Aberrant DNA methylation in gene promoters causes gene silencing and is a common event in prostate cancer development and progression. While commonly identified methylated genes have been analyzed for their potential clinical utility in a variety of cancers, few studies have attempted a genome-wide methylation approach to discover new and possibly improved biomarkers for prostate cancer. In order to identify DNA methylation changes associated with aggressive prostate cancer, we performed a genome-wide analysis of 40 prostate cancers using Agilent human CpG island microarrays. Methylation profiles of candidate genes were validated using quantitative MethyLight technology in an independent series of 219 radical prostatectomies and compared to clinicopathological parameters. The effects of methylation on expression of HOXD3 and HOXD8 and the possible role of HOXD8 in progression of PCa were also investigated. We discovered previously unidentified methylation in the HOXD cluster of genes, namely HOXD3 and HOXD8, as well as TGFβ2 and GENE X as potential prognostic biomarkers. Furthermore, unsupervised clustering of samples by methylation signature indicated ERG oncogene expression as significantly different between clusters. Within the independent cohort, we observed strong correlations between Gleason score (GS) and HOXD3 as well as GENE X, while HOXD3 and HOXD8 methylation were associated with ERG expresson. TGFβ2 was an independent predictor of disease recurrence using Cox multivariate regression analysis. In gene expression studies, both HOXD3 and HOXD8 were elevated in cancers with poor prognosis, while DNA methylation did not correlate with expression levels. Both genes were found to contain alternative transcription start sites, explaining the poor correlation between methylation and expression. Finally, knockdown of HOXD8 expression did not have any effect on viable cells or cell motility in an in vitro model. These results indicate that a panel of novel DNA methylation markers distinguish indolent prostate cancers from aggressive ones, and that expression of HOXD3 and HOXD8 is regulated by mechanisms including, but not dependent on, DNA methylation.

Prostate cancer is prevalent among men and one of the few cancer sites where local therapies currently target the entire organ instead of tumour. MRI holds promise in accurately depicting regions of cancer burden within the prostate gland and guiding tumour-targeted diagnostics and therapeutics. The clinical performance of a novel stereotactic MRI-guided needle navigation system for prostate cancer targeting was evaluated. Mean absolute in-plane stereotactic needle-targeting error for 10 patients was 2.2 mm and mean absolute depth error was 6.5 mm, highlighting a need to improve technical accuracy of the system. Consequently, alternative stereotactic registration techniques were investigated. Metrics of performance were in-plane stereotactic needle-targeting error, depth error, and registration time. A Z-shaped fiducial motif using automated registration performed best in phantom experiments with an in-plane error of 2.0 mm and depth error of 1.0 mm. These results will guide further software and hardware development to improve clinical performance.

Interstitial laser thermal therapy is a novel local approach to treating prostate cancer. During treatment, thermal ablation may occur on the adjacent rectal wall. The aim of this thesis was therefore twofold: to study the effects of rectal cooling on lesion formation, and secondly, to engineer a computer controlled rectal cooling unit. To study the effects of the coolant temperatures and flow rate, thermal simulations were executed, followed by testing the phenomenon using agar gel phantoms which thermally mimic prostate tissue. Further simulations were run using a treatment planning software, which predicted the required coolant temperatures to protect the outer rectal wall while subsequently determining the shape and size of the resulting coagulated lesion at various laser settings. Results suggest that low coolant temperatures and low flow rates cause maximum cooling rates. Furthermore, the shape and size of the coagulated region is affected by coolant temperatures at specific laser powers and positions within the prostate.

Prostate cancer is prevalent among men and one of the few cancer sites where local therapies currently target the entire organ instead of tumour. MRI holds promise in accurately depicting regions of cancer burden within the prostate gland and guiding tumour-targeted diagnostics and therapeutics. The clinical performance of a novel stereotactic MRI-guided needle navigation system for prostate cancer targeting was evaluated. Mean absolute in-plane stereotactic needle-targeting error for 10 patients was 2.2 mm and mean absolute depth error was 6.5 mm, highlighting a need to improve technical accuracy of the system. Consequently, alternative stereotactic registration techniques were investigated. Metrics of performance were in-plane stereotactic needle-targeting error, depth error, and registration time. A Z-shaped fiducial motif using automated registration performed best in phantom experiments with an in-plane error of 2.0 mm and depth error of 1.0 mm. These results will guide further software and hardware development to improve clinical performance.

