Investigating the Role of Protein Arginine ...

[Pages:358]Investigating the Role of Protein Arginine Methyltransferases in Breast Cancer Etiology

Alan James Morettin, B.Sc., M.Sc.

A Thesis Presented to The University of Ottawa

In partial fulfillment of requirements for the degree of

Doctor of Philosophy in

Cellular and Molecular Medicine

? Alan James Morettin, Ottawa, Canada, 2015

ABSTRACT Breast cancer is the most commonly diagnosed cancer amongst Canadian women.

Though numerous treatments are available, in many instances tumours become refractory or recur. Therefore, understanding the biological events that lead to the progression and therapeutic resistance of breast cancer is essential for the development of novel treatment options for this disease. Numerous members of the protein arginine methyltransferase (PRMT) family, which are the enzymes responsible for catalyzing methylation on arginine residues are aberrantly regulated in breast cancer. Hence, understanding the precise contribution of PRMTs to the development and progression of breast cancer is important. This Thesis will present my findings on the alternatively spliced PRMT1 isoform, PRMT1v2, previously identified to be overexpressed in breast cancer cell lines and here shown to promote breast cancer cell survival and invasion. Second, a novel role is ascribed to PRMT6, another PRMT aberrantly expressed in breast cancer. PRMT6 promotes chemoresistance to the drug bortezomib by mediating stress granule formation through down-regulation of eIF4E. Increased stress granule formation in bortezomibresistant cancer cells promotes cell survival. Third, DDX3, a prototypical PRMT substrate which is overexpressed in breast cancer cell lines and stimulates transformation of mammary epithelial cells is a novel substrate of PRMT1, CARM1, and PRMT6. Lastly, TDRD3, a reader/effector of arginine methylation also overexpressed in breast tumours regulates breast cancer cell proliferation, anchorage-independent growth and cell motility and invasion.

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ACKNOWLEDGEMENTS

First, I would like to thank my supervisor Dr. Jocelyn Cote for providing me with the chance to pursue my PhD degree in his laboratory and the opportunity to work on novel, stimulating and innovative projects. I would also like to thank Jocelyn for providing me with the latitude to follow different research angles that may not always have been correct and for his patience in allowing me to learn from my mistakes. Lastly, I would like to thank him for challenging me and having the faith in me that I would meet those challenges.

I would also like to acknowledge my thesis advisory committee members: Dr. Stephen Lee, Dr. David Lohnes, and Dr. Laura Trinkle-Mulcahy. I would like to thank them for providing helpful insight and constructive criticism; facilitating the movement of my research project forward. Their advice will make me a better scientist through allowing me to examine every potential angle to make my research as concrete as possible.

I would like to thank Cote lab members too numerous to mention both past and present for making my graduate experience in Jocelyn's lab as pleasant and as entertaining as possible. I would like to thank those who provided me with technical assistance that I required over the years. Specifically, I would like to thank Dr. Mitch Baldwin for his assistance and advice throughout the years, in addition to our numerous hockey conversations. I would especially like to thank both Helina Tadesse and Emma Bondy-Chorney for their friendship. Only someone who is undergoing the same process

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as you can appreciate the varying degrees of emotions that one endures in this process. I am indebted to both of you.

Lastly, I would like to thank my family for their love and support throughout this process. It is your continual support that gave me the strength and determination to accomplish my goals.

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STATEMENT OF CONTRIBUTION OF COLLABORATORS In Chapter 3 of the dissertation, MCF7 GFP, GFP-PRMT1v1, and GFP-

PRMT1v2 stably expressing cell lines were generated by Isabelle Goulet. Mitch Baldwin performed the characterization of the MCF7 GFP expressing cell lines (Figure 3.12A, 13.12B & 3.12D), motility, invasion and MTT assays (Figure 3.13), immunofluorescence images (Figures 3.16 & 3.19) and -catenin experiments in Figures 3.20 & 3.21. Quantification of the colony scattering assay (Figure 3.17) was performed by Mitch Baldwin, Genevieve Paris and myself.

In Chapter 5, data mining of SILAC mass spectrometry databases was performed by Dr. Laura Trinkle-Mulcahy (Figure 5.2). Mass spectrometry analysis of DDX3 samples immunoprecipitated from HeLa scramble and PRMT6 shRNA expressing cells was performed at the Ottawa Hospital Research Institute Proteomics Core Facility.

In Chapter 6, the colony formation assays in Figure 6.3 were performed by Tyler Murray. The pCMV myc TDRD3 deletion mutants used in Figure 6.6 were generated by Isabelle Goulet. The tail vein injection mouse model (Appendix Iq) was performed by Mitch Baldwin and Genevieve Paris.

