A PROTEOMIC APPROACH FOR THE IDENTIFICATION OF HCC SERUM BIOMARKERS LOW JIAYI (B.Sc. (Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS I would like to thank everyone who has kindly assisted me in this project. This project would not have been possible without their support and encouragement. My deepest gratitude goes to my supervisor, A/P Maxey Chung Ching Ming. I would like to thank him for believing in me, for giving me the freedom to experiment and explore while providing me with appropriate and timely advice. His support and encouragement I could not have done without. I have benefited greatly under his supervision. I thank A/P Lim Seng Gee and Dr Aung Myat Oo for providing, and trusting, me with the tissue and serum samples. I am indebted to the big family in the Protein and Proteomics Centre. In particular, I wish to thank Sandra, Cynthia, Gek San, Teck Kwang, Hwee Tong, Aida, Lifang, Siaw Ling, Qinsong, Justin, and Jason. They taught me many things, engaged in thought-provoking discussions with me and at the same time, extended their friendship. I am thankful for the former, and grateful for the latter. They have all been terrific mentors and wonderful friends. I am fortunate indeed, to have so many mentors. I thank everybody for their selfless and unwavering support. i TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii ABSTRACT vi LIST OF TABLES vii LIST OF FIGURES viii LIST OF ABBREVIATIONS ix 1. INTRODUCTION 1.1 HEPATOCELLULAR CARCINOMA 1.1.1. Hepatocellular Carcinoma 1.1.2. HCC Carcinogenesis 1.1.3. Staging 1.1.4. Aetiology 1.1.5. Chronic HBV Infection 1.1.5.1. Evidence of an Association between Chronic HBV Infection and HCC 1.1.5.2. HBV-induced Carcinogenesis 1.1.6. Liver Cirrhosis 1.1.6.1. Cirrhogenesis and Carcinogenesis 10 13 1.1.7. Diagnosis of HCC 14 1.1.8. Management of HBV-associated HCC 15 1.1.8.1. Prevention 15 1.1.8.2. Antiviral Therapy 16 1.1.8.3. Reversal of Cirrhosis 17 1.1.8.4. Treatment of HCC 17 1.1.8.5. Surveillance of Individuals at Risk 19 ii 1.1.9. Need for Biomarkers that allow Early Cancer Detection 1.1.9.1. The Search for New HCC Biomarkers 1.1.10. The Ideal Biomarker 1.2. TUMOUR IMMUNOLOGY 22 22 24 25 1.2.1. The Humoral Response to Cancer 25 1.2.2. Autoantibodies and Carcinogenesis 27 1.2.3. Anti-Tumour Effects of Autoantibodies 29 1.2.4. Autoantibodies as Biomarkers for Early Cancer Detection 30 1.2.5. Methods in Identifying Autoantibodies 32 1.2.5.1. Serological Identification of Antigens by Recombinant Expression Cloning (SEREX) 32 1.2.5.2. Phage Display 33 1.2.5.3. Protein Microarrays 34 1.2.5.4. SERPA (Serological Proteome Analysis) 36 1.2.6. The Importance of Studying the Proteome 1.3. AIMS OF PROJECT 2. MATERIALS AND METHODS 37 39 40 2.1. LIVER TISSUES, CELL LINES AND HUMAN SERA 40 2.2. SAMPLE PREPARATION 43 2.3. SERPA 43 2.3.1. Two-Dimensional Gel Electrophoresis (2-DE) 43 2.3.1.1. Isoelectric Focusing on IPG (Immobilized pH gradient) Strips 43 2.3.1.2. IPG Strip Equilibration 44 2.3.1.3. Second Dimension Sodium Dodecyl Sulphate – Polyacrylamide Gel Electrophoresis (SDS – PAGE) 45 2.3.2. Western Blot 45 iii 2.3.2.1. Electro-blotting 45 2.3.2.2. Immunodetection 46 2.3.2.3. Colloidal Silver Staining 47 2.3.3. Silver Staining 47 2.3.4. Tandem Mass Spectrometry 48 2.3.4.1. Enzymatic Digestion of Protein Spots 48 2.3.4.2 Matrix-assisted Laser Desorption/Ionization Tandem Time-of-Flight Mass Spectrometry (MALDI-TOF/TOF MS) 50 2.4. ANTIGEN VALIDATION WITH COMMERCIAL ANTIBODIES 51 2.5. PHOSPHOTYROSINE DETECTION 52 AND PHOSPHOSERINE 2.6. GLYCOPROTEIN STAINING 53 2.7. SDS PAGE 54 3. RESULTS 55 3.1. PRELIMINARY STUDIES 3.1.1. The Feasibility of Using a Single Liver Cancer Tissue