Epigenetic Mechanism Involved in the HBV/HCV ...

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Epigenetic Mechanism Involved in the HBV/HCV-Related Hepatocellular Carcinoma Tumorigenesis Liang Rongrui1,6,a, Huang Na2,a, Li Zongfang1,2,3,*, Ji Fanpu4 and Jiang Shiwen5 1

Department of General Surgery, Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, China; Engineering Research Center of Biotherapy and Translational Medicine of Shaanxi Province, Xi’an Jiaotong University, Xi’an, China; 3Key Laboratory of Environment and Genes Related to Diseases of the Education Ministry, School of Medicine, Xi’an Jiaotong University, China; 4Department of Infectious Disease, Second Affiliated Hospital, College of Medicine, Xi’an Jiaotong University, Xi’an, China; 5Mercer Univ, Sch Med, Dept Biomed Sci, Savannah, GA USA; 6Department of Oncology, The First Affiliated Hospital of Soochow University 2

Abstract: Hepatitis B virus (HBV) and hepatitis C virus (HCV) infection were known to be risk factors for HCC, they were suspected to promote its development by eliciting epigenetic changes. However, the precise gene targets and underlying mechanisms have not been elucidated. Epigenetic regulation of gene expression has emerged as a fundamental aspect of cancer development and progression. The molecular mechanisms of carcinogenesis in hepatocellular carcinoma involve a complex interplay of both genetic and epigenetic factors. DNA methylation, post-translational modifications of histone proteins, chromatin remodeling, and noncoding RNAs are four major types of mechanistic layers in the field of epigenetics. HBV infection could affect methylation on p16INK4A, GSTP1, CDH1(E-cadherin), RASSF1A, p21WAF1/CIP1 genes, which may play important roles in the development of HCC. HCV infection was related to aberrant methylation on SOCS-1, Gadd45, MGMT, STAT1 and APC. Other epigenetic alterations included histone proteins, chromatin remodeling, and noncoding RNAs were described in literature. Uncovering the epigenetic alterations of HBV/HCV-induced HCC carcinogenesis could highlight a new strategy for deciphering the mechanism of HCC tumorigenesis and development, as well as a potential diagnostic advantage.

Keywords: Epigenetic, hepatocellular carcinoma, HBV, HCV, DNA methylation and histone modification. INTRODUCTION Hepatocellular carcinoma (HCC) has a low survival rate as the majority of patients present with advanced and late stage disease [1]. Understanding the molecular pathways of HCC is crucial for the development of novel therapies for this highly aggressive cancer [2]. HCC is a well-recognized consequential complication of cirrhosis [3]. Infection by hepatitis B virus (HBV) and hepatitis C virus (HCV) is believed to be the major risk factor associated with the incidence of cirrhosis as well as HCC. Epidemiological studies have provided overwhelming evidence for a crucial role of chronic HBV/HCV infection in the development of HCC. Globally, it is estimated that 350 million people are chronically infected with the HBV [4]. Approximately 25% of chronically HBV-infected individuals will develop HCC [5]. However, the pathogenesis of carcinogenesis of HBV/HCV-associated HCC is still elusive. Moreover, the development of HCC in persons who are persistently infected with HBV/HCV is a growing problem worldwide [6]. Current antiviral therapies are not effective in many patients with these incurable viruses. While genetic alterations are proposed to contribute to the development and progression of HCC, the molecular mechanisms underlying this process remain unclear. Although HBV could integrate viral DNA into the host genome, there is no consistent genetic change associated with hepatocarcinogenesis, and only a handful of genes (such as -catenin, axin, and p53) are known to be frequently mutated in HCC [7]. This has led to research on alternative mechanisms promoting hepatocarcinogenesis other than genetic dysregulation. Epigenetic factors were proved to form the molecular basis of HCC in decades. Epigenetics refers to all stable changes of *Address correspondence to this author at the Department of General Surgery, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, No.157, West 5th Road, Xi'an 710004, Shaanxi Province, PR China; Tel: +86-29-87678006, +86-29-87679508; Fax: +86-29-87678634; E-mail: [email protected] a These authors contributed equally to this work. 1381-6128/14 $58.00+.00

