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Cascade Profiling of the Ubiquitin-Proteasome System in Cancer Anastasiia Rulina

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Cascade Profiling of the Ubiquitin-Proteasome System in Cancer Anastasiia Rulina Cascade profiling of the ubiquitin-proteasome system in cancer Anastasiia Rulina To cite this version: Anastasiia Rulina. Cascade profiling of the ubiquitin-proteasome system in cancer. Agricultural sciences. Université Grenoble Alpes, 2015. English. NNT : 2015GREAV028. tel-01321321 HAL Id: tel-01321321 https://tel.archives-ouvertes.fr/tel-01321321 Submitted on 25 May 2016 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE Pour obtenir le grade de DOCTEUR DE L’UNIVERSITÉ GRENOBLE ALPES Spécialité : Biodiversite du Developpement Oncogenese Arrêté ministériel : 7 août 2006 Présentée par Anastasiia Rulina Thèse dirigée par Maxim Balakirev préparée au sein du Laboratoire BIOMICS dans l'École Doctorale Chimie et Sciences du Vivant Profilage en cascade du système ubiquitine- protéasome dans le cancer Thèse soutenue publiquement le «17/12/2015», devant le jury composé de : M. Damien ARNOULT Docteur CR1, CNRS, Rapporteur M. Matthias NEES Professor Adjunct, University of Turku, Rapporteur M. Philippe SOUBEYRAN Docteur, INSERM, Membre Mme. Jadwiga CHROBOCZEK Directeur de recherche DR1, CNRS, Membre M. Xavier GIDROL Directeur de laboratoire, CEA, Président du jury M. Maxim BALAKIREV Docteur, CEA, Membre, Directeur de Thèse 2 Я посвящаю эту научную работу моей маме, Рулиной Людмиле Михайловне, с любовью и благодарностью. Мне всегда везло с людьми, которые давали мне вдохновение и любовь для занятий биологией, но именно ты заложила во мне те качества, которые помогли мне пройти весь путь от начала и до конца. Именно твоя поддержка, в любых моих начинаниях давали мне ощущение «твердой земли под ногами». 3 4 ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor, Maxim Balakirev for guiding me throughout the thesis. Your expertise and endless enthusiasm helped me to work on this complicated subject. During these 3 years you were always there to discuss and share experience, and I hope I have learned the most I could from you. I would like to express gratitude to all the members of the laboratory, who helped me with the technical part of my thesis. Thank you to Patricia Obeid for introducing me to siRNA screens, as well as for data acquisition and analysis. It was a great pleasure and luck for me to work with such a hard-working, experienced and enthusiastic person. Laurent Guyon, thank you for your job! Your deep and comprehensive data analysis was just priceless for this dissertation. I would also like to thank Frederic Mittler for cell culture, recent Western Blots and IF; Sophie Gerbaud for performing EdU tests and recent IF images; Frederic Kermarreck for qPCRs; Eric Sulpice for helping with FACS analysis. Also I want to show my appreciation to Amandine Pitaval for explaining immunofluorescence microscopy to me and to Vincent Haguet for performing Wound Healing Assay. I am grateful to the members of my “Comité de suivi de thèse” - Jadwiga Chroboczek and François Berger for their time and useful advice in the course of the project. I would like to say “merci” to Nicole Assard. It was a pleasure to share working space with you! I appreciate so much your friendliness and positive attitude, as well as accuracy and professionalism. Thanks to the Eric Sulpice and Stephanie Combe’s team for moral support and useful advice about science and personal life. I am also grateful to our wonderful secretary, Patricia Leclyese for helping with the millions of administrative questions I had. I would like to thank my mother – you have always been a constant source of support. Thanks to all my friends in Grenoble for all the good days we spent together – without you it would be so boring here! Special thanks to Dolega Monika, Brasouskaya Daria, Fefelova Anastasiia, Ilaria Vitulano and Komarynets Olga for the best support during these times. Thank you, Arnaud Lemelle for all the loving, patience and care you give me. 5 6 MOTIVATION AND CONTEXT According to the American Cancer Society, the prostate cancer (PCa) is the most frequent malignancy in men. After lung cancer, the PCa is the second leading oncological cause of death. Despite many years of research, the main current treatments are still surgery, radiation and androgen deprivation therapy. The androgen ablation ultimately leads to the development of a more advanced, hormone-refractory (castration-resistant) form of prostate cancer, which responds poorly to standard chemotherapy and is considered to be terminal. It is