The transcription factors SMAD2 and SMAD3 are the effectors of classical transforming growth factor beta (TGFβ) signalling. This signalling cascade is involved in many cellular processes including proliferation and differentiation and is known to be a potent inhibitor of myogenic differentiation through SMAD3. We have previously shown that retinoic acid (RA) can upregulate SMAD3 in models of adipogenesis and mesenchymal stem cells and that SMAD3 can interact with the bZIP transcription factor C/EBPβ to disrupt its DNA binding. Forced expression of C/EBPβ inhibits myogenic differentiation but the mechanism has not been fully elucidated. Herein we show that RA increases Smad3 expression in myoblasts and that RA treatment antagonizes TGFβ-mediated inhibition of myogenic differentiation. TGFβ treatment increased C/EBPβ expression which was reversed by RA treatment. Further, RA was able to disrupt C/EBPβ occupancy of the Pax7 and Smad2 promoters in myoblasts. Loss of C/EBPβ in primary myoblasts using a conditional knockout model partially protected these cells from the anti-myogenic effects of TGFβ treatment. The TGFβ effector protein SMAD2 is expressed in myoblasts but its specific function in myogenesis has not been determined, as Smad2 knockout models are embryonic lethal. Thus, we created a novel Smad2 conditional knockout model where Smad2 is excised in PAX7-expressing muscle satellite cells. Herein we demonstrate a role for SMAD2 specifically in myogenic fusion. We describe a regeneration defect after acute injury and decreased fiber cross-sectional area at P21 (post-natal day 21) in Smad2cKO muscle, without affecting the numbers of PAX7-positive cells. Further, we reveal a mechanism whereby SMAD2 regulates KLF4 expression and mediates the KLF4-induced increased of the fusion gene Npnt. This work describes the pro-myogenic actions of RA-induced SMAD3 and the novel function of SMAD2 in terminal myogenic differentiation and fusion. This work also discusses future directions, implications and new insights into non-canonical SMAD actions.

Transcription growth factor beta (TGFβ) mediated epithelial to mesenchymal transition (EMT) of lens epithelial cells (LEC) is known to cause posterior capsular opacification (PCO). In this work, I have focused on the TGFβ-induced EMT pathway governed by the cellular actin cytoskeleton dynamics. This study is the first to report the involvement of transcription co-factor myocardin related transcription factor-A (MRTF-A) in TGFβ-induced EMT in the lens. Using rat lens epithelial explants, I have conclusively established that in LECs, TGFβ induces nuclear migration of MRTF-A leading to induction of αSMA expression. Furthermore, I have manipulated the intracellular translocation of MRTF-A indirectly using actin binding drugs and established that inhibiting nuclear migration of MRTF-A reduces αSMA production by the cells. In addition, direct manipulation of MRTF-A using adenoviral vectors carrying modified gene constructs show that presence of functional MRTF-A construct in the nucleus is necessary to trigger αSMA expression by causing EMT. In order to understand the involvement of matrix metalloproteinase -9 (MMP-9) in this specific pathway, explants were treated with an MMP2/9 inhibitor and rhMMP9. I have established that rhMMP-9 does not significantly affect the intracellular migration of MRTF-A. Nevertheless, gene expression studies showed that MMP-9 induces the expression of MRTF-A. Taken together, I believe MMP-9 functions through a feedback mechanism controlling MRTF-A expression in the cell. However, the presence of MMP-9 is necessary but not sufficient for induction in MRTF-A nuclear translocation. Overall, the work presented in this thesis demonstrates for the first time the presence of MRTF-A in LECs and successfully shows that the intracellular translocation of MRTF-A as an integral part of TGFβ induced EMT. Therefore, in the future, MRTF-A may be used as a successful target molecule to inhibit in order to prevent EMT leading to PCO. / Thesis / Doctor of Philosophy (PhD)

La protéine GPR50, qui fait partie de la famille des récepteurs de la mélatonine, est classée, avec une centaine d’autres protéines à sept domaines transmembranaires (7TM), dans la catégorie des récepteurs couplés aux protéines G hétérotrimériques (RCPG) orphelins, c’est-à-dire pour lesquels aucun ligand n’a pu être identifié. De plus en plus d’études montrent que les 7TM peuvent avoir des fonctions indépendantes d’un ligand. C’est le cas de GPR50 qui inhibe les fonctions du récepteur de la mélatonine MT1 en interagissant directement avec lui. Nous avons cherché à identifier d’autres partenaires associés à GPR50 en appliquant la technique de purification par affinité en tandem et avons mis en évidence son interaction avec un récepteur du facteur de croissance Transforming Growth Factor ß (TGFβ), le récepteur de type I (TβRI).Nous décrivons ici