This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Elucidating the role of mitochondrial‑localized hepatocyte growth factor receptor in gastric oncogenesis Sim, Kae Hwan 2016 Sim, K. H. (2016). Elucidating the role of mitochondrial‑localized hepatocyte growth factor receptor in gastric oncogenesis. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/66452 https://doi.org/10.32657/10356/66452 Downloaded on 01 Oct 2021 13:49:56 SGT ELUCIDATING THE ROLE OF MITOCHONDRIAL- LOCALIZED HEPATOCYTE GROWTH FACTOR RECEPTOR IN GASTRIC ONCOGENESIS SIM KAE HWAN SCHOOL OF BIOLOGICAL SCIENCES 2016 ELUCIDATING THE ROLE OF MITOCHONDRIAL- LOCALIZED HEPATOCYTE GROWTH FACTOR RECEPTOR IN GASTRIC ONCOGENESIS SIM KAE HWAN School of Biological Sciences A thesis submitted to the Nanyang Technological University in partial fulfillment of the requirement for the degree of Doctor of Philosophy 2016 Acknowledgement I would like to express my sincere appreciation and thanks to my supervisor Dr. Siu Kwan Sze, for his expert guidance and warm encouragement. He has been a tremendous mentor for me. He has shared his wealth of experience on numerous occasions and provided me with a great learning experience. Moreover, I would like to thank Dr. Ren Yan, Dr. Bamaprasad Dutta and Dr. Sun Yang, for their invaluable discussions and suggestions, which has helped me a lot in this project. I would also like to acknowledge the kind support of my current and former laboratory colleagues, especially Dr. Park Jung Eun, Dr. Hao Piliang, Dr. Sunil Shankar Adav, Dr. Guo Tiannan, Dr. Li Xin, Meng Wei, Qian Jingru, Dr. Cheow Siok Hwee, and Zhang Qi, for extending their helping hand anytime I needed. On the other hand, I would like to convey deep appreciation to my thesis advisory committee members, Dr. Koh Cheng Gee and Dr. Lu Lei, for serving as my committee members even at hardship. They have given me so many brilliant comments and constructive criticism during the annual committee meetings and the conformation of my PhD candidature. In addition, I would like to express my sincere thanks to the laboratory members from Dr. Koh Cheng Gee’s lab, Dr. Li Hoi Yeung’s lab, Dr. Lu Lei’s lab, and Dr. Lin Chun Ling Valerie’s lab. Especially Dr. Ou Sirong, Dr. Weng Ting, Dr. Yeap Szu Ling, and Miss Or Yu Zuan for their kind help in one way or the other in my research work in Nanyang Technological University. I gratefully acknowledge the financial support from the Nanyang Technological University of Singapore in the form of Nanyang Research Scholarship. I am also thankful to the academic and technical staffs at the school of Biological Sciences, who have helped me in my research work. i Last but not least, special thanks go to my family and my girlfriend’s family. Words cannot express how grateful I am to them for all of your selfless support, love and understanding during my PhD study in Singapore. ii Contents Acknowledgement i Contents iii List of figures vii List of tables ix Abbreviations x Abstract xiii Chapter 1 – General Introduction 1.1 Introduction 2 1.1.1 Cancer 2 1.1.1.1 Cancer 2 1.1.1.2 Gastric cancer 4 1.1.2 Receptor tyrosine kinases 6 1.1.3 MET 7 1.1.3.1 MET signaling in cancers 7 1.1.3.2 Mitochondrial localization of MET 8 1.1.4 Mass spectrometry-based quantitative proteomics 9 1.1.4.1 Label-free quantitative proteomic approach 10 1.1.4.2 Labeling based quantitative proteomics approach 11 1.1.5 Objective and overview of project 12 Chapter 2 - Elucidating the molecular mechanism of translocation of hepatocyte growth factor receptor (MET) into the mitochondria in SNU5 gastric cancer cells 2.1 Abstract 15 2.2 Introduction 16 2.2.1 Roles of receptor tyrosine kinases in cancer 16 2.2.2 Dysregulated receptor tyrosine kinases in gastric cancer 17 2.2.2.1 Dysregulation of receptor tyrosine kinases in gastric cancer 17 iii 2.2.2.2 MET in gastric cancer 17 2.2.3 Endocytosis of RTK in cancers 19 2.2.4 Localization of MET in mitochondria 20 2.2.4.1 Knowledge gap 20 2.2.4.2 Specific aims in the study 20 2.3 Materials and methods 22 2.3.1 Chemicals 22 2.3.2 Cell culture 22 2.3.3 Western blotting 23 2.3.4 Mitochondria isolation and purification 23 2.3.5 Bicinchoninic acid protein assay 24 2.3.6 Immunofluorescence 25 2.3.7 Mitochondrial protein digestion 25 2.3.8 LC-MS/MS 26 2.3.9 Data analyses 27 2.3.10 Data annotation 28 2.4 Results 29 2.4.1 Profiling of mitochondrial proteome to uncover the correlation of endocytosis 29 2.4.2 Inhibition of of endocytosis using endocytic inhibitors 31 2.4.3 Time course study of inhibition of endocytosis using different endocytic inhibitors 36 2.4.4 Immunofluorescence analysis of localization of mtMET of SNU5 gastric cancer cells 38 2.5 Discussions 41 2.6 Conclusions and future works 44 Chapter 