Sabe-se que TRAIL é capaz de matar células tumorais de forma seletiva e que TRAIL tem sua expressão reduzida em diversos tumores, porém pouco se sabe sobre os mecanismos responsáveis pela sua inibição. Tendo em vista que a expressão de TRAIL pode ser regulada pelo Ácido Retinóico; que PRAME é capaz de inibir a via do ácido retinóico através da proteína EZH2 e que nós observamos anteriormente que a expressão de TRAIL esta diminuída em pacientes com LMC, nós decidimos investigar a associação entre PRAME, EZH2 e TRAIL na LMC. Nós demonstramos que PRAME, mas não EZH2, tem sua expressão aumentada em células BCR-ABL+ e sua expressão está associada com a progressão da LMC. Alem disto, existe uma correlação positiva entre PRAME e BCR-ABL e negativa entre PRAME e TRAIL nestes pacientes. A inibição da expressão de PRAME ou EZH2 por RNAi induziu um aumento da expressão de TRAIL. Estes dados revelam um novo mecanismo de regulação responsável por diminuir a expressão de TRAIL, e geram novos possíveis alvos para a terapia da LMC e, possivelmente, também para outros tumores. / TRAIL was shown to selectively kill tumor cells. Not surprisingly, TRAIL is down-regulated in a variety of tumor cells, but the mechanism responsible for TRAIL inhibition remains elusive. Because TRAIL can be regulate by retinoic acid; PRAME was shown to inhibit transcription of retinoic acid receptor target genes through the polycomb protein EZH2; and we have found that TRAIL is inversely correlated with BCR-ABL in CML patients, we decided to investigate the association of PRAME, EZH2 and TRAIL in BCR-ABL-positive leukemia. Here, we demonstrate that PRAME, but not EZH2, is up-regulated in BCR-ABL cells and is associated with the progression of disease in CML patients. In addition, PRAME expression is positively correlated with BCR-ABL and negatively with TRAIL in these patients. Importantly, knocking down of PRAME or EZH2 by RNA interference restores TRAIL expression. Our data reveal a novel regulatory mechanism responsible for lowering TRAIL expression and provide the basis of alternative targets for combined therapeutic strategies for CML.

Prostate cancer (PCa) is the most common cancer in American men. Although initial androgen deprivation therapy (ADT) confers a five year survival rate of 99%, the relapse of metastatic and drug resistant PCa (CRPC- Castration-Resistant PCa) continues to account for most deaths. How certain PCa cells develop into CRPC is the key question in the field. In addressing it, attention has focused on epigenetic factors that contribute to CRPC development. Herein we investigated the role and regulation of histone demethylase PHF8 during PCa neuroendocrine differentiation (NED) and progression into CRPC. We utilized bioinformatic analyses and biochemical approaches in PCa/CRPC cell line and mouse models to unravel the following results: First, we discovered that PHF8 post-transcriptionally clusters with cell cycle genes during NED and into CRPC via an AR/MYC/miR-22 regulatory axis. We showed that this axis is dysregulated in CRPC cells to allow enhanced cell proliferation and resistance to the clinical AR antagonist drug Xtandi® (enzalutamide). Second, we revealed that PHF8 is necessary for hypoxia induced NED by demethylating repressive H3K9me2 and H3K27me2, above maintaining active H3K4me3 on select NED genes. Importantly, we unveiled that PHF8 sustains HIF1α expression in CRPC cells via a regulatory role associated with full length AR. Third, we recapitulated the role of PHF8 in vivo by excising its floxed allele in the prostate of TRAMP mice -Transgenic Adenocarcinoma of the Mouse Prostate. We observed that KO of Phf8 lowered tumor burden in part by sustaining Ezh2 expression during NED transition into CRPC. In conclusion, our data implicates PHF8 in multiple oncogenic roles and regulations during PCa NED into CRPC. Our results lay a foundation for understanding the dynamics of histone modifying enzymes during PCa progression and hint at designing small molecule inhibitors against PHF8 as a novel CRPC therapeutic target.

