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The Role of Eif5a Hypusination in Mediating Oncogenic Mtor Signaling

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The Role of Eif5a Hypusination in Mediating Oncogenic Mtor Signaling The role of eIF5A hypusination in mediating oncogenic mTOR signaling Yutian Cai Faculty of Medicine Department of Biochemistry McGill University, Montreal, Quebec, Canada December 2016 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science ©Yutian Cai 2016 TABLE OF CONTENTS TABLE OF CONTENTS ........................................................................................ 2 TABLE OF FIGURES ............................................................................................ 4 PREFACE.............................................................................................................. 5 ACKNOWLEDGEMENTS ...................................................................................... 6 ABSTRACT ........................................................................................................... 7 RÉSUMÉ ............................................................................................................... 9 LIST OF ABBREVIATIONS ................................................................................. 11 CHAPTER 1: INTRODUCTION ........................................................................... 14 1.1 mRNA TRANSLATION ......................................................................... 14 1.1.1 TRANSLATION INITIATION....................................................................... 15 1.1.2 TRANSLATIONAL EFFICIENCY ............................................................... 20 1.1.3 TRANSLATION AND CANCER ................................................................. 25 1.2 POLYSOME FRACTIONATION ............................................................ 27 1.3 mTOR PATHWAY ................................................................................. 30 1.3.1 UPSTREAM ACTIVATORS OF mTOR ...................................................... 31 1.3.2 DOWNSTREAM EFFECTORS OF mTOR IMPLICATED IN TRANSLATIONAL REGULATION....................................................................... 34 1.3.3 PHARMACOLOGICAL INHIBITORS OF mTOR ........................................ 36 1.4 OBJECTIVES ........................................................................................ 39 1.4.1 EUKARYOTIC TRANSLATION INITIATION FACTOR eIF5A .................... 42 1.4.2 DEOXYHYPUSINE HYDROXYLASE/MONOOXYGENASE (DOHH) ........ 47 CHAPTER 2 RESULTS ....................................................................................... 49 2.1 EXPRESSION OF DOHH AND eIF5A mRNA IS UPREGULATED ACROSS A VARIETY OF DIFFERENT CANCER TYPES .............................................. 49 2.2 DOHH IS A DOWNSTREAM TARGET OF THE mTOR PATHWAY ......... 50 2.3 CHANGES IN mTOR SIGNALING DO NOT EXERT A MAJOR EFFECT ON DOHH PROTEIN STABILITY .......................................................................... 51 2.4 DOHH mRNA LEVELS ARE NOT MODULATED BY mTOR SIGNALING .... ......................................................................................................................... 51 2.5 mTOR INCREASES TRANSLATIONAL EFFICIENCY OF DOHH mRNA .... ......................................................................................................................... 51 2.6 DOHH PROTEIN EXPRESSION IS INDEPENDENT OF THE 4E-BP STATUS IN THE CELL .................................................................................... 56 2 2.7 IDENTIFYING THE ROLE OF eIF5A HYPUSINATION IN mTOR DEPENDENT TUMORIGENESIS .................................................................... 57 CHAPTER 3 DISCUSSION ................................................................................. 67 CHAPTER 4 MATERIALS AND METHODS ....................................................... 76 4.1 CELL CULTURE AND DRUG TREATMENTS .......................................... 76 4.2 LENTIVIRUS shRNA AND INFECTIONS .................................................. 77 4.3 CELL LYSIS AND WESTERN BLOTTING ANALYSIS ............................. 77 4.4 POLYSOME PROFILING AND RNA EXTRACTION ................................. 79 4.5 REVERSE TRANSCRIPTASE-QUANTITATIVE PCR (RT-qPCR) AND PRIMERS ......................................................................................................... 80 4.6 SOFT AGAR COLONY FORMATION ASSAY .......................................... 82 CHAPTER 4 REFERENCES ............................................................................... 84 3 TABLE OF FIGURES FIGURE 1.1 A BRIEF OVERVIEW OF TRANSLATIONAL INITIATION IN EUKARYOTES .................................................................................................... 