O câncer de próstata (CaP) é a segunda causa mais comum de câncer em homens. De acordo com o INCA, no Brasil, em 2016, estimam-se aproximadamente 61.200 novos casos de câncer de próstata. Objetivo: Identificar as características demográficas e epidemiológicas, bem como dados do estadiamento tumoral dos pacientes com CaP atendidos na Unidade de Oncologia do Hospital Marcio Cunha na cidade de Ipatinga-MG nos anos de 2012, 2013 e 2014. Metodologia: Trata-se de um estudo retrospectivo e descritivo onde foram analisados 668 prontuários de pacientes, com registro do diagnóstico anatomopatológico, atendidos nos anos de 2012, 2013 e 2014, conforme lista fornecida pela instituição, com diagnóstico de CaP cadastrados com CID-10 - C 61. As variáveis analisadas foram: procedência, ano do diagnóstico, faixa etária, raça autodeclarada, fatores de risco como tabagismo, etilismo, história familiar de CaP, PSA total ao diagnóstico, tipo histológico da biópsia, score de Gleason da biópsia, tipo histológico da peça cirúrgica, score de Gleason da peça cirúrgica. Os dados foram analisados empregando-se estatística descritiva e inferencial, utilizando o software SPSS, versão 19.0. Resultados: A maior incidência de casos de CaP foram provenientes das cidades mais populosas da microrregião de saúde analisada e faixa etária mais prevalente foi entre 61 e 80 anos com prevalência em pardos e brancos e com histórico familiar de 17,2% de parentes de primeiro grau; com o pai em 37,3%, o irmão em 60,8% e filho em 1,9%. Apenas 165 (25,9 %) eram fumantes e 20,8% etilistas. Os níveis de PSA ficaram entre 4,1ng/ e 10ng/ml (49,5%) e quanto maior a faixa etária maiores os valores do PSA. Pacientes pardos apresentaram PSA total mais elevado. Ao avaliarmos se existia relação entre os níveis de PSA total com fatores de risco como tabagismo, etilismo e histórico familiar, somente houve relação estatisticamente significativa com o etilismo. Houve concordância do score de Gleason entre biópsia e peça cirúrgica em 70%, subgraduação em 18,7% e supergraduação em 11,3%. Comparando a idade dos pacientes com Score de Gleason, quanto maior a idade do paciente maior foi o Score de Gleason do material obtido pela biópsia via transretal Pacientes tabagistas e etilistas apresentaram Score de Gleason da peça cirúrgica mais elevados. Conclusão: A concordância entre o Score de Gleason da biópsia e o Score de Gleason da peça cirúrgica foi de 70%; etilistas apresentaram PSA mais elevados; quanto maior foi a faixa etária, mais indiferenciado foi o tumor ( biópsia); pacientes tabagistas e etilistas apresentaram tumores mais indiferenciados na peça cirúrgica; este é o primeiro estudo epidemiológico de CaP desenvolvido na região do Vale do Aço, a caracterização sócio demográfica e as associações aqui encontradas podem contribuir com programas para desenvolver ações de controle do CaP nesta região. / Prostate cancer (PCa) is the second most common cause of cancer in men. According to INCA, in Brazil, in 2016, approximately 61,200 new cases of prostate cancer are estimated. Objective: To identify the demographic and epidemiological characteristics, as well as data on the tumor staging of patients with PCa treated at the Oncology Unit of Hospital Marcio Cunha in the city of Ipatinga-MG in the years of 2012, 2013 and 2014. Methodology: This is a retrospective and descriptive study where 668 patients\' records, with a diagnosis of pathological diagnosis, were analyzed in the years 2012, 2013 and 2014, according to the list provided by the institution, with a diagnosis of PCa registered with ICD-10-C 61. The analyzed variables were: origin, year of diagnosis, age group, self-reported race, risk factors such as smoking, alcoholism, family history of PCa, total PSA at diagnosis, histological type of biopsy, Gleason score of biopsy, histological type of the surgical specimen, Gleason score of the surgical specimen. Data were analyzed using descriptive and inferential statistics, using SPSS software, version 19.0. Results: The highest incidence of PCa cases came from the most populated cities of the analyzed health micro-region and the most prevalent age group was between 61 and 80 years old, with prevalence in brown and whites and with a family history of 17.2% of first-degree relatives degree; With father in 37.3%, brother in 60.8% and son in 1.9%. Only 165 (25.9%) were smokers and 20.8% were alcoholics. PSA levels ranged from 4.1ng / e to 10ng / ml (49.5%) and the higher the age group the higher the PSA values. Brown patients had higher total PSA. When we evaluated whether there was a relationship between total PSA levels and risk factors such as smoking, alcohol consumption and family history, there was only a statistically significant relationship with alcohol consumption. There was concordance of the Gleason score between biopsy and surgical specimen in 70%, subgrade in 18.7% and overdose in 11.3%. Comparing the age of patients with Gleason score, the greater the patient\'s age, the greater the Gleason score of the material obtained by the transrectal biopsy. Smokers and alcoholists presented the highest Gleason score of the surgical specimen. Conclusion: The agreement between the Gleason score of the biopsy and the Gleason score of the surgical specimen was 70%; Higher PSA levels; The longer the age group, the more undifferentiated was the tumor (biopsy); Smokers and alcoholics presented more undifferentiated tumors in the surgical specimen; This is the first epidemiological study of PCa developed in the Vale do Aço region, the socio-demographic characterization and the associations found here can contribute with programs to develop actions of control of PCa in this region.

