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Orientador: Sérgio Luiz Felisbino / Coorientador: Wellerson Rodrigo Scarano / Banca: Raquel Fantin Domeniconi / Banca: Luis Antonio Justulin Júnior / Banca: Renata Carolina Piffer / Banca: Glaura Scatamburio Alves Fernandes / Resumo: Estudos têm sugerido que o excesso de glicocorticoides (GCs) durante períodos críticos do desenvolvimento pode alterar a função reprodutiva. Apesar da função essencial da próstata no sucesso reprodutivo e de sua alta susceptibilidade ao desenvolvimento de lesões com o avançar da idade, o impacto tardio de corticoterapias precoces sobre a homeostase da glândula ainda é desconhecido. No presente estudo, investigamos os efeitos da corticoterapia prenatal (PRE), durante a instalação da puberdade (PU), e sua associação (PRE+PU=REE), sobre a morfofisiologia da próstata senescente. Ratas Wistar prenhes receberam betametasona (0.1mg/kg/dia, i.m.), ou salina, nos dias gestacionais 12, 13, 18 e 19. Os descendentes machos receberam doses de betametasona (7mg/kg/dia, gavage), ou salina, do dia pós-natal 35 ao 50 (PND35-50). Na idade senil (PND300), todos os animais foram eutanasiados, amostras de sangue foram coletadas para dosagens hormonais, e a próstata ventral (VP) foi dissecada e processada para a análise morfológica, bem como para quantificação e localização de proteínas (AR, GR, PAR-4 e PCNA). Reduzidos níveis de testosterona e insulina foram observados no grupo PRE, enquanto apenas a insulina mostrou-se reduzida no grupo PU, e nenhuma redução adicional foi observada em REE. Uma tendência de aumento no índice apoptótico e incidência de ácinos com epitélio metaplásico foi detectada dentre os grupos. A quantificação de proteínas revelou menor expressão de AR no grupo PRE, maior expressão do marcador de proliferação celular (PCNA) no grupo REE, porém, diferença significativa alguma foi observada na expressão do marcador de morte celular por apoptose (PAR-4). A análise da reação imunoistoquímica para o GR indicou uma maior expressão do receptor em células epiteliais dos grupos que receberam betametasona. Com base nos resultados, sugerimos que a corticoterapia com betametasona durante o final da ... / Abstract: Studies have suggested that glucocorticoids (GCs) excess during critical developmental time windows can alter reproductive parameters. Despite of the key function of the prostate in the reproductive success, and its high susceptibility to develop lesions in an age-dependent manner, the impact of early GCs excess on the gland homeostasis is still unknown. In the present study, we have investigated the effects of prenatal (PRE), peripubertal (PU) corticotherapy, and its combination (PRE+PU=REE), on aging prostate's morphophysiology. Pregnant Wistar rats received betamethasone (0.1mg/kg/day, i.m.), or saline, on the gestational days 12, 13, 18 and 19. Male descendents received betamethasone (7mg/kg/day, gavage), or saline, from 35th to 50th postnatal day (PND35-50). Late in life (PND300), all animals were euthanized, blood samples were taken for hormones levels estimation, and the ventral prostate (VP) was excised and processed for morphology evaluation, and for proteins (AR, GR, PAR-4 and PCNA) quantification and localization as well. Lower testosterone and insulin levels were detected in group PRE, while only insulin serum levels was reduced in group PU, and no additional decrease was seen in REE. An increasing trend in the apoptosis index and metaplastic epithelium acini incidence was observed along the treated groups. The protein quantifications showed a decreased AR expression in PRE, higher proliferation marker (PCNA) expression in REE, and no significant difference in the expression of the apoptosis marker PAR-4 was detected among the groups. The immunolocalization of GR indicated a higher receptor expression in epithelial cells of treated groups, when compared to NE. Based on these results, we suggest that the corticotherapy with betamethasone during late pregnancy can program fetal prostate, resulting in altered androgen and glucocorticoids signaling permanently. Peripubertal corticotherapy deflagrated cell ... / Doutor

Prostate cancer represents a serious health concern as the most diagnosed form of cancer in men and the second leading cause of cancer death in the Western world. Current treatments for prostate cancer are non-targeted and result in a number of undesirable, non-specific effects, highlighting the need for novel, targeted therapeutics in the treatment of prostate cancer. Prostate Specific Membrane Antigen (PSMA) offers great promise in the targeting of prostate cancer for imaging and therapy. PSMA is a transmembrane carboxypeptidase with cell surface expression several orders of magnitude higher in cancerous prostatic epithelia than found in other tissue and PSMA is constitutively internalized into cells. The unique expression profile of PSMA and its constitutive internalization offer great value in the targeted delivery of therapeutics to prostate cancer cell. In 2002, two synthetic RNA ligands, aptamers, were selected for their ability to inhibit the enzymatic activity of PSMA. In 2006, the utility of these aptamers in the delivery of cytotoxic siRNA across the cell membrane was demonstrated in vivo using aptamer-siRNA chimeras. However, those experiments were performed by intratumoral injection, and systemic