phenotypic traits that are not coded in the DNA sequence itself [8]. Epigenetic mechanisms can be viewed as an interface between the genome and risk factor/ life style/ environmental influence. Aberrant epigenetic events associated with any of these stressors were shown to have a crucial role in cancer development. Epigenetic alteration included DNA methylation, histone modification and RNA-associated silencing. Disruption of these systems can lead to inappropriate high expression or silencing of genes. Cancer-related epigenetic dysfunction may induce hypomethylation leading to oncogene activation and chromosome instability, hypermethylation resulting in tumor suppressor gene silencing, or chromatin remodeling and RNA-associated silencing [9]. Methylation in DNA has long been recognized as an epigenetic silencing mechanism of fundamental importance [10]. Histone modifications, including acetylation and methylation of conserved lysine residues on the aminoterminal tail domains, have also been defined as epigenetic modifiers and have been studied for years. The role of RNA in posttranscriptional silencing which can lead to mitotically heritable transcriptional silencing by the formation of heterochromatin has attracted much interest. Aberrant epigenetic states may predispose to genetic changes, but genetic changes may also initiate aberrant epigenetic events. Virus infection, especially DNA viruses and retroviruses, which may cause insertion of viral DNA sequence into the host genome, often triggers the host defense mechanism, particularly, DNA methylation machinery, to cause the methylation of foreign movable viral genome. Sequences of HBV [11] or HCV [12] are found integrated in the genome of HCC cells and virusrelated insertional mutagenesis occurs frequently in liver cancers. Therefore, epigenetic dysregulation due to HBV/HCV infection could be of importance in HCC tumorigenesis. In this review, we will focus on epigenetic modifications induced by HBV/HCV infection leading to HCC tumorigenesis. EPIGENETIC EVENTS IN HUMAN MALIGNANCIES Although the genomes carried in all cells are common, only a subset of genes was necessarily expressed at different developmen© 2014 Bentham Science Publishers

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tal stages or with different functions. For different cell types to maintain their identity when they divide, they have to retain a ‘memory’ of which proteins must be produced and, hence, which genes are to be transcribed. The different transcriptional potentials of any particular gene in different cell types are not determined by changes in DNA sequence; therefore, the information specifying these differences is termed ‘epigenetic’. Failure to propagate epigenetic information accurately results in deviations from the normal pattern of gene expression, which in turn can lead to failure of developmental programs and, potentially, to develop cancer. There are four primary types of mechanistic layers in the field of epigenetics: DNA methylation, post-translational modifications of histone proteins, chromatin remodeling, and noncoding RNAs. DNA METHYLATION DNA methylation at the promoter regions is known to be important in both development and human disease, including cancer. DNA methylation-related alterations include three types: hypermethylation, hypomethylation, and loss of imprinting (LOI). DNA hypermethylation occurs at specific regulatory sites in the promoter regions or repetitive sequences [13,14]. It was believed that tumor-specific DNA hypermethylation would play a critical role in cancer development [15-17]. Methylation of CpG dinucleotides is one of the most principal epigenetic mechanisms that governs the transcriptional regulation of genes. Aberrant DNA methylation was the first epigenetic mark to be associated with cancer [18,19]. Mammalian DNA methyltransferases recognize CpG dinucleotides at specific sites within the genome as a “CpG island”. CpG island referred to a CpG-rich region often encompassing the promoter and transcription start site of the associated gene and possesses de novo methylation activity [20]. CpG islands account for only about 1% of the genome and for 15% of the total genomic CpG sites, in contrast, these regions contain over 50% of the unmethylated CpG dinucleotides. There are about 45,000 CpG islands, most of which reside within or near the promoters or first exons of genes and are unmethylated in normal cells, with the exception of CpG islands on the inactive X chromosome in females [21]. In cancer states, hypermethylation of CpG islands in promoter sequences is associated with silencing of tumor suppressor genes and tumor-related genes by subsequent downregulation of mRNA transcript expression [22,23]. Single-gene studies showed that de novo DNA methylation of promoter CpG islands is a frequent alteration in cancer, resulting in transcriptional silencing of dozens, even hundreds, of genes per tumor [24]. Promoter CpG islands are very rarely methylated in normal tissues, while many tissue-specific gains of methylation in cancer were shown [25]. A very important conclusion from single-gene analysis was that many silencing events have a driver function in cancer, or at least significantly modulate the tumor biology. It suggested that, similar to genetic mutation, many DNA methylation events are drivers in tumorigenesis. As well as hypermethylation, DNA hypomethylation was reported to occur in many tumors, particularly in advanced stages, and was generally assumed to be a genome-wide event [26, 27]. DNA hypomethylation signifies one of the major DNA methylation states, in most cases it refers to a relative situation in which there is a decrease from the ‘‘normal’’ methylation level [28]. The mechanism of DNA hypomethylation is still unclear, and, very likely, there is not a single mechanism responsible for demethylation of DNA. Hypermethylation of tumor suppressor gene promoter regions can lead to transcriptional inactivation and the loss of protein expression [29-31]. Also, hypomethylation of the global genome can lead to genomic instability that is exemplified by misalignments, DNA breakage, deletions and duplications during DNA replication, as seen in cancer [32]. This is evident by the fact that almost all of the major human cancers, including colon [33, 34], gastric [35,36], lung [37], liver [38,39], breast [40], bladder [41],

