Le cancer de la prostate (CaP) représente le premier cancer chez l'homme et la seconde cause de mortalité. Alors que la plupart des malades atteints de cancer de la prostate évoluent favorablement (forme indolente), une fraction non négligeable développera une maladie agressive avec l’apparition de métastases. La recherche des biomarqueurs tumoraux peut aider à différencier les CaP dits indolent et les CaP agressifs qui sont responsables du décès des patients. Ils permettront de mieux sélectionner des patients afin d’éviter le surtraitement. La protéine CRIPTO est le premier membre de la superfamille des protéines EGF-CFC. Ce facteur de croissance est largement impliqué dans le développement embryonnaire et s'exprime dans de nombreux types de cancers. Son rôle dans le CaP reste encore non élucidé. L’objectif de mon travail de thèse consiste à examiner le profil d'expression de CRIPTO et d'évaluer son impact potentiel sur l’agressivité du cancer de la prostate.Les résultats ont montré que CRIPTO est exprimé de manière significative dans 37,9% de CaP. Il est absent voire faiblement détecté dans les prostates hyperplasiques bénignes et dans les tissus sains. Nous avons montré ensuite que la surexpression de CRIPTO favorise une transition épithéliale-mésenchymateuse (TEM) associée à l’augmentation de la capacité de migration et de la survie des cellules tumorales. Les voies de signalisation régulées par CRIPTO impliquent l’activation des voies PI3K / AKT et FGFR1 / ERK.De manière très intéressante, les cellules tumorales mésenchymateuses surexprimant CRIPTO secrètent excessivement des vésicules. Nous avons tenté alors de découvrir le rôle de ces vésicules dans la progression du cancer de la prostate. Les vésicules extracellulaires (VEs) purifiés étaient capables de moduler la signalisation du récepteur des androgènes et d’activer la voie du TGFß. Les cellules tumorales prostatiques traitées par ces VEs deviennent plus agressive et acquierent des caractéristiques mésenchymateuses.En conclusion, nos résultats mettent en évidence une nouvelle fonction importante de CRIPTO dans le cancer de la prostate. Nous démontrons également que les cellules tumorales surexprimant ce facteur de croissance secrètent excessivement des vésicules qui participent activement dans la communication intercellulaire et promeuvent la progression du CaP. L’ensemble de nos travaux suggère que le ciblage thérapeutique de CRIPTO et le blocage de la sécrétion des VEs pourraient être des nouvelles approches thérapeutiques innovantes pour le traitement du cancer de la prostate. / Prostate cancer (PCa) remains at the top of the list of the most common malignant tumors and the dominant cause of mortality and morbidity in men worldwide. Detection of tumor biomarkers to aid differentiate indolent from severe PCa cases and well-choose patients at high risk for intensive treatment. The founding member of EGF-CFC protein superfamily, CRIPTO, is widely implicated in embryonic development and is found to be expressed in a wide spectrum of human tumors. As its role in PCa was still unclear, we aimed to investigate expression profile of CRIPTO in PCa and relate its potential impact on prostate malignancy.Prostatic tissues and cell lines, both normal and cancerous, were engaged in experimental studies and design was based on techniques used in biochemistry, cellular and molecular biology.CRIPTO showed to be upregulated in 37.9% of PCas, while being absent or marginally detected in benign conditions. Our results displayed that CRIPTO overexpression promoted epithelial-mesenchymal transition (EMT) associated with enhanced migration capacity and survival under stress conditions due to propensity to stimulate PI3K/AKT and FGFR1/ERK signaling pathways.More interestingly, tumor mesenchymal like cells overexpressing CRIPTO secreted vesicles excessively. Thus we attempted to uncover the role of these vesicles in the progression of PCa. Extracellular vesicles derived from these cells were highly capable to modulate androgen receptor signaling through TGF-ß pathway and rendering the recipient prostatic cells more aggressive by acquisition of mesenchymal features.Our results highlight a new substantial function of CRIPTO in PCa and put in evidence its importance as a new promising target for PCa treatment. Moreover, we emphasize on an original role of mesenchymal extracellular vesicles in the interclonal communication to carry and transfer tumorigenic contents and enhance progression of PCa. This opens new scopes towards better understanding of vesicles secreted by prostate cancer cells and their impact to better cure the disease.

