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L’IMPLICATION DE LA 17ΒETA- HYDROXYSTEROIDE DESHYDROGENASE TYPE 1 ET DE LA TROPOMYOSINE-1 ALPHA DANS LA PROGRESSION ET L’INVASION DES CELLULES CANCÉREUSES DU SEIN Thèse MOUNA ZERRADI Programme de doctorat en biologie cellulaire et moléculaire Philosophiae Doctor (Ph.D.) Québec, Canada © Mouna Zerradi, 2015 L’IMPLICATION DE LA 17ΒETA- HYDROXYSTEROIDE DESHYDROGENASE TYPE 1 ET DE LA TROPOMYOSINE-1 ALPHA DANS LA PROGRESSION ET L’INVASION DES CELLULES CANCÉREUSES DU SEIN Thèse MOUNA ZERRADI Sous la direction de : Sheng Xiang Lin, directeur de recherche Jacques Huot, codirecteur de recherche Résumé Au Canada et partout dans le monde, le cancer du sein est un enjeu de taille en santé publique. L’exposition élevée à l'estradiol (E2) est considérée comme un facteur de risque majeur du cancer du sein. La synthèse de cette hormone est catalysée par la 17β- hydroxystéroïde déshydrogénase (17β-HSD), en particulier la 17β-HSD de type 1 (17β- HSD1) qui utilise le NADPH comme cofacteur et catalyse la conversion de l’estrone (E1) en E2. Le signal d’E2 est médié par le récepteur d’estrogène, une fois ce dernier activé par E2, on constate une stimulation de la croissance et de la prolifération cellulaire. Différents inhibiteurs sont utilisés en clinique pour bloquer la synthèse finale d’E2. Cependant aucun inhibiteur de la 17β-HSD1 n’est encore utilisé en clinique malgré l’importance de cette enzyme dans la conversion de l’E1 en E2. Ceci est certainement dû au fait que les inhibiteurs de la 17β-HSD1 sont des dérivés d’E2 d’où la difficulté d’éliminer complètement l’activité estrogénique non desirée. Dans nos travaux, nous avons tenté d’inhiber l’activité ou l’expression de la 17β-HSD1 pour étudier son effet sur la régulation du profil protéique et génomique, sur le cycle cellulaire et sur l’invasion cellulaire des cellules cancéreuses MCF7 et T47D. Nos résultats montrent que l’inhibition de la 17β-HSD1 module l’expression de diverses protéines impliquées dans la prolifération cellulaire tels que la tumor protein D54, dans le cycle cellulaire tels que la 14-3-3 epsilon et la tumor protein D53, et dans l’invasion cellulaire tels que la nm23. Aussi, l'inhibition de la 17β-HSD1 dans les cellules T47D régule l'expression des gènes impliqués dans le transport (RANBP3L, APOD), la liaison d'ADN (HIST1H2BM), le traitement de l'antigène et la présentation de l'antigène de peptide ou de polysaccharide par l'intermédiaire du CMH de classe II (HLA- DQA2), la régulation transcriptionnelle (TP63) et dans l'adhérence cellulaire (CD36). Au niveau des deux lignées cellulaires utilisées T47D et MCF7, l’inhibition de la 17β-HSD1 diminue la prolifération cellulaire, la formation d’E2, l’invasion et la migration cellulaire et mène aussi à l’arrêt du cycle cellulaire en phase G0/G1. Nos résultats montrent l'importance majeure de l’inhibition de la 17β-HSD1 dans un contexte de cancer du sein estrogéno-dépendant. Le cancer du sein est aussi une pathologie génétique associée à des modifications quantitatives et /ou iii qualitatives des gènes tels que le gène codant pour la tropomyosine (Tm). Il a été démontré que l’expression de la Tm1 est perdue dans les cellules cancéreuses métastatiques du sein. Mon deuxième projet de recherche consistait à étudier l’effet de la phosphorylation de la tropomyosine 1 alpha sur la prolifération, l’adhérence, la formation de colonies et la migration des cellules cancéreuses du sein MDA-MB231. Pour cet effet des transfectants MDA-MB231 stables exprimant soit la Tm-1α sauvage, soit la Tm-1α mutante non- phosphorylable (Ser283Ala) ou encore la Tm-1α mutante pseudophosphorylée (Ser283Glu) ont été générés. Nos résultats montrent que les cellules exprimant la forme phosphomimétique (pseudo-phosphorylée) de la Tm1 S283E/Tm1 sont caractérisées par une adhérence accrue au substrat. Aussi, la migration transendothéliale et la migration dans un essai de cicatrisation de ces cellules est réduite par rapport aux cellules parentales ou ceux exprimant la forme non-phosphorylable de la Tm1 (S283A). En outre, nous avons constaté que les cellules exprimant les mutants S283A forment plus de colonies en agar mou que ceux exprimant les mutants S283E, montrant ainsi que la phosphorylation de la Tm1 sur la Ser283 contribue à ses propriétés anti-tumorales. iv Table des matières Résumé ............................................................................................................................... iii Table des matières............................................................................................................... v Liste des tableaux ............................................................................................................... xi Liste des figures ............................................................................................................... xiii Abréviations .................................................................................................................... xvii Remerciements ............................................................................................................... xxiii Avant-Propos ................................................................................................................ xxvii Chapitre 1 : Introduction générale ...................................................................................... 1 1.1. Le cancer du sein ...................................................................................................... 3 1.2. Cancer du sein hormono-dépendant.......................................................................... 3 1.3. Les enzymes impliquées dans la stéroïdogénèse ...................................................... 