External beam radiation therapy for prostate cancer can result in urinary, sexual, and rectal side effects, often impairing quality of life. A polyethylene glycol-based product, SpaceOAR hydrogel (SOH), implanted into the connective tissue between prostate gland and rectum can significantly reduce the dose received by the rectum and hence risk of rectal toxicity. The optimal way to manage the hydrogel and rectal structures for plan optimization is therefore of interest. A retrospective planning study was completed with 13 patients to examine optimal planning and treatment methods. Computerized tomography (CT) scans were taken pre- and post-SOH implant. Six hypofractionated (60 Gy in 20 fractions) treatment plans were produced per patient using either a structure of rectum plus the hydrogel, termed composite rectum wall (CRW), or rectal wall (RW) as the inverse optimization structure and intensity modulated radiotherapy (IMRT) or volumetric modulated arc therapy (VMAT) as the treatment technique. Dose-volume histogram metrics were compared between plans to determine which optimization structure and treatment technique offered the maximum rectal dose sparing. RW structures offered a statistically significant decrease in rectal dose over CRW structures, whereas the treatment technique (IMRT vs VMAT) did not significantly affect the rectal dose. However, there was improvement seen in bladder and penile bulb dose when VMAT was used as a treatment technique over IMRT. Overall, treatment plans using the RW optimization structure offered the lowest rectal dose while VMAT treatment technique offered the lowest bladder and penile bulb dose. These treatment techniques and optimization structures have now been implemented at BC Cancer - Victoria based on this retrospective study. SOH implant has been shown not to be equally effective in all patients. Determining a priori patients in which the implant will offer most benefit allows for effective management of SOH resources. Several factors have been shown to be correlated to reduction in rectal dose including distance between rectum and planning treatment volume (PTV), volume of rectum in the PTV and change in rectum volume pre- to post-SOH. Several of these factors along with other pre-SOH CT metrics were found via multiple linear regression models to predict reduction of rectal dose using data from 21 patients who received SOH implant. Two high rectal dose metrics were modeled, change in the relative volume receiving 55 Gy and change in the partial high dose integral, integrating over the dose-volume histogram (DVH) from 55 Gy to 60 Gy. Models were also produced to predict pre-SOH RV55Gy. These models offered R-squared between 0.57 and 0.87 with statistical significance in each model. Applying a 3.5% lower limit on pre-SOH RV55Gy removed one third of patients as implant candidates. This may offer a clinically useful tool in deciding which patients should receive SOH implant given limited resources. Predictive models, nomograms and a workflow diagram were produced for clinical management of SOH implant. / Graduate

Le récepteur du mannose 6-phosphate cation indépendant (RM6P-CI) permet l'endocytose puis le transfert de molécules porteuses du marqueur mannose 6-phosphate (M6P) vers les lysosomes. Le RM6P-CI, qui est surexprimé par les cellules de cancer de la prostate, est une cible de choix pour augmenter la délivrance de principes actifs dans ces cellules. Cependant le M6P est dégradé par les phosphatases présentes dans le sérum. Dans le but d'augmenter à la fois la stabilité et l'affinité pour le RM6P-CI, nous avons entrepris la synthèse d'analogues isostères du mannose 6-phosphate. Ces analogues sont fonctionnalisés en position anomère afin de permettre leur greffage sur des nanoparticules de silice mésoporeuse incorporant un photosensibilisateur. L'évaluation biologique a montré un gain d'affinité des nouveaux analogues synthétisés pour le RM6P-CI ainsi qu'une forte augmentation de l'efficacité des nanoparticules fonctionnalisées avec les analogues pour la thérapie photodynamique, in vitro, de cellules de cancer de la prostate. / The cation independant mannose 6-phosphate receptor (CI-M6PR) allows the endocytosis and the transport of mannose 6-phosphate (M6P) bearing molecules toward the lysosomes. The CI-M6PR, which is overexpressed by prostate cancer cells, is a target of interest to increase drug delivery in these cells. However, M6P is sensitive to degradation by phosphatases in the serum. To increase both the stability and the affinity for the CI-M6PR, we synthesized new M6P isosteric analogs. These analogs are functionnalized at the anomeric position to permit their grafting on mesoporous silica nanoparticles incorporating a photosensitizer. The biological evaluation demonstrated an affinity gain of the new analogs for the CI-M6PR as well as an increase of the efficacy of the nanoparticles functionnalized with these analogs for the in vitro photodynamic therapy of cancer prostate cells.

