Cancer cells exploit eIF4E2-directed translation to enhance their proliferation, migration and invasion by Joseph F. Varga A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Molecular and Cellular Biology Guelph, Ontario, Canada © Joseph F. Varga, August, 2016 ABSTRACT Cancer cells exploit eIF4E2-directed translation to enhance their proliferation, migration and invasion Joseph F. Varga Advisor: University of Guelph, 2016 Professor James Uniacke Despite the diversity found in the genetic makeup of cancer, many cancers share the same tumor microenvironment. Hypoxia, an aspect of the tumor microenvironment, causes the suppression of the primary translational machinery. Hypoxic cells switch from using the eukaryotic initiation factor 4E (eIF4E) to using a homologue of eIF4E (eIF4E2), in order to initiate the translation of select mRNAs. This thesis investigates the role of eIF4E2-directed translation in a panel of cancer cell lines during autonomous proliferation, migration and invasion. In this thesis, we show that silencing eIF4E2 abrogates the autonomous proliferation of colon carcinoma. Silencing eIF4E2 in glioblastoma cells resulted in decreased migration and invasion. Furthermore, we link eIF4E2-directed translation of cadherin 22 with the hypoxic migration of glioblastoma. These findings answer questions regarding the biology of cancer and expand the current knowledge of genes exploited during tumor progression. This data also highlights eIF4E2 as a potential therapeutic target. Acknowledgements I would like to express my sincere gratitude to Dr. James Uniacke for taking me on as a graduate student in his laboratory and providing me with the opportunity to contribute to scientific research. I would also like to thank him for his continued support and commitment to my project and also for allowing me to attend several conferences to share my research. From day one, he has challenged me to do my best as a graduate student and was always available if I wanted to chat. To Erin Specker our lab manager, thank you for all of your support and involvement in this project. You were a joy to work with. To my lab mates both past and present; Andrea Brumwell, Sonia Evagelou, Brianna Guild, Sara Timpano, Gaelen Melanson, Dr. Phil Medeiros, Nicole Kelly, Crystal Gong, Shannon Sproul, Lorian Fay, Vincent Lau and Christina Romeo, I am grateful for your friendship and continued support over the years. I am truly privileged to have been part of this laboratory under the guidance of Dr. Uniacke. He has instilled in me a passion for hypoxic research which I hope to continue. To my fellow graduate students; Sherise Charles, Haidun Liu, Ashley Brott, Kathryn Reynolds, Jordan Willis and Richard Preiss, thank you for being part of this journey. To the advisory committee: Dr. Marc Coppolino and Dr. Alicia Viloria-Petit, thank you for your support and insight over the course of my project. Thank you to Cheryl Craag, Dr. Kelly Meckling, Dr. Éva Nagy, Dr. Tony Mutsaers and Dr. Joseph Lam for stimulating and encouraging my passion for scientific research, you gave me the research bug! To my friends and family: thank you for your patience and understanding. To this amazing institution which I have called home for the past six years, the University of Guelph and graduate school, thank you for teaching me so much about myself and life in general. You pushed me to my limits more than once but these two years have been the best years of my life! Cadherins: thanks for keeping everything together. III Declaration of Work Performed Erin Brouwers formed the stable MDA-MB-231, eIF4E2 shRNA knockdowns. Stable U87-MG and HCT-116 eIF4E2 shRNA knockdowns were formed by Dr. James Uniacke at the University of Ottawa and brought to our lab at the University of Guelph. Christina Romeo assisted in some of the Bromodeoxyuridine Assays with U87-MG cell lines. Nicole Kelly and Sonia Evagelou formed the stable U87-MG, CDH22 shRNA knockdowns. The author performed all other experiments. IV List of Figures Figure 1. Schematic of Hypoxia Inducible Factors (HIFs) stimulating the transcription of hypoxic response genes. ......................................................................................................... 9 Figure 2. Schematic diagram of an alternative hypoxic protein synthesis machinery. ........ 13 Figure 3. The proposed hypoxic switch between cadherins during tumor progression. ...... 20 Figure 4. BrdU incorporation in HCT-116 cells under hypoxia and normoxia in serum-free and complete media. ............................................................................................................ 37 Figure 5. BrdU incorporation in MDA-MB-231 cells under hypoxia and normoxia in serum-free and complete media. ........................................................................................ 