haracterization of tumor suppressor genes hDAB2IP and DLEC1 in hepatocellular carcinoma

CHARACTERIZATION OF TUMOR SUPPRESSOR GENES hDAB2IP AND DLEC1 IN HEPATOCELLULAR CARCINOMA QIU GUO-HUA NATIONAL UNIVERSITY OF SINGAPORE 2008 CHARACTERIZATION OF TUMOR SUPPRESSOR GENES hDAB2IP AND DLEC1 IN HEPATOCELLULAR CARCINOMA QIU GUO-HUA (M.SC., China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2008 ACKNOWLEDGEMENTS I would like to express my gratitude to all those who have helped me complete this PhD thesis. Above all, I am deeply grateful to my supervisor, Associate Professor Hooi Shing Chuan, Head of Department of Physiology, National University of Singapore. This work could not have been possible without his patient guidance, valuable discussion and consistent support. I appreciated that Prof. Hooi had a separate meeting with me every Saturday morning, where some good ideas were developed from the insightful conversations. Those times will remain a nice memory for me. I learned a lot from Prof. Hooi about not only science, but English and life as well. More importantly, I would like to express my sincere appreciation to him for letting me complete the PhD study in his lab at my most difficult time. I thank to my previous supervisor Associate Professor Tao Qian in The Chinese University of Hong Kong for his guidance and support when I was in Johns Hopkins Singapore, where I started to study DNA methylation with inspiration. In addition, I wish to extend my appreciation to my colleagues, Puei Nam, Jianjun, Guodong, Mirtha, Baohua and Colyn for their assistance, discussion and friendship. Special thanks to Huangming, Carol and Yuntong, for their effort in this project. I appreciated the administrative assistance and friendship of Ms. Asha Das, Vasantha Nathan, Jenny and Eileen. I would also like to thank the members in my previous lab in Johns Hopkins Singapore, Dr. Wen-Sen Hsieh, Dingxie, Zhaohui, Shiguo, Tzer Jing, Fu Li, John, Vivien, Tan Jing and Cai Yan for their assistance, support and friendship during my PhD study there. I I am grateful as well to the advisory committee members, Associate Professor Yu Qiang and Dr. Linda SH Chuang for beneficial suggestions and comments during my PhD Qualifying Exam. I would like to thank my parents in China for their love and support even though I have been so far away from them since beginning my university studies. I regret that I am not able to spend more time with them. Most importantly, I wish to express my greatest appreciation to my wife Xie Xiaojin, for her love, continuous support, discussion, encouragement and tolerance throughout the duration of my part-time PhD study. Thanks to my lovely daughters, BiQing and Bi-Xin, whom I am proud of. Qiu Guo-Hua National University of Singapore February 2008 II TABLE OF CONTENTS Acknowledgements I Table of Contents III Summary VIII List of Tables X List of Figures XI List of Publications XIII Abbreviations XIV CHAPTER ONE Introduction 1.1 1.2 Overview of Hepatocellular Carcinoma 1.1.1 Incidence and Mortality 1.1.2 Demographic Factors 1.1.3 Etiology of HCC 1.1.4 Host Factors 1.1.5 Diagnosis and Therapeutic Options 13 Genetics of Hepatocellular Carcinoma 19 1.2.1 Microsatellite and Chromosomal Instability 20 1.2.2 Activation of Oncogenes 21 1.2.3 Inactivation of Tumor Suppressor Genes 24 1.2.4 Alteration of Signaling Pathways 29 III 1.3 1.4 Epigenetics of Hepatocellular Carcinoma 1.3.1 DNA Hypomethylation 34 1.3.2 DNA Hypermethylation 36 Approaches to Screen Tumor Suppressor Genes 1.5 31 50 1.4.1 DNA Methylation-based Methods 51 1.4.2 Expression-based Methods 58 Research Objectives 59 1.5.1 MSP-based Approach 60 1.5.2 RT-PCR-based Approach 60 1.5.3 The Objectives of the Thesis 60 CHAPTER TWO Materials and Methods 62 2.1 62 Cell Lines and Cell Culture 2.1.1 HCC Cell Lines 62 