Philosophiae Doctor - PhD / 2018, the International Agency for Research on Cancer (IARC) estimated that prostate cancer (PCa) was the second leading cause of death in males worldwide. The number of deaths are expected to raise by 50 % in the next decade. This rise is attributed to the shortcomings of the current diagnostic, prognostic, and therapeutic biomarkers used in the management of the disease. Therefore, research into more sensitive, specific and effective biomarkers is a requirement. The use of biomarkers in PCa diagnosis and management takes advantage of the genetic alterations and abnormalities that characterise the disease. In this regard, a microRNA, hsa-miR-5698 was identified in a previous study as a differentiating biomarker between prostate adenocarcinoma and bone metastasis. Six putative translational targets (CDKN1A, CTNND1, FOXC1, LRP8, ELK1 and BIRC2) of this microRNA were discovered using in silico approaches. The aim of this study was to analyse via expression profiling and characterization, the target genes of hsa-miR-5698 in order to determine their ability to act as putative dynamic network biomarkers for PCa. The study was conducted using a combined in silico and molecular approach. The in silico part of the study investigated the putative transcriptional effects of hsa-miR-5698 on the promotors of its translational targets, the correlation between hsa-miR-5698 and mRNA expression profiles as well as the co-expression analysis, pathway analysis and prognostic ability of the target genes. A number of computational software were employed for these purposes, including, R Studio, Trident algorithm, STRING, KEGG, MEME Suite, SurvExpress and ProGgene. The molecular part of the study involved expression profiling of the genes in two PCa cell line LNCaP and PC3 via qPCR.

<p>The aims of the thesis were to</p><p>• explore whether radical prostatectomy is beneficial compared with watchful waiting in survival and disease progression</p><p>• find possible effect modifiers</p><p>• evaluate a protocol of multiple biopsies and investigate if men with previous benign prostate biopsies are a group at risk for later prostate cancer</p><p>• inquire into patients’ and clinicians’ experiences of randomization in order to find out what made this study possible to conduct, and thereby contribute to improve randomization in the future</p><p>The background material was a large randomized clinical trial, the Scandinavian Prostatic Cancer Group Study Number 4, or SPCG-4, which was open for inclusion from February 1989 through December 1999. It comprised 695 men in Sweden, Finland and Iceland who had localized prostate cancer and were randomized to either radical prostatectomy or watchful waiting. </p><p>After a mean follow-up time of 6.2 years the first analyses, according to intention-to-treat, showed that radical prostatectomy reduced disease specific mortality, risk of metastases and risk of local progression but did not statistically significantly reduce overall mortality. </p><p>The second analyses confirmed our earlier findings and furthermore, at ten years, radical prostatectomy also statistically significantly reduced overall mortality. Age appeared as an independent effect modifier that will be further investigated.</p><p>A total of 547 men, with a suspicion of prostate cancer that had undergone multiple biopsies, and whose biopsies had benign histology were later compared with the background population to evaluate whether they were a group at risk of developing prostate cancer. Within six years of follow-up, there was no increased risk of prostate cancer.</p><p>Patients as well as clinicians used individual strategies to cope with the situation. The randomizing clinician has to understand the patient’s strategy and his expectations in order to individualize the information accordingly.</p>

<p>Prostasomes are submicron-sized, membrane-bound organelles produced by the epithelial cells of the prostate and normally found in the secretion in the gland ducts. Their physiological role is in the promotion of sperm-function in human reproduction. This thesis contains four papers dealing with the production of prostasomes and some possible applications in clinical urology of the prostasome. </p><p>Paper I and II provided an ultrastructural description of the synthesis, storage and secretion of prostasomes in benign as well as in malignant tissue. Most notable were the extracellular appearances of prostasomes in metastatic lesions whereby the prostasomes become exposed to the immune system of the patient. This supported findings in earlier studies in which patients with advanced prostate cancer had elevated levels of anti-prostasome antibodies. The results of paper III reinforced the view of the prostate-unique origin of the prostasome. In particular, there were no indications in SDS-PAGE patterns or flow-cytometric studies of material from seminal vesicle secretion that it contained components that could be associated