Mon travail de thèse s’est focalisé sur l’étude du rôle des miARN comme modulateurs de l’expression des gonadotropines en réponse à un traitement par la GnRH. Dans un premier temps, nous avons étudié les miARN-132 et -212 fortement induits en réponse à la GnRH dans les cellules gonadotropes. Nous démontrons que l’induction de la production de FSH par la GnRH est dépendante de l’activation des miR-132/212 dans les cellules hypophysaires de rat et dans la lignée cellulaire gonadotrope LβT2. Nous montrons à l’aide de cette lignée que l’élévation de miR-132/212 inhibe l’expression de la lysine déacétylase SIRT1, favorisant ainsi l’acétylation inhibitrice d’un répresseur transcriptionnel de la FSH, conduisant donc à l’activation de l’expression de la FSH (Lannes et al, 2015). J’ai ensuite étudié l’action d’un miARN fortement inhibé en réponse à la GnRH, miR-125b. Nous démontrons que miR-125b bloque la signalisation Gαq/11 en réprimant plusieurs effecteurs de cette voie, sans réguler la voie Gαs. Lors de l'exposition à la GnRH, miR-125b est inactivé par transfert d’un groupement méthyle par l’ARN méthyltransférase NSun2, activée par phosphorylation Gαs/PKA-dépendante. Nous observons que l’induction demiR-132 et de la phosphatase PP1α en réponse à la GnRH dépend de la voie Gαq/11 et est induite par l’inactivation de miR-125b. Nous démontrons que NSun2 est une cible de miR-132 et que la phosphorylation de NSun2 est supprimée par la phosphatase PP1α. Des analyses cinétiques nous ont permis de décrypter le mécanisme de désensibilisation à la stimulation GnRH. Lors de la phase d’induction par la GnRH, l’activation de la voie Gαs/PKA inactive miR-125b permettant une levée d’inhibition de la voie Gαq/11 et par là, l’induction des gènes des gonadotropines, de miR-132 et PP1α. L’activation de ces derniers contribue à l’inactivation de NSun2 et à un retour de miR-125b à son état d'équilibre permettant de nouveau l’inhibition de la voie Gαq/11 et donc de l’expression des gonadotropines (Lannes et al, 2016). Notre étude montre pour la première fois le rôle crucial d’une boucle de régulation entre deux miARN dans le mécanisme de la désensibilisation de la réponse à la GnRH. Le caractère ubiquitaire des acteurs de cette boucle de régulation laisse penser qu’elle pourrait jouer un rôle plus général. Les travaux complémentaires effectués montrent que la GnRH induit la sécrétion de plusieurs miARN. In vitro, nous avons démontré que la GnRH provoque une libération calcium-dépendante de miR-125b et de miR-132 dans le milieu extracellulaire par les cellules gonadotropes. In vivo, nous mettons en évidence chez la rate et la femme, une augmentation sérique des miARN-125b et -132 au moment de la décharge ovulante de LH induite par la GnRH. Ces résultats suggèrent que la cellule gonadotrope hypophysaire est capable d’émettre un message original, sous la forme de miARN, dans la circulation sanguine. Mes travaux de thèse éclairent le rôle clé joué par les miARN dans la réponse de la cellule gonadotrope à la stimulation prolongée à la GnRH. Ils mettent en évidence l’effet bloquant d’un seul miARN, miR-125b sur la signalisation liée à la protéine Gαq/11, une voie activée par nombre de récepteurs. Ils révèlent l’existence d’une boucle de régulation responsable de la désensibilisation à la GnRH mais qui pourrait être plus largement répandue. Enfin, la sécrétion des miARN dans la circulation sanguine induite par la GnRH que nous mettons en évidence pour la première fois ouvre des perspectives particulièrement intéressantes sur la nature du signal généré par les cellules gonadotropes et permet d’envisager l’existence de nouveaux tissus cibles. / GnRH is a hypothalamic neurohormone that stimulates synthesis and release of the pituitary gonadotropins, LH and FSH. Mammalian GnRH receptor lacks a