3 - Quantitative proteomic profiling to identify novel substrates of mitochondria-localized MET (mtMET) SNU5 gastric cancer cells iv 3.1 Abstract 46 3.2 Introduction 47 3.2.1 Gastric cancer 47 3.2.1.1 Gastric cancer 47 3.2.1.2 Advances of genomic and proteomic methods in gastric cancer diagnosis 47 3.2.2 Proteomics and gastric cancer 49 3.2.3 MET: Structure, functions, and dysregulated signaling 50 3.2.4 Specific aims in the study 51 3.3 Materials and methods 52 3.3.1 Chemicals 52 3.3.2 Cell culture 52 3.3.3 Co-IP 53 3.3.4 BCA protein assay 54 3.3.5 Mitochondrial protein digestion 54 3.3.6 Protein digestion and TMT labeling 55 3.3.7 Reverse phase HPLC fractionation 55 3.3.8 Desalting peptide samples 56 3.3.9 LC-MS/MS 56 3.3.10 Data analyses 57 3.3.11 Data annotation 59 3.3.12 In situ proximity ligation assay, microscopy and data handling59 3.4 Results 61 3.4.1 Discovery of novel putative substrates of mtMET using a label-free quantitative strategy 61 3.4.2 TMT labeling of SNU5 mitochondrial proteome under different conditions 70 3.4.3 Protein-protein interaction of mtMET and candidate proteins 91 v 3.4.4 Putative substrates of mtMET in SNU5 gastric cancer cells 93 3.5 Discussions 97 3.6 Conclusions and future works 102 Chapter 4 – Conclusion and future direction 4.1 Concluding remarks and future perspective 104 Reference 108 Appendix A – Publications 130 Appendix B – Conference presentation 131 Supplementary Data 132 vi List of Figures Chapter 1 Figure 1.1: Simple illustration of the initiation and progression of cancer 3 Figure 1.2: Schematic representation of quantitative proteomics methods 10 Figure 1.3: Schematic illustration of the isobaric tagging chemistry of TMT reagent 11 Chapter 2 Figure 2.1: Immunoblotting evidences of MET and phosphor-MET in SNU5 mitochondria 32 Figure 2.2: Immunoblotting evidences showing high purity of mitochondrial fractions 36 Figure 2.3: Immunoblotting evidences showing the expression level of mtMET and phosphor-mtMET under endocytic inhibition 37 Figure 2.4: Fluorescence microscopic imaging showing the effect of dynasore inhibition in SNU5 cells 39 Chapter 3 Figure 3.1: Schematic representation of the experimental design of mtMET co-IP 62 Figure 3.2: Comparison of the identified proteins between IgG-coIPs and MET-coIPs 64 Figure 3.3: Scatter plots showing the normalized emPAI values in the technical triplicate measurements 65 Figure 3.4: Classification analysis of 342 uniquely identified proteins in MET-coIPs 66 Figure 3.5: Protein-protein interactions network extracted from STRING database 68 vii Figure 3.6: Schematic representation of the experimental design to perform the quantitative proteomics analysis of SNU5 mitochondrial proteome using TMT 6-plex isobaric tags 72 Figure 3.7: Physiochemical characteristics of identified protein 73 Figure 3.8: Distribution of the relative expression levels of temporal proteome 63 Figure 3.9: GO analysis illustrates the classes of proteins with significantly reduced expression 76 Figure 3.10: Detection of PLA signals in cytocentrifugation preparations of SNU5 gastric cancer cells using Duolink in situ reagents with two primary antibodies 92 Figure 3.11: Western blot evidences of protein-protein interaction between mtMET and HMGA1 and PKM2 94 Supplementary Data Supplementary Figure 1: Western blot results showing no contamination from other organelles 132 Supplementary Figure 2: RTKs and mitochondrial proteins TOM20 and Bcl-xL sharing sequence which is rich in amino acids (arginine and lysine) of high hydrophobicity and basicity 133 Supplementary Figure 3: Detection of PLA signals in cytocentrifugation preparations of SNU5 gastric cancer cells using Duolink in situ reagents with two primary antibodies (large images) 134 viii List of Tables Chapter 2 Table 2.1: Components of clathrin-mediated endocytosis were identified by mitochondrial proteomic profiling of RTK TKI-sensitive cell lines 30 Chapter 3 Table 3.1: Identification of proteins interacting with MET in co-IP data 69 Table 3.2: Data analysis of identified proteins in different drug treatment of SNU5 mitochondria digests 73 Table 3.3: List of proteins showing significantly reduced protein expression level in the PHA665752_24h (128/126) and Dynasore_24h (130/126) 78 Table 3.4: Annotation of significantly identified proteins in TMT-based quantitative proteomics profiling 83 Table 3.5: Label-free quantitative proteomic approach identified the reduced expression level of HMGA1 and PKM2 upon MET inhibition 96 Supplementary Data Supplementary Table 1: Original co-IP data for 342 significantly identified protein in triplicate MET-coIPs 136 ix Abbreviations 2D-GE Two-dimensional gel electrophoresis ABB Ammonium bicarbonate buffer ACLY ATP citrate