administration will be necessary for use in the clinic. In this thesis, we improve PSMA targeted chimeras to serve as more powerful therapeutics in the treatment of prostate cancer. We optimize existing aptamer-siRNA chimeras for increased potency and stability and improved pharmacokinetics to enable systemic administration. We truncate the PSMA binding aptamers for amenability to large-scale chemical synthesis. With emerging roles for PSMA enzymatic activity in the prostate cancer disease we identify aptamers that are suitable for chemical synthesis and retain inhibitory properties against PSMA. Finally, we assess the use of aptamers as synthetic ligands in the functional inhibition of PSMA mediated motility in prostate cancer. Our results demonstrate the ability of aptamer-siRNA chimeras to specifically kill PSMA-expressing cells with cytotoxic siRNA upon systemic injection. We confirm a newly reported role for PSMA in the promotion of cell motility and demonstrate the ability of aptamers to effectively neutralize PSMA-mediated motility. The results presented within argue strongly for the functional utility of aptamers in the treatment of prostate cancer.

Cancer screening is useful for improving survival rates and treatment outcomes, which is why there are screening recommendations for the most prevalent types of cancer. Despite gains in the reduction of cancer-related mortality rate worldwide in the past few years, the Haitian community continues to experience high mortality rates due to cancer. The prevalence of prostate cancer in the Haitian population is among the highest worldwide at 767 per 100,000, with a mortality rate of 403 per 100,000. One of the causes may be the low prostate cancer screening rate in the Haitian community; however, no studies have been focused on an association between demographic factors within this community and the low prostate cancer screening rate. This study's purpose was to address this gap through a cross-sectional quantitative design using the health belief model as a theoretical framework and a convenience sample of 282 Haitian males. The rate of prostate cancer screening among Haitian immigrants living in Brooklyn was examined based on the demographic variables of age, income, and education. Participants' perceptions regarding prostate cancer screening were also evaluated based on the same variables. Loglinear, and binary logistic regression were used for data analysis. Although education was found to be the strongest and only significant predictor variable for prostate cancer screening participation within the target population, no conclusion could be drawn regarding the effect of the select variables on the participants' perceptions on prostate cancer screening. The implications for this study include increased knowledge for public health promotion initiatives and for those in the Haitian community working to reduce the morbidity and mortality rates due to prostate cancer.

Prostate cancer is the most common cancer occurring in the men in USA and Europe. According to CDC, incidence of Prostate cancer in African American men in the year 2008 was 234.6 cases per 100,000 compared to 150 cases per 100,000 in Caucasian men, reasons for this disparity remain unclear. Castration resistant prostate cancer is an advanced form of prostate cancer with poor survival rates. 10-20% of prostate cancer patients develop metastatic castration resistant prostate cancer (CRPC) within approximately 5 years of follow-up. Androgen deprivation therapy which is at the center of metastatic prostate cancer is often impeded by development of CRPC. Previous studies have demonstrated that prostatic androgen concentration ranging between 10-25 percent in the treated patients versus the untreated could still continue AR signaling. Previous in vitro studies have demonstrated higher tumor homing potential in normal adipose derived mesenchymal stem cells (ADMSC) from African American patient compared to ADMSC derived from a Caucasian patient when grown in prostate cancer cell condition media. This study attempts to exploit this tumortropicity of ADMSC for selective delivery of alpha keto reductases in the metastasized prostate cancer cells to hydrolyse DHT and other androgens into weaker androgens. Enriched ADMSC were plated in a 6 well plate and were co-transfected with transfected with AKRC14 and GFP. Gene expression was confirmed by PCR and WB. ADMSCs are capable of expressing AKR1C14 on transfection with plasmid. Stem cells expressing AKR1C4 open the avenues for furthering therapeutic strategies in metastatic CRPC by hydrolyzing the androgens. / 1 / Manish Ranjan