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ovarian [42,43], and endometrial [44], are characterized by a profound cancer-linked hypomethylation of the genome. More importantly, a significant association was reported between the degree of DNA hypomethylation and the grade/stage of cancer, which offers a firm basis for DNA hypomethylation as a biomarker for the diagnosis and prognosis of cancer [45-49]. Indeed, several studies have demonstrated that DNA hypomethylation is a more informative prognostic marker than tumor stage or grade [47,50]. However, a decrease in DNA methylation, by itself, is not sufficient to address precisely the role of DNA hypomethylation in tumorigenesis because it could simply be a secondary consequence of malignant cell transformation. Loss of imprinting (LOI), which refers to the loss of the differential expression of parental alleles, is often seen in embryonal tumors [51,52]. Genomic imprinting is an epigenetic modification of a specific parental chromosome in the gamete or zygote. Genomic imprinting could lead to parental origin-specific differential expression of the two alleles of a gene in somatic cells of the offspring. Genomic imprinting is a form of non-mendelian inheritance in animals, restricted to mammals, where the ‘imprinted’ genes are expressed uniquely from one allele. Associated with imprinted genes is a regulatory region, the imprinting control region (ICR) or differentially methylated region (DMR), which is methylated on one allele, but not on the other. The regulation of imprinted gene expression is the consequence of a reading of the ICR-DMR methylation status [53]. Genomic imprinting is considered to play a role in human disease and cancer. Thus far, a cluster of imprinting genes has been identified in chromosome 11p15.5, including IGF-II, H19, IPW, and p57kip. LOI of the IGF-II and H19 genes has been found in some embryonal and adult human cancers [54-58]. POST-TRANSLATIONAL MODIFICATIONS OF HISTONE PROTEINS The eukaryotic genome is assembled as a nucleoprotein complex known as chromatin which is a structural polymer consisting of positively charged histone proteins in addition to DNA. It provides a dynamic platform that controls all DNA-mediated processes within the nucleus. Histones are the most abundant proteins bound to DNA in eukaryotic cells and among the most evolutionary conserved proteins known [59]. Each core histone within the nucleosome contains a globular domain which mediates histone-histone interactions and also bears a highly dynamic amino terminal tail approximately 20~35 residues in length and is rich in basic amino acids. The core histone proteins are subjected to a variety of posttranslational modifications in both their unstructured N-terminal tails and their globular domains. This involves acetylation, methylation of lysine and arginine residues, ubiquitylation, SUMOylation of lysines and phosphorylation of serine and threonines [60]. Histone acetylation and deacetylation play an important role in chromatin remodeling and, thus, in gene expression. There is a fine balance between acetylation and deacetylation of histones in normal cells. The enzymes catalyzing these modifications are histone acetyltransferases and histone deacetylases (HDAC), respectively [61]. Histone acetylation is associated with an open chromatin and enhanced transcription, histone deacetylation is associated with closed chromatin and transcriptional repression. Lysine methylation depends on S-adenosylmethionine (SAM or AdoMet) as the methyl donor. Lysine-specific demethylases work in coordination with histone lysine methylases to maintain global histone methylation patterns. Although histone proteins are modified inside the nucleus by many other manners (Such as ubiquitylation, Biotinylation, SUMOylation and phosphorylation), so far only a few modifications have been studied. In conclusion, core histones can be reversibly modified by acetylation, methylation or ubiquitination and these modifications have consequences for gene activation, gene repression, heterochromatization and DNA repair and cancer development.