Les cellules cancéreuses sont capables de réactiver la transition Epithélio-Mésenchymateuse (EMT), mécanisme embryonnaire, pour acquérir une mobilité et une capacité de dédifférenciation. L'EMT conduit à une reprogrammation génétique avec la réactivation d'inducteurs de l'EMT, qui sont en majorité des facteurs de transcription (EMT-TF), et conduit à l'inhibition de miARN. Par ailleurs des stress oncogéniques sont essentiels à la progression tumorale. Le but de mon projet de thèse était de comprendre comment les événements de reprogrammation génétique survenant au cours de l'EMT coopèrent avec des stress oncogéniques dans la transformation tumorale mammaire.Premièrement, un criblage basé sur la coopération oncogénique en soft agar assay, entre les EMT-TFs et les stress oncogénique a été réalisé. Suite à une analyse bioinformatique, différentes signatures d'EMT-TF associés à un stress oncogénique ont été identifiées. Ainsi, par exemple, l'expression de l'EMT-TF Zeb1 et l'EMT-TF GSC sont associés à la délétion du gène suppresseur de tumeur PTEN pour transformer des cellules mammaires immortalisées. Une analyse en immuno-histochimie sur un set de 558 tumeurs du sein triple négatives a validé in vivo la présence d'une corrélation entre l'expression de GSC et l'expression de PTEN. Cependant cette association semble être plus complexe. En effet, l'expression de GSC est négativement associée à l'expression nucléaire de PTEN tandis qu'elle est positivement associée à l'expression de PTEN cytoplasmique. Enfin une analyse sur des métadonnées publiques de cancers telles que le TCGA ou le METABRIC est en cours pour valider ces signatures in vitro et plus largement pour déterminer comment l'EMT ou les signatures associées aux EMT-TF se corrèlent avec les voies oncogéniques classiques. Deuxièmement, une analyse in silico à partir d'algorithmes prédictifs de cibles de miARN, a été réalisée pour sélectionner les miARN capables d'inhiber l'expression de plusieurs EMT-TF. Deux miARN (miR-495 et 590-3p) ont été identifiés ciblant plusieurs membres des 4 principales familles d'EMT-TF (FoxC, Snail, bHLH et ZEB). Des tests in vitro ont été réalisés pour valider ces régulations identifiant Slug comme une cible de miR-590-3p. De plus, l'expression de ces miARN dans des lignées cellulaires mammaires est négativement associée à l'expression des EMT-TF et des marqueurs de l'EMT. Un traitement au TGF, inducteur de l'EMT, diminue leur expression, signifiant potentiellement que ces miARN peuvent négativement réguler l'EMT. En parallèle, plusieurs EMT-TF sont capables de réprimer l'expression de miR-590-3p, agissant directement sur son promoteur, créant ainsi des boucles de régulation. Des études fonctionnelles utilisant des vecteurs d'expression stable de miR-590-3p sembleraient montrer un rôle secondaire de ce miARN dans la régulation de l'EMT car mir-590-3p dérégule des marqueurs secondaires de l'EMT comme la N-cadhérine. Des études de restauration de fonctions sont envisagées pour déterminer quelle est l'importance de ces boucles de régulation dans la progression tumorale mammaire. Plus largement, l'expression des miARN identifiés va être corrélée avec les signatures associées aux EMT-TF et aux voies oncogéniques classiques pour déterminer le lien entre ces trois composants dans la tumorigenèse mammaire. Mes travaux de thèse ont montré qu'il existait un intéractome entre des inducteurs de l'EMT, des stress oncogéniques et des miARNs au cours de la transformation mammaire humaine / Cancer cells are able to reactivate the Epithelio-Mesenchymal Transition (EMT), an embryonic mechanism, to acquire mobility and dedifferentiation capacities. EMT leads to a genetic reprogramming with the reactivation of EMT inductors, mainly transcription factors (EMT-TF) and the inhibition of miRNA. Otherwise, oncogenic stresses are essentials to tumor progression. The aim of my thesis project was to have a better understanding about the cooperation between events of genetic reprogramming occurring during EMT and oncogenic stresses during mammary tumor transformation. First, a screening based on oncogenic cooperation in soft agar assay, between EMT-TFs and oncogenic stresses was performed. Following a bioinformatics analysis, different EMT-TFs signatures associated with an oncogenic stress were identified. Thus, for example, the expression of EMT-TF ZEB1 and GSC were associated with the deletion of tumor suppressor gene PTEN to transform immortalized mammary epithelial cells. An immunohistochemistry analysis on a set of 558 triple negative breast cancers validated in vivo the presence of a correlation between the expressions of GSC and PTEN. However, this association seems to be more complex. Indeed, the expression of GSC is negatively associated with the nuclear expression of PTEN while it’s positively associated with the cytoplasmic expression of PTEN. Finally, an analysis of public metadata on cancer samples as TCGA or METABRIC is ongoing to validate these in vitro signatures and wider to determine how EMT or EMT-TFs associated signatures correlate with classical oncogenic pathways.Secondly, an in silico analysis, from predictive algorithms of miRNA targets, was performed to select miRNA able to inhibit the expression of several EMT-TFs. Two miRNA (miR-495 and miR-590-3p) were identified targeting several members of four principal’s families of EMT-TFs (FOXC, Snail, bHLH and ZEB). In vitro tests were realized to validate these regulations identifying Slug as a target of miR-590-3p. Moreover, these miRNAs expression in mammary cell lines is negatively correlated with EMT-TFs expression and EMT markers. A treatment with TGF-, a major EMT inductor, decreases their expression, potentially meaning that these miRNA can negatively regulate EMT. In parallel, several EMT-TFs are able to repress the expression of miR-590-3p, acting directly on its promotor, thus creating feedback loops. Functional studies using stable expression vector of miR-590-3p suggest a secondary role of this miRNA in the regulation of EMT because miR-590-3p deregulates EMT secondary markers as N-Cadherin. Functions restauration studies are planned to determine how important these feedback loops in mammary tumor progression are. To open the project, expression of these identified miRNA will be correlated with EMT-TF associated signatures and with classical oncogenic pathways to determine the link between these three components in mammary tumorigenesis. My thesis works are shown that there is an interactome between EMT inductors, oncogenic stresses and miRNA during human mammary transformation

