Le déséquilibre HAT/HDAC aurait une influence sur le développement de certains cancers ainsi que dans l’addiction à l’alcool ou à la cocaïne. En inhibant les histones désacétylases, le taux d'acétylation de la chromatine augmente ce qui permet l’accès aux facteurs de transcription et l'expression des gènes. Aujourd'hui, il existe de nombreux inhibiteurs d'HDAC de structures diverses, mais ils ne sont pas spécifiques et présentent des effets secondaires importants. Les inhibiteurs d'HDAC comme le butyrate de sodium ou le MS-275 ont montré une modification de la dépendance à l'alcool chez le rat. MS-275 inhibe principalement la classe I de HDAC et conformément à ces observations nous nous intéressons aux inhibiteurs les plus sélectifs de la classe I tels que le Largazole thiol et le RedFK228. Notre but est de synthétiser de nouveaux cyclodepsipeptides analogues afin d'obtenir un inhibiteur sélectif de la classe I. Les HDAC de la classe I sont Zn-dépendants, ces analogues auront un groupement sulfonylhydrazide ayant une bonne affinité pour l’ion Zn2+ (ZBG). Il sera relié au cyclodepsipeptide par un bras espaceur dont la longueur sera adaptée (n = 2, 3). Une autre pharmacomodulation concerne l'incorporation d’hétérocycles différents (oxazole, thiazole et pyridine). Les inhibitions de ces composés ont pu être testées sur HDAC1, HDAC3 et HDAC6. Un composé a une spécificité pour HDAC3 et un autre a une spécificité pour HDAC1. Les tests sur des rats "binger" permettent de penser que HDAC1 est impliqué dans ce model de consommation et non HDAC3. / The imbalance HAT/HDAC would influence the development of cancers and alcohol or cocaine addiction. HDAC inhibition allows increase of both acetylation rate and gene expression. Today, there are many structurally diverse potent, but non-specific HDAC inhibitors displaying important side-effects. HDAC inhibitors such as sodium butyrate or MS-275 have been shown to alter the alcohol dependence in the rat. MS-275 inhibits mainly class I of HDAC and in line with these observations we are interested in more selective class I inhibitors such as Largazole thiol and RedFK228. Our purpose is to synthesize new cyclodepsipeptides analogues in order to obtain selective class I inhibitor. HDAC class I is a Zn-dependent enzyme and our target molecules have sulfonylhydrazide function as efficient Zinc binding group (ZBG). Additional pharmacomodulations concern the incorporation of different heterocycles (oxazole, thiazole, pyridine) and varying linker lengths (n = 2, 3). Inhibitions of these compounds have been tested on HDAC1, HDAC3 and HDAC6. A compound has specificity for HDAC3 and another has specificity for HDAC1. Tests on rats "binger" suggest that HDAC1 is involved in this model of consumption and not HDAC3.