La formation des trois feuillets embryonnaires primordiaux et leur régionalisation selon les axes embryonnaires sont des étapes cruciales au cours du développement précoce. Dans ce contexte, nous avons montré que XDSCR6, un inducteur du mésoderme et des axes embryonnaires qui présente des propriétés dorsalisantes, interagit physiquement et fonctionnellement avec le facteur de transcription XSTAT3. Au cours des étapes précoces du développement, XSTAT3 est active pendant la gastrulation et l'activation anormale de cette protéine dans la région dorsale induit la ventralisation des tissus embryonnaires. Par ailleurs, nous avons montré que XDSCR6 et XSTAT3 présentent des rôles antagonistes in vivo au cours de la mise en place des axes embryonnaires. Cet antagonisme peut être expliqué par le fait que XDSCR6 régule négativement l'activité transcriptionnelle de XSTAT3 en modulant sa méthylation sur les résidus lysine. L'ensemble de nos résultats a permis de déterminer l'importance cruciale de cette modification post-traductionnelle dans les propriétés ventralisantes de XSTAT3. Par ailleurs, nous avons montré que la méthyltransférase XEZH2 méthyle et active XSTAT3 in vivo. Des travaux antérieurs de l'équipe avaient montré que les propriétés dorsalisantes de XDSCR6 reposent sur sa capacité à inhiber l'activité épigénétique répressive de XEZH2 sur les gènes du mésoderme dorsal. Ainsi, nos travaux suggèrent que XDSCR6 est un modulateur transcriptionnel situé à l'interface entre certains régulateurs chromatiniens et des facteurs de transcription pendant la mise en place des axes embryonnaires. / One of the most challenging questions in developmental biology is to understand how a totipotent zygote differentiates into an organism containing all cell lineages. The formation of the three germ layers and the establishment of embryonic axis are fundamental events during early development. In this context, we demonstrated that XDSCR6, a mesoderm and embryonic axis inducer that exhibits dorsalizing properties, physically and functionally interacts with the transcriptional factor XSTAT3. During early development, XSTAT3 is active throughout gastrulation step and its abnormal activation in dorsal region leads to embryonic tissues ventralization. Furthermore, we showed that XDSCR6 and XSTAT3 have antagonistic roles in vivo during axis formation. This antagonism can be explained by the fact that XDSCR6 negatively regulates the transcriptional activity of XSTAT3 by interfering with its methylation on lysine residues. Moreover, this post-translational modification plays a crucial role in the ventralization abilities of XSTAT3. In a previous study, it has been shown that XDSCR6 negatively regulates the XEZH2 repressive epigenetic activity on dorsal mesoderm genes. Thus, we propose that XDSCR6 is a transcriptional modulator acting between epigenetic regulators and transcriptional factors during embryonic axis formation.

<p>Prostate cancer is (PCa) the second leading cause of cancer death in males in the United State, with 174,650 new cases and 31,620 deaths estimated in 2019. Polo-like kinase 1 (PLK1) has been postulated to have a pro-tumorigenesis function, besides its critical role in regulation of cell cycle, and to be overexpressed in various types of human cancer, including prostate cancer (PCa). However, our understanding remains unclear regarding the pro-tumor properties of PLK1 partially due to a lack of proper animal model. Integrating our recently generated prostate-specific PLK1 knock-in genetically engineered mouse model (GEM) and the transcriptome data of human PCa patients, we identify an oncogenic role of PLK1 in the prostate adenocarcinoma progression, castration resistance and metastatic dissemination. To elucidate the underlying mechanism, we investigate the link between PLK1 and tumor microenvironment in PCa using the transgenic mouse model, and find that PLK1overexpression enable the macrophages polarization towards M2 phenotype via driving the activation of IL4/IL13/STAT6 pathway. These findings first validates PLK1 as a critical oncogene closely associated with PCa progression in vivo, and uncover a novel function of PLK1 to facilitate IL4/STAT6 signaling and M2 macrophage polarization. Importantly, these findings suggest an efficient therapeutic strategy targeting STAT6 for treatment of advanced PCa which usually possessing a high level of PLK1 expression. To further explore the molecular mechanism underlying PLK1-induced PCa progression and resistance to therapy, we turned our eyes to epigenetic modifications. It has been documented that epigenetic deregulation such as histone modification and DNA methylation contributes to PCa initiation and progression. Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of Polycomb-repressive complex 2 (PRC2), plays a critical role in repressing gene expression by tri-methylation of histone 3 at lysine 27 (H3K27me3). Emerging data have demonstrated that there is a link between EZH2 and oncogenesis as EZH2-mediated methylation acts as an important factor in epigenetic silencing of tumor suppressor genes in cancer. Expression of EZH2 is often upregulated in castration-resistant prestate cancer (CRPC), thus EZH2 has been proposed as a target for CRPC. Importantly, it has been demonstrated that EZH2 becomes hyperphosphorylated in CPRC cells. Further, it has been shown that the oncogenic function of EZH2 is usually regulated by the post-translational modifications. PLK1 acting as a serine/threonine kinase to regulate multiple signaling pathways in human cancer, however, whether PLK1 is involved in EZH2 phosphorylation is not known. Herein, we show that Plk1 physically interacts with EZH2 and negatively regulates H3K27 trimethylation (H3K27me3). Furthermore, Plk1 can phosphorylate EZH2 at T144, and Plk1-mediated phosphorylation of EZH2 is involved in inhibiting EZH2 activity toward H3K27me3. More importantly, EZH2 phosphorylation by Plk1 is inhibitory for PRC2-mediated gene repression but required for transcriptional activation toward oncogenesis. Finally, by combination with Plk1 inhibitor BI2536, we show a robust sensitization of EZH2 inhibitors in CRPC cell lines, as well as in CRPC xenograft tumors. Our findings provide a new mechanism to define the oncogenic activity of EZH2 and suggest that inhibition of Plk1-mediated EZH2 activity may provide a promising therapeutic approach for CRPC.</p>

Polycomb repressive complex 2 (PRC2) catalyses the methylation of ‘Lys-27’ of histone H3, leading to transcriptional repression of target genes through its catalytic subunit Enhancer of zeste homolog 1/2 (EZH1/2). PRC2 functions as a critical regulator of stem cells in mouse embryonic and adult tissues. However, the role of PRC2 in human skin remains largely unknown. This study investigated the role of PRC2 in human epidermal homeostasis and hair growth. The expression of EZH2 was elevated in differentiating suprabasal layers of the human epidermis. Consistently, EZH1/2 expression and enzymatic activity was upregulated in differentiating primary human keratinocytes (NHEKs) in vitro. Inhibition of EZH2 and Embryonic ectoderm development (EED) in NHEKs stimulated the expression of differentiation-associated genes, therefore leading to their premature differentiation; while inhibition of EZH1/2 reduced cell proliferation and promoted apoptosis. Silencing of EZH2 in NHEKs induced complex changes in gene expression programmes, including the upregulation of terminal differentiation genes, such as Filaggrin. EZH2 expression was downregulated in aged keratinocytes accompanied with upregulation of senescence-associated genes, p16INK4A and p19INK4D, suggesting EZH2 involvement in epidermal aging. In human anagen hair follicle (HF), EZH2 was detected in stem and progenitor cells; and hair matrix keratinocytes. Silencing EZH2 in HFs accelerated anagen-catagen transition and retarded hair growth accompanied by decreased proliferation and increased apoptosis. Silencing EZH2 in outer root sheath keratinocytes resulted in upregulation of p14ARF and K15, suggesting EZH2 involvement in regulating proliferation and stem cell activity. Thus, this study demonstrates that PRC2-mediated repression is crucial for epidermal homeostasis and hair growth. Modulating the activities of PRC2 in skin might offer a new therapeutic approach for disorders of epidermal differentiation and hair growth.