Introdução: A presença de tecido prostático em pacientes 46,XX portadoras de hiperplasia congênita das supra-renais (HCSR) já foi relatada por alguns autores. Acredita-se que o desenvolvimento deste tecido decorre do estímulo androgênico, através da dihidrotestosterona, sobre as glândulas parauretrais de Skene destas pacientes. Estas glândulas, presentes em todas as meninas, possuem homologia histológica e enzimática com a próstata masculina. Além da presença de tecido prostático nestas pacientes, houve dois relatos de alterações neste tecido, sendo o primeiro o de uma paciente 46,XX portadora de deficiência de 21-hidroxilase, que desenvolveu uma hiperplasia benigna prostática e outra com a mesma deficiência enzimática que aos 62 anos apresentou adenocarcinoma de próstata. Objetivos: 1. Verificar a ocorrência de tecido prostático nas pacientes acima de 6 anos, portadoras da forma clássica de hiperplasia congênita das supra-renais, com cariótipo 46,XX, em acompanhamento no ambulatório de endocrinologia pediátrica do Instituto da Criança; 2. Analisar a sensibilidade e especificidade do antígeno prostático específico (PSA) e da dihidrotestosterona das pacientes com HCSR em relação à detecção do tecido prostático através da ressonância nuclear magnética da região pélvica; 3. Correlacionar os níveis de PSA com os níveis de testosterona e dihidrotestosterona das pacientes portadoras de HCSR. Casuística: Constituiu-se de 32 pacientes 46,XX portadoras da forma clássica de hiperplasia congênita das suprarenais (31 com deficiência de 21-hidroxilase e uma com deficiência de 11- hidroxilase), com uma média de idade de 11,8 +/- 4,2 anos e um grupo controle que incluiu 10 meninas e 10 meninos sem hiperplasia congênita das supra-renais, com média de idade de 9,8 +/- 1,9 anos e 9,3 +/- 2,7 anos respectivamente. Os pacientes do grupo controle faziam acompanhamento no ambulatório de endocrinologia pediátrica por baixa estatura ou puberdade precoce. Métodos: O padrão-ouro utilizado para a detecção de tecido prostático foi a ressonância magnética de região pélvica, que foi realizada em todos os pacientes. Também foram dosados o antígeno prostático específico, a 17-hidroxiprogesterona, o 21-desoxicortisol, o 11- desoxicortisol, a androstenediona, a testosterona e a dihidrotestosterona. Resultados: A detecção do tecido prostático nas ressonâncias de região pélvica foi obtida em 5 pacientes com hiperplasia congênita das supra-renais. O antígeno prostático específico mostrou uma sensibilidade de 100% e especificidade de 88,9% para um ponto de corte de 0,1 ng/mL. A dihidrotestosterona revelou uma sensibilidade de 100% e especificidade de 60% para um ponto de corte de 27,7 ng/dL. Houve uma correlação positiva entre os níveis de PSA com os níveis de T e entre os níveis de PSA e DHT. Conclusões: A ocorrência de tecido prostático nas pacientes 46,XX portadoras de hiperplasia congênita das supra-renais estudadas foi de 15,6%. O PSA mostrou ser um valioso marcador de tecido prostático nas pacientes com HCSR. De acordo com os achados, a pesquisa de tecido prostático deve ser considerada em todas as pacientes portadoras da forma clássica de HCSR / Introduction: The presence of prostatic tissue in 46,XX congenital adrenal hyperplasia (CAH) patients has already been reported. The development of this tissue is due to androgenic stimulation in Skenes paraurethral glands by dihydrotestosterone. These glands have histological and enzymatic homology with the prostate. So far, two cases with alterations of prostatic tissues have been reported: one 46,XX patient with 21-hydroxylase deficiency developed benign prostatic hyperplasia and another with the same enzymatic deficiency, at age of 62 years, had adenocarcinoma of prostate. Objectives: 1.To describe the presence of prostatic tissue in 46, XX patients with the classical form of congenital adrenal hyperplasia 2. To evaluate the sensitivity and specificity of prostatic specific antigen (PSA) and dihydrotestosterone with regard to the detection of prostatic tissue in pelvic MRI. 3. To correlate prostate specific antigen levels with testosterone and dihydrotestosterone levels in girls with congenital adrenal hyperplasia. Patients and Methods: Among our CAH patients followed at our Unit, we selected 32 children and adolescents 46,XX, from 6 to 22 years of age, with the classical form of congenital adrenal hyperplasia (mean age 11.8 +/- 4.2); 31 patients had 21- hydroxylase deficiency and one 11-hydroxylase deficiency. Control group included 10 boys (mean age 9.3 +/- 2.7) and 10 girls (mean age 9.8 +/- 1.9) without CAH. Pelvic MRI (taken as the “gold standard method") was performed in all patients to detect prostatic tissue. Prostate specific antigen, 17-hydroxyprogesterone, 21- deoxycortisol, 11-deoxycortisol, androstenedione, testosterone and dihydrotestosterone were measured in all patients. Results: Five congenital adrenal hyperplasia girls showed image of prostatic tissue on pelvic MRI. Prostate specific antigen had sensitivity and specificity of 100.0% and 88.9%, respectively, taking 0.1 ng/mL as the cutoff level. The dihydrotestosterone had a sensitivity and specificity of 100% and 60%, respectively, with a cutoff level of 27.7 ng/dL. There was a positive correlation between PSA levels and testosterone and dihydrotestosterone levels. Conclusions: The incidence of prostatic tissue in 46,XX patients with the classical form of congenital adrenal hyperplasia was 15.6%. PSA demonstrated to be a good marker of prostatic tissue in these patients. Based on this study, we have to be cautious when treating a girl with the classical form of CAH and the search for prostatic tissue must be considered in every patient

