCaOrgs, interaction between the two, and how any of this may impact on the experience of MWPCa. This thesis aims to address this deficit by drawing on narrative and framing theory to study 201 illness narratives of PCa across time: 140 illness narratives of MWPCa in UK newspapers 1990-2010; 20 with MWPCa interviewed in each of 2000 and 2010; and 21 with advocates around PCaOrgs in 2010. I ask: how have PCaOrgs and the UK print media been a force for change in the UK regarding how PCa is addressed and experienced by MWPCa? And more broadly what does this say about narrative structure and form. My findings indicate that though PCaOrgs and print media told stories of injustice around PCa, the substantive focus of this injustice changed over time—from PCa as “neglected” and “taboo” in the 1990s to other “pockets of injustice” since 2000. While one might expect that this to lessen any interactional difficulty that MWPCa experience in disclosing their illness, my study suggests this may not be so. My findings show how ideas of resonance and dissonance contribute to understanding the recursive and repetitive language around PCa.

Prostate cancer (PCa) is the most commonly diagnosed cancer amongst Western males. PCa progression is strongly linked to steroid receptor signalling, however the modulation of steroid receptor expression in PCa is incompletely understood. Lipocalin 2 (LCN2) is a secreted protein which binds to Fe3+-containing siderophores and was originally identified as part of the innate immune response. LCN2 has been proposed as a potential biomarker for a range of cancers. However, LCN2 effects appear to be tissue specific. LCN2 expression is associated with poor prognosis in breast cancer, but with good prognosis in pancreatic cancer where it has been used therapeutically. The role of LCN2 in prostate cancer is poorly understood, in particular its effects on steroid receptor regulation. To elucidate the role of LCN2 in prostate cancer, the LCN2 gene was ectopically expressed in LNCaP cells to generate the LNCaP-LCN2 cell line. LNCaP-LCN2 cells had elevated androgen receptor expression which was linked to increased levels of KLK3 (PSA). LNCaP-LCN2 cells also had reduced levels of Estrogen receptor α (ERα), but increased expression of ERβ. This was combined with higher levels of E-cadherin, but not to changes in other EMT markers. Reciprocally, LCN2 was suppressed using RNAi in the PC3 cell line to generate PC3-shLCN2 cells. PC3-shLCN2 displayed a distinct change in morphology, with increased cell size and a sub-population of multi-nucleated and highly enlarged cells. PC3-shLCN2 cells had reduced proliferation, and lost the ability to form colonies in a 3D substrate. With regards to steroid receptors, PC3-shLCN2 cells had increased ERα expression, but reduced ERβ expression. This was also combined with a loss of E-cadherin and EGFR. Microarray analysis of PC3-shLCN2 cells identified changes to expression of a wide range of genes including VEGF-R, SPARC and KLK6. Functional grouping of differentially expressed genes suggests that LCN2 in involved in a range of cellular processes including hormone receptor response, Wnt signalling and cell cytoskeletal integrity. Many, but not all genes identified by microarray were responsive to recombinant LCN2 protein indicating a paracrine function for the protein. Treatment of PC3 cells with the iron chelator Deferoxamine resulted in phenotypic changes similar to those found in PC3-shLCN2 cells which suggest that LCN2 functions in part due to intracellular iron regulation. In summary, the data presented in this thesis suggests that LCN2 has both pro- and anti- tumourigenic properties in prostate cancer and that the protein is involved in a much wider range of functions than previously described.

Prostate cancer (PCa) is the most common cancer among men in Western developed countries. While the majority of PCa diagnosed by PSA screening are indolent, advanced and metastatic disease has a significant mortality and morbidity. Bone metastases are extremely common in PCa and identification of bone metastasis associated genes may provide insights into PCa progression and assist in finding new drug targets. However, the genetic study of bone metastases is very limited due to the difficulty of sampling. We performed genome-wide analysis of six fresh frozen PCa bone metastases. We found several alterations commonly present in advanced PCa, including gains at: 1q32.1, broad gains of 8q (MYC, NCOA2), 9q33.2-34.3, 11q13.1-14.1 (CCND1), 12q24.23-24.31, 16p13.3, 16p12.1-11.2 and Xq12-13.1 (AR) as well as losses at: 5q11.1-22.1, 5q14.3-23.1, 6q14.1-22, 8p23.2-p21, 13q13.2-31.1 (RB1), 17p13.1-12 (TP53) and 18q11.1-22.3. Two cases also showed PTEN loss and one sample had deletion indicative of TMPRSS2-ERG fusion. For downstream analysis we concentrated on CCND1 oncogene at 11q13 and FBXL4 at 6q16 as potential drivers of these genomic changes. Using fluorescence in situ hybridisation we found common CCND1 gain and FBXL4 loss in PCa bone metastases (54.5%, 12/22 and 47.8%, 11/23, respectively), much less frequent in primary tumours (7%, 10/142 and 13.8%, 20/145, respectively) and absent in BPH cases (0/55). The expression levels of cyclin D1 protein, coded by CCND1 correlated with CCND1 copy number gain (p < 0.0001) and were higher in metastatic tumours than in primary PCa (p = 0.015), confirming cyclin D1 involvement in advanced PCa. Presence of FBXL4 loss in early stage primary PCa strongly correlated with current PCa prognostic markers and with worse patient survival. Therefore, we propose that FBXL4 may be a tumour suppressor gene in prostate, whose loss in early PCa could be indicative of more aggressive disease. Using in vitro experiments we demonstrated that FBXL4 regulates cells motility and invasion. We confirmed that ERLEC1, an ER lectin involved in ER stress response pathway is a degradation target of FBXL4. As activation of ER stress response pathway is linked to enhanced cell migration and invasion, loss of FBXL4 could be one of the mechanisms by which cancer cells increase their efficiency to respond to stress and to escalate their metastatic potential through stabilisation of ERLEC1. Further studies of FBXL4 - ERLEC1 axis are necessary to establish how they contribute to PCa progression. This knowledge can potentially help to develop novel targeted therapies for aggressive disease harbouring FBXL4 abnormalities.

