Immuno-oncology of human prostate cancer : phenotypical characterization and study of the tumor-derived, androgen-regulated immunosuppressive microenvironment

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.

Análisis del gen adra2a receptor alfa 2a adrenergico en pacientes con trastorno de hiperactividad y déficit de atención

El trastorno de hiperactividad y déficit de atención (THDA), es definido clínicamente como una alteración en el comportamiento, caracterizada por inatención, hiperactividad e impulsividad. Estos aspectos son clasificados en tres subtipos, que son: Inatento, hiperactivo impulsivo y mixto. Clínicamente se describe un espectro amplio que incluye desordenes académicos, trastornos de aprendizaje, déficit cognitivo, trastornos de conducta, personalidad antisocial, pobres relaciones interpersonales y aumento de la ansiedad, que pueden continuar hasta la adultez. A nivel global se ha estimado una prevalencia entre el 1% y el 22%, con amplias variaciones, dadas por la edad, procedencia y características sociales. En Colombia, se han realizado estudios en Bogotá y Antioquia, que han permitido establecer una prevalencia del 5% y 15%, respectivamente. La causa específica no ha sido totalmente esclarecida, sin embargo se ha calculado una heredabilidad cercana al 80% en algunas poblaciones, demostrando el papel fundamental de la genética en la etiología de la enfermedad. Los factores genéticos involucrados se relacionan con cambios neuroquímicos de los sistemas dopaminérgicos, serotoninérgicos y noradrenérgicos, particularmente en los sistemas frontales subcorticales, corteza cerebral prefrontal, en las regiones ventral, medial, dorsolateral y la porción anterior del cíngulo. Basados en los datos de estudios previos que sugieren una herencia poligénica multifactorial, se han realizado esfuerzos continuos en la búsqueda de genes candidatos, a través de diferentes estrategias. Particularmente los receptores Alfa 2 adrenérgicos, se encuentran en la corteza cerebral, cumpliendo funciones de asociación, memoria y es el sitio de acción de fármacos utilizados comúnmente en el tratamiento de este trastorno, siendo esta la principal evidencia de la asociación de este receptor con el desarrollo del THDA. Hasta la fecha se han descrito más de 80 polimorfismos en el gen (ADRA2A), algunos de los cuales se han asociado con la entidad. Sin embargo, los resultados son controversiales y varían según la metodología diagnóstica empleada y la población estudiada, antecedentes y comorbilidades. Este trabajo pretende establecer si las variaciones en la secuencia codificante del gen ADRA2A, podrían relacionarse con el fenotipo del Trastorno de Hiperactividad y el Déficit de Atención.

Oestrogenic and androgenic activity of triclosan in breast cancer cells

As a consequence of its widespread use as an antimicrobial agent in consumer goods, triclosan has become distributed ubiquitously across the ecosystem, and recent reports that it can cause endocrine disruption in aquatic species has increased concern. It is reported here that triclosan possesses intrinsic oestrogenic and androgenic activity in a range of assays in vitro which could provide some explanation for the endocrine disrupting properties described in aquatic populations. In terms of oestrogenic activity, triclosan displaced [H-3]oestradiol from oestrogen receptors (ER) of MCF7 human breast cancer cells and from recombinant human ER alpha/ER beta. Triclosan at 10(-5) M completely inhibited the induction of the oestrogen-responsive ERE-CAT reporter gene in MCF7 cells by 10(-10) M 17 beta-oestradiol and the stimulation of growth of MCF7 human breast cancer cells by 10(-10) M 17 beta-oestradiol. On its own, 1 mu M triclosan increased the growth of MCF7 cells over 21 days. Triclosan also had androgenic activity. It displaced [H-3]testosterone from binding to the ligand binding domain of the rat androgen receptor (AR). Triclosan was able to inhibit the induction of the androgen-responsive LTR-CAT reporter gene in S115 mouse mammary tumour cells by 10(-9) M testosterone and in T47D human breast cancer cells by 10(-8) M testosterone at concentrations of 10(-7) M and 10(-6) M, respectively. Triclosan at 2 x 10(-5) M antagonized the stimulation of the growth of S115+A mouse mammary tumour cells by 10(-9) M testosterone. The finding that triclosan has oestrogenic and androgenic activity warrants further investigation in relation to both endocrine disruption of aquatic wildlife and any possible impact on human health. Copyright (C) 2007 John Wiley & Sons, Ltd.