evident that there is an urgent need for the discovery of alternative targets and the development of new therapeutic approaches for prostate cancer treatment. The design of new therapeutic agents against prostate cancer depends critically on our knowledge of the molecular mechanisms of cancer origin and progression. In 2007 the term “non- oncogene addiction” was proposed by Elledge and co-authors to explain the increased dependency of cancer cells on the function of normal genes. The phenomenon is based on increased cellular stresses experienced by cancer cells (mitotic, proteotoxic, metabolic, etc.), making them more dependent on stress support systems. Among these, the ubiquitin-proteasome system (UPS), as a major mediator of key cellular functions, represents a perfect model for a loss-of-function screen to search for potential drug targets based on non-oncogene addiction. This work was carried out in the Biomics laboratory, which is specialized in functional genome-wide screens and biomarker discovery targeting prostate cancer. In the course of the project, we applied a novel systematic approach to the loss-of-function screen of the UPS. Our strategy was to employ the cascade organization of the UPS and its hierarchical mode of function. Compared to standard genome-wide screens, this "cascade profiling" results in a rather compact and more targeted screen, which facilitates hits identification. Using this approach we have identified components of UPS potentially important for prostate cancer cell viability. 7 8 LIST OF ABBREVIATIONS 2D/3D – two dimensional/three dimensional 7-AAD – 7-Aminoactinomycin D a.a. – amino acid ADAMs – A-disintegrin-and-metalloproteinases AIDS – acquired immune deficiency syndrome AJs – adherens junctions AKT – v-Akt murine thymoma viral oncogene homolog 1 AMACR - alpha-methylacyl-coa racemase AR – androgen receptor ATCC - American Type Culture Collection ATG12 – autophagy 12 ATG8 – autophagy 8 ATM – ataxia telangiectasia mutated bp – base pair BPH – benign prostatic hyperplasia BRCA – breast cancer BSA – bovine serum albumin CAND1 – cullin-associated and neddylation-dissociated 1 Caspase – cysteine-aspartic proteases CHD1 – chromodomain helicase DNA binding protein 1 ChSM – DMEM supplemented with the charcoal/dextran stripped FBS c-Myc – v-Myc avian myelocytomatosis viral oncogene homolog COP1 – constitutive photomorphogenesis protein 1 homolog CR – castration resistant CRL – Cullin-RING-Ligase CUL1-7 – cullins 1-7 DAPI – 4'6-diamidino-2-phenylindole DDR – DNA-damage response DHT - 5α-dihydrotestosterone DMEM – Dulbecco’s Modified Eagle's Medium DMSO – dimethyl sulfoxide DNA – deoxyribonucleic acid DSB – double stranded breaks dsRNA – double-stranded RNA DUB – deubiquitylating enzyme E1 – ubiquitin-activating enzymes E2 – ubiquitin-conjugating enzymes E3 – ubiquitin-protein ligases EdU – 5'-ethyl-2'-deoxyuridine EGFR – epidermal growth factor receptor EMT – epithelial-to-mesenchymal transition ERG – ETS-related gene ETS – v-Ets avian erythroblastosis virus E26 oncogene homolog 1 ETV1-5 - Ets variants 1-5 FACS – fluorescent activated cell sorting FAK – focal adhesion kinase FAs – focal adhesions FAT10 – HLA-F adjacent transcript 10 FAU – Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitously expressed FBS – fetal bovine serum GS – Gleason Score gSD – global Standard Deviation HIF1α – hypoxia-inducible factor-1 alpha IRS-1 - insulin receptor substrate 1 ISG15 – interferon-stimulated gene product of 15 kDa IκB – inhibitor of NF-κB LAP – leukemia associated protein Lys – lysine MDM2 – mouse double minute 2, human homolog of; p53-binding protein 9 miRNA – micro RNA mRNA – messenger RNA MSMB – microseminoprotein, Beta- MW – molecular weight NAE – NEDD8 activating enzyme E1 subunit 1 NEDD8 – neural precursor cell expressed, developmentally down-regulated 8 NF-κB – nuclear factor kappa B NKX3-1 – NK3 Homeobox 1 nt – nucleotide Opti-MEM – reduced serum modification of eagle's minimum essential media p21 – cyclin-dependent kinase inhibitor 1A p27 – cyclin-dependent kinase inhibitor P27 p53 – tumor protein p53 PBS – phosphate buffered saline PBS – phosphate-buffered saline PCa – prostate cancer PCA3 – prostate cancer antigen 3 PDGFRβ – platelet-derived growth factor receptor, beta polypeptide PFA – paraformaldehyde PHD – plant homeo domain PI3K – phosphoinositide 3-kinase PIN – prostatic intraepithelial neoplasia PSA – prostate specific antigen PSMA – prostate-specific membrane antigen PTEN – phosphatase and tensin homolog Rb – retinoblastoma 1 RBR – ring between ring RBX1/2 - RING-box 1/2, E3 Ubiquitin Protein Ligase RdRP – RNA-dependent
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