The diagnostic pathway for prostate cancer involves the blood test prostate specific antigen (PSA) which has high sensitivity but low specificity at age related reference ranges. The resultant clinical consequence is a large number of negative diagnostic studies (transrectal ultrasound guided biopsies - TRUS). There is a need for a secondary screening test to help improve on the current diagnostic pathway. Mechanical markers have been used previously to assess the prostate for disease with numerous ex-vivo reports of differences between benign and malignant prostates. There have been no in-vivo studies with direct elasticity assessment devices for prostate cancer detection. This thesis forms part of work in a collaborative study in conjunction with engineers who have created a microscale device, capable of dynamic elasticity assessment. The specific objectives of this thesis were to a) assess dynamic micromechanical markers for the detection and differentiation of clinically significant from insignificant prostate cancer b) to identify relationships between mechanical and histopathological variables in the ex-vivo and in-vivo environments and c) assess the potential for these markers to differentiate peri-prostatic tissues. A prospective study was set-up with full ethics and management approvals with patients undergoing a systematic mechanical assessment of their prostate using the E-finger device and after prostate excision a systematic ex-vivo mechanical assessment on a calibrated stage. The ex-vivo assessment allowed accurate histopathological and mechanical variable assessment in a controlled environment. 7-Tesla ex-vivo MRI scanning aided in assessing the limitations of mechanical assessment of the prostate. There were clear consistent differences between individual dynamic micromechanical markers for benign and tumour containing measurement areas in both environments. Modelling of these dynamic micromechanical markers yielded encouraging accuracy levels for the detection of prostate cancer and differentiation of significant from insignificant disease. There were associations between individual mechanical markers and important histopathological features associated with cancer (acinar size, tumour volume and reactive stroma). These markers showed promise and utility in the differentiation of prostate from bladder and rhabdosphincter. This work demonstrates the clear potential translational uses for dynamic micromechanical markers in the assessment of the prostate for cancer.

Master of Science / Department of Kinesiology / Brad J. Behnke / Introduction: Prostate tumor arterioles lack functional smooth muscle and have a diminished myogenic response. Previous research has demonstrated an enhanced prostate tumor blood flow and oxygenation associated with the augmented mean arterial pressure during exercise. Thus, we tested the hypothesis that elevations in the heart-to-prostate tumor hydrostatic gradient via adoption of the 70˚ head-up tilt (HUT) body position would enhance perfusion of the prostate tumor, which may improve tumor oxygenation and radiation therapy outcomes (Study I). Based upon those findings, we performed a secondary analysis (Study II) on previously published prostate hemodynamic responses to an identical tilt-test between young and aged animals. Methods: Study I: Dunning Cell AT-1 tumor cells (100,000) were injected into the ventral lobe of the prostate in male Copenhagen rats (4 mo.; n = 7). Four to six weeks after injection blood flow to the prostate tumor, kidneys, and soleus muscle was measured via the fluorescent microsphere technique in the supine and HUT position. Study II: A secondary analysis was performed on blood flow to the prostate (host tissue of the tumor) in young (6 mo.; n =9) and aged (24 mo.; n=7) male Fisher 344 rats from Ramsey et al., 2007 (39) to determine potential age-associated differences in conductance to this tissue. Results: Study I: No significant difference was observed in blood pressure between the two body positions. Compared to the supine posture, there was a significant reduction in blood flow to the soleus muscle. There was no difference in prostate tumor blood flow or vascular conductance between the supine and HUT position. Study II: In response to tilt, there was a significant reduction in prostate vascular conductance in young rats versus that in the supine posture (P<0.05). In the aged animals, there was no difference in prostate vascular conductance with tilt. Discussion: Contrary to our hypothesis, we did not see any significant differences in either blood flow or vascular conductance to the prostate tumor with manipulations in body position. Importantly, we believe this may be an age-associated effect. Given tumors both co-opt existing arterioles from the host tissue that retain vasomotor control and develop new vessels that lack functional smooth muscle, the enhanced vascular resistance in the prostate with young animals during tilt likely contributed to the lack of change in tumor perfusion with body position given the rats from study I were also young. Given the lack of change in vascular conductance in the prostate with tilt in aged animals, future studies should be performed in aged models of prostate cancer, of which currently there are no immunocompetent aged rodent models of prostate cancer.