la formation d’un complexe entre GPR50 et le récepteur TβRI au niveau de la membrane plasmique, avec pour conséquence l’induction d’une activité constitutive du récepteur et des voies de signalisation en aval en l’absence de TGFβ, mais également en l’absence du récepteur TßRII qui est habituellement indispensable pour l’activation de TβRI par phosphorylation. Cette activité constitutive se traduit par la phosphorylation des protéines Smad2 et Smad3, leur intégration dans un complexe avec Smad4, la translocation du complexe dans le noyau et finalement l’activation de la transcription de leurs gènes-cibles. Nous avons décrypté les mécanismes moléculaires de cette activation constitutive en montrant que GPR50 entre en compétition, pour l’interaction avec TβRI, avec le régulateur négatif FKBP12, une protéine inhibitrice de l’activité basale du récepteur en l’absence de ligand. Nous avons identifié dans la queue intracytoplasmique de GPR50 un motif répétitif similaire à la séquence de FKBP12 impliquée dans son interaction avec TβRI , motif qui constitue la base moléculaire de cette compétition.Nous avons étudié les conséquences fonctionnelles de cette activation en surexprimant GPR50 de manière stable dans la lignée cellulaire MDA-MB-231, dérivée d’un cancer de sein. Nous avons observé dans ces cellules des effets pro-migratoires et anti-prolifératifs similaires à ceux causés par l’administration de TGFβ.En conclusion, ce travail décrit un nouveau mode d’activation du récepteur TβRI en l’absence de ligand, mais identifie également une nouvelle fonction indépendante d’un ligand pour le RCPG orphelin GPR50. En perspective de ce travail, nous allons essayer d’identifier des conditions biologiques où cette interaction pourrait prendre place afin de confirmer ces résultats dans un contexte plus physiologique. / During the last years, it became more and more accepted, that orphan G Protein coupled receptors (GPCRs) with a transmembrane spanning heptahelical core (7TM) can have ligand-independent functions. One of those 100 orphan GPCRs is GPR50, a 7TM protein with a long cytosolic domain. Recently, studies revealed ligand-independent functions for GPR50, where it has the capacity to modulate the activity of other proteins upon complex formation. By applying a tandem affinity purification approach we sought to identify further putative interacting partners of GPR50. One of the identified binding partners is the transforming growth factor β (TGFβ) receptor type I (TβRI).The TGFβ-dependent signal transduction pathway of serine/threonine kinases is a pathway with direct signal flow from ligand over the receptor to its substrates, the Smads which translocate into nucleus where they bind DNA and regulate gene expression. An important question concerns the generation of specificity and fine-tuning of TGFβ-dependent signaling. Throughout the years, an important number of proteins which regulate the activity of the TGFβ signal transduction pathway in a positive or negative manner have been identified. Most of them act in a cell-context-dependent manner, allowing the regulation of TGFβ signaling adapted to the particular circumstances.We report here the complex formation of GPR50 and TβRI on the plasma membrane. The consequence of this interaction is the GPR50-mediated induction of a constitutive activation of the TβRI and its downstream signaling in a TGFβ ligand-independent manner. This has been monitored by Smad2/3 phosphorylation, Smad2/3-Smad4 complex formation and their subsequent translocation into the nucleus, where they activate Smad-dependent gene expression. In order to decipher the molecular mechanism that allows this activation, we showed that GPR50 competes with the negative regulator, that prevents leaky TGFβ signaling, the gatekeeping molecule FKBP12, for binding to the TβRI. We identified a motif in FKBP12 involved in the interaction with TβRI with similarities to a motif in GPR50, providing a molecular basis for the replacement of FKBP12 by GPR50 in the TβRI complex. We showed that GPR50 is capable of activating the TβRI even in the absence of the TβRII, which normally is required for activating the TβRI by phosphorylation. This reveals a previously unknown mode of activation of the TβRI in absence of the TGFβ ligand and TβRII. In order to identify the functional consequences of this crosstalk, we studied migration and growth of MDA-MB-231 breast cancer cells stably overexpressing GPR50. In these cells, TGFβ-like pro-migratory and anti-proliferative effects have been observed.Future research will help to identify tissues and biological circumstances, where this crosstalk could take place for putting this novel mode of regulation of TGFβ signaling pathway into a context-dependent-manner. Additionally our work established another ligand-independent task for the orphan 7TM protein GPR50, consolidating its function as binding partner and activity modulator.