2.1.2 Colorectal Cancer Cell Lines 62 2.1.3 Drug Treatment 62 2.1.4 Cell Count 63 2.2 Patients and Tissue Samples 63 2.3 RNA Extraction 64 2.4 2.3.1 Trizol Method 64 2.3.2 RNeasy Mini Kit 64 DNA Extraction 65 2.4.1 Trizol 65 IV 2.4.2 DNeasy Kit 2.5 Reverse Transcription (RT)-PCR 65 66 2.5.1 cDNA Synthesis 66 2.5.2 PCR Reaction 66 2.6 Quantitative Real-time RT-PCR 67 2.7 Bisulfite Treatment 67 2.8 Methylation-specific PCR and Bisulfite Genomic Sequencing 68 2.9 Cloning of DLEC1 Open Reading Frame 68 2.10 Plasmid Construction 68 2.11 Colony Formation 69 2.12 Generation of Stable Clones 69 2.12.1 HepG2 69 2.12.2 HCT116 70 2.13 Cell Proliferation Assay 70 2.14 Cell Cycle Analysis 70 2.15 Luciferase Reporter Assay 71 2.16 Chromatin Immunoprecipitation 71 2.17 Western Blot Analysis 72 2.18 Statistical Analysis 72 2.19 Primers Used in Screening of Tumor Suppressor Genes 72 2.19.1 Methylation-based Method 72 2.19.2 Expression-based Method 73 V CHAPTER THREE Expression and Methylation of hDAB2IP (Paper I) 82 3.1 82 Screening 3.1.1 Rationale 82 3.1.2 Gene Selection 83 3.1.3 MSP 83 3.2 Background of hDAB2IP 84 3.3 Results 86 3.3.1 Differential Expression of hDAB2IPA and hDAB2IPB in Normal Human Tissues 86 3.3.2 Down-regulation and Promoter Hypermethylation of hDAB2IPA in Liver Cancer Cell Lines 87 3.3.3 Hypermethylation of hDAB2IPA in Primary HCC and Correlation with Gene Expression 89 3.3.4 Promoter Methylation of hDAB2IPA and Clinicopathological Correlation 94 3.4 Discussion 94 CHAPTER FOUR Methylation and Effect on Cell Cycle of DLEC1 (Paper II) 99 4.1 Screening 99 4.1.1 Rationale 99 4.1.2 Primers 100 4.1.3 Results 100 4.2 Background of DLEC1 103 4.3 Results 104 4.3.1 DLEC1 Expression is Down-regulated and Correlated to Promoter Methylation in Cell Lines 104 VI 4.3.2 CpG Island Methylation of DLEC1 in HCC Primary Tumors and Clinicopathological Correlation 106 4.3.3 DLEC1 Inhibits Cell Proliferation, Induces G1 Cell Cycle Arrest and Reduces Cell Size 109 4.4 Discussion 112 CHAPTER FIVE Upregulation of p21 by DLEC1 116 5.1 Introduction 116 5.2 Results 118 5.2.1 G1 Phase Arrest in Cell Cycle Induced by DLEC1 118 5.2.2 Growth Inhibition by DLEC1 is Independent of p53, p21 and DNMT3B 119 5.2.3 DLEC1 is a Transcriptional Modulator 120 5.2.4 DLEC1 Upregulates p21 in Stable Clones 122 5.3 Discussion 124 CHAPTER SIX General Discussion and Conclusions 126 Bibliography 130 VII SUMMARY Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. The development of HCC is an intricate multistep process. One of the steps is the inactivation of tumor suppressor genes. However, information about tumor suppressor genes in HCC is relatively scant, compared to other cancers. Therefore, the primary purpose of this study was to identify and characterize tumor suppressor genes in HCC. The first candidate tumor suppressor gene is hDAB2IP, which was screened by an MSP-based approach in HCC. The results showed that of the two isoforms, hDAB2IPA was the predominant one, being expressed in the majority of human normal tissues examined. The expression of hDAB2IPA was silenced or down-regulated but could be restored by 5-aza-2’-deoxycytidine treatment in liver cancer cell lines. The reactivation of hDAB2IPA was due to the promoter demethylation. These results indicate that DNA methylation is involved in the downregulation of hDAB2IPA in HCC cell lines. The correlation between promoter methylation and hDAB2IPA expression was confirmed in eight pairs of matched HCC samples. Furthermore, more than 80% of HCC samples showed hDAB2IPA promoter methylation, compared to 11.5% in the corresponding adjacent normal tissue (p[...]