La Ténascine-X (TNX) est une glycoprotéine architecturale de la matrice extracellulaire. Outre ce rôle, la TNX est également considérée comme une protéine matricellulaire qui est capable de réguler le comportement de cellules normales et tumorales. Toutefois, aucun mécanisme moléculaire et cellulaire ne permettait d'expliquer les effets cellulaires de la TNX, avant notre étude. Au laboratoire, nous avons démontré que le domaine C-terminal de type fibrinogène (FBG) de la TNX était capable d'induire l'activation du Transforming Growth Factor (TGF) bêta latent. En effet, les trois isoformes du TGF bêta sont sécrétées sous la forme de complexes inactifs formés à partir de liaisons non covalentes entre le TGF bêta mature et son propeptide N-terminal LAP (Latency Associated Peptide). Nous avons montré que le domaine FBG de la TNX interagissait physiquement avec le TGF bêta latent, in vitro et in vivo, et induisait un changement de conformation du complexe latent, afin de permettre son activation en une molécule bioactive. De plus, nous avons identifié l'intégrine alpha11 bêta1 comme un récepteur membranaire pour la TNX et nous avons montré que cette intégrine était cruciale pour le processus d'activation du TGF bêta latent par le domaine FBG. Nous avons également démontré que les Méprines alpha et bêta deux protéases de la famille des astacines, pouvaient cliver la TNX, permettant ainsi de libérer des fragments contenant le domaine FBG, capables d'activer le TGF bêta latent. Enfin, nous avons entamé une étude de la pertinence biologique de l'activation du TGF bêta latent par la TNX in vivo en analysant la voie de signalisation du TGF bêta dans des souris déficientes ou non en TNX / Tenascin-X (TNX) is an architectural glycoprotein of the extracellular matrix. Beyond this role, TNX is also considered as a matricellular protein that is able to regulate the behavior of normal and tumor cells. However, no molecular and cellular mechanism has been described to explain TNX cellular effects before our study. In the laboratory, we showed that the C-terminal fibrinogen-like domain (FBG) of TNX was able to induce the latent transforming growth factor (TGF beta activation. Indeed, the three TGF beta isoforms are secreted as inactive complexes formed from non-covalent bonds between the mature TGF beta and its N-terminal propeptide, called LAP (Latency Associated Peptide). We showed that the FBG domain of TNX physically interacted with the latent TGF beta, in vitro and in vivo, and induced a conformational change of the latent complex to allow its activation into a bioactive molecule. Furthermore, we identified alpha1 beta1 integrin as a cell-surface receptor for TNX and showed that this integrin was crucial for the FBG-induced latent TGF beta activation. We also demonstrated that Meprins alpha and beta, two proteases belonging to the astacin family, could cleave the TNX, thereby releasing fragments containing the FBG domain capable of activating latent TGF beta. Finally, we have initiated a study regarding the biological relevance of latent TGF beta activation by TNX in vivo by analyzing the TGF beta signaling pathway in wild type or TNX-deficient mice

Rapidly growing tumor cells outgrow their blood supply resulting in a microenvironment with reduced oxygen and nutrients. Using an in vitro transformation model we found that cancer cells expressing the SV40 ST antigen (+ST cells) are more resistant to glucose deprivation-induced cell death than cells lacking the SV40 ST antigen (−ST cells). Mechanistically, we found that the ST antigen mediates this effect by activating a nutrient-sensing kinase, AMP-activated protein kinase (AMPK). We further show that AMPK mediates its effects, at least in part, by inhibiting mTOR (mammalian target of rapamycin), thereby shutting down protein translation, and by inducing autophagy as an alternate energy source. Resistance to anoikis upon anchorage-deprivation is yet another form of stress tolerated by both normal stem/progenitor cells of various tissues in our body and by cancer cells. Using mammospheres as a model to enrich for stem/progenitor cells we found that mammosphere formation is accompanied with increased activation of AMPK. Concomitant with AMPK activation, we detected increased phosphorylation of the anti-apoptotic protein PED/PEA15. We further demonstrate that AMPK directly interacts with and phosphorylates PEA15 at Ser116, thus establishing PEA15 as a new AMPK target. Thus, our study has identified AMPK-PEA15 signaling as a key component of sphere formation by both normal and cancerous breast tissues. During metastasis, epithelial cells lose attachments to their neighbors, acquire a mesenchymal-like morphology, a process termed as epithelial-mesenchymal transition (EMT) and become motile. Our results indicate that AMPK regulates EMT by both transcriptional and post-translational modification of EMT-inducing transcription factor, Twist. Thus, our study has identified a role for AMPK in nutrient deprivation, anchorage-independent growth, and epithelial-mesenchymal transition involved in metastasis. In addition, we have identified two novel substrates of AMPK, PEA15 and Twist, that may play key roles in cancer progression. Thus, our study suggests that targeting AMPK, or its newly identified substrates, can be explored as possible anti-cancer mechanisms.