Major Histocompatibility Complex Class-II (MHC-II) molecules are critical regulators of adaptive immunity that present extracellular antigens required to activate CD4+ T cells. MHC-II are regulated at the level of transcription by master regulator, the Class II Transactivator (CIITA), whose association with the MHC-II promoter is necessary to initiate transcription. Recently, much research focused on novel mechanisms of transcriptional regulation of critical genes like MHC-II and CIITA; findings that the macromolecular complex of the 26S-proteasome is involved in transcription have been perhaps the most exciting as they impart novel functions to a well studied system. Proteasome is a multi-subunit complex composed of a 20S-core particle capped by a 19S-regulatory particle. The 19S contains six ATPases which are required for transcription initiation and elongation. We demonstrate that 19S ATPase-S6a inducibly associates with CIITA promoters. Decreased expression of S6a negatively impacts recruitment of the transcription factors STAT-1 and IRF-1 to the CIITA due to significant loss in histone H3 and H4 acetylation. S6a is robustly recruited to CIITA coding regions, where S6a binding coordinates with that of RNA polymerase II. RNAi mediated S6a knockdown significantly diminishes recruitment of Pol II and P-TEF-b components to CIITA coding regions, indicating S6a plays important roles in transcriptional elongation. Our research is focused on the ways in which accessibility to and transcription of DNA is regulated. While cancers are frequently linked to dysregulated gene expression, contribution of epigenetics to cancers remains unknown. To achieve metastatic ability, tumors alter gene expression to escape host immunosurveilance. MHC-II and CIITA expression are significantly downregulated in highly metastatic MDA-MB-435 breast cancer cells. This suppression correlates with elevated levels of the silencing modification H3K27me3 at CIITA and a significant reduction in Pol II recruitment. We observe elevated binding of the histone methyltransferase to CIITApIV and demonstrate this enzyme is a master regulator of CIITA gene expression. EZH2 knockdown results in significant increases in CIITA and MHC-II transcript levels in metastatic cells. In sum, transcriptional regulation by the 19S-proteasome and histone modifying enzymes represents novel mechanisms of control of mammalian gene expression and present novel therapeutic targets for manipulating MHC expression in disease.

There are more than 7000 described rare genetic disorders; however, the molecular basis underlying approximately half of these disorders is unknown, and the majority are currently untreatable. Stature and growth abnormalities are a common clinical feature of many rare disorders including: Floating-Harbor syndrome (FHS), a short stature syndrome characterized by delayed osseous maturation, language deficits, and unique dysmorphic facial features; Weaver syndrome, an overgrowth syndrome characterized by advanced osseous maturation, developmental delay, and macrocephaly; and Sotos syndrome with cutis laxa, an overgrowth syndrome with marked tissue laxity in addition to the typical Sotos characteristics of developmental delay, macrocephaly, and a unique facial gestalt. The genetic basis underlying these three rare stature conditions were unknown at the outset of this study. We utilized high-throughput exome sequencing approaches to investigate the molecular etiology of these rare disorders and identified truncating mutations in the final exon of SRCAP as the genetic cause underlying FHS, missense mutations in EZH2 in Weaver syndrome, and novel mutations in the Sotos syndrome gene NSD1 in Sotos syndrome with cutis laxa. Next, we investigated the spectrum of SRCAP mutations in FHS and established the clustering of truncating SRCAP mutations in the final exon as being highly suggestive of a non-haploinsufficiency mutational mechanism in FHS. Finally, global methylation array analysis identified a unique methylation ‘epi-signature’ in FHS individuals, providing further insight into FHS disease mechanism and a diagnostic signature. These studies have delineated the molecular etiology of these three rare stature/growth disorders, furthered our understanding of the associated clinical spectrum, and provided biological insight into disease pathogenesis.