Interstitial laser thermal therapy is a novel local approach to treating prostate cancer. During treatment, thermal ablation may occur on the adjacent rectal wall. The aim of this thesis was therefore twofold: to study the effects of rectal cooling on lesion formation, and secondly, to engineer a computer controlled rectal cooling unit. To study the effects of the coolant temperatures and flow rate, thermal simulations were executed, followed by testing the phenomenon using agar gel phantoms which thermally mimic prostate tissue. Further simulations were run using a treatment planning software, which predicted the required coolant temperatures to protect the outer rectal wall while subsequently determining the shape and size of the resulting coagulated lesion at various laser settings. Results suggest that low coolant temperatures and low flow rates cause maximum cooling rates. Furthermore, the shape and size of the coagulated region is affected by coolant temperatures at specific laser powers and positions within the prostate.

Molecular Imaging provides new aspects in cancer diagnosis and treatment. With the ap-plication of imaging and biological techniques, molecular imaging can monitor molecular and cellular changes of different diseases. To interpret the mechanism of disease, more and more at-tention is focused on the development of new probes for molecular imaging. Magnetic resonance imaging (MRI) is a powerful, non-invasive clinical diagnostic tool with high spatial resolution without the limitation of the depth of tissues. Applications of MRI contrast agents can amply the MRI signal during imaging. Many studies have been devoted to developing targeted MR contrast agents. Proteins and peptides have been widely used for target-ing cancer cells in cancer diagnosis and treatments. GRP, gastrin-releasing peptide, is one of a well-characterized group of mammalian bombesin-like peptides. GRP acts through its cell surface receptors, GRP receptor (GRPR). It has been reported that there is a high density of GRP receptors in the majority of prostate carci-noma. In contrast, the GRPRs are not highly expressed in normal cells of most tissues. Thus, this tumor specific expression pattern provides an advantage for cancer targeting. A novel class of MRI contrast agent was designed by adding the Gd3+ binding sites into a stable host protein, the domain 1 of rat CD2. Due to the unique features of the designed metal binding properties, the protein contrast agent (ProCA1) exhibits more than 10-fold enhanced MRI relaxivity compared to that of the more commonly used Gd-DTPA. The high relaxivity of the designed protein contrast agent largely overcomes the major barrier of low sensitivity of MRI techniques. A peptide of ten amino acids from the C-terminal of GRP was grafted onto ProCA1. GRP-grafted protein contrast agents (ProCA1.GRPs) showed the targeting capability to the GRPRs which are over-expressed on prostate cancer cells. Cell MRI Imaging demonstrated that ProCA1.GRP(52) grafted between Lys51 and Ser52 had better targeting capability than C-terminal one. Administration of ProCA1.GRP into xenograft tumor model enhances the contrast in the GRPR+ prostate tumor under MRI and optical imaging. Study demonstrated a potential application for disease marker targeted MR imaging by using our developed protein contrast agent.

No description available.

As the most frequently diagnosed cancer in North American men, prostate cancer can progress to the androgen independent stage after initial response to androgen ablation therapy. The molecular mechanisms involved in the hormonal progression of prostate cancer are not completely understood. Here, we analyze changes in the transcriptome of prostate cancer cells at different stages of progression to reveal potential mechanisms. Applying Affymetrix GeneChip technology, we identified the transcriptomes in response to stimulation of androgen and PKA pathways in human prostate cancer cells. In addition to PSA, other common target genes were identified. Genes differentially expressed in response to androgen and stimulation of the PKA pathway in vitro were also differentially expressed during hormonal progression in vivo. Upon androgen stimulation, androgen receptor binds to a functional androgen response element within the promoter region of SESN1, a p53 targeted gene, and represses its expression. The expression of SESN1 was induced by castration in LNCaP xenografts, but the expression was eventually suppressed again in the androgen independent stage of prostate cancer. Knockdown of SESN1 promoted the proliferation of prostate cancer cells. Expression patterns of androgen-regulated genes in androgen independent tumours were revealed to be more similar to that from before castration than to the tumors under androgen ablation. The β-catenin, a potent coactivator of the androgen receptor, and Wnt pathway was deregulated in androgen-independent tumours. There was increased nuclear colocalization and interaction of androgen receptor and β-catenin with hormonal progression of prostate cancer. This study provides insight into hormonal effects on prostate cancer and possible pathways involved in the development of androgen independent disease, as well as potential therapeutic targets.