C-terminal tail and is thus not submitted to homologous desensitization. Desensitization of gonadotrope cells to sustained exposure to GnRH relies on post-receptor mechanisms operating at different levels of the Gαq/11-mediated signalling pathway. GnRH was shown to modulate the expression of microRNAs (miRNAs), a new class of signalling regulators composed of small single-stranded RNAs that regulate gene expression at a post-transcriptional level. The purpose of my PhD thesis was to investigate the role of miRNAs in contributing to the regulation of gonadotrope cells by GnRH and notably to its desensitization effect. I first demonstrated that a GnRH-induced rise in miR-132 and miR-212 in rat primary pituitary culture cells and in the LβT2 murine gonadotrope cell line was necessary for efficient stimulation of FSH production. We then showed that the miR-132/212-mediated action of GnRH involved a posttranscriptional decrease of SIRT1, a lysine deacetylase. The lower level of SIRT1 allowed an increase in the acetylated form of FOXO1, a transcriptional repressor of Fshb, leading to its exit from the nucleus and to an increase in FSH expression (Lannes et al, 2015). Then, I focussed on the involvement of miR-125b, a miRNA that was strongly inhibited in response to GnRH. We showed that miR-125b blocked the Gαq/11 signalling pathway, through the repression of several effectors of this pathway, without affecting the Gαs signalling pathway. Upon exposure to GnRH, miR-125b was inactivated by methylation on adenosine by the NSun2 RNA methyltransferase. This later enzyme was activated by a Gαs/PKA-dependent phosphorylation. We observed that the induction of miR-132 and PP1α phosphatase in response to GnRH depends on a Gαq/11 activation allowed by the inactivation of miR-125b. We demonstrated that NSun2 is a target of miR-132 and that phosphorylation of NSun2 is suppressed by PP1α. Kinetic analyses enabled us to decipher the desensitization mechanism to GnRH stimulation. During the induction phase, the Gαs/PKA activation led to lower miR-125b levels, allowing Gαq/11 signalling and hence, transcriptional activation of gonadotropins genes. Co-activation of miR-132 and PP1α contributed to the inactivation of NSun2 and a return of miR-125b back to its equilibrium state leading to Gαq/11 signalling inhibition and therefore, to the arrest of gonadotropins expression (Lannes et al, 2016). Our study shows for the first time the crucial role of a miRNAs regulatory loop in the GnRH-induced mechanism of desensitization. The ubiquitous nature of the actors of this regulatory loop suggests that it may play a more general role. Additional works carried out showed that GnRH induces the secretion of several miRNAs. In vitro, we demonstrated that the GnRH causes a calcium-dependent release of miR-125b and miR-132 in the extracellular medium by the gonadotrope cells. In vivo, we highlighted a miRNA-125b and -132 increase in serum at the time of the ovulating LH surge induced by GnRH in rats and women. These results suggest that the pituitary gonadotropic cell is capable of transmitting an original message in the form of miRNA, into the bloodstream. My PhD work unravels the key role played by miRNAs in the gonadotrope cells response to GnRH-sustained stimulation. They highlight the blocking effect of a single miRNA, miR-125b on the Gαq/11 signalling, a pathway activated by many other membrane receptors. They indicate the existence of a regulation loop responsible for the desensitization to GnRH but which could be more widespread. Finally, the secretion of miRNAs in blood flow induced by the GnRH that we show for the first time opens up particularly interesting perspectives on the nature of the signal generated by the gonadotrope cells and allows to consider the existence of new target tissues.

