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Accounting for 14% of all new cancer diagnosis in the United States, prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer related death in the United States. Prognosis for patients diagnosed with metastatic disease is especially poor, since no effective treatments have been developed (1). In this study, we examined the expression and function of POU5F1B, a protein-encoding pseudogene of the homeodomain transcription factor Oct4, in prostate cancer. POU5F1B is located at 8q24, a "gene desert" containing numerous alleles associated with prostate cancer risk. A recent study has indicated that a number of these risk alleles are correlated with POU5F1B expression and prostate cancer susceptibility. The role of POU5F1B in prostate cancer carcinogenesis and progression, however, is not known. In our study, we found that POU5F1B expression is upregulated in prostate cancers and highly overexpressed by high grade (Gleason ≥8) and metastatic prostate cancers. We cloned POU5F1B from prostate cancer cell lines, which contains prostate cancer risk associated SNPs, including a missense mutation inside the homeobox DNA binding domain, to study the functional effects of POU5F1B overexpression in prostate cancers. Here, we report that POU5F1B from prostate tumor encodes functional proteins that exhibit gene transactivation activity comparable to its parent gene, Oct4. Further, we report that POU5F1B overexpression in prostate cancer cell lines increases prostate cancer cell proliferation, migration, anchorage independent growth, and drug resistance in vitro and tumor xenograft growth in vivo. Conversely, shRNA mediated knockdown of endogenous POU5F1B expression in prostate cancer cells inhibit cell proliferation in vitro and tumor growth in vivo, as well as prolong tumor free survival in animal models. The data provide compelling evidence that POU5F1B is an important mediator of prostate cancer progression. We further examined the molecular mechanism behind POU5F1B driven prostate cancer progression. Our studies found that POU5F1B overexpression suppresses E-Cadherin expression at both mRNA and protein levels. Our studies further found POU5F1B overexpression in prostate cancer cells increases Wnt1, TCF1, and TCF4 expression, as well as increased Wnt/β-Catenin signaling - indicating the induction of epithelial-to-mesenchymal transition (EMT) in POU5F1B overexpressing cells(2). Consistently, qPCR analysis found that POU5F1B overexpression significantly increased the expressions of numerous EMT related genes and prostate cancer stem cell markers. Functional studies further confirmed that the transactivation activity of Nanog, another stem cell related transcription factor, is dramatically increased in POU5F1B overexpressing cells. Taken together, our data strongly suggests that POU5F1B overexpression drives prostate cancer progression through the induction of EMT and conferment of stem-cell properties to tumor cells. In summary, our data demonstrated that POU5F1B is overexpressed in prostate tumors, especially high-grade and metastatic tumors, and is a functional driver of prostate cancer progression by inducing EMT in prostate cancer cells. Our study also showed that POU5F1B can potentially be targeted to treat prostate cancer. Based on our findings, depletion of POU5F1B may reduce the risk of metastatic disease or tumor recurrence when used with concurrent therapies in early state tumors and may attenuate treatment resistance in diseases at advanced stages.

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Affecting one in every eight Canadian men, prostate cancer is the most common type of cancer among males. As with other forms of cancer, men with prostate cancer are much more likely to develop comorbid depression than the general population without cancer diagnoses. Depression negatively affects these men’s quality of life and increases mortality rates among cancer patients. Therefore, effective therapies to manage depression in this unique subpopulation are needed. This project sets out to assess the efficacy of glutamate-targeting drugs as antidepressants. The major excitatory neurotransmitter in the central nervous system, glutamate is released in excessive quantities by cancer cells. It is thought that this abundance of glutamate leads to excitotoxicity and neurodegeneration, affecting neurons in important regions of the brain relating to mood and mood regulation. A validated mouse model of depression was established using RM1 murine prostate cancer cells. This model was then used to test the properties of three drugs: sulfasalazine (SSZ), (S)-4-carboxyphenylglycine ((S)-4-CPG), and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydro-benzo[f]quinoxaline-7-sulfonamide (NBQX). Results show that these drugs were able to improve depressive-like behaviours and symptoms to varying degrees, at least partially reversing the negative effects of tumours. This project showed that disrupting glutamate release and/or signaling could be an effective approach for an antidepressant therapy or adjuvant in prostate cancer patients. / Thesis / Master of Science (MSc) / Prostate cancer affects one in every eight Canadian men. Cancer patients are at a much higher risk of developing depression than the rest of the population. Unfortunately, current antidepressants are limited in their ability to improve depressive symptoms in cancer patients. Therefore, this project sets out to identify new options for treating depression in prostate cancer patients. Glutamate is a signalling molecule that is released in abundance by cancer cells and is largely responsible for communication between neurons in the central nervous system. This project showed that limiting the amount of glutamate released by cancer cells and limiting glutamate-based signaling improves depressive-like symptoms in mice with prostate cancer tumours. These results suggest that targeting glutamate could be an effective antidepressant therapy in the cancer population.