De plus en plus d’inhibiteurs de kinase (IKs) sont testés dans le cancer de la prostate qui représente chez l’homme un enjeu de santé publique majeur de par son incidence (1er cancer) et sa mortalité (4ème cancer). Les essais cliniques pour évaluer l'efficacité des IKs dans cette indication ont donné des résultats mitigés malgré la présence de leurs cibles pharmacologiques dans les tumeurs de prostate (VEGF, EGFR, CMET..), pouvant faire penser que l’inefficacité serait en partie liée à la molécule elle-même et à sa pharmacocinétique/pharmacodynamie. En effet, les IKs sont sujets à un métabolisme et un transport intense via des enzymes de phase I et II et des transporteurs contrôlés pour la majorité par le récepteur nucléaire PXR (Pregnane X Receptor, gène NR1I2). En plus d’être abondamment exprimé dans le foie et le long du tractus gastro-intestinal, PXR est également exprimé dans certaines tumeurs épithéliales et pourrait être impliqué dans la résistance aux chimiothérapies par augmentation du catabolisme et de l’efflux de ces agents anticancéreux. A ce jour une seule étude a révélé l’expression de PXR dans le cancer de la prostate sans en avoir évalué l’impact sur la réponse aux traitements utilisés dans cette indication. En collaboration avec le Pr G. Fromont, nous avons observé dans une cohorte de 449 patients que l’expression de PXR était plus fréquemment retrouvée dans les cancers résistants à la castration et les métastases, par rapport aux cancers cliniquement localisés dans lesquels l’expression de PXR était corrélée avec le stade TNM et le score ISUP. Ces résultats confirment donc l’intérêt d’étudier le rôle que peut jouer PXR et les gènes du métabolisme et du transport qu’il régule, dans la sensibilité aux IKs dans les cancers de la prostate.Nous avons mesuré l’expression de PXR et de ses gènes cibles dans les lignées de cancer de la prostate 22RV1, LnCap, PC3 et DU145. Les résultats montrent une expression significative des enzymes et transporteurs responsables de la détoxication des IKs mais une faible expression de PXR liée à des phénomènes d’hyperméthylation NR1I2 dans nos lignées Cela nous a conduit à établir des modèles de surexpression stable de PXR dans lesquels l’agoniste SR12813 est capable d’induire l’activité transcriptionnelle de ce xénorécepteur, indiquant la compétence métabolique de ces lignées. À l'aide de ces modèles, nous avons démontré que la surexpression de PXR module la réponse à l’erlotinib, le dasatinib, le dabrafénib et l’afatinib démontrant que PXR joue un rôle fonctionnel dans la sensibilité à ces IKs. Nous avons également démontré que certains inhibiteurs avaient des propriétés agonistes de PXR, notamment le dabrafénib qui montre un effet agoniste plus marqué que le composé de référence SR12813, ce qui n’a jamais été démontré. Cette découverte originale nous a conduit à engager une collaboration pour tenter de cristalliser le complexe PXR/dabrafénib et à tester l’hypothèse que l’induction de l’activité PXR pouvait entraîner une modification du métabolisme et/ou du transport d’autres médicaments co-administrés. Or, nous avons observé dans la lignée 22RV1 un effet additif entre le dabrafénib et le tramétinib, une combinaison approuvée dans le traitement du mélanome, qui devient antagoniste lorsque PXR est surexprimé, résultat qui va effectivement dans le sens de notre hypothèse même s’il reste à démontrer que cet effet est bien lié à une altération du métabolisme de ces IKs, ce que nous sommes en train d’évaluer en dosant les métabolites de ces IKs. L’ensemble de nos données pourraient servir de rationnel biologique dans le choix des IKs ou de leurs combinaisons à tester avec les hormonothérapies et chimiothérapies déjà utilisés dans le traitement du cancer de la prostate, afin de potentialiser la réponse tumorale. / More and more kinase inhibitors (KIs) are tested in prostate cancer that represents a major health issue in men with its incidence and mortality rates. Clinical trials to evaluate KIs efficacy in prostate cancer gave disapointing results depsite the presence of KIs pharmacological targets in prostate tumors (VEGF, EGFR, CMET..), suggesting that inefficiency of these drugs would be at least in part linked to the inhibitor itself or its pharmacodynamics/pharmacokinetics parameters. Indeed KIs are metabolized and transported via phase I and II enzymes that are mainly controlled by the xenoreceptor PXR (Pregnane X Receptor, gène NR1I2). It is mainly expressed in liver and gastro-intestinal tract but also in epithelial tumors. PXR is also involved in the resistance to chemotherapies by increasing the catabolism and the efflux of these anticancer agents. To date only one study evaluated PXR expression in prostate cancer without evaluating its impact on treatment efficacy. In collaboration with Pr G. Fromont we analyzed a cohort of 449 prostate tumors and observed that PXR was more frequently detected in castration resistant or metastatic tumors as compared to clinically localized forms in which PXR expression was significantly correlated with TNM and ISUP Score. These results confirmed the interest to study the potential role of PXR and its target genes in the sensitivity to kinase inhibitors in prostate cancer models.We measured the expression of PXR and its target genes in prostate cancer cell lines 22RV1, LnCap, PC3 and DU145. The results showed that enzymes and transporters involved in KI detoxification was significantly expressed in these cells whereasPXR was poorly expressed due to hypermethylation of NR1I2 in our cells. This lead us to develop specific prostate cancer cell models stably overexpressing PXR in which transcriptional activity of PXR can be induced by its known agonist SR12813 further indicating that prostate cancer cells are metabolically competent. Using these models we showed that PXR overexpression modulates the sensitivity of 22RV1 cells to erlotinib, dasatinib, dabrafenib and