Identification of genes and pathways linking cancer metabolism to cell surface dynamics through protein N-glycosylation by Xiangyuan Ma A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto © Copyright by Xiangyuan Ma 2014 Identification of genes and pathways linking cancer metabolism to cell surface dynamics through protein N-glycosylation Xiangyuan Ma Master of Science Department of Molecular Genetics University of Toronto 2014 Abstract N-glycosylation is a co-translational modification that covalently attaches oligosaccharide to and regulates proper folding, trafficking, and surface residency of secreted proteins. MGAT5 encodes a glycosyltransferase that synthesizes β1,6GlcNAc-branched N-glycans in medial Golgi. MGAT5 is frequently up-regulated in and associated with the progression of multiple types of human carcinomas. In order to identify Mgat5 interacting genes and signaling pathways, immortalized mouse embryonic fibroblast (MEF) cell lines that are Mgat5 wild-type and null were established on p53 null genetic background. Pooled lentiviral shRNA drop-out screens revealed that mTOR pathway was essential in the wild-type MEFs, whereas signaling dependency shifts to RAS-MEK-ERK pathway was observed in the null cells. Metabolite profiling of the MEFs and human cell lines with MGAT5 knock-down showed drastic changes in tricarboxylic acid cycle metabolites and amino acids upon MGAT5 disruption. Together, these approaches permitted a global survey of genetic and metabolic changes that occur when MGAT5 is disturbed. ii Acknowledgements I would like to express my utmost gratitude to my co-supervisors, Dr. James W. Dennis and Dr. Jason Moffat, for your constant guidance and encouragement over my graduate study. Your passion in pursuing science, patience, and attention to details inspires me, and invigorates me in the future days to come. I would like to extend my gratitude to my committee members, Dr. Johanna Rommens and Dr. Sean Egan, for your constructive advices and criticisms over my project, as well as your helps when I needed most outside academia life. I would also like to thank all of the Dr. Moffat Laboratory and Dr. Dennis Laboratory members, as well as people I worked in collaboration with. Without your help, completion of this project would not have been possible. Last but not least, I am greatly indebted to my parents, Mrs. Xiaoping Chen and Mr. Weijv Ma, for giving my life and your constant love. iii Table of Contents Abstract ........................................................................................................................................... ii Acknowledgements ........................................................................................................................ iii List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii List of Appendices ....................................................................................................................... viii Abbreviations ................................................................................................................................. ix CHAPTER 1 INTRODUCTION .................................................................................................... 1 1.1 Protein N-glycosylation and MGAT5 .............................................................................. 1 1.2 The N-glycan lattice model .............................................................................................. 5 1.3 Glucose and glutamine metabolism, the Warburg effect ................................................. 7 1.4 Hexosamine biosynthesis pathway................................................................................... 9 1.5 Ras and PI3K signaling .................................................................................................. 10 1.6 Summary of the work ..................................................................................................... 12 CHAPTER 2 MATERIALS AND METHODS ........................................................................... 13 2.1 Generation and immortalization of the mouse embryonic fibroblast cell lines, PCR genotyping ................................................................................................................................. 13 2.2 Cell culture, virus production, and MEF immortalization ............................................. 14 2.3 L-PHA and PI staining for flow cytometry .................................................................... 14 2.4 Lentiviral shRNA drop-out Screen ................................................................................ 15 2.5 Measuring MGAT5 expression by RT-PCR .................................................................. 16 2.6 Metabolite profiling........................................................................................................ 17 2.7 Pathway Enrichment and correlation study.................................................................... 18 CHAPTER 3 IDENTIFICATION OF MGAT5 INTERACTING GENES AND SIGNALING PATHWAYS ................................................................................................................................ 19 3.1 Summary ........................................................................................................................ 19 3.2 Introduction .................................................................................................................... 19 3.3 Generation of the Mgat5 MEF cell lines ........................................................................ 21 3.4 Pooled shRNA screens reveal genetic interaction between Mgat5 and mTOR signaling pathway ..................................................................................................................................... 24 3.5 Discussion ...................................................................................................................... 31 iv CHAPTER 4 MASS SPECTROMETRY ANALYSIS OF MGAT5 INTERACTING METABOLITES ........................................................................................................................... 34 4.1 Summary ........................................................................................................................ 34 4.2 Introduction .................................................................................................................... 34 4.3 Mgat5+/+ MEF cells contained higher level of glycolysis, TCA cycle, HBP, and PPP intermediates ............................................................................................................................. 36 4.4 Human cell lines with Mgat5 knock-down showed higher metabolite levels................ 39 4.5 Multiple metabolites displayed strong correlation with MGAT5 disruption ................. 52 4.6 Discussion ...................................................................................................................... 57 CHAPTER 5 DISCUSSION ......................................................................................................... 60 5.1 Mgat5 wt and null MEF cell line displayed different gene essentiality profile ............. 60 5.2 Signaling dependency shift from PI3K pathway in Mgat5 wt to MAPK in Mgat5 null MEFs ………………………………………………………………………………………… 62 5.3 Mgat5 deletion in MEF cell line and MGAT5 knock-down in human cell line caused metabolic reprogramming but of opposite trend ....................................................................... 64 5.4 Statistical tests identified key metabolites in glycolysis, TCA cycle, and amino acid metabolism to be differentially regulated in MGAT5 knock-down human cell lines .............. 66 5.5 Conclusions .................................................................................................................... 68 Reference ...................................................................................................................................... 69 Appendices.................................................................................................................................... 80 v List of Tables Table 3.1. Gene Set Enrichment Analysis of dGARP score ......................................................... 29 Table 4.1. List of metabolites showing statistically significant difference in at least two of the human cell lines ............................................................................................................................ 49 Table 4.2. Pearson’s correlation test of metabolite levels with remaining MGAT5 activity ....... 53 vi List of Figures Figure 1.1. N-glycan branching pathway in multicellular organisms............................................. 2 Figure 1.2. N-glycan-Galectin lattice model................................................................................... 6 Figure 3.1. PCR genotyping of the null and the wt MEFs............................................................ 22 Figure 3.2. Serial passage of the null and wt MEFs ..................................................................... 23 Figure 3.3. Surface L-PHA staining of the null and wt MEFs ....................................................