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 University of Guelph, 2016
Advisor: 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.
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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
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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
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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 TAF
Epidermal Growth Factor Receptor Tumor Angiogenesis Factor
- HIFs
- Hypoxia-Inducible Factors
- ATP
- Adenosine triphosphate
- HRE
- Hypoxia Response Element
ARNT PHDs pVHL EPO
Aryl Hydrocarbon Receptor Nuclear Translocator Prolyl-4-hydroxylase Domains Von Hippel-Lindau Tumor Suppressor Protein Erythropoietin
- EIFs
- Eukaryotic Initiation Factors
eIF4E eIF4E2 4EBP RBM4 mTOR IRES
Eukaryotic Translation Initiation Factor 4E Eukaryotic Translation Initiation Factor 4E Family Member 2 4E Binding Proteins RNA-Binding Protein-4 Mammalian Target of Rapamycin Internal Ribosome Entry Site
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- PAR-CLIP
- Photoactivable Ribonucleoside-Enhanced Crosslinking and
Immunoprecipitation
- rHRE
- RNA Hypoxia Response Element
Platelet Derived Growth Factor Receptor Alpha Epithelial to Mesenchymal Transition E-cadherin
PDGFRA EMT CDH1 CDH22 CDH11 ECM
Cadherin 22 Osteoblast-cadherin Extracellular Matrix
MMPs RCC
Matrix Metalloproteinases Renal Cell Carcinoma
- 4E-T
- Eukaryotic Translation Initiation Factor 4E Transporter
Chromosome Maintenance 1 Protein Homologue Bromodeoxyuridine
CRM1 BrdU PDVF PBS-T GAPDH PBS
Polyvinylidene Difluoride Phosphate Buffered Saline with Tween 20 Glyceraldehyde 3-phosphate dehydrogenase Phosphate Buffered Saline
- IGF1-R
- Insulin-Like Growth Factor Receptor 1
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L1CAM BCAM
L1 Cell Adhesion Molecule Basal Cell Adhesion Molecule
- Metadherin
- MTDH
ADAM11 ADAM12
ADAM metallopeptidase domain 11 ADAM metallopeptidase domain 21
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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
1.1 Cancer and Personalized Medicine..........................................................................................1
1.2 The Hypoxic Tumor Microenvironment and the Hallmarks of Cancer ...................................3
1.3 The Hypoxia Inducible Factor Regulatory Pathways...............................................................6
1.4 Differential Roles of HIF-1α and HIF-2α in Tumor Pathogenesis and Disease Progression ...10
1.5 Protein Synthesis and Translational Control under Hypoxic Stress.......................................11
1.7 The Role of Hypoxia in Tumor Migration and Invasion ........................................................17
1.8 Targeting the Hypoxic Tumor Microenvironment and eIF4E2 in Cancer Therapy...............23
1.9 Experimental Objectives........................................................................................................26
Chapter 2 - Materials and Methods ................................................................................................29 2.1: Cell Culture .............................................................................................................................29 2.2: Creation of Stable Cell Lines....................................................................................................29 2.3: Western Blotting......................................................................................................................30 2.4: Bromodeoxyuridine Assay .......................................................................................................31 2.5: Scratch Wound Migration Assay .............................................................................................32 2.6: Boyden Chamber Migration Assay ..........................................................................................32 2.7: Matrigel Invasion Assay...........................................................................................................32 2.8: RNA extraction........................................................................................................................33 2.9: Quantitative real-time PCR (qPCR) ........................................................................................33 2.10: Statistical Analyses.................................................................................................................34 Chapter 3 – Results.........................................................................................................................35
3.1: HCT-116 cells depend on eIF4E2 for their autonomous proliferation ......................................35 3.2: Inhibition of eIF4E2 increases the migration of HCT-116 cells ................................................40 3.3: eIF4E2 is required by U87-MG cells for their hypoxic migration and invasion in vitro............42 3.4: Inhibition of CDH22 impairs U87-MG migration but not invasion in vitro ..............................48 3.6: U87-MG cells do not depend on CDH22 for their proliferation................................................57
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Chapter 4 – Discussion....................................................................................................................59 Chapter 5 – Summary.....................................................................................................................68 References ......................................................................................................................................70
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Chapter 1 - Introduction
1.1 Cancer and Personalized Medicine
Cancer is recognized as one of the leading causes of mortality and morbidity worldwide, with 14 million cases and 8.2 million cancer-related deaths in 2014 (World Cancer Report, 2014). Cancer includes more than 100 distinct diseases with diverse risk factors and epidemiology, making it one of the most complex diseases of modern day. It is characterized by the rapid, uncontrolled proliferation of abnormal cells that are able to metastasize to distant organs and invade tissues (Stratton et al., 2009). During normal tissue homeostasis, there is persistent crosstalk between normal cells and the surrounding environment, which keeps cellular proliferation at bay. However in cancer, this system goes awry. Cancer cells gain the ability to proliferate independently due to the accumulation of mutations within their genomes. These mutations, combined with a chaotic tissue microenvironment, drive the development of cancer. The development of cancer is an evolutionary process, where mutant cells compete for space and nutrients, evade the immune system and disperse to new organs, forming colonies (Merlo et al., 2006). This evolutionary process is strongly supported by the tumor microenvironment (Stratton et al., 2009).