The mechanism by which prostate cancer progresses from androgen dependence (AD) to androgen independence/castration resistance (AI/CR) is currently a major focus of prostate cancer-related research. Prostate cancers that progress to a state of AI/CR are typically resistant to most standard types of treatments. Due to its primary role in driving normal prostate cell growth and proliferation, the androgen receptor (AR) is believed to play a key role in progression. Coregulators, or any proteins which may either enhance or abrogate AR activity, are considered to be one of the potential mechanisms by which AR function may become impaired. Cyclin G-associated kinase (GAK) was initially identified as a potential coregulator of AR in a Tup 1 repressed transactivation system. A LNCaP cDNA library was screened for proteins which interacted with the NH2-terminus of AR. GAK was isolated from three independent library clones using two different AR baits (AR 1-549 and AR 1-646). This interaction was confirmed via GST pulldown and coimmunoprecipitation experiments, and preliminary luciferase assays suggested that GAK activates AR in a hormone dependent manner. In this study, my objectives were to validate GAK’s role as a coregulator of AR and to determine if overexpressing GAK affects progression to AI. In vitro luciferase assays whereby GAK was either overexpressed or knocked down in both LNCaP and PC3 cells did not significantly affect AR activity. Xenograft experiments utilizing a doxycycline (DOX) inducible lentiviral LNCaP-GAK overexpressing stable cell line demonstrated that while GAK may not play a significant role in modulating AR activity, it may adopt a more subtle role enhancing tumour take and tumour volume growth rate in vivo. While these results could not confirm GAK to be a direct coregulator of AR, it is entirely possible that GAK may influence prostate cancer progression, albeit indirectly. Recent publications report a growing amount of evidence suggesting GAK’s involvement in the critical cellular process of clathrin coated vesicle endocytosis, the dysregulation of which could potentially indirectly affect AR regulated genes.

Les métalloprotéinases matricielles (MMPs) jouent un rôle crucial dans le développement malin des tumeurs. Elles dégradent la matrice extracellulaire (MEC) permettant ainsi la migration des cellules cancéreuses vers d'autres foyers. Ce processus d'invasion tumorale est appelé "processus métastasique". La MMP-9, appelée aussi gélatinase B, est fortement exprimée lors de ce processus. Son expression peut être régulée à différents niveaux via des voies de signalisation intracellulaires, notamment au niveau de l'expression du gène, mais aussi lors de la transcription, la stabilité de son ARNm, la traduction ou la sécrétion de la protéine. Des agents anti-tumoraux sont en cours de développement ciblant les différentes étapes du développement cancéreux. Le PCK3145, un peptide synthétique, dérivé de la PSP94, dirigé contre le cancer de la prostate avancé et métastasé est au stade d'essais cliniques en phase II. Ce peptide réduit chez la souris immunodéficiente la croissance des xénogreffes de tumeurs prostatiques humaines en diminuant les concentrations plasmatiques de la MMP-9. Nos travaux visent à élucider in vitro les mécanismes moléculaires impliqués dans la régulation des fonctions de la MMP-9 via l'effet anti-métastasique du PCK3145. Dans un premier temps, nous avons étudié l'interaction de la MMP-9 avec la surface de la cellule cancéreuse. Nous démontrons que le PCK3145 inhibe la sécrétion de la MMP-9 et déclenche le processus de clivage de CD44 de la surface cellulaire via une cascade de signalisation intracellulaire impliquant la GTPase RhoA. Dans un second lieu, nous avons étudié le mécanisme d'action du PCK3145 menant à l'inhibition de la sécrétion de la MMP-9. Nous démontrons que cette inhibition requiert le récepteur de la laminine à 67 kDa et est dépendante de l'activation de la voie des MAPKinases. Le PCK3145 entraine la diminution de la sécrétion de la MMP-9 via HuR, une protéine intracytoplasmique qui stabilise l'ARNm de la MMP-9 en se liant à sa séquence riche en AU. Les propriétés anti-métastasiques du PCK3145 peuvent être dirigées contre d'autres types de tumeurs, autres que le cancer de la prostate, liés à l'augmentation de la MMP-9 et à la dégradation de la MEC lors d'un processus métastasique. Par ailleurs, l'identification du mode d'action du PCK3145 via le récepteur de la laminine à 67 kDa permet de cibler des cancers dont le taux de ce récepteur est particulièrement élevé telle que la leucémie. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Cancer de la prostate, Métastase, Migration, Adhésion, MMP-9, HuR, ERK1/2, RhoA, CD44.