afatinib, demonstrating that PXR plays a functional role in the sensitivity to KIs. We also demonstrated that several KIs were PXR agonists, including dabrafenib that displayed enhanced agonistic properties as compared to SR12813, a result that was never published before. This original finding led us to engage the cristalization of PXR/dabrafenib complex and to test whether induction of PXR could lead to an alteration of metabolism and transport of other drugs that are co-administered. In this line we have observed that in 22RV1 cells the additive effect of the combination of dabrafenib with trametinib that is already approved in the treatment of melanomas, became antagonistic when PXR was overexpressed in these cells. This result is supporting our hypothesis though we still need to demonstrate that this effect is linked to a change in drugs metabolism, which is currently under investigation by the measurement of the known metabolites of these KIs.Altogether, our data could serve as rational basis for the choice of kinase inhibitors or their potential combinations that could be tested in further clinical trials alone or in association with hormone therapies or with chemotherapies that are currently prescribed in the treatment of advanced prostate cancers, in order to potentiate tumor response.

Le chondrosarcome (CHS), cancer du cartilage est une tumeur chimio- et radiorésistance dont le seul traitement efficace reste la chirurgie. Une prodrogue vectorisée et activable en hypoxie, ICF05016, est actuellement développée par l’UMR 1240, et évaluée en préclinique comme potentielle alternative théra-peutique pour ce cancer. La structure de cette molécule regroupe i) une moutarde cytotoxique, ii) un vecteur ammonium quaternaire chargé positivement possédant un tropisme pour l’aggrécane, protéoglycane majoritaire de la matrice extracellulaire tumorale, iii) une gâchette de type 2-nitroimidazole permettant une activation sélective en situation d’hypoxie, une des caractéristiques principales du CHS.Ces travaux de thèse ont consisté à pharmaco-moduler cette prodrogue bispécifique ICF05016 en modifiant la position du vecteur ainsi que la nature de l’agent cytotoxique. Ainsi sept prodrogues vectorisées ont été synthétisées présentant une chaine vectrice N,N,N-triméthylpropylaminium soit en C-4, soit en N-1 du cycle imidazole. Leur activation par réduction chimique, mimant l’hypoxie, ainsi que leur affinité pour l’aggrécane ont été confirmées in tubo par des analyses de RMN 31P et de SPR, toutefois elles se sont avérées non sélectives en termes de cytotoxicité (CI50 comprises entre 15 et 1 µM, et ce, quelles que soient les conditions d’oxygénation) et faiblement sensibles à une bio-réduction enzymatique. La fonctionnalisation par un vecteur ammonium quaternaire de la gâchette 2-nitroimidazole annihile donc l’activation en hypoxie des prodrogues.Cette stratégie a ensuite été étendue au cancer de la prostate en remplaçant le vecteur ammonium quaternaire par un ligand de type urée affin pour l’antigène membranaire spécifique de la prostate (PSMA). La première molécule synthétisée, qui possède un espaceur triazole, a démontré une affinité pour le récepteur PSMA, par une étude de compétition avec un radioligand, ainsi qu’une activation in tubo par bioréduction enzymatique. Toutefois aucune cytotoxicité n’a été constatée sur les lignées LNCaP-Luc et PC3-Luc. Une seconde molécule combinant un espaceur triazole avec une séquence peptidique identifiée pour la molécule PSMA-617, actuellement en cours d’essai clinique, est actuellement développée mais sa synthèse doit être optimisée, notamment au niveau de l’étape de cycloaddition 1,3-dipolaire. / Chondrosarcoma (CHS), the malignant tumor of the cartilage, is a chemo- and radio-resistant cancer. Surgical resection is still considered the mainstay of treatment of this pathology. A dual targeted hypoxia-activated prodrug, ICF05016 was developed by the UMR 1240 and evaluated in preclinical studies as a potential therapeutic alternative for CHS. The latter is a nitroheteroaryl-based compound designed as follows: a phosphorodiamidic mustard functionalized with a quaternary ammonium (QA) used as targeting function, and a 2-nitroimidazole group to trigger fragmentation and then release the bis-alkylating mustard anion by bioreduction under hypoxic conditions, chemical hallmark of CHS.This project deals with the pharmacomodulation of ICF05016, more specifically by modification of the position of the targeting moiety as well as the nature of the cytotoxic agent. Seven QA-targeted prodrugs have been synthesized with N,N,N-trimethylpropylaminium tethered to the imidazole either in the C-4, or the N-1 position. These prodrugs were cleaved in vitro under chemical reductive conditions, which mimic in vivo hypoxia conditions. In addition, the binding of these derivatives to aggrecan was highlighted by surface plasmon resonance. In vitro assays on human CHS cells (H-EMC-SS) demonstrated quite equivalent cytotoxicities, whatever the oxygen conditions used and their evaluation as substrate of an oxygen-insensitive nitroreductase revealed the almost total lack of activation. A QA targeting moiety grafted on the trigger seems to alter hypoxia activation.New prodrugs with prostate specific membrane antigen (PSMA)-targeting ligand were synthesized to extend this HAP strategy to prostate cancer. The first tested compound, having a triazole spacer, presented selective affinity for PSMA in an in vitro binding experiment as well as activation under enzymatic reduction. However, no cytotoxicity was observed on LNCaP-Luc and PC3-Luc cells. The synthesis of a prodrug combining the spacer of PSMA-617, currently in clinical trial, and a propyltriazole moiety, was initiated but the 1,3-dipolar cycloaddition still need to be optimized.