Epigenetic Mechanism in HBV/HCV-derived HCC

CHROMATIN REMODELING AND NONCODING RNAS Eukaryotic genomes are packaged into chromatin, which creates a natural barrier against access to DNA during transcription, replication, repair and recombination. Nature has evolved elaborate mechanisms to dynamically modulate chromatin structure, including chromatin remodeling by ATP-dependent complexes, covalent histone modifications, utilization of histone variants and DNA methylation. Dynamic modulation of chromatin structure, that is, chromatin remodeling, is a key component in the regulation of gene expression, apoptosis, DNA replication/repair and chromosome condensation/segregation. Disruption of these processes is intimately associated with human diseases, including cancer [62]. Change in chromatin structure is accompanied by changes in DNA accessibility to nucleases, several factors such as DNaseI and restriction enzymes and the affected nucleosomal stretch of DNA are termed as hypersensitive [63]. The packaging of genomic DNA into chromatin regulates access to regulatory proteins and other processes that use DNA as template, such as DNA replication, cell cycle progression, recombination and repair. Consequently, alleviating nucleosomal repression through remodeling of chromatin is mandatory to potentiate gene expression [64]. ATP-dependent chromatin remodelers are large multiprotein complexes containing an ATPase subunit that belongs to the SWI2-SNF2 (switching-sucrose nonfermenting) subfamily. These complexes use ATP hydrolysis to remove histones or to reconfigure the nucleosomes, increasing the accessibility of DNA elements to the regulatory proteins. ATPdependent chromatin-remodeling enzymes, which are highly conserved in organisms from yeast to humans, are similar to the SNF2 (sucrose non-fermenting 2) family of DNA translocases and all contain a catalytic ATPase subunit [65]. A very important recent development has been the realization that inactivation of SWI/SNF can play a critical causal role in the development of human cancers. SNF5 for instance, is one of the core subunits required for the ATPdependent remodeling activity of the SWI/SNF complex. SNF5 might prevent tumorigenesis by regulating cell proliferation, controlling cell-cycle progression, maintaining chromosomal stability and participating in DNA-damage repair [66]. In recent years, moreover, noncoding RNAs (ncRNAs) have been attracting the interest of many researchers. Accumulated evidence suggests that ncRNAs are strongly involved in cancer. For example, altered expression of microRNAs (miRNAs) is a common feature of malignancies [67,68], and epigenetic mechanisms are strongly involved in the dysregulation of miRNAs in cancer [69]. The discovery of small noncoding RNAs has opened the door to a previously unexplored world of posttranscriptional regulation that affects virtually every facet of developmental and cellular biology, these small RNAs are believed to regulate more than 60% of whole human genes [70]. Loss or amplification of miRNA genes by gross cytogenetic abnormalities or minute molecular aberrations has been observed in a variety of human malignancies and more than half of the tumor-related genes were proved to be located in such regions. The consequence of such aberrations is altered expression of these small noncoding regulatory genes that influence the levels of various protein coding genes, which could play key roles in survival, proliferation and differentiation programs in sorts of cells. HBV-DERIVED EPIGENETIC ALTERATION RELATED TO HCC TUMORIGENESIS HBV infection is one of the many factors having been linked to the development of HCC. The HBV genome is a partially doublestranded relaxed circular DNA molecule about 3200 nt in length. After attachment to the hepatocyte, the HBV genome moves to the nucleus and assumes a highly stable conformation, called covalently closed circular DNA (cccDNA). Since virus genome may cause disruption of the host genome by insertion mutations and chromosomal rearrangements, cells infected by HBV can be predisposed into cancer [72]. A number of cellular genes could be down-