La transition épithélio-mésenchymateuse (TEM) est un processus physiologique décrit dans le développement embryonnaire et chez l'adulte au cours de la cicatrisation. La TEM est également détournée dans le contexte pathologique au cours de l'invasion tumorale et les mécanismes moléculaires qui la contrôlent font à ce jour l'objet d'intenses investigations. Cette étude décrit le rôle du gène de la lignée germinale NANOS-3 dans la régulation de l'invasion tumorale broncho-pulmonaire associée à la TEM. Nous démontrons que l'expression de Nanos-3 est corrélée à l'agressivité des cancers bronchiques non à petites cellules (CBNPC) humains in vivo et qu'il est surexprimé pendant la TEM induite in vitro. De plus, la surexpression de Nanos-3 dans les lignées tumorales bronchiques augmente leurs capacités invasives in vitro en induisant la TEM alors que son inhibition induit l'effet opposé et promeut la transition mésenchymo-épithéliale (TME). Au cours de cette étude, nous rapportons également des mécanismes à la fois transcriptionnels et post-transcriptionnels de régulation des cibles de Nanos-3. Ainsi, nous montrons que Nanos-3 réprime la transcription du gène CADHERINE-E indépendamment des facteurs de transcription des familles Snail et ZEB. Nous décrivons également que la protéine Nanos-3 co-immunoprécipite avec certains ARNm de ses cibles et, plus particulièrement, qu'elle est capable de réguler la longueur de la queue poly-(A) du transcrit codant pour une de ses cibles majeures : la Vimentine. En parallèle, par des méthodes d'études in silico et in vitro, nous démontrons une localisation à la fois cytoplasmique et nucléaire de Nanos-3 ainsi que son accumulation nucléolaire. Enfin, nous mettons en évidence que la réexpression ectopique de Nanos-3 dans le contexte tumoral pourrait être attribuée à une dérégulation des mécanismes épigénétiques physiologiquement mis en place dans les cellules somatiques adultes. Ainsi, cette étude démontre le rôle de Nanos-3 dans l'acquisition d'un phénotype invasif par les cellules tumorales bronchiques et décrit un nouveau mécanisme de régulation de la TEM dépendant de la longueur de la queue poly-(A) de certains ARNm. / The Epithelial-Mesenchymal Transition (EMT) is a basic cellular process used by embryo to generate different tissues or in adult during wound healing. EMT is also misappropriated by cancer cells during the first step towards metastasis. Molecular mechanisms driving EMT during tumor progression are extensively studied and post-transcriptional regulations of EMT-associated genes emerge as major and promising field in oncology. Here we report a dual post-transcriptional and transcriptional regulation of EMT-associated genes by the NANOS-3 germline gene during lung tumor invasion. We show that the Nanos-3 expression in vivo correlates with aggressiveness of human non-small cell lung carcinomas (NSCLC) and that Nanos-3 is upregulated in cells which undergo an EMT in our in vitro EMT-inducible models. Moreover, Nanos-3 overexpression in human NSCLC cell lines enhances their invasive abilities by EMT regulation while its silencing induces the opposite effect leading to a Mesenchymal-Epithelial Transition (MET). Molecular investigations indicate that Nanos-3 controls its targets by either transcriptional or post-transcriptional mechanisms. We show that Nanos-3 represses E-CADHERIN transcription independently of Snail and ZEB transcription factor families. Moreover, we also find that mRNAs of post-transcriptionally regulated targets are co-immunoprecipitated with the Nanos-3 protein and that Nanos-3 regulates the length of the 3' poly-A tail of VIMENTIN mRNA. This dual mechanism of EMT regulation by Nanos-3 is to be related to the specific subcellular localization of Nanos-3 in both cytoplasm and nucleus associated with a nucleolus accumulation as shown by in vitro and in silico experiments. Finally, we demonstrate an epigenetic regulation of NANOS-3 gene expression in lung cell lines, thus supporting that its ectopic expression could be attributed to an epigenetic machinery deregulation in cancer cells.Thus, here we demonstrate a new innovative role for Nanos-3 in the acquisition of an invasive phenotype by lung tumor cells and we describe a novel mechanism of post-transcriptional regulation of EMT via the control of the mRNA poly-A tail length.

La progression tumorale, qui conduit à la formation de métastases, est le résultat de profondes modifications des différents niveaux de régulation de l'expression des gènes comme la transcription ou l'épissage alternatif. Au cours de ma thèse, j'ai étudié le rôle de DDX5 et DDX17, deux ARN hélicases qui, au cours de la progression tumorale, sont impliquées dans la régulation transcriptionnelle, l'épissage alternatif et la biogénèse des microARNs. Pour cela, j'ai utilisé deux modèles de progression tumorale : le modèle murin 4T1, composé de cellules cancéreuses qui présentent des propriétés métastatiques différentes, et les cellules humaines MCF10A qui, après traitement au TGF-beta, sont capables de réaliser la transition épithélio-mésenchymateuse, un processus de trans-différenciation qui contribue à la formation des métastases. Dans le modèle 4T1, j'ai montré que Ddx17 et Ddx5 contribuent à l'invasivité des cellules tumorales en contrôlant des programmes transcriptionnels et d'épissage alternatif. Plus précisément, j'ai démontré que Ddx5 et Ddx17 favorisent l'agressivité des cellules cancéreuses en régulant l'épissage des variants de l'histone macroH2A1 qui, à leur tour, contrôlent l'expression de gènes impliqués dans la progression tumorale. Dans le modèle MCF10A où la transition épithélio-mésenchymateuse peut être induite sous TGF-beta, j'ai montré que DDX5 et DDX17 orchestrent dynamiquement des programmes transcriptionnels et d'épissage. Le travail effectué pendant ma thèse met en évidence l'importance des ARN hélicases DDX5 et DDX17 comme régulateurs clés de la progression tumorale, et souligne le rôle de l'épissage alternatif lors de la progression tumorale. De plus, ce travail met l'accent sur l'importance d'intégrer les différents niveaux de régulation de