The transforming growth factor beta (TGFβ) is a cytokine that regulates a plethora of cellular processes such as cell proliferation, differentiation, migration and apoptosis. TGFβ signals via serine/threonine kinase receptors and activates the Smads to regulate gene expression. Enigmatically, TGFβ has a dichotomous role as a tumor suppressor and a tumor promoter in cancer. At early stages of tumorigenesis, TGFβ acts as a tumor suppressor by exerting growth inhibitory effects and inducing apoptosis. However, at advanced stages, TGFβ contributes to tumor malignancy by promoting invasion and metastasis. The pro-tumorigenic TGFβ potently triggers an embryonic program known as epithelial-mesenchymal transition (EMT). EMT is a dynamic process whereby polarized epithelial cells adapt a mesenchymal morphology, thereby facilitating migration and invasion. Downregulation of cell-cell adhesion molecules, such as E-cadherin and ZO-1, is an eminent feature of EMT. TGFβ induces EMT by upregulating a non-histone chromatin factor, high mobility group A2 (HMGA2). This thesis focuses on elucidating the molecular mechanisms by which HMGA2 elicits EMT. We found that HMGA2 regulates a network of EMT transcription factors (EMT-TFs), such as members of the Snail, ZEB and Twist families, during TGFβ-induced EMT. HMGA2 can interact with Smad complexes to synergistically induce Snail expression. HMGA2 also directly binds and activates the Twist promoter. We used mouse mammary epithelial cells overexpressing HMGA2, which are mesenchymal in morphology and highly invasive, as a constitutive EMT model. Snail and Twist have complementary roles in HMGA2-mesenchymal cells during EMT, and tight junctions were restored upon silencing of both Snail and Twist in these cells. Finally, we also demonstrate that HMGA2 can epigenetically silence the E-cadherin gene. In summary, HMGA2 modulates multiple reprogramming events to promote EMT and invasion.

