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BECN1 is a haploinsufficient tumor suppressor gene that is monoallelically deleted or epigenetically silenced in many human cancers. In breast cancer, 40% of tumors exhibit monoallelic deletion of Beclin 1. Additionally, low Beclin 1 mRNA expression is observed in aggressive breast cancer subtypes and reduced expression is an independent predictor of overall patient survival. The role of Beclin 1 in cancer has almost exclusively been attributed to its function in autophagy. However, our lab demonstrated an alternative role for Beclin 1 in the regulation of growth factor receptor signaling that could contribute to cancer. The goal of my thesis project was to investigate the molecular basis by which Beclin 1 regulates breast tumor growth and progression in vivo. Using in vivo models, I discovered that Beclin 1 promotes endosomal recruitment of hepatocyte growth factor tyrosine kinase substrate (HRS), which is necessary for sorting receptors to intraluminal vesicles for signal silencing and degradation. Beclin 1-dependent recruitment of HRS results in the autophagy-independent regulation of endocytic trafficking and degradation of the epidermal growth factor (EGFR) and transferrin (TFR1) receptors. When Beclin 1 expression is low, endosomal HRS recruitment is reduced and receptor function is sustained to drive tumor proliferation. An autophagy-independent role for Beclin 1 in regulating tumor metabolism was also observed. Collectively, my results demonstrate a novel role for Beclin 1 in impeding tumor growth by coordinating the regulation of growth promoting receptors. These data provide an explanation for how low levels of Beclin 1 facilitate tumor proliferation and contribute to poor cancer outcomes, independently of autophagy.

L’immunologie anti-tumorale et l’immunothérapie ont connu dernièrement de grandes avancées avec la mise en évidence du processus d’immuno-surveillance et le développement de plusieurs approches vaccinales. Il n’en demeure pas moins que l’induction d’une réponse immunitaire anti-tumorale se traduit peu par l’éradication de la tumeur. Comme phénomène dynamique et interactif, la réponse cytotoxique anti-tumorale implique les effecteurs cytotoxiques et les cibles tumorales; pourtant, le microenvironnement tumoral et sa plasticité influent largement sur l’efficacité de celle-là. Avec l’appui de récentes données expérimentales, il apparaît crucial de prendre en compte la susceptibilité tumorale à la lyse par les effecteurs cytotoxiques anti-tumoraux, notamment les lymphocytes T cytotoxiques (CTLs), et plus particulièrement dans un contexte de plasticité cellulaire. Ainsi, le principal objectif de mes travaux de thèse est de saisir le rôle de la Transition Épithélio-Mésenchymateuse (EMT) dans la susceptibilité des cellules tumorales à la lyse par les CTLs dans des modèles cellulaires de cancer du sein. Nos résultats montrent que l’EMT est capable d’induire une diminution de la susceptibilité des cellules mésenchymateuses à la lyse spécifique. Elle engage de ce fait de multiples acteurs. Tout d’abord, dans les deux modèles d’étude, il s’avère que l’EMT est capable de réguler négativement l’expression de la molécule HLA-A2. Ensuite, dans le premier modèle expérimental, nous avons établi que l’EMT induit une altération de la signalisation au niveau de la synapse immunologique. De plus, le régulateur de l’autophagie, Becline 1, joue un rôle crucial dans l’induction de la diminution de la sensibilité à la lyse par les lymphocytes T-CD8+ suite à l’induction de l’EMT. Dans le deuxième modèle d’étude, le mécanisme mis en jeu par l’EMT pour réguler la susceptibilité des cellules mésenchymateuses à la lyse par les CTLs se manifeste dans l’induction du facteur de transcription inducteur des propriétés de cellules souches cancéreuses, le KLF4 ainsi que via la régulation négative de l’expression du miR-7. Ensemble, ces résultats élucident de nouveaux mécanismes d’échappement des cellules tumorales malignes à la lyse par les lymphocytes T-CD8+ suite à l’induction de l’EMT. Cette étude soutient ainsi l’importance du ciblage des facteurs de transcription inducteurs de l’EMT et responsables de la plasticité cellulaire afin de neutraliser leur fonction. Cela pourrait aider à construire une nouvelle stratégie pour mieux contrôler l’échappement des cellules tumorales invasives à la lyse spécifique et in fine pour garantir une immunothérapie plus efficace contre le cancer. / The anti-tumor immunology and immunotherapy have recently undergone major breakthroughs, with the identification of immune surveillance process and the