l'expression des gènes (transcription, épissage, microARN) pour une compréhension globale de la progression tumorale / Tumor progression leading to the formation of metastases result from deep modifications of gene expression programs at several levels, including transcription and splicing. During my PhD, I investigated the role in tumor progression of DDX5 and DDX17, two highly related multifunctional DEAD box RNA helicases that are involved in transcription and splicing as well as in microRNA biogenesis. For this purpose, I used two breast cancer models of tumor progression : the 4T1 mouse model composed of cancer cells that exhibit different metastatic properties and MCF10a human cells that undergo epithelial-to-mesenchymal transition upon Tgf-beta treatment, a trans-differentiation process contributes to metastasis formation. In the 4T1 mouse model, I showed that Ddx17 and Ddx5 contribute to tumor-cell invasiveness by controlling both transcriptional and splicing programs. More specifically, I demonstrated that Ddx5 and Ddx17 promote cancer cells aggressiveness by regulating the splicing of the macroH2A1 histone which in turn impacts on the expression of genes implicated in tumor cell invasiveness. In the Tgf-beta induced epithelial-to-mesenchymal trans-differentiation model, I showed that DDX5 and DDX17 dynamically orchestrate transcription, microRNA and splicing programs. The work performed during my PhD highlights the importance of DDX5 and DDX17 RNA helicases as key regulators of tumor progression in breast cancer, and also underlines the role of alternative splicing during tumor progression. Furthermore, this work emphasizes the importance of integrating the different layers of the gene expression process (transcription, splicing, microRNA) for a comprehensive understanding of tumor progression

Le TGFbeta intervient dans la régulation de nombreux processus cellulaires comme la prolifération, la différenciation, la migration, l'apoptose, du développement embryonnaire jusqu'à la vie adulte. Le TGFbeta est aujourd'hui bien décrit pour son rôle de suppresseur de tumeur de par ses activités anti-prolifératives et pro-apoptotiques, en particulier sur les cellules épithéliales. Cependant, au cours de la progression tumorale, le TGFbeta devient un promoteur de tumeur en favorisant l'angiogenèse, l'échappement de la tumeur vis-à-vis du système immunitaire et en induisant la transition épithélio-mésenchymateuse. Après fixation du ligand TGFbeta , le complexe de récepteurs active les protéines cytoplasmiques Smad2 et Smad3 qui s'associent à Smad4 pour former le complexe transcriptionnel qui se transloque alors dans le noyau pour réguler la transcription de nombreux gènes cibles. Récemment, la protéine TIF1gamma a été décrite pour intervenir dans la régulation négative de la voie du TGFbeta , en monoubiquitinant Smad4 ou en interagissant avec Smad2/3 en compétition avec Smad4. Cette voie de signalisation devant être finement contrôlée pour cibler son action en fonction du contexte cellulaire, nous analysons ici la régulation des interactions fonctionnelles entre la voie canonique du TGFbeta et la protéine TIF1gamma. Dans cette étude, nous montrons que TIF1gamma agit comme un régulateur négatif des fonctions de Smad4 dans la voie de signalisation du TGFbeta au cours du processus de transition épithélio-mésenchymateuse et au cours de la différenciation terminale des cellules épithéliales mammaires et de la lactation. Nous étudions également la SUMOylation de TIF1gamma comme nouveau niveau de régulation de la réponse cellulaire au TGFbeta . Nous avons ainsi caractérisé les sites fonctionnels de SUMOylation de TIF1gamma et montré que cette modification post-traductionnelle inhibe la formation du complexe transcriptionnel Smad et est nécessaire pour réguler temporellement la résidence de Smad4 au niveau du promoteur de gènes cibles du TGFbeta . Nos résultats montrent le rôle important de la SUMOylation de TIF1gamma dans la régulation de la transition épithélio-mésenchymateuse induite par le TGFbeta . En conclusion, notre travail met en avant le rôle majeur de TIF1gamma dans la régulation de la réponse transcriptionnelle au TGFbeta . De plus, nous montrons que la SUMOylation de TIF1gamma est nécessaire à son activité répressive sur Smad4 / The cytokine TGFbeta regulates several cellular processes such as proliferation, differentiation, migration and apoptosis, from embryonic development to adulthood. TGFbeta is well described for its tumor suppressor role through antiproliferative and proapoptotic activities, in particular in epithelial cells. During tumor progression however, TGFbeta becomes a tumor promotor, favoring angiogenesis, immune suppression and inducing the epitheliomesenchymal transition. Binding of TGFbeta ligand to its receptors activate cytoplasmic messenger Smad2 and Smad3 to complex with Smad4 and shuttle into the nucleus to regulate TGFbetatarget genes expression. Recently, TIF1gamma has been described as a new negative regulator of TGFbeta signaling, through monoubiquitination of Smad4 or direct competition with Smad4 to bind activated Smad2/3. This signaling pathway has to be finely tuned to target an action dependent on a cellular context, which is why we analyze here the regulation of functional interactions between the TGFbeta canonical signaling and TIF1gamma. In this study, we show that TIF1gamma acts as a negative regulator of Smad4 functions in TGFbetasignaling during the epithelio-mesenchymal transition and during terminal differentiation of mammary epithelial cells and lactation. We are also interested in studying TIF1gamma SUMOylation as additional level of regulation of cell response to TGFbeta. Thus we characterized four functional SUMOylation sites in TIF1gamma and we found that this post-translational modification inhibits the formation of Smads transcriptional complex and is needed to temporally restrict Smad4 residence on the promoter of TGFbetatarget genes. Our results show the critical role of TIF1gamma SUMOylation in the regulation of TGFbeta- induced epithelio-mesenchymal transition. As a conclusion, our study unveils the major role of TIF1gamma in the regulation of TGFbeta transcriptional responses. Moreover, we show that TIF1gamma requires SUMOylation to exert its repressive activity on TGFbetasignaling