Le développement embryonnaire est un processus hautement contrôlé où différentes voies de signalisation se coordonnent pour la construction d'un organisme. L'une des principales voies de signalisation impliquées dans ce processus est la voie canonique Wnt. La longue quête pour comprendre la cascade de signalisation Wnt/β-catenine a révélé que la réponse transcriptionelle induite par le signal Wnt/β-catenine est dépendante du contexte, ou compétence, cellulaire. Peu de choses sont connues sur les évènements moléculaires qui influencent cette compétence cellulaire. Dans les embryons de X. laevis Wnt/β-catenine est le signal inducteur pour l'Organisateur de Spemann. On ne sait pas ce qui limite l'activité Wnt dans ce territoire et par voie de conséquence la taille de l'Organisateur. Les résultats présentés dans ce manuscrit de thèse montrent que le facteur de transcription Barhl2 affecte le développement de l'organisateur de Spemann. Nous démontrons que Barhl2 inhibe l'activité Wnt via son interaction avec le corépresseur Groucho et le facteur de transcription Tcf, et mobilise l'activité de Hdac1 qui agit sur la structure chromatinienne. En utilisant des expériences in vitro et in vivo sur des cellules en culture et des embryons de Xénope nous démontrons que la régulation de Barhl2 sur les activités Groucho-Tcf est maintenue pendant l'embryogenèse et joue un rôle dans le confinement des progéniteurs neuraux dans le cerveau. Ensemble, nos résultats fournissent un mécanisme nouveau et important agissant sur le contrôle de l'activité transcriptionelle Wnt et la compétence des cellules à répondre à ce signal. / Embryonic development is a highly controlled process where different signaling pathways participate into the elaboration of an organism. One of the main signaling pathways is the Wnt canonical pathway. The long-lasting search to understand Wnt/β-catenin transduction cascade revealed that the net transcriptional read out of Wnt/β-catenin signaling is highly dependent on the cellular context. In X. laevis embryos Wnt/β-catenin signaling is the informative signal for the Spemann Organizer induction. However little is known on what limits Wnt activity in this territory and consequently the size of the Spemann Organizer. The results presented in this manuscript provide evidence that the evolutionarily conserved transcription factor Barhl2 limits the development of the Spemann organizer. In this territory Barhl2 inhibits Wnt activity via its interaction with the co-repressor Groucho and the transcription factor Tcf. It participates to the recruitment of the chromatin remodeling enzyme, Hdac1 that represses the expression of Spemann organizer genes. Using a Xenopus tropicalis Tcf reporter line we demonstrate that Barhl2 inhibitory effect on Groucho-Tcf activities is maintained during embryogenesis and plays a role in the confinement of neural progenitors in the brain. Together, our results provide a new and important mechanism for the control of Wnt transcriptional activity.

No description available.

Muscle invasive bladder cancer (MIBC) has a poor prognosis. Currently, therapy consists of radical radiotherapy or cystectomy with or without chemotherapy. The average age of patients with MIBC is high and older patients are less able to tolerate surgery or chemoradiation due to their impaired physical fitness and generally poor renal function. There is an urgent need to find new treatment regimes that are both tolerable and effective. The aims of this project were to investigate the radiosensitising effects of the histone deacetylase (HDAC) inhibitor panobinostat in bladder cancer cell lines, with the ultimate goal of proposing a novel radiosensitising therapy for MIBC, and to study the effects of panobinostat on the major DNA double strand break (DSB) repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ), to determine the predominant pathway targeted and to look further upstream at effects on the MRE11/RAD50/NBS1 (MRN) complex. The HDAC inhibitor panobinostat was found to be toxic in the low nanomolar range and significant radiosensitising effects were demonstrated at doses lower than IC50 in all the bladder cell lines studied. The radiosensitising effect of panobinostat was not influenced by TP53 status, which is generally regarded as an important determinant of bladder cancer response to radiotherapy. In the “synthetic lethality” bladder cancer cell model, panobinostat predominantly targets the HR pathway, reportedly the only proficient DNA repair pathway in MIBC. HR proteins RAD51 and CtIP were downregulated upon panobinostat treatment in a dose-dependent manner. Upstream, MRE11 and NBS1 proteins were also targeted by panobinostat, with levels slightly decreased in RT112 and T24 cells and in CAL29 cells post-ionising radiation. In summary, the HDAC inhibitor panobinostat was shown to be an efficient radiosensitiser in bladder cancer cells at low toxic doses and to predominantly target the HR pathway. These findings are promising and may contribute towards establishing a novel combination therapy of panobinostat with IR for MIBC patients.