development of several vaccine approaches. However, the fact remains that the induction of an antitumor immune response is still not effective enough. Certainly, the antitumor cytotoxic response is a dynamic and interactive phenomenon, involving cytotoxic effectors and tumor targets, but its effectiveness is considerably influenced by the tumor microenvironment and its plasticity. Recent studies support the importance of taking into account the tumor susceptibility to lysis by anti-tumor cytotoxic effectors, notably Cytotoxic T Lymphocytes (CTLs), especially in a context of cellular plasticity. On the grounds of these studies, this research aims at understanding the role of Epithelial to Mesenchymal Transition (EMT) in the susceptibility of tumor cells to CTLs mediated lysis in different models of breast cell carcinoma. Our results reveal that EMT is able to induce a decrease in the susceptibility of mesenchymal cells to specific lysis. It calls therefore multiple actors. First, in both study models, it turns out that the EMT is able to downregulate the expression of HLA-A2 molecule. Then, in the first experimental model, we show that EMT induces an alteration of signalling at the immunological synapse. Moreover, the regulator of autophagy, Beclin 1, plays a crucial role in the induction of reduced susceptibility to lysis by T-CD8+ lymphocytes following induction of EMT. In the second experimental model, we show that the mechanisms used by EMT to regulate the susceptibility of mesenchymal cells to lysis by CTLs involve the induction of the transcription factor inducing cancer stem cells properties, KLF4, as well as the downregulation of miR-7 expression. Together, these results shed light on new mechanisms used by malignant tumor cells to escape to lysis by T-CD8+ lymphocytes following the induction of EMT. Thus, this study advocates the importance of targeting transcription factors, which are inducers of EMT and responsible for cellular plasticity, in order to neutralize their function. These insights may prove useful for the development of new strategies aimed at better controlling the escape of invasive tumor cells to specific lysis, and ultimately ensuring a more effective immunotherapy against cancer.

The mammary gland is a dynamic organ that undergoes the majority of its development in the postnatal period in four stages; mature virgin, pregnancy, lactation, and involution. Every stage relies on tightly regulated cellular proliferation, programmed cell death, and tissue remodeling mechanisms. Misregulation of autophagy, an intracellular catabolic process to maintain energy stores, has long been associated with mammary tumorigenesis and other pathologies. We hypothesize that appropriate regulation and execution of autophagy are necessary for proper development of the mammary ductal tree and maintenance of the secretory epithelia during late pregnancy and lactation. To test this hypothesis we examined the role of two genes during development of the mammary gland. Beclin1 (Becn1) is an essential autophagy gene. Since the Becn1 knockout model is embryonic lethal, we have generated a Becn1 conditional knockout (cKO). We used two discrete mammary gland-specific Cre transgenic lines to interrogate the role of BECN1 during development. We report that MMTV-CreD; Becn1fl/fl mice have a hyper-branching phenotype and WAP-Cre; Becn1fl/- mice are unable to sustain a lactation phase. Becn1 mutants exhibit abnormal glandular morphology during pregnancy and after parturition. Moreover, when autophagy is chemically inhibited in vitro, mammary epithelial cells have an increased mean number of lipid droplets per cell. MTOR inhibits autophagy upstream of BECN1; we looked higher in the regulatory pathway for regulatory candidates. It has been well characterized that Tuberous sclerosis complex 1 (TSC1), in a heterodimer with its primary binding partner TSC2, inhibits MTOR signaling via inhibition of RHEB. Using the Tsc1 floxed model we generated a mammary gland specific Tsc1 cKO and found that these mice phenocopy the Becn1 cKO mice, including a gross lactation failure. Tsc1 cKO glands have altered morphology, retained lipid droplets in secretory epithelia, and an overall increase in MTOR signaling. We show that TSC1 and BECN1 are interacting partners, and that the interaction is nutrient responsive. These results suggest that Becn1 and Tsc1 are necessary for proper mammary gland development and differentiation. Furthermore, we have demonstrated a novel murine protein-protein interaction and an important link between regulation of MTOR pathway and regulation of autophagy in a developmental context.

