IDENTIFICATION AND FUNCTIONAL VALIDATION OF CALDESMON AS A POTENTIAL GASTRIC CANCER METASTASIS-ASSOCIATED PROTEIN HOU QIAN NATIONALUNIVERSITY OF SINGAPORE 2013 IDENTIFICATION AND FUNCTIONAL VALIDATION OF CALDESMON AS A POTENTIAL GASTRIC CANCER METASTASIS-ASSOCIATED PROTEIN HOU QIAN B Sc (Hons.), NTU A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONALUNIVERSITY OF SINGAPORE 2013 iii DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously _ Hou Qian 31 July 2013 iv ACKNOWLEDGEMENTS I wish to thank those people who have assisted me throughout my PhD project First of all, I would like to express my greatest gratitude to my supervisor, A/P Maxey Chung Ching Ming who has provided me the wonderful opportunity to perform the research in his lab With his constant guidance, help and advice for the past years, this project was made possible I have benefited tremendously during the journey of my PhD under his supervision I would like to thank Dr Tan Hwee Tong for his invaluable mentoring, discussion and help during the past years, thank Dr Tony Lim and Ms Avery Khoo from Singapore General Hospital for the collaboration work done with their mentoring and help I would also like to thank our lab’s former research assistants Ms Cynthia Liang and Ms Tan Gek San for their guidance and assistance I am also greatly indebted to Dr Lin Qingsong and Mr Lim Teck Kwang for helping me in 2-D LC MALDI-TOF/TOF MS analysis for my samples My seniors:Dr Zubaidah, Mr Vincent Lau, Mr Hendrick Loei have helped me in my lab techniques My labmates: Qifeng, Seow Chong, Wu Wei, Yee Jiun have been wonderful colleagues and friends in our journeys towards PhD Here I would like to express my gratitude to them My parents have encouraged me to pursue a postgraduate degree and I would like to thank them for their support I would also like to thank my husband Lichuan who has been with me through this wonderful journey v JOURNAL PUBLICATION AND CONFERENCES ATTENDED Published: Hou Q, Tan HT, Lim KH, Lim TK, Khoo A, Tan IB, Yeoh KG, Chung MC (2013) Identification and Functional Validation of Caldesmon as a Potential Gastric Cancer Metastasis-associated Protein J Proteome Res., 12(2):980-90 Conference poster presented in 6th International Conference on Structural Biology & Functional Genomics, December 6-8, 2010 Hou Q, Tan HT, Lim TK, Chung MC Unraveling the Molecular Basis of Gastric Cancer Metastasis by Comparative Proteome Analyses of Gastric Cancer Cell Lines Conference poster presented in FAOBMB Student Symposium, October, 2011 Hou Q, Tan HT, Lim KH, Chung MC Role of Fascin and Caldesmon in Gastric Cancer Metastasis Conference poster presented in YLLSoM 2nd Annual Graduate Scientific Congress, 15 February, 2012 (Best Poster Presentation Award) Hou Q, Tan HT, Lim KH, Chung MC Role of Fascin and Caldesmon in Gastric Cancer Metastasis Conference poster presented in the 11th Annual HUPO World Congress, 913 September, 2012 Hou Q,Tan HT; Lim TK,Lim KH, Chung MC Identification of Caldesmon as a Potential Gastric Cancer Metastasis Regulator by Comparative Proteome Analyses of Gastric Cancer Cell Lines vi Conference poster presented in Singapore Gastric Cancer Consortium 6th Annual Scientific Meeting, 25 – 26 July, 2013 Hou Q, Tan HT, Lim KH, Lim TK, Khoo A, Tan IB, Yeoh KG, Chung MC Identification and Functional Validation of Caldesmon as a Potential Gastric Cancer Metastasis-associated Protein vii Table of Contents ACKNOWLEDGEMENTS v JOURNAL PUBLICATION AND CONFERENCES ATTENDED vi ABSTRACT xi LIST OF TABLES xiii LIST OF FIGURES xiv LIST OF ABBREVIATIONS xvi CHAPTER INTRODUCTION 1.1 GASTRIC CANCER 1.1.1 Gastric Cancer Epidemiology 1.1.2 Gastric Cancer Risk Factors 1.1.3 Gastric Cancer Histological classifications 1.1.4 Screening and Diagnostic Tools for Gastric Cancer 1.1.5 Treatment Options for Gastric Cancer 1.1.6 Molecular Patterns and Biomarkers for Gastric Cancer 1.2 CANCER METASTASIS 