Differential crosstalk between estrogen receptor (ER) alpha and ER beta and the thyroid hormone receptor isoforms results in flexible regulation of the consensus ERE

Crosstalk between nuclear receptors is important for conversion of external and internal stimuli to a physiologically meaningful response by cells. Previous studies from this laboratory have demonstrated crosstalk between the estrogen (ER) and thyroid hormone receptors (TR) on two estrogen responsive physiological promoters, the preproenkephalin and oxytocin receptor gene promoter. Since ERa and ERb are isoforms possessing overlapping and distinct transactivation properties, we hypothesized that the interaction of ERa and b with the various TR isoforms would not be equivalent. To explore this hypothesis, the consensus estrogen response element (ERE)derived from the Xenopus vitellogenin gene is used to investigate the differences in interaction between ERa and b isoforms and the different TR isoforms in fibroblast cells. Both the ER isoforms transactivate from the consensus ERE, though ERa transactivates to a greater extent than ERb. Although neither of the TRb isoforms have an effect on ERa transactivation from the consensus ERE, the liganded TRa1 inhibits the ERa transactivation from the consensus ERE. In contrast, the liganded TRa1 facilitates ERb-mediated transactivation. The crosstalk between the TRb isoforms with the ERa isoform, on the consensus ERE, is different from that with the ERb isoform. The use of a TRa1 mutant, which is unable to bind DNA, abolishes the ability of the TRa1 isoform to interact with either of the ER isoforms. These differences in nuclear receptor crosstalk reveal an important functional difference between isoforms, which provides a novel mechanism for neuroendocrine integration.

Ligand induced interaction of thyroid hormone receptor beta with its coregulators

Thyroid hormones exert most of their physiological effects through two thyroid hormone receptor (TR) subtypes, TR alpha and TR beta, which associate with many transcriptional coregulators to mediate activation or repression of target genes. The search for selective TR beta ligands has been stimulated by the finding that several pharmacological actions mediated by TR beta might be beneficial in medical conditions such as obesity, hypercholesterolemia and diabetes. Here, we present a new methodology which employs surface plasmon resonance to investigate the interactions between TR beta ligand binding domain (LBD) complexes and peptides derived from the nuclear receptor interaction motifs of two of its coregulators, SRC2 and DAX1. The effect of several TR beta ligands, including the TR beta selective agonist GC-I and the TR beta selective antagonist NH-3, were investigated. We also determined the kinetic rate constants for the interaction of TR beta-T3 with both coregulators, and accessed the thermodynamic parameters for the interaction with DAX1. Our findings Suggest that flexibility plays an important role in the interaction between the receptor and its coregulators. and point out important aspects of experimental design that should be addressed when using TR beta LBD and its agonists. Furthermore, the methodology described here may be useful for the identification of new TR beta ligands. (C) 2008 Elsevier Ltd. All rights reserved.

Analysis of Agonist and Antagonist Effects on Thyroid Hormone Receptor Conformation by Hydrogen/Deuterium Exchange