Timely detection of prostate cancer (PCA) with prostate-specific antigens (PSA) and digital rectal examinations (DRE) are essential in optimizing incidence, minimizing prevalence, and reducing mortality rates. Given the low levels of participation in cancer screening, this study was conducted to examine the factors men consider when deciding whether to screen for PCA in Nigeria. A cross-sectional, online-based survey of 180 consenting Nigerian men 50 years old and older was carried out. Logistic regression analysis and descriptive statistics were used to analyze the data. Based on the data, there was a moderate positive association between the health belief model constructs and DRE/PSA screening intentions, which were statistically significant (p < 0.05). The results also demonstrated that there were no statistically significant associations between previous screening and age, previous screening and ethnicity, and previous screening and education among men in the sample (all p > 0.05). Of the 180 men surveyed, 29% (n = 53) had been screened for PCA before, while 76% (n = 137) reported no health insurance. Factors significantly associated with screening included income, insurance, and family history of PCA (all p < 0.05). Cancer fatalism, pain, and embarrassment were the most common barriers to screening reported. Focused interventions that help healthcare providers identify barriers quickly could improve screening outcomes. The implications for positive social change from this study include an increase in PCA screening, positive screening intentions, and a decrease in PCA mortality rate among men in Nigeria.

Le récepteur des androgènes (RA) est la principale cible thérapeutique du cancer de la prostate (CaP) métastatique. Bien que cette thérapie soit initialement efficace, les effets sont transitoires. De nombreux mécanismes peuvent expliquer la progression du CaP vers un stade de résistance à la castration, telles les modifications du RA. Des données récentes ont montré que les variants constitutifs RA-Q641X et RA-V7, caractérisés par la perte du domaine de liaison au ligand, étaient associés à l’expression de marqueurs mésenchymateux. L’étude de la régulation de la N-cadhérine a mis en évidence que si le RA sauvage et les variants constitutifs se liaient tous deux aux éléments de réponse du gène codant, seuls les derniers étaient associés à une augmentation de l’acétylation de l’histone H4, marque positive de la transcription. Le RNA-seq a révélé que leur expression était aussi corrélée à la régulation de sets de gènes spécifiques incluant des facteurs de transcription dont certains ont déjà été caractérisés en cancérologie.En ce qui concerne le RA-T576A, porteur d’une mutation faux-sens, les données ont révélé une séquence consensus de liaison à l’ADN moins conservée pour le RA sauvage que pour ce mutant et l’importance du 11ème nucléotide des éléments de réponse. De plus, cette mutation a semblé impacter le transcriptome du RA. Ce travail met en évidence le comportement distinct des variants du RA et aide à mieux comprendre leurs modes d’action en décrivant leurs activités transcriptionnelles. / The androgen receptor (AR) is the main therapeutic target in metastatic prostate cancer (PCa). Although this therapy is initially effective, the effects are transient. Many mechanisms can explain PCa progression toward castration resistance including abnormalities in the AR. Recent data have shown that constitutive AR (e.g AR-Q641X and AR-V7), which have lost the ligand binding domain, were associated with the induction of mesenchymal marker expression. The study of N-cadherin regulation highlighted that while both constitutive AR and wild type AR bound to response elements located in the encoding gene, only the AR variants were associated with an increase of H4 acetylation, a positive transcription mark. RNA-seq revealed that their expression was also correlated to specific sets of genes regulation, including transcription factors and genes involved in migration, AR regulation, and therapeutic resistance.Concerning AR-T576A, which hold a missense mutation, data revealed a less conserved consensus sequence for the wild type AR than for this mutant and highlighted the importance of the 11th nucleotide of the response element for AR recruitment to DNA. Plus, this mutation seemed to impair AR transcriptome. This work highlights the distinct AR variants’ behavior and helps to understand their mode of action by depicting their transcriptional landscapes.