Prostate cancer is the second leading cause of cancer-related death in men in the Western world. Deregulation of transforming growth factor β (TGFβ) signaling pathway is frequently detected in prostate cancer and contributes to tumor growth, migration, and invasion. In normal tissue and the early stages of cancer, TGFβ acts as a tumor suppressor by regulating proliferation, differentiation, and apoptosis. In later stages of cancer, TGFβ acts as a tumor promoter by inducing angiogenesis, tumor invasion, and migration. Thus, it is important to investigate the molecular mechanisms behind the tumor-promoting effects of TGFβ, which is the topic of this thesis.   The tumor necrosis factor receptor–associated factor 6 (TRAF6) controls non-canonical TGFβ signals due to its enzymatic activity, causing polyubiquitination of the cell membrane–bound, serine/threonine kinase TGFβ type I receptor (TβRI) and its subsequent cleavage in the extracellular domain by tumor necrosis factor a–converting enzyme (TACE) in a protein kinase C ζ (PKCζ)-dependent manner. TRAF6 also recruits the active g-secretase complex to the TβRI, resulting in a second cleavage in the transmembrane region and the liberation of the TβRI intracellular domain (TβRI-ICD), which enters the nucleus, where it associates with the transcriptional co-regulator p300. In Paper I, the aim was to elucidate by which mechanisms TβRI-ICD enters the nucleus. We found that the endocytic adaptor protein APPL1 interacts with TβRI and PKCζ. APPL proteins are required for TβRI translocation from endosomes to the nucleus via microtubules in a TRAF6-dependent manner. Moreover, APPL proteins are important for TGFβ-induced cell invasion, and high levels of APPL1 are detected by immunohistochemistry in prostate cancer. Finally, we demonstrated that the APPL1–TβRI complex visualized with the in situ proximity ligation assay (PLA) correlates with Gleason score, indicating that it might be a novel prognostic marker for aggressive prostate cancer. In Paper II, the aim was to explore by which mechanisms TGFβ causes activation of the AKT pathway, which regulates migration and therapy resistance of cancer cells. We found that the E3 ligase activity of TRAF6 induces Lys63-linked polyubiquitination of p85α upon TGFβ stimulation, resulting in plasma membrane recruitment, Lys63-linked polyubiquitination, and subsequent activation of AKT. Moreover, the TRAF6 and PI3K/AKT pathway were found to be crucial for the TGFβ-induced migration. Importantly, we demonstrated, by PLA, a correlation between Lys63-linked polyubiquitination of p85α and aggressive prostate cancer in tissue sections from patients with prostate cancer. In Paper III, the aim was to investigate the mechanisms for TGFβ-induced activation of PKCζ and the role of PKCζ in tumor regression. We found that TRAF6 caused Lys63-linked polyubiquitination of PKCζ. By using two novel chemical compounds that inhibit PKCζ, we demonstrated that PKCζ is crucial for prostate cancer cell survival and invasion. In Paper IV, the aim was to investigate further the target genes for the nuclear TβRI-ICD-APPL1 complex identified in Paper I. We provide evidence that APPL proteins and the TGFβ signaling pathway are important for cell proliferation. In summary, the results reported in this thesis suggest the potential usefulness of the identified signaling components of the tumor-promoting effects of TGFβ as drug targets and biomarkers for aggressive prostate cancer.

Low to moderate level of chronic arsenic exposure contributes to cardiovascular ailments including heart disease and aneurysms. Current research on the etiology and progression of cardiovascular disease focuses mainly on adult which fails to capture the developmental origins of cardiovascular disease. Thus, disruption in morphogenetic events during early development may initiate and pattern the molecular programming of cardiovascular ailments in adulthood. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. Coronary vessel development is initiated by precursor cells that are derived from the epicardium. Epicardial derived cells undergo proliferate, migrate, and differentiate into several cardiac cell types which are the cellular components of the coronary vessels. The key cellular event occurs in this process is the epithelial to mesenchymal transition (EMT), which can also be utilized by endocardial cushion cells to form aortic and pulmonary valves. The TGFβ family of ligands and receptors are essential for developmental cardiac EMT and coronary smooth muscle cell differentiation. Whether arsenic has any impact on TGFβ mediated cardiovasculogenesis is not known. Monomethylarsonous acid [MMA(III)] is the most potent metabolite of inorganic arsenic and has been shown to partly account for arsenic induced toxicity. The fetus is exposed to relatively higher levels of MMA (III) as compared to adults probably due to deficiency in methylation of transferred inorganic arsenic from the placenta. However, the developmental toxicity of MMA (III) has not yet been studied. In this study, we exploit a novel cardiac progenitor cell line to recapitulate epicardial EMT in vitro and to study developmental toxicity caused by arsenicals. We show that chronic exposure to low level of arsenite and MMA (III) disrupts developmental EMT programming in epicardial cells causing deficits in cardiac mesenchyme production. The expression of EMT program genes is also decreased in a dose-dependent manner following exposure to arsenite and MMA (III). Smad-dependent TGFβ2 canonical signaling and the non-canonical Erk signaling pathways are abrogated as detected by decreases in phosphorylated Smad2/3, Erk1/2 and Erk5 proteins. There is also loss of nuclear accumulation of p-Smad and p-Erk5 due to arsenical exposure. These observations coincide with a decrease in vimentin positive mesenchymal cells invading three-dimensional collagen gels. However, arsenicals do not block TGFβ2 stimulated p38 activation. Additionally, smooth muscle cell differentiation, which is proven to be governed by p38 signaling in epicardial cells, also remains intact with arsenical exposure. Overall these results show that arsenite and MMA (III) are strong and selective cardiac silencers. The molecular mechanisms of arsenical toxicity on TGFβ-Smad signaling in epicardial cells is further explored. A relatively high level of acute arsenical exposure rapidly depletes phosphorylated nuclear Smad2/3. Restoration of the nuclear accumulation of Smads can be achieved by inhibiting the expression or activation of Smad specific exportins suggesting that arsenicals augment Smad nuclear exportation. Abrogated Smad signaling caused by arsenicals is associated with severe deficits in EMT during mouse epicardium and chick endocardial cushion development. Thus progenitor cell outgrowth, migration, invasion and vimentin filament reorganization are significantly inhibited in response to arsenical exposure. Disrupted Smad nuclear shuffling is probably caused by zinc displacement on the MH-1 DNA binding domain of Smad2/3. Thus zinc supplementation restores both nuclear content and transcriptional activities of Smad2/3. Rescued TGFβ-Smad signaling by zinc also contributes to cellular transformation and mesenchyme production in embryonic heart explants. LINE1 (L1) retrotransposons are a group of mobile DNA elements that shape the genome via novel epigenetic controls. Although expression of L1 is required for early embryo implantation and development, abnormally elevated L1 is shown to inhibit embryonic cells from transforming and differentiating during organogenesis. Cellular redox signaling, which is regulated by antioxidant responsive elements (AREs), has been shown to play a key role in L1 activation and retrotransposition. However, whether L1 can be induced by the cellular oxidative stress caused by arsenic is not known. We provide evidence showing that L1 ORF-1 and ORF-2 mRNA levels are both up-regulated by arsenic. Nuclear accumulation of L1 ORF-2 is observed in response to 30 min arsenic exposure, which may lead to active retrotransposition events in the genome. Transcriptional activity of L1 is regulated by Nrf2 as mutations in ARE regions within the L1 promoter and Nrf2 silencing using siRNA both significantly inhibit L1 transcriptional activity. Nrf2 overexpression together with arsenic exposure creates synergic induction in L1 promoter activity suggesting that arsenic mediated L1 activation is partially Nrf2 dependent. Taken together, these findings reveal a molecular mechanism responsible for arsenic cardiac toxicity and define a novel genetic toxic effect of arsenic during embryonic heart development.