Patients with prostate cancer (PC) are at high risk of developing migratory thrombosis compared to healthy individuals. This is due to the haemostatic abnormality as a result of the presence of cancer, and is referred to as Trousseau’s syndrome. Trousseau's syndrome leads to increased mortality among cancer patients, and is considered the second cause of death after cancer itself. We investigated the role of microvesicles (MVs), which are circular membrane compartments shed from cancer as well as from healthy cells, in the development of Trousseau’s syndrome. We compared the pro-coagulant activities between MVs derived from PC cell lines with different oncogenic and metastatic characteristics, using chromogenic assays to determine their thrombin generation. Microvesicles from the more aggressive DU145vIII and more metastatic PC3-MLN4 show increased thrombin generation compared to MVs derived from DU145 and PC3. We also compared thrombin generation in MVs extracted from plasma of PC patients of various cancer stages. MVs from PC patients with a metastasized tumour had increased thrombin generation compared to patients with localized tumours. Finally, we transfected the CHO cell line with the human protease-activated receptor 1 (hPAR1), the principal receptor of thrombin. PC MVs led to the activation of PAR1 in CHO (hPAR1), indicating thrombin generation. Our in vitro studies suggest a potential role of PC MVs in the migratory thrombosis observed in Trousseau’s syndrome, due to their independent ability to generate active thrombin. We also demonstrated that thrombin generation of PC-derived MVs correlated with the oncogenic and metastatic characteristics of prostate cancer. / Thesis / Master of Science (MSc)

Aim: Several studies have tried to quantify overdiagnosis of prostate cancer with Prostate-specific antigen(PSA) screening, but estimates vary widely. This study aims to evaluate the degree of overdiagnosis of prostate cancer with 10 or 14 follow-up years after the stop of screening in Finland. Methods: We selected 80379 men aged 55-69 years who were randomized to a screening or a control arm, distinguishing four birth cohorts: 1941-44,1937-40, 1933-36 and 1929-32. The first PSA screening test occurred during1996-1999. Men without detected as prostate cancer in the previous screening would be invited to the next screening 4 years later. The estimate of overdiagnosis is the ratio of the cumulative excess incidence to the cumulative incidence of prostate cancer in the screened group after the year-specific incidence became stable. Results: The patterns of all incidences in these four cohorts have not become stable yet, and the difference of cumulative incidence in the current longest follow- up years is the best estimate of overdiagnosis so far. Conclusion: Overdiagnosis rates of prostate cancer in people who received screening in Finland was estimated as 2.27%,15.4%, 11.4%, and 10.2% for 1929-32, 1933-36,1937-40, and 1941-44 cohorts, respectively. / Thesis / Master of Science (MSc)

Yes / The aim of this study was to assess the relationship between pre-prostatectomy urinary Engrailed-2 (EN2), a transcription factor secreted by prostate cancer cells, with tumour volume and pathological characteristics in resected prostate specimens. First pass urine samples (10 ml) without prior prostatic massage were collected and stored at –80°C. EN2 levels were measured using an enzyme-linked immunoabsorbent assay. Tumour volume in the prostatectomy specimens was determined histologically. 57 men undergoing RP in one urological cancer network were evaluated. EN2 was detected in 85% of RP patients. EN2 correlated with tumour volume (but not total prostatic volume) in a linear regression analysis, with increasing pathological T stage and margin positivity. Using three “cutoff levels” of tumour volume (0.5 ml, 1.3 ml and 2.5 ml) to define “significant disease”, men with “significant disease” had markedly higher levels of urinary EN2 (p < 0.001 for each cut off level). Levels of urinary EN2 may be useful in predicting tumour volume in men with prostate cancer by potentially identifying men with small volume “insignificant” disease. This study justifies a larger multicentre evaluation of urinary EN2 levels as a biomarker of PC significance using cancer volume, pathological and PSA criteria.