38 Figure 6. BrdU incorporation in U87-MG cells under hypoxia and normoxia in serum-free and complete media. ............................................................................................................ 39 Figure 7. Inhibition of eIF4E2 enhances the migration of HCT-116 cells. ............................ 41 Figure 8. Inhibition of eIF4E2 impairs the hypoxic migration of U87-MG cells. ................. 43 Figure 9. Re-introduction of exogenous eIF4E2 rescues the loss of migration observed in eIF4E2-depleted cells. .......................................................................................................... 44 Figure 10. U87-MG cells harbouring shRNA against eIF4E2 exhibit less migration. ......... 45 Figure 11. Inhibition of eIF4E2 impairs the hypoxic invasion of U87-MG cells................... 47 Figure 12. CDH22 protein accumulates in hypoxia but not the mRNA. ............................... 50 Figure 13. The addition of neutralizing antibody against CDH1 and CDH22 impairs the hypoxic migration of U87-MG wild-type cells. ................................................................. 51 Figure 14. The addition of neutralizing antibody against CDH22 reduced the migration and invasion of U87-MG cells. ................................................................................................... 52 Figure 15. Depletion of CDH22 reduces the hypoxic migration of U87-MG cells. ............... 54 V Figure 16. Inhibition of CDH22 impairs the hypoxic migration of U87-MG cells and enhances invasion. ............................................................................................................... 55 Figure 17. Morphology of CDH22-depleted cells under normoxia. ....................................... 56 Figure 18. Depletion of CDH22 in U87-MG cells does not affect proliferation under hypoxia or normoxia. ......................................................................................................................... 58 VI List of Tables Table 1. eIF4E2-dependence of genetically distinct human cancer cell lines on autonomous proliferation, migration and invasion…………………………………………………………73 Table 2. CDH22-dependence of the U87-MG cell line on proliferation, migration and Invasion………………………………………………………………………………………….76 VII List of Abbreviations EGFR Epidermal Growth Factor Receptor TAF Tumor Angiogenesis Factor HIFs Hypoxia-Inducible Factors ATP Adenosine triphosphate HRE Hypoxia Response Element ARNT Aryl Hydrocarbon Receptor Nuclear Translocator PHDs Prolyl-4-hydroxylase Domains pVHL Von Hippel-Lindau Tumor Suppressor Protein EPO Erythropoietin EIFs Eukaryotic Initiation Factors eIF4E Eukaryotic Translation Initiation Factor 4E eIF4E2 Eukaryotic Translation Initiation Factor 4E Family Member 2 4EBP 4E Binding Proteins RBM4 RNA-Binding Protein-4 mTOR Mammalian Target of Rapamycin IRES Internal Ribosome Entry Site VIII PAR-CLIP Photoactivable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation rHRE RNA Hypoxia Response Element PDGFRA Platelet Derived Growth Factor Receptor Alpha EMT Epithelial to Mesenchymal Transition CDH1 E-cadherin CDH22 Cadherin 22 CDH11 Osteoblast-cadherin ECM Extracellular Matrix MMPs Matrix Metalloproteinases RCC Renal Cell Carcinoma 4E-T Eukaryotic Translation Initiation Factor 4E Transporter CRM1 Chromosome Maintenance 1 Protein Homologue BrdU Bromodeoxyuridine PDVF Polyvinylidene Difluoride PBS-T Phosphate Buffered Saline with Tween 20 GAPDH Glyceraldehyde 3-phosphate dehydrogenase PBS Phosphate Buffered Saline IGF1-R Insulin-Like Growth Factor Receptor 1 IX L1CAM L1 Cell Adhesion Molecule BCAM Basal Cell Adhesion Molecule MTDH Metadherin ADAM11 ADAM metallopeptidase domain 11 ADAM12 ADAM metallopeptidase domain 21 X Table of Contents ABSTRACT ..................................................................................................................................... II Acknowledgements ......................................................................................................................... III Declaration of Work Performed ..................................................................................................... IV List of Figures .................................................................................................................................. V List of Tables ................................................................................................................................. VII List of Abbreviations .................................................................................................................... VIII Chapter 1 - Introduction ...................................................................................................................1