Angiogenesis is the formation of new blood vessels by sprouting from pre-existing ones. Transforming growth factor-beta (TGFβ) promotes angiogenesis and is a known inducer of endothelial-mesenchymal transition (EndMT), a process whereby endothelial cells become fibroblastic and motile. We hypothesize that TGFβ-induced EndMT enables endothelial cells to detach from the mature vessel and migrate to form the sprout that becomes a new vessel during angiogenesis. This study characterized EndMT in response to TGFβ +/- vascular endothelial growth factor (VEGF). Bovine aortic endothelial cells (BAEC) were stimulated with TGFβ +/- VEGF for prolonged periods. Confocal imaging and immunoblotting analyses revealed the strongest EndMT response at 5 ng/ml of TGFβ after 144 hours of exposure. A three-dimensional collagen model of angiogenesis revealed a potential relationship between EndMT and blood vessel sprouting. These results suggest that EndMT induction in BAECs requires high concentrations and prolonged exposure to TGFβ and is not significantly influenced by VEGF. / NSERC

Chondroitin sulphate proteoglycans (CSPGs) from the glial scar inhibit axonal regeneration following spinal cord injury. CSPG expression can be induced by transforming growth factor β (TGFβ), which suggests that inhibition of TGFβ may reduce CSPG levels. Astrocytes were treated with cyclic AMP (cAMP), which reduced TGFβ signaling protein Smad2 in astrocytes. However, cAMP-treated astrocytes showed strong neurocan expression following TGFβ treatment, which suggests that TGFβ may mediate CSPG expression through non-Smad pathways. Smad2 or Smad4 were knocked down in astrocytes using siRNA and TGFβ-induced neurocan, brevican and aggrecan expression were still observed, indicating that Smad signaling is not required for CSPG expression. Administration of a PI3K/Akt inhibitor produced significant reductions in neurocan, brevican and aggrecan expression in astrocytes, which suggests that PI3K/Akt pathway mediates CSPG expression. Erk1/2 inhibitor treatment did not reduce CSPG expression significantly. Targeting non-Smad signaling pathways may therefore be effective strategies to reduce CSPG expression following injury.

Breast cancer is a disease that will be diagnosed in about 1 in 10 women throughout their lifetime. The majority of breast cancers are originated from the epithelial cells of the mammary ducts, and this occurrence can be due to several factors including hereditary and acquired mutation. There are several major breast cancer subtypes, including estrogen receptor-α (ERα)-positive, HER2-enriched and triple-negative (TNBC). Patients diagnosed with ER+ tumors are generally treated with estrogen blockers (e.g., tamoxifen, letrozole and fulvestrant). Patients with HER2+ tumors are commonly administered with drugs that block HER2 signaling (e.g., trastuzumab) or inhibit HER2’s tyrosine kinase activity (e.g., lapatinib). For patients with TNBC, chemotherapies such as taxanes and anthracyclines are standard of care therapies. However, for each breast cancer subtype, a significant number of patients develop resistance to these therapies and eventually die from metastasis, a process which accounts for ~90% of breast cancer mortality. Currently, metastatic breast cancer is incurable, and the short median survival of 3 years for patients with metastatic breast cancer has not significantly changed in over 20 years. Therefore identification of new molecules that are involved in breast cancer metastasis and development of more precisely targeted therapeutic strategies are urgently needed to improve the clinical outcome for this disease. The transforming growth factor pathway beta (TGFβ) pathway has been show to play a key role in metastasis through induction of epithelial-mesenchymal transition (EMT), cell migration and invasion. Over more than a decade, this pathway has been studied across several cancers and it is now better established that it has context-dependent tumor suppressive and oncogenic qualities. In the early stages of breast cancer, TGFβ pathway is a suppressor of benign and early stage tumor growth. However, as disease progresses and corresponding levels of TGFβ ligands become elevated, a “switch” will take place and promote oncogenic phenotypes like EMT and cancer cell stemness which drive metastasis. Long noncoding RNAs (lncRNAs) are an emerging subclass of RNA molecules in cancer biology. LncRNAs are >200nt and can influence target gene expression locally in “cis”, or along a distant chromosome in “trans”, through various mechanisms and interactions with other biological molecules. The contribution of TGFβ-regulated lncRNAs to associated phenotypes like EMT and cancer cell stemness has not been very well studied. The aim of this doctoral dissertation is to address the functional and mechanistic roles of lncRNAs in these processes. Using a well-established TGFβ-induced EMT model (e.g., mouse mammary epithelial cell NMuMG treated with TGFβ, we have identified 3 conserved lncRNAs (lncRNA-HIT, WDFY3-AS2 and TIL) that are significantly upregulated upon TGFβ-induced EMT. They all mediate TGFβ-induced EMT, cell migration and invasion. Overexpression of these lncRNAs is frequently detected during the breast cancer progression and is associated with high grade and late stage of breast cancer as well as metastatic lesion. We have also demonstrated that lncRNA-HIT positively regulates HOXA13 through “cis” mechanism and that WDFY3-AS2 induces WDFY3 and STAT3 expression at mRNA level by direct interaction with hnRNP-R. Interestingly, TIL stimulates C-MYC protein but not mRNA expression by promoting Akt phosphorylation of NF90 leading to its translation from the nucleus to cytosol where NF90 binds to C-MYC mRNA and enhances C-MYC translation. Importantly, we have shown that knockdown of lncRNA-HIT and WDFY3-AS2 significantly reduces breast cancer growth and lung metastasis in orthotopic breast cancer model. These findings indicate that these TGF-induced lncRNAs play critical role in EMT, metastasis, and are relevant in human patient tumors. Therefore, it is important to consider utilizing these molecules for clinical applications like diagnosis, monitoring recurrence, predicting a response to therapy, and even as a direct target for therapeutic intervention.

