UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE Sience et Vie de Strasbourg U 1109 THÈSE présentée par : Alexia ARPEL soutenue le : 06 Décembre 2013 pour obtenir le grade de : Docteur de l’université de Strasbourg Discipline/ Spécialité : Pharmacologie DEVELOPPEMENT PRECLINIQUE DE PEPTIDES THERAPEUTIQUES TRANSMEMBRANAIRES APPLIQUES AU TRAITEMENT DU CANCER DU SEIN THÈSE dirigée par : Monsieur BAGNARD Dominique MCU, université de Strasbourg Monsieur LAQUERRIERE Dominique Professeur, université de Strasbourg RAPPORTEURS : Monsieur SCHNEIDER Dirk Professeur, université de Mainz Monsieur BENTIRES-ALJ Mohamed Group Leader, FMI AUTRES MEMBRES DU JURY : Madame RIO Marie-Christine Professeur, université de Strasbourg Monsieur HUBERT Pierre CR, Aix-Marseille université Alexia Arpel DEVELOPPEMENT PRECLINIQUE DE PEPTIDES THERAPEUTIQUES TRANSMEMBRANAIRES APPLIQUES AU TRAITEMENT DU CANCER DU SEIN Résumé Le domaine transmembranaire des récepteurs membranaires est aujourd’hui considéré comme essentiel dans l’activation et la régulation des voies de signalisation sous-jacentes. Ceci est tout particulièrement le cas pour neuropiline-1 et -2 (NRP1/2), et ErbB2, trois récepteurs impliqués dans la croissance tumorale. Notre laboratoire a initialement démontré qu’un peptide ciblant le domaine transmembrane du récepteur NRP1, bloque l’oligomérisation de ce récepteur et provoque ainsi l’inhibition de la prolifération/migration des cellules tumorales et l’angiogenèse in vivo. L’objectif principal de ce travail de thèse était d’élargir cette stratégie aux récepteurs membranaires NRP2 et ErbB2, et ce, dans le contexte du cancer du sein. Mes travaux montrent que ces peptides inhibent la pousse tumorale et les métastases associées dans différents modèles de cancer du sein. Les effets anti-tumoraux peuvent s’expliquer par les propriétés anti- angiogéniques et anti-prolifératives des peptides démontrées in vitro et in vivo. J’ai également disséqué le mécanisme d’action du peptide ErbB2 et montré que le peptide inhibiteur de NRP2 induit des effets secondaires rédhibitoires (promotion des métastases osseuses). Dans l’ensemble, mes recherches valident le potentiel thérapeutique de cette stratégie peptidique et renforce l’idée d’un développement clinique de ces composés. D’une terre inconnue à une terre d’espoir, le cœur de la membrane est incontestablement une nouvelle source d’inspiration pour le développement des médicaments de demain. Cancer du sein, Peptides transmembranaires, Neuropiline-1, Neuropiline-2, HER2, Métastases, Etude préclinique, Imagerie Résumé en anglais The role of transmembrane domains (TMD) in membrane receptor activation and regulation is nowadays appearing as a key step of cell signaling. This has been indeed evaluated for neuropilin-1 and -2 (NRP1/2) and ErbB2 receptors, three membrane receptors whose signaling has clearly been implicated in tumorigenesis. Our team had demonstrated that a synthetic peptide blocking the transmembrane domain of NRP1 blocked NRP1-dependent signaling leading to the inhibition of glioma cell proliferation/migration and tumor associated angiogenesis in vivo. The major goal of this thesis project was to extend this novel strategy to NRP2 and ErbB2 in the breast cancer context. Thus, I was able to demonstrate for the first time that the use of peptides, inhibiting the TMD of these receptors, was able to inhibit tumor growth and related metastases in vivo, in three different breast cancer mouse models that I have developed in the laboratory. These results were supported by in vitro experiments demonstrating anti-proliferative and anti-angiogenic properties of these peptides. Besides, I was able to dissect the mechanism of action of the peptide targeting ErbB2 receptor in vitro and in vivo, and I provided data excluding NRP2 as a target because of an unexpected promotion of bone metastasis. Altogether, my data offer convincing evidences to further develop MTP-ErbB2 and MTP-NRP1 peptides as novel therapeutic compounds for patients suffering metastatic cancers. From terra incognita to the exploration of a world of hope, the heart of the membrane is becoming a new promising estate for drug design. Breast cancer, Transmembrane peptides, Neuropilin-1, Neuropilin-2, HER2, Metastasis, Preclinical studies, Imaging This project would not have been possible without the support of many people. Many thanks to my PhD jury members for their insightful comments. I would like to first thank Marie-Christine RIO for accepting the role as president of my jury and internal reviewer and for her time evaluating my work. Thanks to Mohamed Bentires-alj and Dirk Schneider, external reviewers, for their time to judge my work. Last but not least, Pierre Hubert, examiner, for the great and very nice technical discussions during my PhD. I am also deeply thankful to my thesis director Dominique Bagnard, who supervised my thesis. I had a great time working with you and I really appreciated your human and technical qualities in any way. You lead me onto many interesting and challenging paths and you were always available for me, even on Sundays and even when you were overwhelmed with your courses, start up, exams… Thank you for your patience which was really needed time to time to handle me and for being an open person to ideas. I have learned a lot from you. Thank you, for the wonderful time we had together with the Dream B “family” team (wine and cheese, crêpes party, marches gourmandes…)! I could not have imagined having a better advisor and mentor for my PhD! I would also like to express my thanks to Patrice Laquerrière my thesis co-director for all your contributions th during these three years and specially to have helped me the 24 of December with the CT acquisitions of my animals. Thanks to Gertraud Orend who offered guidance and support during my PhD thesis. My greatest appreciation and friendship goes to the Dream B “family” team. Thanks to the lovely dream ladies first. Thanks to my soul sister Edwige, chatouny. You have learned so much to me! I have spent so abundant super moments with you, we had so much fun together, it was great. I loved the moon walking in the animal house and the body building sessions together! Also, thanks