<p>The main focus of my thesis has been to develop protocols for generating antigen-specific cytotoxic T lymphocytes (CTLs) and T helper cells (T_H) for adoptive transfer to treat cytomegalovirus (CMV) disease and prostate cancer. CMV viremia is a severe complication in immunocompromised stem cell transplanted patients. Prostate cancer is a leading cause of death for men in Western countries. Although different in nature, CMV-infected cells and prostate cancer cells can both be eliminated through specific activation of the adaptive immune system. </p><p>To generate CMV pp65-specific T cells, I utilized dendritic cells (DCs) modified with an HLA-A*0201/pp65_495-503 peptide, a recombinant adenovirus coding for pp65, <i>in vitro</i> transcribed pp65 mRNA and a recombinant pp65 protein. Peptide stimulation yielded large numbers of peptide-specific CD8⁺ T cells with high lytic activity while adenovirus or mRNA stimulation resulted in the expansion of CTLs against multiple pp65 epitopes. The recombinant protein activated primarily CD4⁺ T_H cells. Stimulation with DCs co-modified with pp65 mRNA and pp65 protein simultaneously generated both pp65-specific CTLs and T_H cells. Such T cells would cover all pp65 epitopes while avoiding potential virus related biohazards. The mRNA/protein combinatory approach can be used to stimulate T cells <i>ex vivo</i> from virtually all stem cell donors for adoptive T cell transfer. </p><p>I have identified two immunogenic HLA-A*0201-restricted peptide epitopes from the prostate tissue antigen TARP. Repeated stimulations with TARP peptide-pulsed DCs yielded up to 20% TARP-directed CD8⁺ T cells even when starting from undetectable frequencies (<0.01%). The T cells could be sorted to 99% purity and expanded 1000-fold with retained specificity and activity. We also detected TARP-directed CD8⁺ T cells in the blood of prostate cancer patients. Therefore, TARP seems to have potential as antigen in DC vaccination or adoptive T cell therapy of prostate cancer. </p>

The main focus of my thesis has been to develop protocols for generating antigen-specific cytotoxic T lymphocytes (CTLs) and T helper cells (TH) for adoptive transfer to treat cytomegalovirus (CMV) disease and prostate cancer. CMV viremia is a severe complication in immunocompromised stem cell transplanted patients. Prostate cancer is a leading cause of death for men in Western countries. Although different in nature, CMV-infected cells and prostate cancer cells can both be eliminated through specific activation of the adaptive immune system. To generate CMV pp65-specific T cells, I utilized dendritic cells (DCs) modified with an HLA-A*0201/pp65495-503 peptide, a recombinant adenovirus coding for pp65, in vitro transcribed pp65 mRNA and a recombinant pp65 protein. Peptide stimulation yielded large numbers of peptide-specific CD8+ T cells with high lytic activity while adenovirus or mRNA stimulation resulted in the expansion of CTLs against multiple pp65 epitopes. The recombinant protein activated primarily CD4+ TH cells. Stimulation with DCs co-modified with pp65 mRNA and pp65 protein simultaneously generated both pp65-specific CTLs and TH cells. Such T cells would cover all pp65 epitopes while avoiding potential virus related biohazards. The mRNA/protein combinatory approach can be used to stimulate T cells ex vivo from virtually all stem cell donors for adoptive T cell transfer. I have identified two immunogenic HLA-A*0201-restricted peptide epitopes from the prostate tissue antigen TARP. Repeated stimulations with TARP peptide-pulsed DCs yielded up to 20% TARP-directed CD8+ T cells even when starting from undetectable frequencies (&lt;0.01%). The T cells could be sorted to 99% purity and expanded 1000-fold with retained specificity and activity. We also detected TARP-directed CD8+ T cells in the blood of prostate cancer patients. Therefore, TARP seems to have potential as antigen in DC vaccination or adoptive T cell therapy of prostate cancer.

Background: The secretory epithelial cells of the prostate gland use sophisticated vehicles named prostasomes to relay important information to sperm cells in semen. This prostasome-forming and secretory ability of the epithelial cells is also preserved in poorly differentiated prostate cancer cells. Aim: The aim of this thesis was to examine different characteristics of prostasomes, especially those derived from malignant prostate cells, linked to their potential role in diagnosis and prognostication of prostate cancer. Results: Serum samples of prostate cancer patients contained autoantibodies against seminal prostasomes in a higher concentration than did control sera. These autoantibodies were most frequently directed against 25 prostasome-associated proteins, but no one was prostate specific. Clusterin was one of the most frequently occurring prostasomal proteins. Elevated titers were however seen in both patients´ and control sera. Clusterin turned out to be a major antigen of seminal prostasomes. No prostate specific or prostate cancer specific protein was discovered upon proteomic analysis of prostasomes deriving from malignant cells of vertebral metastases of prostate cancer patients. Human chromosomal DNA was identified in both seminal prostasomes and PC-3 cell prostasomes and strong evidence existed that the DNA was localized inside the prostasomes. Four out of 13 DNA clones of seminal prostasomes featured gene sequences (31%). The corresponding figures for PC-3 cell prostasomes were 4 out of 16 clones (25%). Conclusions: Prostasomes are immunogenic and give rise to serum autoantibodies. The most frequently occurring autoantibodies were directed against 25 prostasomal proteins but none of these was exclusively prostate specific. Thirty different proteins were identified in prostate cancer metastasis-derived prostasomes but none of these proteins was prostate cancer specific. Human chromosomal DNA was identified in prostasomes of both normal and malignant cell origin.