O câncer de pâncreas é considerado um dos adenocarcinomas mais agressivos, ocupando o quarto lugar de mortes devidas a câncer, isto é dado principalmente a seu desenvolvimento silencioso e a sua complexidade genética, tornando-o de difícil detecção. Consequentemente, o diagnóstico desta doença ocorre apenas em fase tardia, quando o tratamento é apenas para fins paliativos. As anomalias genéticas mais frequentes no câncer de pâncreas invasivo estão relacionadas à ativação por mutações do gene KRAS e à inativação dos genes supressores de tumor CDKN2A, TP53, SMAD4 e BRCA2. Além destas conhecidas vias de sinalização, fatores de crescimento como o TGF-? e EGF também apresentam papel fundamental na progressão e metástase do câncer de pâncreas. Interessantemente, TGF-? e EGF são também indutores do processo denominado transição epitelial-mesenquimal (EMT), onde células epiteliais normais, durante a embriogênese, ou células cancerosas durante a progressão tumoral e metástase, perdem seus contatos intracelulares e adquirem caráter migratório. Desta forma a EMT, induzida por altos níveis de TGF-? e/ou EGF, é considerada como um dos mecanismos de progressão tumoral em adenocarcinomas. No presente estudo, foram estudados os proteomas e o fosfoproteoma da EMT do PanCa. Assim, células de câncer de pâncreas PANC- 1 foram induzidas à EMT em cultura com os fatores de crescimento TGF-?1 ou TGF-?2 e EGF, e após a indução, marcadores moleculares e propriedades funcionais de migração e invasão foram confirmados. Duas condições de indução da EMT foram estabelecidas, para as quais foram desenvolvidas as análises proteômicas quantitativas. A abordagem foi baseada em marcação isotópica de células em cultura (SILAC), em conjunto com fracionamento celular e de proteínas intactas, e cromatografia líquida acoplada à espectrometria de massas para identificação de proteínas em larga escala. No total, aproximadamente 5.000 proteínas foram identificadas, e a maioria delas quantificadas com precisão nas duplicatas experimentais. Foram selecionadas 37 proteínas com expressão diferencial estatisticamente significativa nos experimentos proteômicos, as quais participam principalmente em processos de biogênese, adesão e apoptóticos. A análise de redes de interação revelou que as proteínas alteradas estavam principalmente localizadas em vias de sinalização que controlam processos de organização da matriz extracelular, splicing alternativo e regulação da apoptose. A análise do fosfoproteoma foi feita usando TGF-?1 como agente indutor da EMT nas células PANC-1, usando a estratégia ERLIC para o enriquecimento de fosfopeptídeos. No total, foram identificados 5.965 fosfopeptídeos não redundantes, correspondendo a um total de 2.250 fosfoproteínas analisadas, sendo quantificadas 2.053 ao menos em duas replicatas, e destas foram identificados 61 fosfopeptídeos regulados pertencentes a 55 fosfoproteínas, relacionados com processos de regulação do mRNA e vias de sinalização ligadas à adesão celular. Em conclusão, nosso estudo elucida potenciais novos alvos para inibição da EMT, controle da metástase, ou para auxiliar no diagnóstico da doença, quando devidamente validada. / Pancreatic cancer kills more than 200 thousand people worldwide every year. Also, pancreatic cancer is considered one of the most aggressive adenocarcinomas and difficult to diagnose since it develops silently and presents a high genetic complexity. Consequently, the diagnostic is often late, when the pancreatic cancer has already metastasized and the treatment has only palliative purposes. The most frequent genetic alterations observed in pancreatic cancer are related to mutations in KRAS oncogene and CDKN2A, TP53, SMAD4 and BRCA2 tumor suppressor genes. In addition to these known frequent mutations, growth factors such as TGB- ? and EGF play important roles in pancreatic cancer progression and metastasis. Interestingly, TGB- ? and EGF are also inducers of the Epithelial to Mesenchymal Transition (EMT), in which epithelial cells lose their intracellular contacts and acquire migratory capacities. Therefore, EMT is considered one of the mechanisms responsible for tumor progression and metastasis in adenocarcinomas in addition of being correlated to the process of generating cancer stem cells. In our present study, pancreatic cancer cell line PANC-1 was induced to EMT by using growth factors TGF-?1 or TGF-?2 and EGF. Both molecular and functional properties, such as invasion and migration, were evaluated in PANC-1 cells undergoing EMT and confirm the induction. Two conditions of EMT induction were properly established and in-depth quantitative proteomic analysis based on stable isotope labeling in cell culture (SILAC) followed by cellular and protein fractionation were assessed. In total, 5.000 proteins were identified and most of them were accurately quantified in duplicate experiments. Thirty-seven proteins were selected as differentially expressed with statistical significance, and were related mainly with biogenesis, adhesion and apoptosis processes. Interaction network analysis showed that regulated proteins were predominantly participating in signaling pathways linked to extracellular matrix organization, alternative splicing and apoptosis regulation. Phosphoproteome analysis was done using TGF-?1 as EMT-inductor agent on PANC-1 cells and ERLIC strategy for phosphopeptides enrichment. In total, were identified 5.965 non-redundant phosphopeptides corresponding to approximately 2.250 analyzed phosphoproteins, thereof 2.053 were quantified in at least two replicates. At comparison, were identified 61 regulated phosphopeptides belonging to 55 phosphoproteins, which were related with mRNA regulation processes and signialing pathways linked to cellular adhesion. In conclusion, our study highlighted potential new targets for EMT inhibition, metastasis control or to help in pancreatic cancer diagnosis, when careful validated.