with a production of prostasomes. </p><p>Some possible clinical functions of the prostasomes were investigated in paper IV. Exposure of prostasomes to the immune system through mechanical and thermal trauma to the prostate did not induce an evident formation of anti-prostasome autoantibodies. Furthermore, the serum levels of anti-prostasome antibodies registered by assays with preparations of prostasomes from seminal plasma as antigen did not correlate with existing prostate cancer. Seminal prostasomes seemed not to function as substitute markers for prostate cancer in the test kit used. A possible explanation could be underestimated differences in antigen properties between seminal or prostate gland-derived prostasomes and prostasomes from tumor tissue.</p>

1,25-(OH)2 vitamin D3 inhibits cell proliferation of a variety of cancers including prostate. In the human prostate cancer cell line LNCaP, 1,25-(OH)2 vitamin D3-mediated growth inhibition is attributed to cell cycle G1 accumulation which correlates with a robust decrease of cyclin-dependent kinase 2 (CDK2) activity and pronounced relocalization of CDK2 into the cytoplasm. Nuclear targeting CDK2 blocks the 1,25-(OH)2 vitamin D3-mediated growth inhibition and cell cycle G1 accumulation. Further, the nuclear targeted CDK2 blocks 1,25-(OH)2 vitamin D3-mediated inhibition of CDK2 activity and nuclear exclusion in LNCaP cells. Therefore, CDK2 cytoplasmic relocalization is the key mechanism for 1,25-(OH)2 vitamin D3 effects. Since cyclin E is important for CDK2 nuclear localization and activation, 1,25-(OH)2 vitamin D3 may exert its effects through regulation of cyclin E. Cyclin E but not a cyclin E mutant deficient in CDK2 binding reverses 1,25-(OH)2 vitamin D3-mediated antiproliferation which suggests the involvement of cyclin E as a mechanism. However, the studies showed no effects of 1,25-(OH)2 vitamin D3 on cyclin E levels, intracellular localization or binding to CDK2. In order to develop a model for studying 1,25-(OH)2 vitamin D3-mediated antiproliferative effects, LNCaP vitD.R cell line, a vitamin D resistant LNCaP derivative, was generated by continuously culturing of LNCaP cells in medium supplemented with 10 nM 1,25-(OH)2 vitamin D3 for over 9 months. The initial characterization of this cell line showed complete resistance to 1,25-(OH)2 vitamin D3-mediated effects. Analysis of vitamin D regulation of VDR target gene expression revealed that vitamin D resistance in LNCaP vitD.R cells was not due to deregulation of VDR signaling. HDAC inhibitor Trichostatin A (TSA) did not confer sensitivity of LNCaP vitD.R cells to vitamin D treatment suggested the resistance to 1,25(OH)2 vitamin D3 effect of LNCaP vitD.R cells is not due to histone deacetylase remodeling of the chromatin structure which leads to inhibition of gene transcription. While the partial sensitization of LNCaP vitD.R cells to 1,25(OH)2 vitamin D3 effect by demethylation reagent 5-Aza-2¡¯-deoxycytidine treatment suggested a set of genes involved in 1,25(OH)2 vitamin D3-mediated antiproliferative effects is silenced via hypermethylation in LNCaP vitD.R cells. These results suggested LNCaP vitD. R cell line is a useful tool and further studies to elucidate the genes involved in this effect will help uncover the mechanisms of 1,25(OH)2 vitamin D3-mediated antiproliferative effects.

Most patients undergoing androgen deprivation therapy relapse eventually and progress to androgen-independent (AI) prostate cancer. Although the mechanisms underlying progression to AI prostate cancer are not well understood, studies suggest that androgen receptor (AR) is still required for AI prostate cancer. Our lab found that Vav3, a Rho GTPase guanine nucleotide exchange factor (GEF), is up-regulated during the progression of androgen-dependent human prostate cancer cells to androgen-independence in vivo and in cell-based experiments. Since Vav3 significantly increases ligand-dependent AR transcriptional activity and this action requires the Vav3 pleckstrin homology (PH) domain but not Vav3 GEF activity, we explored the role of the Vav3 PH domain in ligand-dependent AR coactivation by Vav3. We found that targeting the Vav3 PH mutant into nuclei but not the plasma membrane restored Vav3 PH mutant in AR coactivation. Targeting Vav3 to the plasma membrane eliminated the capacity of Vav3 to coactivate AR. In agreement with nuclear targeting of Vav3 via its PH domain, chromatin immunoprecipitation assays showed that Vav3 enhancement of AR transcriptional activity was accompanied by Vav3 recruitment to AR transcriptional complexes at an AR target gene enhancer. Further, Vav3 increased AR occupancy at the target gene enhancer upon androgen treatment and this may underlie the capacity of Vav3 to enhance AR transcriptional activity. Because Vav3 can also be activated by growth factors (GFs) and GFs activate AR in the absence of androgen (ligand-independent), we investigated the crosstalk between Vav3 and GF activation of AR