The tumor microenvironment includes a heterogeneous mix of malignant, and stromal cells such as cancer-associated fibroblasts, immune cells and endothelial cells (Hanahan & Coussens, 2012). During the development of a tumor, cancer cells are exposed to a variety of environmental stressors, including: oxidative stress, acidosis, starvation and hypoxia among others. Unlike normal cells, cancer cells are able to overcome these environmental stressors due to the accumulation of beneficial mutations. Stress within the tumor microenvironment also contributes to selective pressure on specific populations of cancer cells. This is a crucial aspect of tumor
1development, which is dependent on two continuous processes: the heritability of genetic variation in single cells and natural selection acting on the resultant phenotypic diversity due to the accumulation of mutations (Stratton et al., 2009). By continuously selecting for advantageous phenotypes, cells acquire the ability to autonomously proliferate and survive more effectively than their neighbours (Stratton et al., 2009).
Understanding the processes behind tumor development is one of the main goals of cancer research. In medicine, the understanding of these processes fuels the advancement of prevention, detection and treatment (Chin, Andersen, & Futreal, 2011). Recent advances in genomics have contributed significantly to cancer research and modern day medicine. Genomic advancements have made it possible to quickly identify genetic variants that underlie a specific cancer phenotype and have also contributed to the development of novel cancer therapies that are highly precise. These therapies are unique to each individual patient and are known as personalized medicine. Currently, cancer therapy is shifting from cytotoxic anticancer drugs to genotype-directed therapy (Mendelsohn, 2016). This approach has proven to be successful and has improved treatment outcomes in several cancers (Bai, Staedtke, & Riggins 2011; Sequist et al. 2011; Schroth et al., 2011). In metastatic colorectal cancer for example, RAS mutations actually predicted a lack of response in patients who received the commonly used drug panitumumab, an epidermal growth factor receptor (EGFR) inhibitor. However, in tumors that harboured a wildtype-RAS, panitumumab was very effective and increased overall survival by 10.5 months (Tabernero et al., 2013). Despite the advancements in genomic sequencing and molecular profiling, cancer is still considered a complex disease due to molecular heterogeneity and phenotypic diversity within the total tumor cell population. Molecular heterogeneity and phenotypic diversity are influenced by genetic and non-genetic factors (Marusyk, Almendro, & Polyak, 2012).
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1.2 The Hypoxic Tumor Microenvironment and the Hallmarks of Cancer
Regardless of their tissue of origin or genetic makeup, solid tumors share a feature commonly known as the hypoxic tumor microenvironment. Due to the rapid uncontrolled proliferation of abnormal cells, tumors rapidly outgrow their vasculature, creating pockets of hypoxia. Hypoxia is universal across solid tumor microenvironments, since the diffusion limit of oxygen is only 150 μm from a blood vessel in human tissue (Possible & For, 1955). In tumors more than 1 mm in diameter, an oxygen gradient exists as a result of restricted diffusion. The core of the tumor is necrotic, the intermediate layer surrounding the core is hypoxic and the outermost layer, which is surrounded by vascular networks of capillaries, is oxygenated (Li et al., 2007; Brahimi-Horn, Chiche, & Pouysségur, 2007). Therefore, over the course of tumor development, the cells within the microenvironment are exposed to varying amounts of oxygen.
In an attempt to combat hypoxia, cancer cells undergo a process known as angiogenesis, in which new blood vessels are formed upon pre-existing vasculature (Risau, 1997). This process was first described by Judah Folkman in 1971 and is now recognized as one of the hallmarks of cancer (Folkman et al., 1971; Hanahan & Weinberg, 2011). As a pioneer of angiogenesis, Folkman isolated a tumor factor that is responsible for angiogenesis and named it Tumor Angiogenesis Factor (TAF) (Folkman et al., 1971). Based upon his previous work, he also suggested that the inhibition of TAF may arrest tumor growth. Prior to this, Folkman et al. demonstrated that when perfused organs were isolated from mice and injected with melanoma cells, tumors would form but would only reach a maximum size of 1 – 2 mm3 and never became vascularized (Folkman et al., 1963). However, when the same tumor cells were implanted into organs within mice, the tumors grew well beyond 1 – 2 mm3 and established a vascular network (Folkman et al., 1963).