Les carcinomes cortico-surrénaliens (CCS) sont des tumeurs malignes rares de mauvais pronostic et pour lesquelles les options thérapeutiques efficaces sont inexistantes. Il est donc indispensable de comprendre les mécanismes moléculaires impliqués dans le développement des CCS, afin d’améliorer leur prise en charge. Les deux altérations les plus fréquentes dans les CCS sont une surexpression du facteur de croissance IGF2 et l'activation constitutive de la voie Wnt/β-caténine. Le laboratoire a mis en évidence le rôle oncogénique de la β-caténine à l'aide d'un modèle murin (souris ΔCat) présentant une activation constitutive de la β-caténine dans la cortico-surrénale. Toutefois, la faible pénétrance du phénotype malin suggère la nécessité d'autres altérations pour la progression tumorale. L’objectif initial de ma thèse était de tester le pouvoir oncogénique de IGF2, seul ou en association avec l’activation constitutive de la β-caténine. Les modèles de surexpression de Igf2 dans la cortico-surrénale nous ont permis de montrer que Igf2 n'initie pas le développement de tumeurs cortico-surrénaliennes. Dans un contexte d'activation de la β-caténine, la surexpression de Igf2 favorise le développement tumoral à des stades tardifs. Toutefois la formation de tumeurs malignes reste un évènement rare. Ces résultats suggèrent donc que la surexpression de Igf2 et l'activation de la β-caténine ne sont pas suffisantes dans notre modèle pour induire le développement de CCS. Une analyse rétrospective des données de transcriptome nous a permis de mettre en évidence une surexpression de l’oncogène putatif et histone méthyl-transférase EZH2, qui est associée à un mauvais pronostic. Mes travaux in vitro suggèrent que EZH2 est surexprimé en réponse à une surexpression des facteurs E2F et qu’il pourrait être impliqué dans le contrôle de la prolifération, de l'apoptose et de certaines caractéristiques tumorales des cellules cortico-surrénaliennes humaines H295R. Des inhibiteurs pharmacologiques étant disponibles, EZH2 pourrait constituer une cible thérapeutique intéressante pour le traitement des CCS. En parallèle de ces travaux, nous avons cherché à identifier les mécanismes impliqués dans la zonation du cortex surrénalien. Au cours du renouvellement tissulaire, les cellules acquièrent d’abord une identité glomérulée puis fasciculée. L'identité de la zone glomérulée repose en partie sur l'activité de la voie Wnt/β-caténine. Cette voie de signalisation induit l'expression de gènes essentiels à l'identité de cette zone et inhibe l'identité fasciculée. La différenciation fasciculée des cellules doit donc reposer en partie, sur l'inhibition de cette voie de signalisation. Nous avons donc émis l'hypothèse que la signalisation PKA, activée dans la zone fasciculée par la liaison de l'ACTH à son récepteur MC2R, s'oppose à l'activité de la β-caténine pour permettre la différenciation fasciculée. A l'aide d'approches pharmacologiques et génétiques, nous avons pu mettre en évidence que l'activation de la PKA inhibe la voie Wnt/β-caténine dans le cortex surrénalien et que ceci est à l'origine d'une perte de la zone glomérulée au profit d'une expansion de la fasciculée. L'effet de la PKA sur la voie Wnt résulte au moins en partie de l'inhibition de l'expression de Wnt4 en réponse à l'activation de la PKA. En effet une diminution d'expression de Wnt4 est observée en réponse à l'activation de la PKA dans la cortico-surrénale et l'invalidation de Wnt4 spécifiquement dans le cortex induit un phénotype proche de celui observé lors de l'activation de la PKA. Au delà des mécanismes moléculaires de la zonation, nous avons également montré que l’effet inhibiteur de la PKA sur la signalisation Wnt était capable de s’opposer aux effets oncogéniques de la β- caténine dans la cortico-surrénale. Ces observations pourraient s’avérer pertinentes, la voie de signalisation ACTH/PKA étant inhibée dans les CCS. / Adrenocortical carcinoma (ACC) is a rare tumour associated with poor prognosis and for which, efficient therapeutic approaches are not available. It is therefore essential to understand the molecular mechanisms involved in CCS development in order to improve their clinical management. The two most frequent alterations in ACC are overexpression of IGF2 and constitutive activation of β-catenin. Our lab has previously demonstrated the oncogenic activity of β-catenin in the adrenal cortex by developing a mouse model of constitutive β-catenin activation (ΔCat mice). However, the low malignant progression in ΔCat mice suggests that other alterations are necessary for acquisition of