The Defender against apoptotic cell death (DAD1) is a negative regulator of programmed cell death that was initially identified in the temperature sensitive tsBN7 cell line. It has been shown to be an essential subunit of oligosaccharyltransferase and is localized to the Endoplasmic reticulum (ER) in a normal physiological state. However, our data suggests that DAD1 localizes outside of the cell and alters the apoptotic signaling via FAS ligand to give cancer cells a survival advantage. Although the mechanism is poorly understood, increased expression of DAD1 has been associated with increasing Gleason score in prostate cancer (PCa) patient tumors. Based on the aforementioned evidence, our study attempts to unravel cellular localization and the underlying mechanisms by which DAD1 mediates prostate cancer cell survival, and explore its potential as a biomarker in prostate cancer. As evidenced by qRTPCR, immunocytochemistry, immunohistochemistry, Co-ip, ELISA, and immuno-blot analysis, cancer cells down regulate the expression of the binding partner of DAD1 responsible for retention of DAD1 in the ER, which allows DAD1 to exit the ER and be exocytosed. The exocytosed DAD1 binds to FAS L and prevents apoptotic signaling. Treatment with DAD1 antibody induces significantly higher cell death in prostate cancer cells compared to the non-tumorigenic cells. Combination of DAD1 antibody with currently used chemotherapeutic agents like Docetaxel and Doxorubicin can be used to achieve higher cell death at a lower dose of these drugs to minimize side effects. Further, our immunohistochemistry data in tumor microarray suggests that DAD1 could serve as a potential biomarker marker in PCa. In addition to the tissue, we also examined the expression of DAD1 in prostate cancer patient serum samples using sandwich ELISA; our results indicate DAD1 is a more sensitive and specific prognostic marker for prostate cancer compared to PSA. Our data suggests that localization of DAD1 outside of the cells is crucial to the survival of PCa cells and this phenomenon can be exploited to specifically target prostate cancer cells in therapy and serve as a biomarker in prostate cancer. / 1 / Nobel Bhasin

Estrogens maintain the appropriate androgen-estrogen balance for normal regulation of the structure and function of the male reproductive tract, including the prostate gland. This research investigated viability of cells and expression of selected genes in prostate carcinoma cells (PC-3) exposed to bisphenol A (BPA), an estrogen-like substance present in a number of plastic materials. PC-3 cells are able to metabolize BPA at concentrations below 100 µM. BPA exposure at concentrations between 1nM and 100 µM does not increase or significantly reduce cell viability of these cells. Although the genes investigated in this study (GSTP1 and MGMT) did not show a significant change in expression following in vitro exposure to BPA, the positive control ethinyl estradiol (EE2) caused an increase in GSTP1 expression at mRNA level. These results indicate that BPA does not affect the viability of prostate cells, and motivate a need for further research to identify other genes that could be affected by BPA.

Prostate cancer, one of the most common cancers among men, can be relatively harmless or extremely aggressive. The most widely used biomarker for the disease, the PSA test, is not independently diagnostic or prognostic of prostate cancer. One of the main challenges of prostate cancer research is to find reliable and effective prognostic biomarkers that will predict cancer recurrence following surgery, in order to identify clinically significant prostate cancer and improve management of the disease. In recent years, microRNAs (miRNAs) have been identified as master regulators of cellular processes, and dysregulated miRNAs have been associated with cancer development and progression. The intent of my PhD research program was to uncover novel miRNAs that contribute to prostate cancer pathogenesis in order to assess their potential as predictors of clinical progression. By analyzing a large cohort of primary prostate cancer samples, we have discovered that microRNA-221 (miR-221) is associated with metastasis and biochemical recurrence in prostate cancer, and is downregulated in TMPRSS2:ERG fusion gene- positive tumors. In addition, we have determined that microRNA-182 (miR-182) is overexpressed in prostate cancer and is associated with increased metastasis and clinical progression by targeting a tumors suppressor Forkhead box O1 (FOXO1). Overall, this work introduces novel candidate miRNA genes and downstream targets that are aberrantly expressed in more aggressive prostate cancer, and presents a potentially significant role for miRNAs as prognostic biomarkers that are associated with clinical progression, and perhaps aids in defining how miRNAs might one day serve as anti-cancer therapeutic agents.