As neoplasias mamárias apresentam um grande desafio tanto para a medicina humana, quanto para a medicina veterinária. Esses tumores apresentam ampla heterogeneidade intertumoral e intratumoral, dificultando assim a busca por tratamentos eficazes. Recentemente, pesquisadores tem voltado sua atenção para uma população de células que apresentam características muito semelhantes as células-tronco. São as chamadas células iniciadoras de tumores (CITs). Estas são descritas como as principais responsáveis por falhas nas quimioterapias e no surgimento de recidivas tumorais, devido ao seu potencial tumorigênico, de auto-renovação e de resistência a drogas antineoplásicas. Entretanto, o estudo dessas células é limitado pelas dificuldades no isolamento e na caracterização pós-enriquecimento dessas células, devido à perda do fenótipo em modelos in vitro. Sendo assim, vários estudos estão buscando maneiras alternativas de enriquecer essa população. Uma das maneiras mais utilizadas, baseia-se na indução do processo de transição epitélio-mesenquimal, através da superexpressão de fatores de transcrição como SNAI1, SLUG, ZEB1 e ZEB2. Sendo assim, nós objetivamos expressar de maneira exógena os fatores de transcrição SLUG e ZEB1 em células oriundas de carcinomas mamários caninos, caracterizar seus efeitos nessas células e observar se esses fatores de transcrição seriam capazes de induzir o fenótipo de CIT. Primeiramente, quatro amostras de carcinomas mamários caninos foram analisados quanto sua morfologia e os níveis de expressão gênica de quatro fatores de transcrição associados a transição epitélio-mesenquimal: SLUG, STAT3, ZEB1 e ZEB2. Após, nós selecionamos duas dessas amostras (CC-20E e CL-28E), que apresentavam morfologia típica de células epiteliais e baixa expressão dos fatores de transcrição citados acima e expressamos de maneira exógena e de forma estável os fatores de transcrição SLUG e ZEB1, através do processo de transdução lentiviral. Entretanto, apenas a transdução com os plasmídeos contendo a região codificante de SLUG foi eficiente. Sendo assim, nós avaliamos os efeitos da expressão exógena de SLUG nas células CC-20E e CL-28E, quanto a alteração de morfologia e expressão de filamentos intermediários como citoqueratina, vimentina e actina. Além disso, nós avaliamos se a expressão exógena de SLUG poderia regular a expressão de outros genes associados a EMT, além de genes supressores de tumores, alvos de SLUG. Por fim, nós avaliamos se a expressão exógena de SLUG poderia induzir ao fenótipo de CITs, verificando se havia alteração na sensibilidade das células aos quimioterápicos doxorrubicina e paclitaxel, além de avaliar o potencial tumorigênico e de auto-renovação dessas células em cultivos de baixa aderência. A expressão exógena de SLUG nas células CC-20E e CL-28E, não induziu a alterações na morfologia epitelial das células. Entretanto, as células alteraram sua disposição em monocamada no cultivo, formando tipos de túbulos semidiferenciados, característicos do processo de EMT híbrido ou parcial. Além, disso, houve um equilíbrio entre a expressão dos filamentos intermediários de citoqueratina e vimentina nas células, além do aumento na expressão dos genes CDH1 (E-caderina) e CDH2 (N-caderina), resultado que sustentou a indução de EMT parcial. O processo de EMT parcial induziu maior resistência ao quimioterápico paclitaxel, além de potencializar a tumorigenecidade e a capacidade de auto-renovação das células em cultivos de baixa aderência. Sendo assim, no presente estudo, nós obtivemos um cultivo com características que mimetizam as CITs, demonstrando que os processos que induzem esse fenótipo são semelhantes tanto na espécie canina, quanto em humanos, sustentando a hipótese de que neoplasias mamárias caninas podem servir como modelo para o estudo das CITs e, consequentemente, do desenvolvimento neoplásico de tumores sólidos. / Mammary neoplasms present a major challenge for both human and veterinary medicine, due to intertumoral and intratumoral heterogeneity, hindering the search for effective treatments. Recently, researchers has highlighted a population of cells with features very similar to stem cells. Known as Tumor-Initiating Cells (TICs), they are described as the main responsible for chemotherapy failures and tumor recurrence, due to their tumorigenic potential, self-renewal ability and drug resistance. The study of TICs is limited mainly by their difficult isolation owing to specific markers absence, and furthermore, cells lose their phenotype when placed in vitro. Therefore, several studies are seeking for alternatives to enrich this population in regular cultures. One way is based on the epithelial-mesenchymal transition induction through of transcription factors overexpression, such as SNAI1, SLUG, ZEB1 e ZEB2. So, the aim of this study was to overexpresse the SLUG and ZEB1 transcription factors in a cell culture derived from canine mammary carcinomas, evaluate its effects and observe whether these transcription factors would be capable of inducing the TIC phenotype. First, four canine mammary carcinomas cell cultures were analyzed for their morphology and gene expression levels of four transcription factors associated with epithelial-mesenchymal transition: SLUG, STAT3, and ZEB1 ZEB2. After, we selected two samples (CC-20E and CL-28E) with typical morphology of epithelial cells and low expression of the transcription factors mentioned above. We then overexpress, stably, the transcription factors SLUG and ZEB1 by lentiviral transduction, However, only SLUG transduction was efficient. Then, we evaluated the effects of SLUG overexpression in CC-20E and CL-28E cells as the change of morphology, expression of intermediate filaments as cytokeratin, vimentin and actin. In addition, we evaluated whether SLUG overexpression could regulate the expression of other EMT-associated genes as well as tumor suppressor genes, and assessed evaluated the tumorigenic potential and self-renewal of these cells in low adherence cultures. Finally, we assessed whether SLUG overexpression could induce drug resistance through doxorubicin and paclitaxel sensivity assay. The SLUG overexpression did not induce modification in epithelial cell morphology, however, cells changed their arrangement in monolayer culture, inducing the semidifferentiated tubules, typical of hybrid or partial EMT process. In, addition, there was a balanced expression between cytokeratin and vimentin, possibly explained by an increase in CDH1 expression (E-cadherin) and CDH2 (N-cadherin) typical of partial EMT. Furthermore, the partial EMT generated cells presenting paclitaxel resistance, and enhanced the tumorigenic potential and self-renewal capacity of the cells on low adherent plates. Thus, in this study, we obtained a cell culture exhibiting features that mimics the TICs, demonstrating the mechanisms which regulate this phenotype are similar in dogs and humans, supporting the hypothesis that canine mammary carcinomas are a great model for the study of TICs and solid tumors development.