to you: UR, oritoo, my little sister at the very beginning, than my twin sister and lastly my big sister (Aurore ya plus d’images!! HELP). You are a great person to work with; I like the madness in you and I can always count on you. We have spent so much good moments! I loved the many apéros we shared together (especially when the red wine color would bridge to the beauty spot)! Thanks to Nur1, justone, you brought a nice and “cool” atmosphere in the lab, thanks for all the help given to me during the thesis defense stressful period. I would like to thank the Dream boys team. Bobitto, my twin brother. The one I had on the phone every evening before an oral presentation, asking always the same questions, and you would always answered again and again. Thank you for your great support from the very beginning, during hard moments, and up to the end! It was lovely to work with you day after days. Lionelitto, also a great member of my Dream B sibship. You were always there to help me at any time of the day, thank you very much for that! Thank you G.I. JOE, Miku for your investment in the confection of weapons for my thesis film! You are really reliable and it was really nice working with you. Thank you Gégé, the endless postdoc of our lab. Thanks for all the knowledge given to me, you are best than any library or search engines. Thank you for being a great “grandpa” taking care of all these children around you. Just one thing you should improve is your way to throw brains and red cells. Last but least Guy! A OUAI! Thank you for being always in a good mood and thank you for all your wise advices in immunology. Thanks to this wonderful team for which at the beginning their names could not be recalled at first place and now will be in my mind forever! I wish you all the best. Thanks to Thibaut, Amore mio. I am truly appreciative of all that you have done for me over the years. You are a pillar of strength through all my ups and downs. Thanks for your patience, thanks for being there, thanks for being extremely reliable, thanks for being a great dad. I feel blessed to be part of your life; it is a pleasure to wake up every day by your side. A few words for my little Jeanne, you’re a real sunshine! A new great journey is beginning for the three of us, everything is already fantastic can’t wait for the future to come. And finally, parents, family, extended family, and numerous friends especially the “COM” who make my world. You have endured this long process with me, always offering support and love. Thanks to all the people who contributed in some way to the work described in this thesis, all the fellow lab mates and persons in my INSERM unit. Thanks again very much to all of you, Alexia, alexiittooo, pikpik. TABLE OF CONTENTS ABBREVIATIONS ................................................................................................................................ 4 I) BREAST CANCER ............................................................................................................................ 7 I.1) FACTS AND STATISTICS ................................................................................................................ 7 I.2) ANATOMY..................................................................................................................................... 8 I.3) MAMMARY GLAND DEVELOPMENT ................................................................................................ 8 I.4) BREAST CANCER RISK FACTORS .................................................................................................... 9 I.5) THE PROCESS OF TUMORIGENESIS ............................................................................................. 10 I.6) DIAGNOSIS ................................................................................................................................ 11 I.7) CLASSIFICATION OF BREAST CANCER ......................................................................................... 12 I.7.1) Histological classification.................................................................................................................. 12 I.7.2) Tumor grade ............................................................................................................................................ 12 I.7.3) Tumor stage (TNM) ............................................................................................................................. 13 I.7.4) Receptor status ...................................................................................................................................... 13 I.7.5) Molecular classification ..................................................................................................................... 14 I.7.5.1) Normal-like subtype.................................................................................................................... 14 I.7.5.2) Luminal A and Luminal B subtype ....................................................................................... 14 I.7.5.3) Her2 subtype .................................................................................................................................. 14 I.7.5.4) Claudin-low subtype ................................................................................................................... 15 I.7.5.5) Basal-like subtype ........................................................................................................................ 15 I.8) TREATMENT .............................................................................................................................. 15 I.8.1) Surgery ....................................................................................................................................................... 16 I.8.2) Chemotherapy ........................................................................................................................................ 16 I.8.2.1) Taxanes .............................................................................................................................................. 