malignancy. The initial aim of my thesis was to test the oncogenic potential of IGF2 alone or associated with constitutive β-catenin activation. We showed that overexpression of Igf2 in the adrenal cortex does not trigger adrenal cortex tumourigenesis. In a context of constitutive β-catenin activation, overexpression of Igf2 promotes tumour development at later stages. However the formation of malignant tumours remains a rare event. These data suggest that the overexpression of Igf2 and constitutive activation of β-catenin are not sufficient to trigger malignant tumour progression. Retrospective analysis of available ACC transcriptome data highlighted overexpression of the putative oncogene and histone methyltransferase EZH2 in ACC, which was associated with poor prognosis. My in vitro studies suggest that EZH2 is overexpressed in response to overexpression of E2F transcription factors and that it could be involved in control of proliferation, apoptosis and oncogenic capacities of adrenocortical carcinoma cells H295R. Interestingly, the availability of pharmacologic inhibitors suggests that EZH2 could be a novel therapeutic target for the treatment of ACC. In parallel, we sought to identify the mechanisms involved in zonation of the adrenal cortex. During adrenal cortex renewal, cells first differentiate as glomerulosa before switching to fasciculata as they move within the cortex. Establishment of glomerulosa identity relies on the Wnt/β-catenin pathway, which induces expression of genes involved in glomerulosa differentiation and inhibits fasciculata identity. These data suggest that β-catenin has to be inhibited in order to allow the lineage conversion from glomerulosa to fasciculata. We thus postulated that PKA signalling pathway, which is triggered by ACTH binding to its receptor MC2R in zona fasciculata, played a role in repressing Wnt/β-catenin signalling to allow fasciculata differentiation. Using pharmacologic and genetic models, we have shown that PKA inhibits β-catenin signalling, which leads to loss of zona glomerulosa and expansion of zona fasciculata. The inhibitor effect of PKA on β-catenin pathway could be the result of decreased expression of Wnt4. Indee, a decrease of Wnt4 expression is observed in response to PKA activation and inactivation of Wnt4 in the adrenal cortex phenocopies PKA activation. We have also shown that PKA inhibits oncogenic effects of β-catenin in the adrenal cortex. The observation of decreased ACTH/PKA signalling in ACC suggests that this inhibition could be relevant to human adrenal tumour development.

No description available.

Background: Recent evidence suggests that the prostate cancer (PCa)-specific up-regulation of certain genes such as AMACR, EZH2, PSGR, PSMA and TRPM8 could be associated with an aberrant expression of non-coding microRNAs (miRNA). Methods: In silico analyses were used to search for miRNAs being putative regulators of PCa-associated genes. The expression of nine selected miRNAs (hsa-miR-101, -138, -186, -224, -26a, -26b, -374a, -410, -660) as well as of the aforementioned PCa-associated genes was analyzed by quantitative PCR using 50 malignant (Tu) and matched non-malignant (Tf) tissue samples from prostatectomy specimens as well as 30 samples from patients with benign prostatic hyperplasia (BPH). Then, correlations between paired miRNA and target gene expression levels were analyzed. Furthermore, the effect of exogenously administered miR-26a on selected target genes was determined by quantitative PCR and Western Blot in various PCa cell lines. A luciferase reporter assay was used for target validation. Results: The expression of all selected miRNAs was decreased in PCa tissue samples compared to either control group (Tu vs Tf: -1.35 to -5.61-fold; Tu vs BPH: -1.17 to -5.49-fold). The down-regulation of most miRNAs inversely correlated with an up-regulation of their putative target genes with Spearman correlation coefficients ranging from -0.107 to -0.551. MiR-186 showed a significantly diminished expression in patients with non-organ confined PCa and initial metastases. Furthermore, over-expression of miR-26a reduced the mRNA and protein expression of its potential target gene AMACR in vitro. Using the luciferase reporter assay AMACR was validated as new target for miR-26a. Conclusions: The findings of this study indicate that the expression of specific miRNAs is decreased in PCa and inversely correlates with the up-regulation of their putative target genes. Consequently, miRNAs could contribute to oncogenesis and progression of PCa via an altered miRNA-target gene-interaction.