No description available.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

<p>The androgen receptor (AR) signaling axis is a well-established therapeutic target in prostate cancer, due to its central role in tumor maintenance and progression. Although patients respond initially to androgen deprivation therapies and AR antagonists, they invariably progress to a castration-resistant state. Consequently, there is an unmet need for agents that target the AR signaling axis in a unique manner. </p><p>Histone deacetylase (HDAC) inhibitors repress AR signaling and prostate cancer growth in cellular and xenograft models. However, HDAC inhibitors also induce epithelial to mesenchymal (EMT) and neuroendocrine differentiation, both of which are associated with prostate cancer progression and aggressiveness. Given that 18 different HDAC isoforms have been identified in humans, and non-selective or Class I (HDAC1, 2, 3, and 8) HDAC inhibitors have been used in most of these studies, the relative contribution of individual HDAC isoforms to AR transcriptional activity and prostate cancer pathophysiology remains to be elucidated. The overarching goals of this study were to (1) determine the role of individual Class I HDACs in AR transcriptional activity and prostate cancer growth, (2) identify selective HDAC inhibitors that have reduced adverse profiles to the treatment of prostate cancer, and (3) identify potential HDAC-interacting proteins that regulate AR target gene transcription and prostate cancer growth. </p><p>Using genetic knockdown studies and pharmacological inhibitors with isoform selectivity, we identified that HDAC3 was required for AR transcriptional activity and proliferation in cellular models of androgen-sensitive and castration-resistant prostate cancer (CRPC). Additionally, we found that RGFP966, an HDAC3-selective inhibitor, attenuated the growth of a xenograft model of CRPC. Furthermore, non-selective HDAC inhibitors induced EMT and neuroendocrine markers in prostate cancer cells, but RGFP966 treatment did not. These studies provide rationale for selective inhibition of HDAC3 for the treatment of CRPC, and could provide an explanation for the lack of success using non-selective HDAC inhibitors in clinical trials for prostate cancer.</p><p>We also assessed the role of REV-ERB alpha, an HDAC3-interacting protein, in the regulation of AR transcriptional activity and prostate cancer growth. Using siRNA knockdown studies, REV-ERB inhibitors, and overexpression studies, we concluded that REV-ERB alpha; was required for AR target gene induction and prostate cancer growth, including models of CRPC. These studies also provide rational for targeting REV-ERB alpha; for the treatment of CRPC.</p><p>Taken together, these studies identify two novel targets in the HDAC signaling axis for the treatment of prostate cancer: HDAC3 and REV-ERB alpha. Our studies provide greater insight into AR transcriptional regulation and prostate cancer pathophysiology.</p> / Dissertation

Le pancréas est impliqué dans la digestion alimentaire et le contrôle glycémique. Cette diversité fonctionnelle repose sur des facteurs transcriptionnels et épigénétiques qui assurent une régulation coordonnée de l’identité et de la fonction des cellules. Ma thèse vise à caractériser le rôle de certains de ces facteurs. Nous avons précédemment montré le rôle de Hnf1b dans le développement du pancréas. Nous montrons que son inactivation post-natale chez la souris induit de graves défauts canalaires ainsi qu’une pancréatite associée à des néoplasies intra-épithéliales. La pathologie MODY5 est liée à des mutations de HNF1B. Nous avons analysé un modèle murin du MODY5 généré par insertion d’une mutation humaine chez la souris. Notre étude montre que ces souris constituent un bon modèle de la maladie. Elles sont intolérantes au glucose et présentent une réduction du volume total des gros ilots et des cellules bêta. Sur le plan exocrine, elles développent une pancréatite. Nos analyses suggèrent une étiologie embryonnaire. Antérieurement, notre équipe a montré le rôle des HDACs dans le développement du pancréas. Basés sur l’analogie des voies qui régulent développement et régénération du pancréas, nous avons étudié le rôle des HDACs dans la régénération pancréatique. Nous montrons que l’inactivation de HDAC3 sensibilise le tissu acinaire à l’inflammation et abroge sa régénération. Nos études dévoilent l’implication de nouveaux mécanismes moléculaires dans la survenue de la pancréatite, du cancer du pancréas et du diabète. Ces données pourront contribuer à l’identification de nouvelles cibles thérapeutiques pour le traitement ou la prévention des pathologies du pancréas. / The pancreas is crucial for food digestion and glycemic control. Its functional diversity is based on transcriptional and epigenetic factors which ensure the regulation of cellular identity and function. My thesis aims to characterize the role of some of these factors. We previously showed that Hnf1b is required for pancreas development. We show here that Hnf1b post-natal inactivation in mouse leads to strong ductal defects and pancreatitis associated with intraepithelial neoplasia. In human, HNF1B heterozygous mutations cause the MODY5 pathology. We analyzed a unique MODY5 mouse model generated by insertion of human mutation in mouse. We show that these mice constitute a valuable model of the disease. They are glucose intolerant and display reduced total volume of large islets and beta-cells. The exocrine compartment is also altered as mice develop a pancreatitis phenotype. Our analyses suggest an embryonic etiology. Previously, our team revealed HDAC roles in pancreas development. Pancreas regeneration and pancreas development sharing common molecular pathways, we investigated the role of HDACs in pancreas regeneration. We found that HDAC3 inactivation sensitizes acinar tissue to inflammation and prevents its regeneration. Our studies reveal new molecular mechanisms implied in the development of pancreatitis, pancreatic cancer and diabetes. These data could contribute to the identification of new therapeutic targets for the treatment of pancreatic pathologies.