6 1.3.1. L’aromatase ...................................................................................................................... 6 1.3.2. La stéroïde sulfatase ......................................................................................................... 7 1.3.3. La 17β-HSD1 Type 1 ....................................................................................................... 8 1.4. Mode d’action d’E2 ................................................................................................ 11 1.4.1. La voie ER-dépendante .................................................................................................. 11 1.4.1.1. La voie génomique classique ................................................................................... 11 1.4.1.2. La voie génomique non classique ............................................................................ 12 1.4.1.3. La voie non génomique ........................................................................................... 12 1.4.2. La voie ER-indépendante ............................................................................................... 13 1.5. Récepteur d’E2 : ER ............................................................................................... 14 1.6. Les anti-estrogènes.................................................................................................. 15 1.6.1. Le tamoxifène et ses analogues ...................................................................................... 16 1.6.2. Les anti-estrogènes purs ................................................................................................. 19 1.7. Cancers non hormonaux dépendants : les oncogènes et les suppresseurs de tumeurs .............................................................................................................................. 20 1.8. La tropomyosine ..................................................................................................... 21 1.8.1. Les isoformes de la tropomyosine ................................................................................. 24 1.9. La relation entre la tropomyosine et le cytosquelette d’actine ............................... 27 1.10. Le niveau d’expression de la tropomyosine dans les tumeurs humaines ................ 27 v 1.11. Les modifications post-traductionnelles de la tropomyosine .................................. 28 1.12. Les mécanismes de régulation de la tropomyosine................................................. 29 1.13. Les hypothèses de recherche ................................................................................... 30 1.14. Les objectifs de travail ............................................................................................ 32 Chapitre 2: Differential proteome of estrogen-dependent breast cancer cells by 17β- HSD type 1 inhibition and knockdown: modulation of nm23-H1 expression, cell cycle and cell invasion ............................................................................................................... 33 Summary ........................................................................................................................... 37 Résumé en français ........................................................................................................... 39 1. Introduction ............................................................................................................. 41 2. Materials and methods ............................................................................................ 44 2.1. Cell culture ..................................................................................................................... 44 2.2. Plasmid construction and stable transfection .................................................................
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  • Cell Division & Proliferation

    Cell Division & Proliferation

    ptglab.com 1 ANTIBODIES FOR CELL BIOLOGY CELL DIVISION & PROLIFERATION www.ptglab.com 2 Antibodies For Cell Biology: Cell Division & Proliferation Front & Back Cover: Immunohistochemistry of paraffin-embedded human skin tissue slide using Involucrin Antibody (55328-1-AP) at a dilution of 1:50 (10x objective). ptglab.com 3 WELCOME Foreword Cell division and proliferation are processes essential to life; they are processes that underpin growth and development and tissue maintenance. Cell division is the process by which cells must partition their DNA and cell contents appropriately into new daughter cells. This intricate mechanism enables cell proliferation, which must be tightly regulated in balance with cell loss through apoptosis or necrosis. These two processes can go wrong, leading to pathological proliferation, and in so doing form the basis of neoplastic disease. Throughout this catalog you will find hundreds The processes of cell division and proliferation of Proteintech®* antibodies relevant to cell require a wealth of proteins, as demonstrated division and proliferation. They cover mitosis by the long list of components above; if you and meiosis, DNA replication, transcription cannot identify an antibody for your target and translation, as well as protein expression, protein in these pages, please visit our website folding and trafficking and nutrient sensing at ptglab.com to search our full catalog of over and metabolism. There are also antibodies 10,000 pre-made antibodies. We’re confident against cell cycle control proteins featured; you will find the antibody you’re looking for. for instance you will find a poster dedicated to BRCA signaling, a pathway deregulated in many cancers.