Le VIH-1, ou Virus de l’ImmunoDéficience Humain de type I, est un pathogène intracellulaire dont le cycle de réplication dépend entièrement des machineries cellulaires. Des interactions entre les protéines virales et cellulaires sont donc essentielles pour l’achèvement de chaque étape du cycle de multiplication du VIH-1. Comprendre comment le virus VIH-1 détourne la machinerie cellulaire à son profit est un élément clé pour le combattre. L’objectif de mon travail de thèse était d’explorer le rôle de la protéine Bécline-1 dans la formation et la libération des particules virales. Cette protéine, composant du complexe phosphatydylinositol-3-Phosphate-kinase III (PI3K-III), est impliquée dans l’autophagie, le trafic intracellulaire et la cytocinèse.Au cours de ma thèse, nous avons développé les outils d’ARN interférence afin de décrypter le rôle de Bécline-1 dans le cycle réplicatif du VIH-1. De manière très intéressante, nous avons mis au jour un nouveau rôle de Bécline-1 lors des étapes précoces de la mitose, jamais décrit, ni publié à ce jour. Un mauvais déroulement de la mitose induit de l’instabilité génomique pouvant conduire à la mort cellulaire ou à une transformation maligne. Au cours de cette thèse, nous avons établi que l’extinction de Bécline-1 induit un défaut d’attachement des chromosomes, via leurs kinétochores, aux microtubules, conduisant à un blocage des cellules en prométaphase. Cette extinction provoque un défaut d’organisation du kinétochore en diminuant le recrutement des protéines CENP-E, CENP-F et ZW10 au niveau de cette structure protéique. Nous avons également montré une interaction directe entre Bécline-1 et Zwint-1, une protéine du kinétochore, jouant un rôle essentiel pour l’accrochage des microtubules aux kinétochores. Enfin, nous avons montré que ce nouveau rôle de Bécline-1 dans l’alignement des chromosomes en mitose est indépendant de son association avec ses partenaires du complexe PI3K-III et de son rôle dans l’autophagie.Le second volet de ma thèse a porté sur l’étude du rôle de Bécline-1 au cours du cycle réplicatif du virus. Nos résultats montrent que cette protéine est nécessaire à l’établissement des phases tardives du cycle viral. Nos expériences de production virale montrent que l’extinction de Bécline-1 provoque une forte accumulation des sous-produits de la protéine Gag du virus dans les cellules, et réduit la libération des particules virales dans le milieu extracellulaire. Par immunofluorescence, nous observons que des composants viraux s’accumulent massivement à la membrane sous l’effet de l’extinction de Bécline-1 dans les cellules. Par ailleurs, ces résultats sont dépendants de l’expression du facteur de restriction BST2 dans la cellule. Ces derniers résultats ouvrent d’importantes perspectives quant au rôle de Bécline-1 et du PI3K-III sur le cycle de réplication du VIH-1.L’ensemble de ces travaux démontre l'importance de la protéine Beclin-1, pour le maintien de l'intégrité du génome pendant la mitose et pour le cycle de réplication du VIH 1. / HIV-1, or Human Immunodeficiency Virus type 1, is an intracellular pathogen whose replication cycle entirely depends on cellular machineries. Interactions between the viral and cellular proteins are therefore essential to the completion of each step of HIV-1's multiplication cycle. Understanding how the HIV-1 virus uses the cellular machinery to its own benefit is a key element to combat it. The objective of my thesis work was to explore the role of the Beclin-1 protein in the formation and the release of viral particles. This protein, a component of the phosphatydylinositol-3-Phosphate-kinase III (PI3K-III) complex, is involved in autophagy, intracellular trafficking and cytokinesis. Throughout my thesis researches, we have developed the RNA interference tools in order to decipher the role of Beclin-1 in HIV-1's replicative cycle. Very interestingly, we have found out a new role of Beclin-1 in the early stages of mitosis which had never been described nor published before. A bad execution of mitosis induces genomic instability which can lead to cell death or malignant transformation. During this thesis work, we have demonstrated that the extinction of Beclin-1 causes a defect