17 I.8.2.2) Anthracyclines ............................................................................................................................... 17 I.8.3) Endocrine .................................................................................................................................................. 18 I.8.4) Targeted therapy................................................................................................................................... 19 I.8.4.1) Inhibition of proliferative signaling..................................................................................... 19 I.8.4.1.1) Targeting ErbB family ........................................................................................................ 19 I.8.4.1.2) Targeting ErbB signaling .................................................................................................. 22 I.8.4.2) Interfering with DNA repair pathways: Synthetic lethality by inhibiting poly- ADP-ribose polymerase (PARP) in breast cancer susceptibility gene 1 and 2 (BRCA1, BRCA2) deficient cells ................................................................................................................................ 22 I.8.4.3) Anti-angiogenic therapies ........................................................................................................ 23 II) THERAPEUTIC TARGETS IN BREAST CANCER: VALIDATED AND FUTURE TARGETS ............................................................................................................................................................. 25 1 II.1) HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR 2 (HER2) .................................................. 25 II.1.1) Signaling network platform ........................................................................................................... 25 II.1.2) Roles during embryogenesis and adulthood ......................................................................... 26 II.1.3) Functions in the mammary gland ................................................................................................ 28 II.1.4) Protein Regulation .............................................................................................................................. 29 II.1.5) Variants, homologue and nomenclature .................................................................................. 29 II.1.6) Transcriptional targets ..................................................................................................................... 30 II.1.7) Validated target in breast cancer and MBC ............................................................................ 31 II.2) NEUROPILINS: NEUROPILIN-1 (NRP1) AND NEUROPILIN-2 (NRP2) ..................................... 32 II.2.1) Signaling network platform ........................................................................................................... 32 II.2.2) Roles during embryogenesis and adulthood ......................................................................... 34 II.2.3) Neuropilins in the mammary gland............................................................................................ 35 II.2.4) Neuropilins regulation ...................................................................................................................... 36 II.2.5) Spliced variants .................................................................................................................................... 37 II.2.6) Novel targets for breast cancer and metastatic breast cancer ...................................... 38 III) TERRA INCOGNITA: TRANSMEMBRANE DOMAINS (TMD) AND THE PEPTIDIC STRATEGY ........................................................................................................................................ 42 III.1) TRANSMEMBRANE DOMAIN .................................................................................................... 42 III.1.1) Once upon a time: Glycophorin A (GPA) ................................................................................ 42 III.1.2) Transmembrane domain of HER2 ............................................................................................. 43 III.1.3) Transmembrane domain of neuropilins................................................................................. 44 III.1.4) Principles of TM signaling and dimerization motifs ......................................................... 44 III.1.5) Importance of TMD for protein function ................................................................................ 46 III.1.6) Strategies for studying TMD-TMD interaction within the cell membrane ............ 47 III.1.6.1) Computational approaches: molecular dynamic modeling .................................. 47 III.1.6.2) Reporter assay: BACTH system .......................................................................................... 49 III.1.6.3) FRET: Förster Resonance Energy Transfer measurements ................................. 49 III.2) INTERFERING PEPTIDE STRATEGY .......................................................................................... 50 III.2.1) Hypothesis ............................................................................................................................................ 50 III.2.2) A prophetic view slow to materialize ...................................................................................... 50 IV) THESIS OBJECTIVES ................................................................................................................ 53 V) MATERIAL AND METHODS ..................................................................................................... 55 V.1) MMTV-NEUNT MOUSE MODEL ............................................................................................... 55 V.2) IMAGING: COMPUTERIZED TOMOGRAPHY (CT) ....................................................................... 56 V.3) CELL LINES ............................................................................................................................... 58 VI) TO EVALUATE THE POTENTIAL OF THE MTP-ERBB2 PEPTIDE TO ANTAGONIZE ERBB2, A VALIDATED TARGET IN BREAST TUMOR GROWTH AND METASTASIS ........ 59 2