Thyroid hormone receptors (TRs) are ligand-gated transcription factors with critical roles in development and metabolism. Although x-ray structures of TR ligand-binding domains (LBDs) with agonists are available, comparable structures without ligand (apo-TR) or with antagonists are not. It remains important to understand apo-LBD conformation and the way that it rearranges with ligands to develop better TR pharmaceuticals. In this study, we conducted hydrogen/deuterium exchange on TR LBDs with or without agonist (T(3)) or antagonist (NH(3)). Both ligands reduce deuterium incorporation into LBD amide hydrogens, implying tighter overall folding of the domain. As predicted, mass spectroscopic analysis of individual proteolytic peptides after hydrogen/deuterium exchange reveals that ligand increases the degree of solvent protection of regions close to the buried ligand-binding pocket. However, there is also extensive ligand protection of other regions, including the dimer surface at H10-H11, providing evidence for allosteric communication between the ligand-binding pocket and distant interaction surfaces. Surprisingly, C-terminal activation helix H12, which is known to alter position with ligand, remains relatively protected from solvent in all conditions suggesting that it is packed against the LBD irrespective of the presence or type of ligand. T(3), but not NH(3), increases accessibility of the upper part of H3-H5 to solvent, and we propose that TR H12 interacts with this region in apo-TR and that this interaction is blocked by T(3) but not NH(3.) We present data from site-directed mutagenesis experiments and molecular dynamics simulations that lend support to this structural model of apo-TR and its ligand-dependent conformational changes. (Molecular Endocrinology 25: 15-31, 2011)

Expressão gênica de bcl2, receptor de estradiol e receptores de progesterona A e B em endométrio eutópico e ectópico de pacientes inférteis sem e com endometriose

A endometriose caracteriza-se pela presença de tecido endometriótico – glândulas e estroma – fora da cavidade uterina. A doença acomete superfícies peritoniais da pélvis, ovários e septo rectovaginal, sendo mais comum a endometriose pélvica. Embora os dados sobre a prevalência exata de endometriose sejam incertos, as evidências indicam uma prevalência em torno de 5 a 10% das mulheres em idade reprodutiva. Dor, dismenorréia, dispareunia e infertilidade são sintomas freqüentes da doença. Além de apresentar dependência aos hormônios sexuais, a endometriose está associada a um conjunto de desordens de diferentes etiologias, sendo considerada uma doença multifatorial de caráter genético e imune. Neste sentido, a carência de respostas imunológicas adequadas, aliada a um provável desequilíbrio entre os processos de proliferação e apoptose celular, pode ter um papel importante na instalação e manutenção das lesões endometrióticas. O objetivo deste trabalho foi caracterizar a expressão de genes relacionados aos processos de proliferação e diferenciação celular em implantes endometrióticos e endométrio eutópico de mulheres inférteis sem e com endometriose, através da análise semiquantitativa por RT-PCR. Foram estudadas 34 pacientes consultando por infertilidade e submetidas à laparoscopia diagnóstica. Quinze pacientes (31,7 ± 1,5 anos) apresentavam endometriose e 19 (32,9 ± 0,9 anos) foram consideradas controles inférteis sem endometriose. Os dois grupos apresentaram IMC e SHBG similares. As biópsias de endométrio foram realizadas, em fase folicular, nas pacientes do grupo controle (C) e nas pacientes com endometriose (endométrio eutópico (EUT) e ectópico (ECT)). A presença do mRNA para os receptores de estrogênio e progesterona detectada nos 3 grupos estudados, sustenta a existência de sensibilidade tecidual local aos hormônios sexuais. Nas condições experimentais do presente estudo não foi observada diferença estatística na expressão gênica de bcl2 entre os grupos. A expressão do gene do ER endometrial também foi similar entre os grupos controle, eutópico e ectópico. Contudo, o endométrio ectópico apresentou níveis de mRNA mais elevados para PR-A (0,84  0,02 UA) em relação aos grupos controle e eutópico (0,60  0,04 UA e 0,63  0,04 UA; p < 0,05). A expressão gênica do PR-B não foi estatisticamente diferente entre os grupos do presente trabalho. Porém, houve forte correlação negativa entre a expressão dos genes bcl2 e PR-B nas amostras de endométrio sem e com endometriose (r = - 0,795 e p = 0,018), sugerindo que um papel anti-proliferativo do PR-B possa ser operativo neste modelo de estudo.