as an Antigen Source 55 55 3.2. THE SEARCH FOR CIRRHOSIS- AND HCCASSOCIATED AUTOANTIBODIES 58 3.3. ANTIGEN VALIDATION WITH COMMERCIAL ANTIBODIES 77 3.4. THE SEARCH FOR POST-TRANSLATONAL MODIFICATIONS IN THE CIRRHOSIS- AND HCCASSOCIATED AUTOANTIGENS 82 3.4.1. The Search for Phosphorylated Autoantigens 82 3.4.2. The Search for Glycosylated Autoantigens 82 3.5. THE SEARCH FOR DIFFERENTIALLY-REGULATED AUTOANTIGENS 85 4. DISCUSSION 4.1. PRELIMINARY STUDIES 88 88 iv 4.1.1. A Single Liver Cancer Tissue as the Antigen Source 88 4.2. THE SEARCH FOR CIRRHOSIS- AND HCCASSOCIATED AUTOANTIBODIES 89 4.2.1. Reliability of Protein Identities 89 4.2.2. Cirrhosis- and HCC-associated Autoantibodies 90 4.2.2.1. The TAA Panel 90 4.2.2.2. The TAA Panel: Criteria for a Screening Test 92 4.2.2.3. The Autoantigens: Overlap with Other HCC Studies 93 4.2.2.4. Biological Properties of the Autoantigens 94 4.2.2.4.1. Proteins Involved in Signaling 96 4.2.2.4.2. Chaperones 98 4.2.2.4.3. Enzymes 100 4.2.2.4.4. Proteins with Other Functions 101 4.2.2.5. Non-Specific Autoantigens 103 4.2.2.6. Summary 104 4.3. ISSUES TO CONSIDER 105 4.3.1. Types of Sera Analyzed 105 4.3.2. Expression Levels of Autoantigens in Cirrhotic Tissues 107 4.4. FUTURE PROSPECTS 108 4.4.1. Validation of Results 108 4.4.2. Other Applications of Autoantibodies 109 4.4.3. Other Lines of Studies 110 5. CONCLUSION 111 6. REFERENCES 112 7. APPENDIX I v ABSTRACT Hepatocellular carcinoma (HCC), generally known as primary liver cancer, is the fifth most common malignancy in the world. It is also the third leading cause of cancerrelated deaths worldwide, with a mortality rate comparable to its incidence rate. This high mortality rate can be significantly lowered if diagnosis is made early and curative treatments are provided in time. Since 80% of HCCs arise from a cirrhotic background, the detection of cirrhosis can aid risk stratification for early HCC detection. Early biomarkers of cirrhosis and HCC are therefore urgently needed. In this work, we aim to identify cirrhosis- and HCC-associated autoantibodies that can serve as biomarkers in the early detection of HCC. Autoantibodies against tumourassociated antigens have been detected in cancer patients’ sera. These autoantibodies are elicited during early carcinogenesis, and are possibly the earliest cancer biomarkers that can be detected in sera. Hence, they facilitate the development of non-invasive serological tests for early cancer detection. In this study, tumour proteins were separated by 2-DE before being transferred onto PVDF membranes and probed with patient or control sera. The immunoreactive profiles were compared and twelve cirrhosis- and HCC-associated antigenic spots were detected and identified by tandem mass spectrometry. In addition, their identities were independently verified by commercial antibodies. These autoantigens were also analyzed to determine if they were differentially regulated or post-translationally modified by either phosphorylation or glycosylation. Six of these autoantigens can potentially form a biomarker panel for the detection of cirrhosis and HCC. In conclusion, this study identified a distinct repertoire of cirrhosis- and HCC-associated autoantibodies that can potentially enable early HCC diagnosis. vi LIST OF TABLES TABLE PAGE 1.1 The TNM staging of HCC 1.2 Stage grouping of HCC 1.3 Child’s-Pugh grading of severity of liver disease 12 1.4 Diagnostic criteria for Hepatocellular carcinoma 15 2.1 Clinical characteristics of 15 patients with liver disease 41 2.2 Clinical characteristics of 12 patients with liver disease 42 3.1 MS/MS data of the 12 immunoreactive spots 70 3.2 Summary of the Autoantibodies