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TABLE OF CONTENTS

ABSTRACT................................................................................... ii ACKNOWLEDGEMENTS.................................................................. iii STATEMENT OF CONTRIBUTION OF COLLABORATORS...................... v TABLE OF CONTENTS..................................................................... vi LIST OF FIGURES AND ILLUSTRATIONS............................................ ix LIST OF ABBREVIATIONS............................................................... xiii

1. CHAPTER 1: INTRODUCTION........................................................ 1 1.1 Arginine Methylation............................................................ 1 1.2 Regulation of Arginine Methylation.......................................... 2 1.3 Biological Roles of Arginine Methylation.................................... 7 1.4 Arginine Methylation and Breast Cancer..................................... 11 1.5 Protein Arginine Methyltransferase 1......................................... 13 1.6 Protein Arginine Methyltransferase 6......................................... 18 1.7 Stress Granules................................................................... 24 1.8 Arginine Methylation and RNA Binding Proteins........................... 31 1.9 DEAD-box RNA Helicases..................................................... 32 1.10 DDX3.............................................................................. 33 1.11 DDX3's Roles in RNA Metabolism............................................ 33 1.12 DDX3 and Cancer............................................................... 39 1.13 Tudor Domain Containing Proteins............................................ 41 1.14 TDRD3............................................................................ 45 1.15 TDRD3 and Breast Cancer...................................................... 50 1.16 Rationale for Study............................................................... 50

2. CHAPTER 2: MATERIALS AND METHODS........................................ 54 2.1 Cell Lines.......................................................................... 54 2.2 Establishment of Stably Expressing Cell Lines.............................. 54 2.3 Antibodies........................................................................ 54 2.4 Affinity Purification of PRMT1v2 Antibody................................ 55 2.5 siRNA Transfection............................................................. 56 2.6 Transfection of DNA Plasmids................................................ 57 2.7 DNA Constructs................................................................. 57 2.8 Site-Directed Mutagenesis...................................................... 58 2.9 RNA Isolation.................................................................... 58 2.10 Reverse Transcription Polymerase Chain Reaction......................... 59 2.11 Trypan Blue Exclusion Assay.................................................. 59 2.12 MTT Assays...................................................................... 60 2.13 Flow Cytometry and Annexin V Staining.................................... 60 2.14 Cell Motility and Invasion Assays.............................................. 61 2.15 Colony Scatter Assay............................................................... 61 2.16 Immunofluorescence............................................................. 62 2.17 Protein Purification................................................................ 62 2.18 In Vitro Methylation Assays.................................................... 63

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2.19 In Vivo Methylation Assays..................................................... 64 2.20 Protein Isolation from Mammalian Cells..................................... 64 2.21 Immunoprecipitation and Immunoblotting................................... 64 2.22 GST Pulldown Assay............................................................ 65 2.23 CAP-binding Assay............................................................. 66 2.24 Mass Spectrometry.............................................................. 66 2.25 Statistical Analysis.............................................................. 67

3. RESULTS................................................................................... 68

Chapter 3: Protein Arginine Methyltransferase 1 Isoform 2 Promotes Breast Cancer Cell Survival and Invasiveness......................................................... 68

3.1 Characterization of a PRMT1v2 Specific Antibody......................... 68 3.2 PRMT1v2 Depletion Affects Breast Cancer Cell Growth and Viability. 76 3.3 Depletion of PRMT1v2 Induces Apoptosis.................................. 81 3.4 PRMT1v2 Depletion Inhibits Breast Cancer Cell Motility and

Invasion........................................................................... 91 3.5 Overexpression of PRMT1v2 Promotes Breast Cancer Cell Motility

and Invasion...................................................................... 97 3.6 Overexpression of PRMT1v2 Alters Breast Cancer Cell Morphology... 109 3.7 PRMT1v2 Repress -catenin Expression Resulting in Increased Cell

Invasion........................................................................... 112

Chapter 4: Elucidating the Role of PRMT6 in Chemoresistance through its Function in Stress Granule Formation..................................................... 125

4.1 Depletion of PRMT6 Inhibits Bortezomib-Induced Stress Granule Formation......................................................................... 125

4.2 Re-Introduction of PRMT6 Rescues Bortezomib-Induced Stress Granule Formation.............................................................. 139

4.3 Overexpression of PRMT6 does not Alter Bortezomib-Induced Stress Granule Formation...................................................... 144

4.4 PRMT6 Protein Expression is Induced upon Bortezomib Treatment.... 147 4.5 PRMT6 Depletion Inhibits Formation of the eIF4E-eIF4G1 Complex.. 157 4.6 PRMT6 Depletion Results in Decreased Cellular Survival in

Bortezomib-Resistant Cancer Cells........................................... 163

Chapter 5: Arginine Methylation of the DEAD-box RNA Helicase DDX3........... 167

5.1 DDX3 is an Arginine Methylated Protein.................................... 167 5.2 PRMT1, CARM1, and PRMT6 Methylate DDX3, In Vitro............... 170 5.3 PRMT6 Methylates DDX3 Arginine Residues 585 and 587, In Vitro... 189 5.4 PRMT6 Methylates DDX3, In Vivo........................................... 190 5.5 Characterizing the Interaction between PRMT6 and DDX3.............. 204

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Chapter 6: Investigating the Role of TDRD3 in Breast Cancer......................... 215 6.1 TDRD3 Protein Expression is not Altered in Breast Cancer Cell Lines.............................................................................. 215 6.2 TDRD3 Depletion Inhibits Breast Cancer Cell Proliferation............. 218 6.3 TDRD3 Depletion Inhibits Anchorage Independent Growth............. 218 6.4 TDRD3 Depletion Impedes Breast Cancer Cell Motility and Invasion........................................................................... 221 6.5 TDRD3's Promotion of Breast Cancer Cell Invasion Properties is Dependent on its Interaction with the RNA Binding Protein FMRP.... 226

4. CHAPTER 7: DISCUSSION............................................................ 233 5. CONCLUSIONS........................................................................... 274 6. APPENDIX I............................................................................... 275 7. APPENDIX II.............................................................................. 309 8. LITERATURE CITED.................................................................... 310 9. CURRICULUM VITAE.................................................................. 341

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