20 FIGURE 1.2 SHEMATIC REPRESENTATION OF POLYSOME PROFILING TECHNIQUE ....................................................................................................... 29 FIGURE 1.3 THE SCHEMATIC REPRESENTATION OF THE mTOR SIGNALING NETWORK ...................................................................................... 36 FIGURE 1.4 mTOR INHIBITORS SUPPRESS TRANSLATION OF A SUBSET OF mRNAS THAT ENCODE PROTEINS INVOLVED IN AMINO ACID METABOLIC PROCESSES INCLUDING DOHH ................................................ 41 FIGURE 1.5 A SCHEMATIC REPRESENTATION OF THE HYPUSINATION REACTION .......................................................................................................... 46 FIGURE 2.1 EXPRESSION OF DOHH AND EIF5A MRNA IS UPREGULATED ACROSS A VARIETY OF DIFFERENT CANCER TYPES ................................. 59 FIGURE 2.2 DOHH IS A DOWNSTREAM TARGET OF THE mTOR PATHWAY ............................................................................................................................. 60 FIGURE 2.3 CHANGES IN MTOR SIGNALING DO NOT EXERT A MAJOR EFFECT ON DOHH PROTEIN STABILITY ......................................................... 61 FIGURE 2.4 DOHH MRNA LEVELS ARE NOT MODULATED BY mTOR SIGNALING ......................................................................................................... 62 FIGURE 2.5 MTOR INCREASES TRANSLATIONAL EFFICIENCY OF DOHH mRNA .................................................................................................................. 63 FIGURE 2.6 DOHH PROTEIN EXPRESSION IS INDEPENDENT OF THE 4E-BP STATUS IN THE CELLS ..................................................................................... 65 FIGURE 2.7 DOHH IS REQUIRED FOR THE NEOPLASTIC GROWTH OF HCT116 PTEN-/- CELLS ...................................................................................... 66 4 PREFACE This thesis was written based on the Guidelines for Thesis Preparation from the Faculty of Graduate Studies and Research of McGill University. Our lab manager Shannon McLaughlan initiated the project and helped me optimize the experimental conditions throughout the project. Also, our collaborator Dr. Ola Larsson at Karolinska Institute in Sweden helped to develop the R script for polysome analysis. 5 ACKNOWLEDGEMENTS I first wish to thank my supervisor Dr. Ivan Topisirovic for his patience, guidance, and expertise in science. Moreover, I wish to thank him for giving me the opportunity to attend R programming workshop in Sweden and broaden my knowledge in bioinformatics. I would also like to thank my colleagues in the lab Valentina Gandin, Shannon McLaughlan, Marie Cargnello and Laura Hulea for teaching me the experimental techniques and helping me to develop critical thinking. Last but not the least, I would like to thank all my friends and family for the moral support I needed throughout the master study. 6 ABSTRACT Protein synthesis (or mRNA translation) is a major step in the regulation of gene expression and one of the most energy consuming processes in the cell. eIF5A is activated by a unique post-translational modification called hypusination (modification of Lys50 on eIF5A to hypusine), which is catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). eIF5A hypusination is thought to play a significant role in translation, but little is known about its regulation. Our recent genome-wide interrogation of the translatome identified DOHH mRNA as a potential translational target of the mechanistic/mammalian target of rapamycin (mTOR). We were able to validate these findings and demonstrate that treatment with mTOR inhibitors, rapamycin and torin1 causes a substantial decrease in DOHH protein levels. Using proteasome inhibitors and measuring mRNA levels by RT-qPCR, we concluded the mTOR neither controls stability of DOHH protein nor the transcription of the corresponding gene. We next conducted polysome-profiling experiments using mTOR inhibitors, which revealed that mTOR regulates DOHH expression at the level of translation. Since both mTOR and eIF5A hypusination are often found to be upregulated in cancer, and we established the link between mTOR and eIF5A hypusination, we hypothesized the DOHH/eIF5A pathway at least in part mediates oncogenic effects of mTOR. Using soft agar assay, we demonstrated 7 that DOHH depletion was sufficient to attenuate anchorage-independent growth induced by hyperactivated mTOR signaling. These findings suggest that DOHH and consequently eIF5A hypusination may play a major role in mTOR-driven oncogenesis. 8 Résumé La synthèse des protéines (ou traduction des ARN messagers) est une étape clé de la régulation de l’expression des gènes dont la dérégulation est associée au développement de nombreuses pathologies humaines telles que le cancer. Le facteur d’initiation de la traduction eIF5A est activé par hypusination, une modification post-traductionnelle
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