and found Vav3 strongly enhanced AR transcriptional activity induced by GFs. GEF function and the downstream Rho GTPase, Rac1 were required for constitutively active (Ca) Vav3 activation of AR, which differs from Vav3 activation of AR in the presence of androgen. We also investigated the possible signal pathways contributing to AR activation by Ca Rac1. Ca Rac1 caused ligand-independent activation of AR in part through MAPK/ERK signaling and conferred prostate cancer growth in the absence of androgen in cell culture, soft agar and mouse tumor xenografts. Thus, our findings indicate that Vav3 activates AR in the presence or absence of ligand through two distinct mechanisms, which supports a versatile regulatory effect of Vav3 in AR signaling and prostate cancer progression.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the most active metabolite of vitamin D, inhibits the proliferation of a variety of cell types including adenocarcinoma of the prostate. The primary mechanism for the antiproliferative effects of 1,25-(OH)2D3 in prostate cancer cells is inhibition of G1 to S phase cell cycle progression. While 1,25-(OH)2D3-mediated growth inhibition requires the vitamin D receptor (VDR), a ligand activated transcription factor, expression of functional VDR is not sufficient. To define target genes that might participate in the antiproliferative actions of 1,25-(OH)2D3, we developed a derivative of the human prostate cancer cell line, LNCaP, which retains transcriptionally active VDRs but unlike parental LNCaP cells, is not growth inhibited by 1,25-(OH)2D3. Gene expression profiling of these resistant cells (termed VitD.R) compared to control LNCaP cells revealed two novel 1,25-(OH)2D3-inducible genes, GADD45G and MIG6. The expression of GADD45G and MIG6 genes was induced by 1,25-(OH)2D3 in LNCaP but not in the resistant VitD.R or in ALVA31 cells, human prostate cancer cells that exhibit natural resistance to growth inhibition by 1,25-(OH)2D3 despite expression of functional VDR. Ectopic expression of GADD45G but not MIG6 in either LNCaP or ALVA31 cells resulted in accumulation of cells in G1 and inhibition of proliferation equal to or greater than that caused by 1,25-(OH)2D3 treatment. While GADD45G is induced by androgens in prostate cancer cells, up-regulation of GADD45G by 1,25-(OH)2D3 was not dependent on androgen receptor signaling further refuting a requirement for androgens/androgen receptor in vitamin D-mediated growth inhibition in prostate cancer cells. These data introduce two novel 1,25-(OH)2D3-regulated genes and establish GADD45G as a growth inhibitory protein in prostate cancer. Further, defects in vitamin D-mediated induction of GADD45G may underlie vitamin D resistance in prostate cancer cells. We previously demonstrated that the antiproliferative actions of 1,25-(OH)2D3 in prostate cancer cells are associated with decreased CDK2 activity and increased stability of the cyclin dependent kinase inhibitor (CKI) p27KIP1. We defined a novel mechanism that may underlie these antiproliferative effects, 1,25-(OH)2D3 -mediated cytoplasmic relocalization of CDK2, which would provide a unifying mechanism for the observed effects of 1,25-(OH)2D3 on CDK2 and p27. In the present study, we investigated the role of CDK2 cytoplasmic relocalization in the antiproliferative effects of 1,25-(OH)2D3. CDK2 was found to be necessary for prostate cancer cell proliferation. In contrast, while p27KIP1 is induced by 1,25-(OH)2D3, this CKI was completely dispensable for 1,25-(OH)2D3-mediated growth inhibition. Reduction of CDK2 activity by 1,25-(OH)2D3 was associated with decreased T160 phosphorylation, a residue whose phosphorylation in the nucleus is essential for CDK2 activity. Since cyclin E is important for nuclear translocation of CDK2, we investigated cyclin E effects on 1,25D-mediated growth inhibition. Ectopic expression of cyclin E blocked 1,25-(OH)2D3-mediated cytoplasmic relocalization of CDK2 and all antiproliferative effects of 1,25-(OH)2D3, yet endogenous levels of cyclin E or binding to CDK2 were not affected by 1,25-(OH)2D3. Similarly, knockdown of the CDK2 substrate retinoblastoma (Rb), which causes cyclin E up-regulation, resulted in resistance to 1,25-(OH)2D3 mediated growth inhibition. VitD.R cells did not exhibit 1,25-(OH)2D3-mediated cytoplasmic relocalization of CDK2. Importantly, targeting CDK2 to the nucleus of LNCaP cells blocked G1 accumulation and growth inhibition by 1,25-(OH)2D3. These data establish central roles for CDK2 nuclear-cytoplasmic trafficking and uncoupling of VDR in the regulation of antiproliferative target genes in the mechanisms of 1,25-(OH)2D3-mediated growth inhibition in prostate cancer cells. Since 1,25-(OH)2D3 continues to be evaluated for its chemotherapeutic and chemopreventative potential, elucidating the mechanism of 1,25-(OH)2D3 antiproliferative effects is critical in the determination of 1,25-(OH)2D3 responsiveness and the design of individualized treatment strategies.