Chaque année, environ 23 300 Canadiens se font diagnostiquer un cancer de la prostate. Malheureusement, 10% d'entre eux en mourront, représentant la troisième cause de décès par cancer chez les hommes. La recherche sur ce sujet est alors bien importante pour améliorer la compréhension de ce cancer afin de développer de nouvelles approches thérapeutiques qui ciblent le métabolisme des cellules. La prostate est une glande du système reproducteur mâle ayant un métabolisme unique. Cet organe possède un cycle de Krebs tronqué, de manière à pouvoir produire et sécréter un métabolite important, le citrate. Toutefois, lors de la tumorigenèse, ce profil métabolique est complètement reprogrammé pour favoriser la croissance des cellules tumorales et le citrate n'est non plus sécrété, mais redirigé vers le cycle de Krebs afin de produire de l'énergie. Toutefois, comment ce programme métabolique fonctionne dans la prostate, ainsi que comment les cellules tumorales piratent le métabolisme du citrate, restent encore bien mal compris et cela a été le sujet de mes travaux. Pour ce faire, j'ai initialement travaillé à l'optimisation de modèles d'organoïde de prostate, qui sont les premiers modèles à reproduire, *ex vivo*, le programme sécrétoire de citrate de la prostate. Après validation de ces modèles, tant chez la souris que chez l'humain, par immunohistochimie, immunofluorescence et métabolomique, j'ai ensuite travaillé à optimiser les méthodes de transductions lentivirales des organoïdes en culture primaire. Grâce à une méthode de modification génétique par shRNA, plusieurs enzymes et transporteurs ont été ciblés afin de comprendre leur rôle et les voies métaboliques impliquées dans la production et la sécrétion du citrate. Nous avons en premier lieu validé la répression d'expression par qRT-PCR et par immunobuvardage de type Western, puis effectué des essais enzymatiques ainsi que des tests de prolifération cellulaire. Nous avons notamment identifié l'enzyme isocitrate déshydrogénase 1 (IDH1) comme étant essentielle pour le métabolisme des cellules tumorales. Mes travaux contribuent à mettre en évidence IDH1 comme nouvelle cible thérapeutique contre le cancer de la prostate. Globalement, les travaux présentés dans ce mémoire permettront une meilleure compréhension du métabolisme de la prostate saine et de sa reprogrammation durant la tumorigenèse. À terme, nous espérons que ces découvertes mèneront au développement de nouvelles approches thérapeutiques ciblant le métabolisme tumoral. / Every year, approximately 23,300 Canadians are diagnosed with prostate cancer. Sadly, 10% of them will dieof it, representing the third leading cause of cancer death in men. Research on this subject is therefore vital to improve our understanding of this cancer, and to develop new therapeutic approaches that target cell metabolism. The prostate is a gland of the male reproductive system with a unique metabolism. This organ has a truncated Krebs cycle, so that it can produce and secrete an important metabolite, citrate. However, during tumorigenesis, this metabolic profile is completely reprogrammed to promote tumor cell growth, and citrate is no longer secreted, but redirected to the Krebs cycle for energy production. However, how this metabolic program works in the prostate, and how tumor cells hack citrate metabolism, is still poorly understood and was the subject of my work. To do so, I initially worked on optimizing prostate organoid models, which are the first models to reproduce, *ex vivo*, the citrate secretory program of the prostate. After validating these models by immunohistochemistry, immunofluorescence and metabolomics, I then worked on optimizing lentiviral transduction methods for organoids in primary culture. Using a shRNA-mediated genetic modification method, several enzymes and transporters were targeted in order to understand their role and the metabolic pathways involved in citrate production and secretion. We first validated expression repression by qRT-PCR and Western immunoblotting, followed by enzymatic and cell proliferation assays. In particular, we identified the enzyme isocitrate dehydrogenase 1 (IDH1) as essential for tumor cell metabolism. My work is helping to identify IDH1 as a new therapeutic target for prostate cancer. Overall, the work presented in this thesis will lead to a better understanding of healthy prostate metabolism and its reprogramming during tumorigenesis. Ultimately, we hope these findings will lead to the development of new therapeutic approaches targeting tumor metabolism