Le cancer de la prostate est une pathologie impliquant des facteurs divers comme l'hérédité, l'appartenance ethnique mais aussi des facteurs environnementaux. En effet, il a été démontré que certains micronutriments dont les phyto-oestrogènes contenus dans l'alimentation pouvaient avoir un rôle protecteur vis-à-vis de cette pathologie. Ces molécules seraient capables de moduler les mécanismes épigénétiques observés dans le cancer de la prostate. L'objectif de ce travail a été de déterminer si les phyto-oestrogènes du soja pouvaient induire la réversion de la méthylation d'oncosuppresseurs impliqués dans la cancérogenèse prostatique et par quelles voies moléculaires. Nos études, in vitro, réalisées sur des lignées continues de cancer de la prostate (DU-145, PC-3 et LNCaP) ont montré dans un premier temps, une diminution de la méthylation des gènes GSTP1, RASSF1A, EPHB2 et BRCA1 et une augmentation des protéines correspondantes, suite au traitement par la génistéine (40μM) et la daidzéine (110μM) pendant 48H. Dans un deuxième temps, une étude comparative entre l'effet des phyto-oestrogènes et l'oestradiol sur la méthylation de l'ADN d'un panel de 24 gènes a permis de mettre en évidence une régulation des mécanismes épigénétiques par les phyto-oestrogènes via la voie des Récepteurs aux oestrogènes. En conclusion, les phyto-oestrogènes agissent sur les mécanismes épigénétiques dans la cancérogenèse prostatique laissant supposer que ces molécules pourraient jouer un rôle préventif dans cette pathologie. / Prostate cancer is a disease caused by a multiple interacting factors such as family history of prostate cancer, age and ethnic origin. Environnemental factors play also a role in prostatic carcinogenesis events. Indeed, several studies have reported the efficiency of nutrients such as phytoestrogens to possess anticancer properties. It has been reported that these compounds may have the ability to induce the reversion of epigenetic modifications observed in prostate cancer cells. The aim of this work was to determine if soy isoflavone could reverse the DNA methylation of oncosuppressor which are hypermethylated in prostate cancer and through which metabolic pathways. Our in vitro studies were carried out on tree prostate cancer cell lines: DU-145, PC-3 and LNCaP. The qualitative and quantitative studies performed demonstrated a decrease of methylation percentage of GSTP1, RASSF1A, EPHB2 and BRCA1 after soy isoflavone treatment. In a second step, a comparative study between the effect of phytoestrogens and estradiol on the DNA methylation of a panel of 24 genes was performed. Our results has highlighted that the regulation of epigenetic mechanisms by phytoestrogens may be mediated via the estrogen receptor pathway. In conclusion, phytoestrogen act on epigenetics mechanisms on prostate carcinogenesis suggesting that these molecules may play a role in the prevention of this pathology.

Le cancer de la prostate est le plus fréquent et représente la troisième cause de mortalité par cancer chez l’homme en France. Outre la théorie de la génétique dans le développement des cancers, l’implication des modifications épigénétiques au cours de la carcinogenèse prostatique ne fait plus aucun doute. L’épigénétique est définie comme l’étude des modifications de l’expression des gènes qui sont transmissibles lors de la mitose et/ou la méiose, mais ne découlent pas de modifications dans la séquence de l’ADN (Berger et al., 2009). Autrement dit, les altérations épigénétiques sont capables de moduler le niveau d’expression des gènes en agissant sur la chromatine. Depuis quelques années, l’attention des chercheurs porte de plus en plus sur l’implication des modifications épigénétiques dans la carcinogenèse prostatique. Une activité anormale d’un ou de plusieurs acteurs épigénétiques entraîne des modifications de profil d’expression des gènes pouvant être associées à la carcinogenèse. Dans le cancer de la prostate, on retrouve ainsi une forte activité des ADN méthyltransférases (DNMT1) et une dérégulation des modificateurs d’histones, notamment de l’histone méthyle transférase EZH2, associée à la répression de la transcription de certains gènes (Gillio-Tos et al., 2012; Gravina et al., 2013; Koh et al., 2011; Yu et al., 2007). En plus de ces facteurs épigénétiques, des facteurs environnementaux et plus particulièrement la nutrition intervient dans les processus de carcinogenèse. La thématique du laboratoire a été longtemps axée sur la nutrition et le développement des cancers. En effet, des études sur les effets préventifs des phyto-oestrogènes du soja dans la carcinogenèse prostatique ont largement été abordées (Adjakly et al., 2011). Les phyto-oestrogènes du soja ont montré leur effet déméthylant sur les promoteurs des gènes suppresseurs de tumeurs. L’hypothèse de ces travaux a été établie du fait de la faible incidence du cancer de la prostate retrouvée dans les pays asiatiques où une forte consommation de soja a été remarquée. Les micronutriments contenus dans le soja auraient ainsi la capacité de moduler les modifications épigénétiques dans le cancer de la prostate (Vardi et al., 2010). Dans le cadre de cette thèse, nous nous sommes focalisés sur les modifications des histones notamment la méthylation des histones qui reste peu étudiée dans le cancer de la prostate. Premièrement, nous avons étudié le rôle de la triméthylation de la lysine 27 de l’histone H3 (H3K27me3) dans l’implication et la progression du cancer de la prostate. La deuxième partie a consisté en l’identification de nouveaux marqueurs pronostiques et épigénétiques liés à la méthylation des histones afin d’établir un profil épigénétiques des tumeurs prostatiques. Nous avons également évoqué l’effet des phyto-oestrogènes du soja sur la modulation des modifications épigénétiques dans le cancer de la prostate et leurs rôles protecteurs dans le développement tumoral. / Prostate cancer is the most common and represents the third leading cause of cancer death in men in France. In addition to the theory of genetics in the development of cancers, the involvement of epigenetic changes during prostate carcinogenesis is no