Prostate cancer is the leading non-cutaneous malignancy affecting men in the United States. One in every six men will be affected by prostate cancer. Due to the high incidence of prostate cancer, there is a need to develop biomarkers capable of identifying tumors from benign prostatic lesions. miRNAs are small molecules that regulate protein translation and impact cellular integrity when dysregulated. It is widely thought that miRNAs have the potential to serve as biomarkers. This study utilizes a unique combinatorial analysis of miRNA dysregulation to identify key miRNAs involved in prostate tumor initiation, progression and metastasis. Numerous dysregulated miRNAs potentially influence cancer development. A unique bioinformatically driven, network based approach was used to rank potential miRNAs that drive tumor progression. This study showed that miRNAs preferentially regulate highly connected proteins and transcription factors that affect numerous downstream targets. Thus dysregulation of a single highly connected miRNA could severely impact homeostatic maintenance of the tissue. In combination with miRNA profiling of a cancer cell progression model, the utilization of laser captured microdissection was used to separate cancer specific microRNA portraits from background differences arising from stroma cells, lymphocytes, and remaining normal epithelial cells. Integration of miRNA profiles with information gathered using networks biology and targeted proteomics resulted in the identification of a key miRNA that affects prostate cancer development and may be useful as a novel biomarker for identification/ staging of prostate cancer. Human miR-125b was identified as a potential miRNA suppressor of tumor formation. Previous work has identified miR-125-b as the post-transcriptional regulator of the ErbB2/ ErbB3 growth factor receptor family. Loss of miR-125b drives up expression of ErbB2/ ErbB3 activating downstream PI3K/AKT and RAS oncogene pathways. The level of miR-125b decreases 3-5-fold between benign and tumor epithelium. Further, miR-125b decreases during the development of prostatic intraepithelial neoplasia, which is regarded as an early indicator of prostate cancer. Thus miR-125b may be an ideal marker of early changes indicative of cancer. Restoration of miR-125b into highly tumorigenic, metastatic cells reduces mobility and invasion of underlying tissues. Taken together these data show miR-125b is a tumor suppressor in the healthy prostate.

microRNA dependent gene expression regulation has roles in diverse processes such as differentiation, proliferation and apoptosis. Therefore, deregulated miRNA expression has functional importance for various diseases, including cancer. miR-125b is among the commonly downregulated miRNAs in breast cancer cells . Therefore we aimed to characterize the effects of miR-125b expression in MCF7 breast cancer cell line (BCCL) to better understand its roles in tumorigenesis. Here, we investigated mir-125 family members

La découverte récente d'adipocytes bruns fonctionnels chez les humains adultes a conduit à envisager leur utilisation afin d’augmenter la dépense énergétique dans de potentiels traitements contre l'obésité et les maladies associées. Par ailleurs, des ilots d’adipocytes bruns, appelés adipocytes "brite" (brown in white), émergent dans le tissu adipeux blanc après une exposition au froid ou une stimulation des récepteurs β3-adrénergiques. En utilisant les cellules hMADS, nous avons identifié plusieurs miARNs régulés pendant le « britening ». miR-125b et let-7i ont des niveaux d’expression plus bas dans les adipocytes « brites ». Des analyses fonctionnelles utilisant un « mimic » de miR-125b ou un inhibiteur ont révélé que miR-125b agit comme un frein sur le « brunissage » des cellules hMADS en altérant leur respiration ainsi que leur contenu mitochondrial. In vivo, nous avons montré que miR-125b et let-7i sont moins exprimés dans le tissu adipeux brun par rapport au tissu adipeux blanc. La stimulation des récepteurs β3-adrénergiques ou l'exposition au froid induit une diminution d’expression des miARNs dans les deux tissus et est associée à l'activation du tissu adipeux brun et au recrutement des adipocytes « brites ». Nous avons constaté que l’injection de miR-125b ou let-7i dans le tissu adipeux blanc sous-cutané inhibait l’expression de gènes du « brunissage » induite par la stimulation de la voie β3-adrénergique. En conclusion, nos observations ont montré que miR-125b et let-7i jouaient un rôle important dans la modulation des adipocytes « brites » et des adipocytes « bruns » en ciblant l’expression de gènes mitochondriaux et en diminuant la biogenèse mitochondriale. / The recent discovery of functional brown adipocytes in adult humans has led to the consideration of their use to increase energy expenditure in the treatment of obesity and associated metabolic disorders. Furthermore, in rodents and humans, islands of thermogenic adipocytes, termed “brite” (brown in white) adipocytes, emerge within white adipose tissue after cold exposure or β3-adrenergic receptor stimulation. Using hMADS cells, we identified several miRNAs regulated during “britening” including miR-125b and let-7i which showed lower levels in brite adipocytes. Functional analysis using miR-125b mimic or miR-125b inhibitor transfection revealed that miR-125b-5p acts as a brake of the browning of hMADS cells by impairing respiration rate as well as their mitochondrial content. miR-125b and let-7i levels were lower in brown compared to white adipose tissue. In vivo, we showed that both miRNAs levels were down regulated in mice sub-cutaneous white and brown adipose tissues upon β3-adrenergic receptors stimulation or cold exposure, which is associated with BAT activation and brite adipocyte recruitment. We found that injection of both miRNA mimics in subcutaneous white adipose tissue inhibited β3-adrenergic-induced brown adipocyte markers expression. Altogether, our observations showed that miR-125b and let-7i played an important role in the modulation of brite and brown adipocytes function targeting oxygen consumption and mitochondrial gene expression.