Advanced prostate cancer (PrC) is the second leading cause of death from cancer in US males. Advanced PrC cells are initially androgen-sensitive and thus androgen ablation therapy causes tumors to undergo regression and fall into a remission phase where residual cells remain dormant while androgen levels remain very low. Unfortunately, this phase usually lasts 3 to 5 years prior to tumor relapse, where the tumor cells re-grow in the absence of androgens. This form of the disease is aggressive and invariably fatal. In this study, we investigated the effects of a combination of herbal extracts on various stages of PrC, androgen-dependent and castrate-resistant, using CWR22 and CWR22Rv1 cells, respectively. Zyflamend, a commercially available product consisting of 10 different herbal extracts, had been shown to reduce pre-malignant forms of PrC in clinical trials. We expanded these earlier experiments by using Zyflamend in a model of advanced PrC. Our initial results indicated that Zyflamend could repress androgen-sensitive and castrate-resistant (androgen-insensitive) prostate tumor growth. Using a cell model for castrate-resistant PrC, Zyflamend inhibited the growth of CWR22Rv1 cells by increasing the expression of the cell cycle inhibitors p21 and p27. These effects were mediated via hyperacetylation of histone 3 through the suppression of class I and II histone deacetylases (HDACs) and an induction of CBP/p300 histone acetyl transferase activity. The latter effect was mediated by the upregulation/activation of Erk-1/-2 and Elk-1. Zyflamend also inhibited androgen receptor expression, its downstream gene target, prostate specific antigen (PSA), and increased cell death by inducing apoptosis as indicated by caspase 3 activity and PARP cleavage. The reduction of androgen receptor was confirmed in CWR22Rv1 xenograft tissues. Our results suggest the extracts of this herbal combination inhibits castrate-resistant prostate cancer cell growth epigenetically and by coordinately affecting androgen receptor signaling pathways involved in cell growth/death. In an androgen-dependent PrC tumor xenograft model, Zyflamend reduced the growth of CWR22-derived tumors and enhanced the responsiveness of tumor cells to hormone ablation. Zyflamend potentiated the regression of PrC cells and their sensitivity to androgen deprivation. These results suggest that Zyflamend may be an effective adjuvant when used with hormone ablation therapy.

The aims of the thesis were to • explore whether radical prostatectomy is beneficial compared with watchful waiting in survival and disease progression • find possible effect modifiers • evaluate a protocol of multiple biopsies and investigate if men with previous benign prostate biopsies are a group at risk for later prostate cancer • inquire into patients’ and clinicians’ experiences of randomization in order to find out what made this study possible to conduct, and thereby contribute to improve randomization in the future The background material was a large randomized clinical trial, the Scandinavian Prostatic Cancer Group Study Number 4, or SPCG-4, which was open for inclusion from February 1989 through December 1999. It comprised 695 men in Sweden, Finland and Iceland who had localized prostate cancer and were randomized to either radical prostatectomy or watchful waiting. After a mean follow-up time of 6.2 years the first analyses, according to intention-to-treat, showed that radical prostatectomy reduced disease specific mortality, risk of metastases and risk of local progression but did not statistically significantly reduce overall mortality. The second analyses confirmed our earlier findings and furthermore, at ten years, radical prostatectomy also statistically significantly reduced overall mortality. Age appeared as an independent effect modifier that will be further investigated. A total of 547 men, with a suspicion of prostate cancer that had undergone multiple biopsies, and whose biopsies had benign histology were later compared with the background population to evaluate whether they were a group at risk of developing prostate cancer. Within six years of follow-up, there was no increased risk of prostate cancer. Patients as well as clinicians used individual strategies to cope with the situation. The randomizing clinician has to understand the patient’s strategy and his expectations in order to individualize the information accordingly.