longer in doubt. Epigenetics is defined as the study of changes in the expression of genes that are transmissible during mitosis and / or meiosis, but do not result from modifications in the DNA sequence. In other words, epigenetic alterations are able to modulate the level of gene expression by acting on chromatin. In recent years, the attention of researchers has increasingly focused on the involvement of epigenetic modifications in prostatic carcinogenesis. Abnormal activity of one or more epigenetic actors leads to changes in gene expression patterns that may be associated with carcinogenesis. In prostate cancer, there is thus a strong activity of DNA methyltransferases (DNMT1) and a deregulation of histone modifiers, especially histone methyl transferase EZH2, associated with the repression of the transcription of certain genes (Gillio- Tos et al., 2012, Gravina et al., 2013, Koh et al., 2011, Yu et al., 2007). In addition to these epigenetic factors, environmental factors and more particularly nutrition is involved in the processes of carcinogenesis. The work of the laboratory has long focused on nutrition and cancer development. Indeed, studies on the preventive effects of soy phytoestrogens in prostate carcinogenesis have been widely discussed (Adjakly et al., 2011). The soy phytoestrogens showed their demethylating effect on the promoters of tumor suppressor genes. The hypothesis of this work was established because of the low incidence of prostate cancer found in Asian countries where high consumption of soybeans was noticed. The micronutrients contained in soy thus have the ability to modulate epigenetic modifications in prostate cancer (Vardi et al., 2010). In this thesis, we focused on histone modifications including histone methylation, which remains poorly studied in prostate cancer. First, we investigated the role of trimethylation of histone H3 lysine 27 (H3K27me3) in the involvement and progression of prostate cancer. The second part consisted of the identification of new prognostic and epigenetic markers related to histone methylation in order to establish an epigenetic profile of prostate tumors. We also discussed the effect of soy phytoestrogens on the modulation of epigenetic changes in prostate cancer and their protective roles in tumor development.

Prostate cancer is the most common non-skin cancer in America, and the most commonly diagnosed cancer among males. When metastatic, the disease can ultimately be incurable. Consequently, alternative strategies to current treatments are sought, especially in the area of immunotherapy. Vaccine immunotherapy using a specific antigen, such as prostate specific antigen (PSA) seeks to stimulate both the innate and adaptive immune system to destroy tumor cells in the body. PSA is an ideal target antigen given that it has a narrow distribution in tissues and is expressed in virtually all prostate cancer cases. An adenovirus encoding for PSA (Ad-PSA) can be used to deliver the genomic data encoding for PSA production and secretion to the target cell. This type of viral gene delivery system has already been shown to have the potential to stimulate anti-tumor activity. To enhance this activity and increase transfection efficiency, we proposed the combination of a viral system with a non-viral system, in the form of a cationic polymer such as poly(ethyl)enimine (PEI) or chitosan. Cationic polymers complex with the negatively charged adenovirus to form nanoparticles that can be used in gene delivery. Delivery in nanoparticle form can give enhanced uptake by the antigen-presenting cells necessary to initiate the targeted immune response. To further augment this response, previous research has shown that CpG sequences act as an adjuvant to enhance the efficacy of the Ad-PSA vaccines' tumor protection. CpG delivered in particulate form has also been shown to be more effective than delivery in solution. The objective of this proposal was to test the hypothesis that co-delivery of this targeted viral/non-viral gene delivery system will enhance tumor protection in a mouse model of prostate cancer. Using the OVA model antigen system, we found that the adenovirus encoding OVA (AdOVA), coupled with the polymer PEI, enhanced tumor protection in vivo compared to AdOVA alone. To move towards our therapeutic model, these experiments were repeated using chitosan as the cationic polymer carrier, delivering AdOVA, and incorporating CpG into some particles. In this set of experiments, we found that AdOVA + CpG gave the best tumor protection in challenge studies. AdOVA + chitosan + CpG showed a decrease in protective levels and numbers of antigen-specific immune cells. Further experiments focused on elucidating the mechanisms by which chitosan and CpG modulate the immune response. Using the therapeutic AdPSA model, chitosan was not found to enhance tumor protection or numbers of antigen-specific immune cells. Additional experiments found that this depression was not due to problems with viral infectivity or secretion due to chitosan complexation. A series of kinetics studies were performed which showed that peak levels of effector T cells were present 14 days later in AdPSA + CpG immunized mice than in AdPSA alone. This delayed effect may explain the increased levels of protection in AdPSA + CpG mice, and be useful in future vaccine design concerning the timing of peak response.