against the listed autoantigens 72 3.3 General biological properties of the autoantigens 73 3.4 MS/MS data of proteins that reacted with normal and patient sera 76 4.1 Autoantigens that make up a TAA panel that enable early detection of HCC as well as risk stratification of HCC patients 92 4.2 Criteria for Screening Tests 93 vii LIST OF FIGURES FIG. PAGE 1.1 Development of HBV-associated HCC 1.2 BCLC staging and treatment strategy for HCC patients 19 1.3 Diagnostic algorithm for hepatic nodule detected in a cirrhotic liver by ultrasound 20 3.1 Outline of preliminary experiments 56 3.2 Preliminary Western blot results 57 3.3 Outline of the SERPA approach in identifying cirrhosis-and HCC-associated autoantibodies 60 3.4 Summary of the different types of sera analyzed by the SERPA approach 61 3.5 Western blot analysis of HCC tissue lysate probed against human serum 62 3.6 A comparison of the immunoreactivity of each autoantigen with patient and normal sera 66 3.7 Location of cirrhosis- and HCC-specific antigens on a silverstained 2-D gel 68 3.8 Moderately differentiated HCC tissue lysate probed only with secondary antibody (anti-human IgG) 69 3.9 Antigen validation with Western blot using commercial antibodies 78 3.10 HCC tissue lysate probed with anti-phosphoserine and antiphosphotyrosine antibodies respectively 83 3.11 2-D gels of HCC tissue lysate stained first with Pro-Q Emerald glycoprotein stain, then with Sypro Ruby total protein stain 84 3.12 Expression levels of Cirrhosis- and HCC-associated Autoantigens in Moderately differentiated HCC tissue lysates 86 viii LIST OF ABBREVIATIONS 2-DE Two-dimensional gel electrophoresis ACN Acetonitrile AFP Alpha fetoprotein CAPZ1 F-actin capping protein alpha-1 subunit cDNA Complementary DNA CHAPS 3-[(3-cholamidopropyl)dimethylaminonio]-1-propanesulphonate DCP des-γ-carboxy prothrombin DMEM Modified Eagle medium DTT Dithiothreitol ECL Enhanced Chemiluminescence EDTA Ethylenediaminetetraacetic acid ELISA Enzyme linked immunosorbent assay ES1 ES1 protein homolog FBS Fetal bovine serum FH Fumarate hydratase GPC-3 Glypican-3 HBc Hepatitis B core protein HBsAg Hepatitis B virus surface antigen HBV Hepatitis B virus HBx Hepatitis B virus protein X HCC Hepatocellular carcinoma HCV Hepatitis C virus HSC70 Heat shock cognate 71 kDa protein HSP60 Heat shock protein 60 IAA Iodoacetamide IgG Immunoglobulin G IPG Immobilized pH gradient IPI International Protein Index MALDI-TOF/TOF MS Matrix-assisted laser desorption/ionization tandem time-offlight mass spectrometry NH4HCO3 Ammonium bicarbonate PCBP1 Poly(rC)-binding protein ix Hanash, S. 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II [...]... of HCC One is the late presentation of HCC, where the dearth of symptoms at the early stage of the disease results in detection of cancer only when at an advanced stage (Usatoff and Habib, 2002) Another is the paucity of curative treatments for late-stage HCC Consequently, 1 in most cases, by the time diagnosis is made, no curative treatment is available (Hoofnagle, 2004) 1.1.2 HCC Carcinogenesis HCC. .. that can justify its replacement (Brown and Scharschmidt, 1999; Lopez, 2005) In light of this, there is an impetus to find new biomarkers that are more sensitive and specific for HCC and that can detect HCC early 1.1.9.1 The Search for New HCC Biomarkers In light of the limitations of AFP as a HCC biomarker, researchers have been actively searching for alternative biomarkers A plethora of potential biomarkers. .. that could code for HCC- associated antigens Uemura et al (2003) uncovered 27 TAAs Le Naour et al (2002) identified 8 TAAs, but only 1 (autoantibody against a novel truncated form of