... identify candidate tumor suppressor genes in HCC and then to investigate the methylation status of these genes The thesis will focus on the candidate tumor suppressor genes hDAB2IP and DLEC1 in HCC and their molecular mechanisms in tumorigenesis Prior to presenting my results, I will make an overview of HCC and then review our understanding of HCC at the molecular level, including the alterations of oncogenes,... silences tumor suppressor genes and thus enhances the process of tumorigenesis Therefore, a greater understanding of the mechanisms underlying DNA methylation of tumor suppressor genes is extremely important for unraveling the possible roles of tumor suppressor genes in the progression from normal to tumor cells This has direct complication for early tumor detection and disease monitoring (Jablonka and. .. tumor suppressor genes and their related genes during hepatocarcinogenesis 2 1.1 Overview of Hepatocellular Carcinoma Hepatocellular carcinoma (HCC) is one of the primary hepatic neoplasms that arise from hepatocytes, the major cell type of the liver (Farazi and DePinho, 2006) It is the most common type of malignant primary liver cancer, representing 75-90% of all cases in most countries (McGlynn and. .. screening of tumor suppressor genes in 3p21.3 by RT-PCR 74 Table 3-1 Methylation status of candidate tumor suppressor genes in HCC 84 Table 3-2 Primer sequences used in Paper I 87 Table 3-3 Clinicopathological features and hDAB2IPA promoter methylation 93 Table 4-1 Primers used in Paper II 104 Table 4-2 Clinicopathological features and DLEC1 promoter methylation 108 Table 5-1 DLEC1 induces G1 arrest in. .. McGlynn and London, 2005) Multiple processes are involved in the HBV-induced hepatocarcinogenesis The HBV genome encodes several viral proteins which are essential to its life cycle and its direct involvement in the functional processes, including inactivation of p53 by HBx binding, host–viral interactions (induction of oxidative stress), sustained cycles of necrosis–inflammation–regeneration and targeted... Differential expression of hDAB2IPA and hDAB2IPB in normal human tissues 88 Figure 3-3 Expression and DNA methylation of hDAB2IPA m2a in liver cancer cell lines 90 Figure 3-4 Correlation between downregulation and promoter hypermethylation of hDAB2IPA in liver tissue samples 92 XI Figure 4-1 RT-PCR screening of genes or ESTs in 3p21.3 101 Figure 4-2 Correlation between CpG island methylation and DLEC1 expression... proposed that tumor usually arises from cells that have accumulated multiple genetic abnormalities, including the chromosomal instabilities, alterations of tumor suppressor genes and proto-oncogenes (Fearon and Vogelstein, 1990) .Tumor suppressors are inactivated by ‘loss -of- function’, thus causing the loss of control over cell growth, while protooncogenes are constitutively activated through ‘gain -of- function’,... 4-3 Methylation analysis of DLEC1 in HCC primary tumors 107 Figure 4-4 Inhibition of cell growth by DLEC1 in vitro 110 Figure 4-5 DLEC1 overexpression inhibits proliferation, reduces cell size and induces G1 arrest in cell cycle 111 Figure 5-1 The growth inhibition by DLEC1 is independent of p53, p21 and DNMT3B 119 Figure 5-2 DLEC1 is a transcriptional modulator 121 Figure 5-3 DLEC1 activates p21 transcription... cell lines The correlation between hypermethylation and expression was also demonstrated in ten pairs of HCC and adjacent normal tissues (t-test, p . CHARACTERIZATION OF TUMOR SUPPRESSOR GENES hDAB2IP AND DLEC1 IN HEPATOCELLULAR CARCINOMA QIU GUO-HUA NATIONAL UNIVERSITY OF SINGAPORE 2008 2 CHARACTERIZATION. abnormalities, including the chromosomal instabilities, alterations of tumor suppressor genes and proto-oncogenes (Fearon and Vogelstein, 1990) .Tumor suppressors are inactivated by ‘loss -of- function’,. cancer in the world. The development of HCC is an intricate multistep process. One of the steps is the inactivation of tumor suppressor genes. However, information about tumor suppressor genes in