Le mésothéliome pleural est un cancer de la plèvre provoqué principalement par linhalation de fibres damiante. Nous avons émis lhypothèse que la dérégulation de lexpression génique est un paramètre important du développement de cette maladie. Or, les histones désacétylases (HDACs) peuvent jouer le rôle de répresseur transcriptionnel en modifiant la conformation de la chromatine. Dans ce contexte, nous avons étudié lactivité anticancéreuse du valproate, un inhibiteur dHDAC, en combinaison avec différents types de traitements utilisés en chimiothérapie. Nous avons démontré leffet synergique entre la chimiothérapie et le valproate dans des lignées cellulaires et dans des biopsies isolées à partir de patients. Nous avons étudié les processus impliqués dans lapoptose et révélé limplication des caspases, des espèces oxygéno-réactives et le rôle important de la protéine Bid. Nous avons ensuite réalisé une étude transcriptomique par microdamiers dans le but de mieux caractériser les mécanismes impliqués. Enfin, nous avons démontré lefficacité du valproate dans un modèle préclinique murin. Ces recherches ont permis la mise en place dun essai clinique de deuxième ligne sur des patients réfractaires à une première chimiothérapie.

<p>Reversible acetylation has emerged as an important post-translational modification that rivals phosphorylation in regulating chromatin structure and gene expression. Acetylation of histone is associated with transcriptional activation while deacetylation is linked to transcriptional repression. Moreover, histone deacetylase inhibitors induce growth arrest, differentiation and apoptosis of cancer cells and therefore appear to be promising anti-tumor agents. While transcriptional deregulation is thought to be the main mechanism underlying their therapeutic effects, the exact mechanisms and targets by which HDAC inhibitors, which are mostly non-specific, achieve their anti-tumor effects remain poorly understood. In other words, it is not known which and how each HDAC members are involved in supporting tumor growth.</p><p>In this thesis, I have showed that HDAC6, a cytoplasmic localized and cytoskeleton-associated deacetylase, is required for efficient oncogenic transformation and tumor formation. I have found that HDAC6 expression is induced upon oncogenic Ras-induced transformation in both human somatic cells and murine fibroblasts. Conversely, murine fibroblasts deficient in HDAC6 are more resistant to both oncogenic Ras and ErbB2-dependent transformation, indicating a critical role for HDAC6 in oncogene-induced transformation. Supporting this hypothesis, inactivation of HDAC6 in several human cancer cell lines effectively impairs anchorage-independent growth <em>in vitro</em> and their ability to form tumors in immunocompromised mice. I have demonstrated that the impairment of anchorage independent growth in HDAC6 deficient cells is associated with increased anoikis and mechanistically a defect in activation of the AKT and ERK kinase cascades. Additionally, <em>HDAC6 </em>null mice are more resistant to two-stage chemical carcinogen-induced skin tumors. Finally, I have demonstrated that the tumor-promoting effect of HDAC6 is probably mediated through the molecular chaperon Hsp90. While Hsp90 is known to be deacetylated by HDAC6 and has been implicated in stabilizing Raf-1 and ErbB2, I have found that suppression of HDAC6 impairs the stability of Raf-1 and the association between Hsp90 and ErbB2.</p><p>In conclusion, my work provides the first experimental evidence that of all the HDAC members, the cytoplasmic deacetylase HDAC6 is required for efficient oncogenic transformation, indicating that reversible acetylation plays a critical role in regulating cellular functions of non-histone non-nuclear cytoplasmic proteins that contributes to malignant transformation. Furthermore, this work identifies HDAC6 as an important component underlying the anti-tumor effects of HDAC inhibitors.</p> / Dissertation