Prostate cancer is the most common cancer affecting men today. Therefore, there is a strong need for accurate biomarkers and successful therapeutic treatments. A novel approach combining a computationally built protein-protein interaction network of proven microRNA protein targets with high throughput proteomics identified ErbB2 and ErbB3 as key proteins in prostate cancer. These results coupled with microRNA array screening of an androgen-independent prostate cancer progression model, substantiated by single microRNA analysis, suggested miR125b as a key tumor suppressor contributing to prostate cancer progression. miR125b expression was shown to be substantially increased in the non-tumorigenic P69 cell line compared to its highly tumorigenic, metastatic M12 variant. Luciferase reporter gene assays including the entire 3’UTR of either ErbB2 or ErbB3 revealed a 2.8- and 2.4-fold decrease (respectively) compared to control vector. Thus, this combinatorial approach has suggested an additional microRNA and its target involved in prostate tumor progression.

Cancer may arise as a result of deregulation of oncogenes and/or tumor suppressors. Although much progress has been made for the identification of such cancer related genes, our understanding of the complex tumorigenesis pathways is still not complete. Therefore, to improve our understanding of how certain basic mechanisms work in normal and in cancer cells, we aimed to characterize two different breast cancer related genes. First part of the study focused on subcellular localization USP32 (Ubiquitin Specific Protease 32) to help understand the function of this uncharacterized gene. USP32 is a member of deubiquitinating enzymes (DUBs) and the gene maps to a gene rich region on 17q23. Genes on 17q23 are known to undergo amplification and overexpression in a subset of breast cancer cells and tumors. DUBs are known to be implicated in a variety of cellular functions including protein degradation, receptor endocytosis and vesicle trafficking. Therefore to elucidate the function of USP32, we localized the full length USP32 protein fused to GFP, in HeLa cells, using Fluorescence Protease Protection (FPP) assay and confocal microscopy. Results suggested a Golgi localization for USP32 as confirmed by co-localization study via BODIPY-TR, a Golgi specific marker. Additional investigations to find the role of USP32 in Golgi will further clarify the function of this candidate oncogene. Second part of the study focused on a potential tumor suppressor. For this purpose, we functionally characterized miR-125b, a microRNA gene as a potential tumor suppressor in breast cancer. microRNAs are regulators of gene expression and their deregulation is detected in cancer cells. miR-125b is reported as a down regulated microRNA in breast cancers. In this study, we investigated the expression, function and possible targets of miR-125b in breast cancer cell lines (BCCLs). Our results revealed a dramatic down regulation of miR-125b in a panel of BCCLs. Restoring the expression of miR-125b in low miR-125b expressing cells decreased the cell proliferation and migration as well as cytoplasmic protrusions, detected by staining of actin filaments. While connection of miR-125b and cell motility based on ERBB2 targeting has been reported earlier, here we present data on ERBB2 independent effects of miR-125b on cell migration in non-ERBB2 overexpressing breast cancer cells. Our results showed involvement of a miR-125b target, ARID3B, in cell motility and migration. Our findings showed miR-125b to be an important regulator of cell proliferation and migration in ERBB2 negative breast cancer cells, possibly through regulating multiple targets.