calreticulin) is commonly induced in HCC Zhou et al (2005) identified HCC- 22-5 as a TAA Takashima et al (2006) identified 4 TAAs Li et al (2008) identified 6 TAAs These TAAs remain to be validated 1.1.10 The Ideal Biomarker... tumour-associated autoantibodies can also serve as biomarkers for diagnosing HCC Two of the more established HCC- associated TAAs are p53 (Raedle et al, 1998; Soussi, 2000) and p62 (Lu et al, 2001; Zhang and Chan, 2002) Many other TAAs have been discovered For 23 instance, Stenner-Liewen et al (2000) found 19 distinct antigens associated to HCC, of which 3 are novel Wang et al (2002) identified 55 cDNA sequences... (Pang et al., 2008) Aside from the four more established new HCC serum biomarkers discussed above, many other serum biomarkers have been found Some examples are gamma-glutamyl transferase, alpha-l-fucosidase, transforming growth factor-beta-1, insulin-like growth factor-II and golgi protein 73 (Pang et al., 2008; Wright et al., 2007; Zhou et al., 2006) Tumour-associated autoantigens (TAAs) and their corresponding... implementation of the HBV vaccination program in Taiwan 1.1.8.2 Antiviral Therapy Once chronic HBV infection is established, treatment efforts should focus on preventing the progression to cirrhosis There are several antiviral therapy options available, utilizing interferon-α and the nucleoside analogs lamivudine and adefovir dipivoxil However, these antiviral therapies are not suitable for every patient... disease-specific mortality was measured instead of all-cause mortality so the impact of the surveillance program may not be completely represented Various cohort and meta-studies have concluded that screening for HCC can identify tumours at an early stage, resulting in more patients having a higher chance of receiving curative treatment, thereby prolonging survival rates (McMahon et al., 2000; Oka et al.,... DNA damage characterized by gene amplification, deletion or mutation hastens the rate of carcinogenesis and contributes to the formation of dysplastic hepatocytes, and eventually, dysplastic nodules, resulting in the emergence of HCC Despite efforts to elucidate the molecular pathology leading to HCC development, no genetic predisposition for HCC has been found In fact, the molecular profile of HCC. .. increases the specificity and sensitivity for diagnosing HCC (Pang et al., 2008) Glypican-3 (GPC-3) is another serum biomarker that has been widely studied It can be detected in 40 – 53 % of HCC patients and is not detectable in healthy individuals (Capurro et al., 2003; Nakatsura et al., 2003; Pang et al., 2008) Combination testing of both GPC-3 and AFP can increase sensitivity for diagnosing HCC (Pang... 1.1.8 Management of HBV-associated HCC 1.1.8.1 Prevention The best ‘treatment’ for HBV-associated HCC is prevention If chronic HBV infection is not established, HBV-associated HCC would be a non-issue (Lok, 2004) Fortunately, a vaccine for HBV is available, and the implementation of universal HBV vaccination programs has reduced HBsAg carrier rates and HCC incidence 15 significantly This is irrefutably . Tanaka et al., 2006; Twu et al., 1993) ; and upregulating amongst other signaling pathways, the Ras-Raf-MAPK signal transduction pathway, the JAK/STAT pathway and the protein kinase B pathway. symptoms at the early stage of the disease results in detection of cancer only when at an advanced stage (Usatoff and Habib, 2002). Another is the paucity of curative treatments for late-stage HCC. . Summary of the different types of sera analyzed by the SERPA approach 61 3.5 Western blot analysis of HCC tissue lysate probed against human serum 62 3.6 A comparison of the immunoreactivity