The interplay between cancer cells and the extracellular matrix (ECM) remains a critical regulator of both normal tissue organization and cancer cell invasion. Proteins that function as ECM receptors function to link the cell with the ECM. Abberations in either the structure of the ECM or the expression of ECM receptors leads to disrupted interaction and downstream signaling effects. Dystroglyan (DG) is an ECM receptor that is expressed in a variety of tissue types and functions to mediate sarcolemma stability, epithelial polarity, and is critical in the early formation of basement membranes. However, DG has primarily been studied in muscle where loss of its function is linked to a host of muscular dystrophies. In the epithelium, the role of DG remains enigmatic. While DG has repeatedly been shown to lose function during cancer development and progression, the mechanism and functional consequence of its loss are currently unknown. In order to increase our understanding of DG in cancer development, we analyzed its expression and glycosylation, a functional requirement for DG, in a range of prostate cancer cell lines. Previous work has shown DG to be downregulated in prostate cancer, but the mechanism by which this occurs has remained largely unclear. We found that DG expression is maintained while its glycosylation was heterogeneous in the cell lines. Further investigation revealed that lines with hypoglycosylated DG strongly associated with the loss of expression of the glycosyltransferase LARGE2. Further this enzyme is frequently downregulated in human cancers and appears to serve as a required enzyme in DG glycosylation within prostate epithelium. This is the first work to demonstrate the functional requirement of LARGE2 for DG, and the only work to implicate loss-of-function of LARGE2 in cancer progression. To determine whether loss of LARGE2 is found in other tumor types, we analyzed human clear cell renal cell carcinoma (ccRCC) samples by immunohistochemistry and via in silico analysis with the Cancer Genome Atlas (TCGA). Our work demonstrated a frequent and significant downregulation of LARGE2 expression and its association with DG hypoglycosylation. Additionally, we found the loss of LARGE2 strongly associated with increased mortality. Thus, we again demonstrated a functional requirement of LARGE2 but also found a clinical correlate with increased mortality. Finally, we examined the functional outcome of DG hypoglycosylation or loss of expression in both a mouse model of prostate cancer and a variety of cell lines models. We found that while loss of DG expression does not increase prostate cancer growth or metastasis in one model of cancer, loss of its glycosylation does seem to mediate downstream metabolic changes within cells. The mechanism for this change remains unclear. In summary, these studies have contributed to our understanding of DG glycosylation and function in both prostate and renal carcinoma. Additionally, we have shown a novel mechanism by which DG glycosylation is lost with downregulation of LARGE2 expression. Finally, while we were unable to demonstrate a clear mechanism by which signaling changes arose, we were able to demonstrate a strong correlation between DG hypoglycosylation and increased mortality in ccRCC. These insights could be used to improve treatment of multiple cancer types as our understanding of DG function continues to improve.

C-Terminal glycine-extended prohormones are enzymatically converted to á-amidated peptides, by peptidylglycine á-amidating monooxygenase (PAM). PAM is a bifunctional enzyme with two catalytic domains: peptidylglycine á-hydroxylating monooxygenase (PHM) and peptidylglycine peptidylglycineaminoglycolate lyase (PAL). PAM has a significant role in the proliferation of androgen-independent prostate cancer. Thus, the inhibition of PAM could halt cancer growth. Hippurate and hippurate analogs were used as lead compounds for developing inhibitors for PAM. The hippurate analogs exhibiting the highest affinity to PAM (lowest inhibition constant) did inhibit the growth of human androgen-independent prostate cancer DU 145 cells.