in the attachment of chromosomes to microtubules through their kinetochores, leading to the locking of cells in prometaphase. This extinction provokes a defect in the organization of the kinetochore by diminishing the recruitment of the CENP-E, CENP-F and ZW10 proteins at the level of this protein structure. We have also found out that there is a direct interaction between Beclin-1 and Zwint-1, a protein of the kinetochore that plays an essential role in the attachment of the kinetochores to the microtubules. Finally, we have demonstrated that this new role of Beclin-1 in the alignment of the chromosomes during mitosis is independent of both its association with its partners from the PI3K-III complex and its role in autophagy. The second strand of my thesis dealt with the role of Beclin-1 in the virus replicative cycle. Our results show that this protein is essential to the setting up of the late phases of the viral cycle. Our experiments of viral production have shown that the extinction of Beclin-1 causes a high accumulation of the by-products of the virus' Gag protein in the cells, and reduces the release of viral particles in the extracellular medium. By immunofluorescence, we detected a massive accumulation of viral components on the membrane as a result of the extinction of Beclin-1 in the cells. Besides, these results depend on the expression of the BST2 restriction factor in the cell. These latter results open up significant prospects regarding the role of Beclin-1 and that of the PI3K-III complex in HIV-1's replication cycle. All these researches demonstrate the importance of the Beclin-1 protein in two mechanisms that allow the maintenance of genomic integrity during mitosis and the HIV-1 replication cycle.

Early in infection, HIV crosses the blood-brain barrier and induces neuropathology. Viral presence in the CNS coupled with secretion of neurotoxic proteins causes neuroinflammation, glial dysfunction, excitotoxicity, and neuronal death. Despite advances in combined antiretroviral therapy, HIV-infected patients present with a spectrum of cognitive and psychomotor deficits collectively referred to as HIV-associated neurological disorders (HAND). A subset of HAND patients abuses drugs such as opiates like heroin and morphine show an exacerbation and rapid progression of HIV neuropathology; however, the mechanisms of this synergy are not well understood. Autophagy is a lysosomal degradative process which eliminates and recycles cytosolic components and is implicated in facilitating HIV-1 replication in the CNS and periphery, and in Tat-induced neurodegeneration. When a key initiator of autophagy Beclin 1 was silenced using siRNAs, there was a marked reduction of HIV-1 replication in human microglia and astrocytes and the corresponding inflammatory response. As such, the goal of the current study is to determine if diminished Beclin 1 is neuroprotective against Tat and morphine-induced neurodegeneration using heterozygous Beclin 1 (Becn1+/-) mice. Examination of Tat and morphine-induced inflammatory molecule secretion revealed that Becn1+/- mixed astrocyte and microglia (glia) exhibited attenuated secretion of cytokine IL-6 and chemokines RANTES and MCP-1 compared to control (C57BL/6J) glia, an effect mediated through the μ-opioid receptor. Dysregulation of autophagy-related gene expression and excessive intracellular calcium accumulation were limited in Becn1+/- glia. When determining the effects of Tat-and morphine co-exposure on neuronal survival in vitro, we found Becn1+/- neurons were particularly sensitive to injury, excitotoxicity, and toxic exposures; however, when C57BL/6J neurons were exposed to conditioned media of C57BL/6J and Becn1+/- glia treated with Tat and morphine, neurons treated with Becn1+/- supernatant had better outcomes than those treated with C57BL/6J conditioned media. Furthermore, despite minimal difference between strains in locomotor assessment, we observed significantly greater striatal neuron losses in adult C57BL/6J mice exposed to intrastriatal Tat-and systemic morphine compared to Becn1+/- mice. Our studies demonstrate the potential of targeting Beclin 1 in glia for the prevention of Tat and opiate-induced CNS dysfunction.