Le cancer de la prostate est le cancer le plus fréquemment diagnostiqué chez les hommes canadiens et la troisième cause de décès relié au cancer. Lorsque diagnostiqué à un stade précoce de la maladie, le cancer de la prostate est traité de manière curative par chirurgie et radiothérapie. Par contre, les thérapies actuelles ne peuvent éradiquer la maladie lorsqu’elle progresse à des stades avancés. Ces thérapies, comme la chimiothérapie et l’hormonothérapie, demeurent donc palliatives. Il est primordial d’optimiser de nouvelles thérapies visant l’élimination des cellules cancéreuses chez les patients atteints des stades avancés de la maladie. Une de ces nouvelles options thérapeutiques est l’immunothérapie. L’immunothérapie du cancer a fait des progrès considérables durant les dernières années. Cependant, les avancements encourageants obtenus lors d’essais précliniques ne se sont pas encore traduits en des résultats cliniques significatifs. En ce qui concerne le cancer de la prostate, les résultats négligeables suivants des interventions immunothérapeutiques peuvent être causés par le fait que la plupart des études sur le microenvironnement immunologique furent effectuées chez des modèles animaux. De plus la majorité des études sur l’immunologie tumorale humaine furent effectuées chez des patients atteints d’autres cancers, tels que le mélanome, et non chez les patients atteints du cancer de la prostate. Donc, le but central de cette thèse de doctorat est d’étudier le microenvironnement immunologique chez les patients atteints du cancer de la prostate afin de mieux définir les impacts de la tumeur sur le développement de la réponse immunitaire antitumorale. Pour réaliser ce projet, nous avons établi deux principaux objectifs de travail : (i) la caractérisation précise des populations des cellules immunitaires infiltrant la tumeur primaire et les ganglions métastatiques chez les patients atteints du cancer de la prostate; (ii) l’identification et l’étude des mécanismes immunosuppressifs exprimés par les cellules cancéreuses de la prostate. Les résultats présentés dans cette thèse démontrent que la progression du cancer de la prostate est associée au développement d’un microenvironnement immunosuppressif qui, en partie, est régulé par la présence des androgènes. L’étude initiale avait comme but la caractérisation du microenvironnement immunologique des ganglions drainant la tumeur chez des patients du cancer de la prostate. Les résultats présentés dans le chapitre III nous a permis de démontrer que les ganglions métastatiques comportent des signes cellulaires et histopathologiques associés à une faible réactivité immunologique. Cette immunosuppression ganglionnaire semble dépendre de la présence des cellules métastatiques puisque des différences immunologiques notables existent entre les ganglions non-métastatiques et métastatiques chez un même patient. La progression du cancer de la prostate semble donc associée au développement d’une immunosuppression affectant les ganglions drainant la tumeur primaire. Par la suite, nous nous sommes intéressés à l’impact de la thérapie par déplétion des androgènes (TDA) sur le microenvironnement immunologique de la tumeur primaire. La TDA est associée à une augmentation marquée de l’inflammation prostatique. De plus, les protocoles d’immunothérapies pour le cancer de la prostate actuellement évalués en phase clinique sont dirigés aux patients hormonoréfractaires ayant subi et échoué la thérapie. Cependant, peu d’information existe sur la nature de l’infiltrat de cellules immunes chez les patients castrés. Il est donc essentiel de connaître la nature de cet infiltrat afin de savoir si celui-ci peut répondre de manière favorable à une intervention immunothérapeutique. Dans le chapitre IV, je présente les résultats sur l’abondance des cellules immunes infiltrant la tumeur primaire suivant la TDA. Chez les patients castrés, les densités de lymphocytes T CD3+ et CD8+ ainsi que des macrophages CD68+ sont plus importantes que chez les patients contrôles. Nous avons également observé une corrélation entre la densité de cellules NK et une diminution du risque de progression de la maladie (rechute biochimique). Inversement, une forte infiltration de macrophages est associée à un plus haut risque de progression. Conjointement, durant cette étude, nous avons développé une nouvelle approche informatisée permettant la standardisation de la quantification de l’infiltrat de cellules immunes dans les échantillons pathologiques. Cette approche facilitera la comparaison d’études indépendantes sur la densité de l’infiltrat immun. Ces résultats nous ont donc permis de confirmer que les effets pro-inflammatoires de la TDA chez les patients du cancer de la prostate ciblaient spécifiquement les lymphocytes T et les macrophages. L’hypothèse intéressante découlant de cette étude est que les androgènes pourraient réguler l’expression de mécanismes immunosuppressifs dans la tumeur primaire. Dans le chapitre V, nous avons donc étudié l’expression de mécanismes immunosuppressifs par les cellules cancéreuses du cancer de la prostate ainsi que leur régulation par les androgènes. Notre analyse démontre que les androgènes augmentent l’expression de molécules à propriétés immunosuppressives telles que l’arginase I et l’arginase II. Cette surexpression dépend de l’activité du récepteur aux androgènes. Chez les patients castrés, l’expression de l’arginase II était diminuée suggérant une régulation androgénique in vivo. Nous avons observé que l’arginase I et l’arginase II participent à la prolifération des cellules du cancer de la prostate ainsi qu’à leur potentiel immunosuppressif. Finalement, nous avons découvert que l’expression de l’interleukin-8 était aussi régulée par les androgènes. De plus, l’interleukin-8, indépendamment des androgènes, augmente l’expression de l’arginase II. Ces résultats confirment que les androgènes participent au développement d’une microenvironnement immunosuppressif dans le cancer de la prostate en régulant l’expression de l’arginase I, l’arginase II et l’interleukin-8. En conclusion, les résultats présentés dans cette thèse témoignent du caractère unique du microenvironnement