10 1.2.1 TNM Staging of Tumor, Lymph Node Metastasis 10 1.2.2 Metastasis Is a Multi-Step Process 12 1.2.3 Molecular Features of Metastasis 14 1.3 PROTEOMICS: A HIGH-THROUGHPUT METHOD IN UNDERSTANDING THE MECHANISMS OF CANCER 16 1.3.1 Proteomics in Cancer Marker Discovery 16 1.3.2 Proteomics Techniques: Gel-based platforms 18 1.3.3 Proteomics Techniques: LC-based platforms 19 1.3.4 Proteomics in Gastric Cancer Biomarker Discovery 21 1.4 OBJECTIVES 22 Chapter MATERIAL AND METHODS 24 2.1 CELL CULTURE 24 2.2 QUANTITATIVE PROTEOMICS USING ITRAQ 26 2.2.1 Cell Lysates Preparation 26 viii 2.2.2 iTRAQ Labeling 26 2.3 TWO-DIMENSIONAL LIQUID CHROMATOGRAPHY 28 2.4 MALDI-TOF/TOF MS 28 2.5 MS DATA ANALYSIS 29 2.6 WESTERN BLOTTING 30 2.7 IMMUNOCYTOCHEMISTRY, TISSUE IMMUNOHISTOCHEMISTRY 31 2.7.1 Immunocytochemistry (ICC) Sample Preparation 31 2.7.2 Tissue Immunohistochemistry (IHC) Sample Preparation 32 2.7.3 Immunostaining 32 2.8 SIRNA-MEDIATED CALDESMON KNOCKDOWN 33 2.9 REVERSE-TRANSCRIPTION POLYMERASE CHAIN REACTION (RT-PCR) FOR IDENTIFICATION OF CALDESMON ISOFORMS 34 2.9.1 RNA Isolation 34 2.9.2 cDNA synthesis 34 2.9.3 Reverse-Transcription Polymerase Chain Reaction (RT-PCR) 34 2.10 STABLE OVER-EXPRESSION OF CALDESMON 38 2.11 CELL ASSAYS 38 2.11.1 Cell Proliferation Assay 38 2.11.2 Wound Healing Assay 39 2.11.3 Transwell Migration Assay and Matrigel Invasion Assay 39 2.11.4 Cell Attachment Assay 39 2.12 CO-IMMUNOPRECIPITATION 41 2.12.1 Caldesmon Co-Immunoprecipitation 41 2.12.2 Silver Staining 41 2.12.3 In-Gel Tryptic Digestion 42 CHAPTER RESULTS 44 3.1 ITRAQ ANALYSIS OF METASTATIC VERSUS PRIMARY GASTRIC CANCER CELL PROTEOMES 44 3.2 VERIFICATION OF SELECTED TARGETS WITH WESTERN BLOTTING 52 3.3 VERIFICATION OF CALDESMON AND FASCIN EXPRESSION WITH IMMUNOCYTOCHEMISTRY 57 3.4 CALDESMON AND FASCIN EXPRESSION IN PAIRED LYMPH NODE METASTASIS AND PRIMARY GASTRIC CANCER TISSUES 59 ix 3.5 TISSUE MICROARRAY ANALYSIS OF CALDESMON AND FASCIN EXPRESSION 64 3.6 KNOCKDOWN OF CALDESMON EXPRESSION WITH RNA INTERFERENCE 68 3.7 REVERSE-TRANSCRIPTION PCR IDENTIFIED CALDESMON ISOFORM AS THE UBIQUITOUS EXPRESSED ISOFORM 73 3.8 STABLE OVER-EXPRESSION OF CALDESMON IN AZ521 CELL LINE 76 3.9 CO-IMMUNOPRECIPITATION IDENTIFIED CALDESMON-INTERACTING PROTEINS 81 CHAPTER DISCUSSION 89 4.1 GASTRIC CANCER CELL LINES AS MODEL FOR PROTEOME PROFILING 89 4.2 DIFFERENTIALLY EXPRESSED PROTEINS IN GASTRIC CANCER METASTASIS 90 4.2.1 Up-Regulated Protein Functional Groups 90 4.2.2Down-Regulated Protein Functional Groups 92 4.3 FASCIN UP-REGULATION IN GASTRIC CANCER METASTASIS 94 4.4 CALDESMON DOWN-REGULATION IN GASTRIC CANCER METASTASIS 96 4.5 CALDESMON IN TUMOR STROMA OF GASTRIC CANCER PATIENTS 98 4.6 CALDESMON MAY BE INVOLVED IN GASTRIC CANCER METASTASIS BY REGULATING THE ACTIN CYTOSKELETON AND INVADOPODIA 100 4.7 CALDESMON INTERACTING PROTEINS IDENTIFIED BY COIMMUNOPRECIPITATION 103 CHAPTER CONCLUSION 107 CHAPTER FUTURE STUDIES 109 REFERENCES 111 x ABSTRACT Gastric cancer is the fourth most common cancer in the world and the second most common cause of cancer-related death The rate of metastasis is high in gastric cancer patients During cancer metastasis progression, the tumor cells underwent a plethora of molecular and morphological changes to become motile and invasive for the malignant transformation Thus discovery of the molecules that were involved in metastasis progression is critical for early diagnosis and prognosis for gastric cancer patients In this study, we aim to identify biomarkers for gastric cancer metastasis using a quantitative proteomics approach The proteins extracted from a panel of gastric cancer cell lines, two derived from primary cancer (AGS, FU97) and two from lymph node metastasis (AZ521, MKN7) were labeled with iTRAQ (8-plex) reagents and analyzed by 2D - LC MALDI-TOF/TOF MS In total, 641 proteins were identified with at least a 95% confidence Using cutoff values of >1.5 and