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Les catécholamines induisent la synthèse d’AMPc par une stimulation des récepteurs β-adrénergiques et contrôlent ainsi la fonction cardiaque en activant une pléiade de voies de signalisation intracellulaires. Les protéines Epac sont des facteurs d’échange pour les petites protéines G et sont directement activés par l’AMPc. Devant l’importance de la voie β-adrénergique dans la physiopathologie cardiaque et dans le but de mieux comprendre la régulation des processus cellulaires dépendants de l’AMPc dans le cœur, il apparaît essentiel de caractériser le rôle des facteurs d’échange Epac dans le myocarde. Dans une première partie, cette étude démontre que les effets de Epac sur l’hypertrophie des cardiomyocytes ventriculaires de rats nouveaux nés requièrent les GTPases H-Ras et Rap2B. Epac active la voie PLC/IP3/Ca2+ qui est nécessaire pour l’activation de H-Ras. Au niveau transcriptionnel, Epac induit l’export nucléaire de HDAC4 permettant l’activation d’un programme génique d’hypertrophie. Dans une deuxième partie, cette étude révèle l’implication de Epac1 dans l’hypertrophie des cardiomyocytes in vivo, chez la souris. La délétion de Epac1 protège du remodelage cardiaque induit par l’activation prolongée des récepteurs β-adrénergiques et améliore la fonction cardiaque. La surexpression de Epac1 spécifiquement dans le myocarde entraîne une hypertrophie des cardiomyocytes. Par ailleurs, la voie β-AR/Epac1 induit l’accumulation de protéines ubiquitinylées et provoque l’activation du processus d’autophagie in vitro et in vivo. L’autophagie protège des effets délétères de la voie β-adrénergique/Epac en participant à l’élimination des agrégats protéiques et en contrant les effets hypertrophiques de Epac1. Ces résultats ouvrent de nouvelles perspectives pour le traitement de l’hypertrophie et de l’insuffisance cardiaque. / Catecholamines regulate cardiac function by stimulating β-adrenergic receptors (β-AR), leading to cAMP production and activation of a multiplicity of signaling pathways. Epac proteins are exchange factors for small G proteins which are directly activated by cAMP. Given the importance of the β-adrenergic pathway in cardiac physiopathology, it becomes essential to characterize functions of Epac protein in myocardium. In a first part, this study shows that H-Ras and Rap2B GTPases are involved in Epac-induced neonatal rat cardiac myocytes hypertrophy. Epac induces activation of the PLC/IP3/Ca2+ pathway which is necessary for H-Ras activation. At the transcriptional level, Epac causes HDAC4 nuclear export leading to activation of a hypertrophic gene program. In a second part, this study reveals implication of Epac1 in cardiac hypertrophy in vivo. Deletion of Epac1 in mice protects from cardiac remodeling induced by chronic isoproterenol infusion and enhances cardiac function. Cardiac specific overexpression of Epac1 in mice induces cardiac myocytes hypertrophy. Interestingly, β-AR/Epac1 pathway triggers ubiquitinated proteins accumulation and activation of autophagy both in vitro and in vivo. By eliminating aggregates and by counteracting hypertrophic effects of Epac, autophagy protects from deleterious effects of the β-AR/Epac pathway. These results open news insights into the treatment of cardiac hypertrophy and heart failure.