Dans les années 2000, la Dre Johanne Tremblay et son équipe identifient un gène régulé négativement par le calcium extracellulaire dans les glandes parathyroïdiennes de rat hypertendu (SHR). Initialement nommé Hypertension-related calcium regulated gene (HCaRG), puis COMM domain-containing 5 (COMMD5), ce gène codant pour une petite protéine de 24,67 kDa fait partie d’une famille de 10 protéines ayant une structure carboxy-terminale homologue nommée domaine COMM. De nombreux rôles ont été associés à cette famille de protéines et l’analyse expressionnelle dans différents types de cancers montre une modulation, laissant penser qu’elles auraient un rôle oncogénique ou suppresseur de tumeurs. Les études ont démontré que COMMD5 entraine une maturation des jonctions cellulaires, une diminution de la prolifération et favorise la migration cellulaire. La surexpression de COMMD5 dans les tubules proximaux de rein accélère la réparation suite à un dommage aigu en limitant d’une part la prolifération tout en favorisant la migration et la re-différenciation cellulaire. Partant de ces observations, nous avons focalisé nos études sur le développement du carcinome à cellules rénales, une pathologie affectant 300 000 personnes chaque année dans le monde. L’hypothèse que nous avons émise était que COMMD5 puisse potentiellement jouer un rôle anti-oncogénique dans le cancer du rein en contrôlant la prolifération et la différenciation cellulaires. Afin de vérifier notre hypothèse, nous avons étudié le rôle de COMMD5 dans le maintien de l’intégrité épithéliale des cellules via la régulation de la transition épithélio-mésenchymateuse (EMT) et le contrôle du récepteur du facteur de croissance épidermique (EGFR). Nos résultats ont démontré que COMMD5 est diminuée dans les carcinomes rénaux et est corrélée avec la survie des patients. La présence du facteur de transcription induit par l’hypoxie 1 (HIF1α), exprimé dans la majorité des tumeurs solides rénales a induit une diminution de COMMD5. La perte de COMMD5 dans les cellules de tubules proximaux de reins humains (HK-2) a favorisé la dé-différenciation et la tumorigénicité des cellules, médiées par l’activation de la transition épithélio-mésenchymateuse. De plus, cette perte de COMMD5 a entrainé également une réorganisation du cytosquelette d’actine ayant pour conséquence la dérégulation endosomale du récepteur de l’EGF et favorisant une activation prolongée. Dans les carcinomes rénaux, la surexpression de COMMD5 a diminué la prolifération cellulaire suivie d’une re-différenciation grâce à deux mécanismes. D’une part, COMMD5 a régulé négativement la protéine HIF1α, induisant ainsi une transition mésenchymo-épithéliale (MET), tout en séquestrant le facteur de transcription SNAIL dans le cytoplasme. D’autre part, COMMD5 contrôle négativement l’expression transcriptionnelle des récepteurs ErbB par une hyperméthylation de leurs promoteurs. Dans son ensemble, les résultats innovant de cette thèse démontrent que COMMD5 est un gène ayant des caractéristiques anti-oncogéniques en contrôlant la différenciation cellulaire via le mécanisme de transition épithélio-mésenchymateux et la régulation de l’expression des récepteurs ErbB. / In the 2000s, Dr. Johanne Tremblay and her team identified a gene that was negatively regulated by extracellular calcium in hypertensive rat parathyroid glands. Originally named Hypertension-related calcium regulated gene (HCaRG) and renamed COMM Domaincontaining 5 (COMMD5), this gene encoding a small protein of 24.67 kDa is part of a family of 10 proteins sharing a homologous structure in the carboxy-terminal position named COMM domain. Many roles have been associated and expressional analysis of different types of cancer shows a modulation suggesting that they have an oncogenic or tumor suppressor roles. Studies have shown that COMMD5 induces maturation of the cell junctions, decreased cell proliferation and promotes migration. The overexpression of COMMD5 in the renal proximal tubules accelerates repair by promoting cell proliferation and ultimately induces cell migration and redifferentiation after acute injury. Based on these observations, we focused on the development of renal cell carcinoma, a disease affecting 300,000 people each year worldwide. Our hypothesis is that COMMD5 plays a tumor suppressor role in kidney cancer by controlling cell proliferation and differentiation. To test our hypothesis, we investigated the role of COMMD5 in maintaining the epithelial integrity of cells through the regulation of epithelial to mesenchymal transition (EMT) and the control of epidermal growth factor receptors (EGFR). The results showed that COMMD5 is decreased in kidney carcinomas resulting of a great negative indicator of the survival prognostic. The presence of hypoxia-inducible factor 1a (HIF1α), expressed in the majority of solid tumors, leads to a decrease of COMMD5 in the proximal tubule cells (HK-2). Inhibition of COMMD5 promotes dedifferentiation and tumorigenicity of cells mediated by epithelial to mesenchymal transition. The loss of COMMD5 induces a reorganization of the actin cytoskeleton resulting in endosomal dysregulation of the EGFR receptor and promoting its activation. In renal cell carcinoma, COMMD5 overexpression decreases cell proliferation and induces their redifferentiation by two mechanisms: firstly, COMMD5 induces an inhibition of the HIF1α protein expression resulting in a mesenchymal to epithelial transition and sequesters the SNAIL transcription factor in the cytoplasm; secondly, COMMD5 negatively regulates the transcriptional expression of the ErbB receptors. Taken together, these results of this thesis show that COMMD5 is a gene showing tumor suppressor characteristics by controlling cellular differentiation and by regulating the expression of ErbB receptors.