immunologique chez les patients atteints du cancer de la prostate. Nos travaux ont également permis d’établir de nouvelles techniques basées sur des logiciels d’analyse d’image afin de mieux comprendre le dialogue entre la tumeur et le système immunitaire chez les patients. Approfondir les connaissances sur les mécanismes de régulation du microenvironnement immunologique chez les patients atteint du cancer de la prostate permettra d’optimiser des immunothérapies mieux adaptées à éradiquer cette maladie. / Prostate cancer is the most frequently diagnosed cancer in Canadian men and the third cause of cancer related death. When diagnosed at an early stage, prostate cancer can be effectively cured by surgery and radiotherapy. However, current therapies do not eradicate the advanced stages of the disease. Treatment of prostate cancer via chemotherapy or hormonotherapy remains palliative. It is thus essential to optimize novel therapies whose goal is to eliminate tumor cells in patients with advanced prostate cancer. One such approach is immunotherapy. Cancer immunotherapy has made important strides in recent years. The encouraging progress observed in pre-clinical trials has nonetheless not translated to significant results in the clinical setting. Concerning prostate cancer, the limited clinical efficacy of current immunotherapeutic protocols could be explained by the lack of studies directly evaluating the immunological microenvironment in prostate cancer patients and not in animal models or in patients afflicted by other malignancies, such as melanoma. Thus, the fundamental goal of this Ph.D. thesis is to study the immunological microenvironment in prostate cancer patients in order to better understand the impact of the tumor on the development of the anti-tumoral immune response. To realize this project, we established two main working objectives: (i) to precisely characterize the immune cell populations in tumor draining lymph nodes (LNs) and in the primary tumor of prostate cancer patients; (ii) to identify and to study the immunosuppressive pathways expressed by prostate cancer cells. The results detailed in this thesis demonstrate that prostate cancer progression is associated with the development of an immunosuppressive microenvironment, which is regulated, in part, by the presence of androgens. The initial study was based on the characterization of the immunological microenvironment of tumor draining LNs of prostate cancer patients. The results presented in chapter III allowed us to demonstrate that metastatic lymph nodes displayed cellular and histopathological evidence associated with a reduced immunological reactivity. This LN immunosuppression seemed to be dependant on the presence of metastatic cells as we noted significant immunological differences between non-metastatic and metastatic lymph nodes of the same patient. Prostate cancer progression was thus associated with the development of an immunosuppressive state, which affected tumor-draining lymph nodes. Next, we studied the impact of androgen deprivation therapy (ADT) on the immunological microenvironment of the primary tumor. Following ADT, there is a marked augmentation in intra-prostatic inflammation. Immunotherapeutic protocols currently evaluated in clinical trials are targeted at hormone refractory patients, which have received and failed ADT. However, very little information is available regarding the nature of the post-ADT immune infiltrate. Thus, it becomes essential to understand whether this post-ADT infiltrate could positively react to immunotherapy. In chapter IV, I present the results of the quantification of the immune cell abundance within the primary tumor. In patients who have received ADT prior to surgery, there was an elevated density of CD3+ and CD8+ T lymphocytes as well as CD68+ macrophages compared to control patients. We also observed an inverse correlation between the NK cell density and the risk of disease progression (biochemical recurrence). Conversely, an elevated macrophage infiltration was associated with a higher risk of progression. Furthermore, for this study, we developed a novel computerized approach allowing for the standardization of the quantification of immune cell infiltrate. This approach could facilitate the interpretation of results from independent studies on the density of immune cells within pathological specimens. This study confirmed that the pro-inflammatory impact of androgen deprivation therapy in prostate cancer patients target specifically the T lymphocyte and macrophage populations. The interesting hypothesis arising from this study was that androgens could positively regulate the expression of immunosuppressive pathways within the primary tumor. In chapter V, we evaluated the immunosuppressive mechanisms expressed by prostate cancer cells and regulated by androgens. Our analysis demonstrate that androgens increase the expression of molecules with immunosuppressive properties, such as arginase I and arginase II in an androgen receptor dependent manner. This androgen regulated expression of arginase II was also observed in prostate cancer patients treated by ATD. We observed that arginase I and arginase II participate in prostate cancer cell proliferation as well as in their immunosuppressive potential. Finally, we discovered that interleukin-8 expression was also regulated by androgens. Moreover, interleukin-8, independently of androgens, increased the expression of arginase II. Altogether, these results confirmed that androgens participate in the development of an immunosuppressive microenvironment in prostate cancer by regulating the expression of arginase I, arginase II and interlukin-8. In conclusion, the results presented in this thesis attest to the unique character of the immunological microenvironment in prostate cancer patients. Our work has also allowed to establish novel software-based analysis methods in order to better understand the dialogue between the tumor and the immune system. Further understanding of the regulatory pathways involved in the immunological microenvironment will allow for the optimization of immunotherapies better suited to eradicate prostate cancer.