Les échanges entre les cellules tumorales et le microenvironnement jouent un rôle essentiel dans le développement des tumeurs. Dans ce contexte, la nouvelle famille de métalloprotéases, les protéines ADAM, constituent aujourd’hui des régulateurs majeurs de la progression tumorale en agissant sur la biodisponibilité des médiateurs de la communication cellulaire que sont les cytokines, chimiokines et facteurs de croissance. Au sein de cette famille, ADAM12 est la plus associée au cancer. Elle possède la particularité de jouer un rôle dans la signalisation cellulaire, de façon indépendante de son activité métalloprotéase, notamment dans les voies de signalisation du TGFβ. Notre étude montre pour la première fois un rôle pour la forme membranaire d'ADAM12 dans l'induction de la transition épithélio-mésenchymateuse (EMT), un processus essentiel à l'invasion tumorale dont le TGFβ est un inducteur majeur. Cet effet est médié par l'activation des voies de signalisation du TGFβ, impliquant les protéines SMAD3, AKT et ERK et requiert le domaine cytoplasmique d'ADAM12L mais pas son domaine catalytique. L'activation de ces voies de signalisation pourrait impliquer une relocalisation d'ADAM12L au sein de plates-formes de signalisation dans les radeaux lipidiques. Par ailleurs, nous avons montré qu'ADAM12L interagit avec les protéines ZO-1 et ZO-2, des protéines des jonctions serrées, et pourrait favoriser leur désassemblage au cours de l'EMT. Nos travaux ont permis de mettre en évidence une nouvelle fonction pour ADAM12L dans l'EMT, par un mécanisme impliquant une modulation des signaux régulant ce processus. Une meilleure compréhension de la dynamique de ces mécanismes moléculaires pourrait permettre de développer de nouvelles thérapies ciblées pour lutter contre la progression tumorale. / Communication between tumoral cells and the microenvironnement plays an essential role in the developpement of tumors. In that context, the new family of metalloproteases, the ADAM proteins, are major regulators of the tumoral progression by acting on the bioavaibility of importants mediators of cellular communication as cytokines and growth factors. Among this family, ADAM12 is the most associated with cancer. It has been shown to mediate signaling pathways by a process independant of its metalloproteasis activity, in particular for TGFβ signaling. This study show for the first time a role for the membrane form of ADAM12 in the induction of epithelial to mesenchymal transition (EMT), a essential process involved in tumor invasion, whom TGFβ is a main inducer. This effect is mediated by the activation of TGFβ signaling pathways, SMAD3, AKT and ERK and require the cytoplasmic tail of ADAM12L but not its catalytic activity. Activation of these pathways could involve a relocalisation of ADAM12L in special signaling platform in lipid rafts. Moreover, we have shown that ADAM12L interact with ZO-1 and ZO-2, two proteins of tight junctions, and could facilitate their desassembling during EMT. This work underscore for the first time a new function of ADAM12L in EMT, by a mecanism invovlving a modulation of signals regulating this process. A better understanding of the dynamic of these molecular mecanisms could allow the developpement of new targeted therapies to fight against tumoral progression.