Polymorphisms in the AR and PSA Genes as Markers of Susceptibility and Aggressiveness in Prostate Cancer

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Tissue changes in senescent gerbil prostate after hormone deprivation leads to acquisition of androgen insensitivity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Progesterone (PR), Oestrogen (ER-alpha and ER-beta) and Oxytocin (OTR) Gene Expression in the Oviduct and Uterus of Pregnant and Non-pregnant Bitches

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

NF-kB overexpression and decreased immunoexpression of AR in the muscular layer is related to structural damages and apoptosis in cimetidine-treated rat vas deferens

Background: Cimetidine, histamine H2 receptors antagonist, has caused adverse effects on the male hormones and reproductive tract due to its antiandrogenic effect. In the testes, peritubular myoid cells and muscle vascular cells death has been associated to seminiferous tubules and testicular microvascularization damages, respectively. Either androgen or histamine H2 receptors have been detected in the mucosa and smooth muscular layer of vas deferens. Thus, the effect of cimetidine on this androgen and histamine-dependent muscular duct was morphologically evaluated.Methods: The animals from cimetidine group (CMTG; n=5) received intraperitoneal injections of 100 mg/kg b.w. of cimetidine for 50 days; the control group (CG) received saline solution. The distal portions of vas deferens were fixed in formaldehyde and embedded in paraffin. Massońs trichrome-stained sections were subjected to morphological and the following morphometrical analyzes: epithelial perimeter and area of the smooth muscular layer. TUNEL (Terminal deoxynucleotidyl-transferase mediated dUTP Nick End Labeling) method, NF-kB (nuclear factor kappa B) and AR (androgen receptors) immunohistochemical detection were also carried out. The birefringent collagen of the muscular layer was quantified in picrosirius red-stained sections under polarized light. The muscular layer was also evaluated under Transmission Electron Microscopy (TEM).Results: In CMTG, the mucosa of vas deferens was intensely folded; the epithelial cells showed numerous pyknotic nuclei and the epithelial perimeter and the area of the muscular layer decreased significantly. Numerous TUNEL-labeled nuclei were found either in the epithelial cells, mainly basal cells, or in the smooth muscle cells which also showed typical features of apoptosis under TEM. While an enhanced NF-kB immunoexpression was found in the cytoplasm of muscle cells, a weak AR immunolabeling was detected in these cells. In CMTG, no significant difference was observed in the birefringent collagen content of the muscular layer in comparison to CG.Conclusions: Cimetidine induces significant damages in the epithelium; a possible antiandrogenic effect on the basal cells turnover should be considered. The cimetidine-induced muscle cells apoptosis confirms the susceptibility of these cells to this drug. The parallelism between enhanced cytoplasmic NF-kB immunolabeling in the damaged muscular tissue and muscle cell apoptosis suggests that this drug may avoid the translocation of NF-kB to the nucleus and interfere in the control of NF-kB-mediated smooth muscle cell apoptosis. The decreased immunoexpression of ARs verified in the damaged muscular tissue reinforces this possibility. © 2013 Koshimizu et al.; licensee BioMed Central Ltd.

Androgen deprivation from pre-puberty to peripuberty interferes in proteins expression in pubertal and adult rat epididymis

Few studies have focused on experimental testosterone deprivation in immature animals. Therefore, this study used sexually immature rats aiming to evaluate the testes and epididymis histology and proteins expression in these organs on PND50 and 75, after premature antiandrogen exposure, from PND21 to 44. Although the androgen deprivation from pre-puberty up to peripuberty did not alter the histological organization of the testes and epididymis either at puberty or at adulthood, the treatment impaired the expression of specific proteins in epididymal tissue at puberty and adulthood (androgen receptor, calmodulin, Rab11A). These changes may be related to impaired epididymal function, sperm quality and fertility capacity as observed in a previous study. Further studies are necessary to better investigate the molecular mechanisms involved in the impairment on reproductive competence of male rats after precocious hormonal injury. © 2013 Elsevier Inc.

Expressão dos transcritos do receptor de LH em células da granulosa de fêmeas nelore submetidas ou não à superestimulação ovariana

Pós-graduação em Medicina Veterinária - FMVZ

Thyroid Hormone Stimulates the Proliferation of Sertoli Cells and Single Type A Spermatogonia in Adult Zebrafish (Danio rerio) Testis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Avaliação da expressão proteica de PTEN, MDM2, P53 e AR em lesões proliferativas da próstata canina

Pós-graduação em Medicina Veterinária - FCAV