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regulated or upregulated by HBx, as indicated by oligonucleotide microarray analysis [73,74]. However, these dysregulations were difficult to be elucidated by the known genetic regulators. Several studies have reported a strong correlation between HBV infection and epigenetic alteration of tumor suppressor genes, expression of which is frequently downregulated in HCC by hypermethylation. Aberrant epigenetic alterations in the host cellular genome have been proposed as possible mechanisms resulting in the development and progression of HCC associated with HBV infection [75]. HBV-MEDIATED ABERRANT DNA METHYLATION DNA methylation occurs in the early stage of cancer development, including HCC. Recently, methylation has been identified as a novel host defense mechanism, and methylation of viral DNA leads to downregulation of HBV gene expression. In HBV infection-associated HCC, HBV encoded protein X (HBx) was found to upregulate expression of the DNA-methyltransferase (DNMT) genes through its transcriptional transactivation property [76], HBx may also directly interact with DNMT’s, directing their recruitment at specific genes and thus affecting their methylation and expression. Thus, the corresponding down-regulation of several important cellular genes has been attributed to the DNMT-mediated methylation of the target genes [77]. A detailed analysis of DNA methylation in the HBV genome in liver samples of patients at different stages of hepatocarcinoma development and in in vitro infected hepatocytes was executed to found discrete CpG sites in the HBV genome that are recurrently hypermethylated in cancer but not in chronic hepatitis tissue [78]. To date, several aberrant DNA methylations were reported which would take charge to HCC tumorigenesis. p16INK4A Tumor suppressor gene p16INK4A is one of the key cell cycle regulators in Rb pathway, a tumor suppressor signaling [79]. Loss of p16INK4A was seen with tumor progression whereas aberrant p53 expression was frequent in undifferentiated tumor cells [80]. In HCC, deficiency of the p16INK4A/CDK4/Rb pathway is a frequent molecular event, and transferring the p16INK4A gene into cancer cells can induce cell cycle arrest and apoptosis, suggesting that p16INK4A is a good target for cancer gene therapy [81]. Several studies have shown that p16INK4A is frequently inactivated (~60%) in HBVrelated HCC tumor specimens due to extensive CpG methylation within the promoter region. A quantitative analysis of the methylation status of CpG island loci on 133 DNA samples from histologically confirmed cirrhotic nodules, low-grade dysplastic nodules (LGDN), high-grade dysplastic nodules (HGDN), and the recently introduced early hepatocellular carcinoma (eHCC), and progressed HCCs (pHCC) in HBV-infected livers using real-time PCR-based MethyLight method had demonstrated a remarkable stepwise increase in p16INK4A methylation levels. This gene was almost exclusively methylated in neoplastic lesions. Moreover, the data have shown that methylation of p16INK4A appeared in LGDN and significantly increased in HGDN, eHCCs, and pHCCs whereas absent in normal or cirrhotic livers [82], indicating that p16INK4A methylation is an early event in hepatocarcinogenesis, appearing from the level of LGDN. Another experiment investigated the p16INK4A methylation status in five cell lines with or without HBVinfection to clarify whether the high frequency of hypermethylation is related toHBV infection. The data showed that HBV-infected cell lines were exclusively methylation-positive, suggesting that p16INK4A methylation is associated with HBV infection and is an early event in hepatocarcinogenesis [83]. It was reported that replication and/or integration of HBV may contribute to high rate of p16 INK4A methylation in hepatocarcinogenesis, indicating that persistent HBV infection may be associated with high rate of p16INK4A methylation, and involved in the development of HCC [84]. Additional investigation has shown that the expression of hepatitis B virus X protein (HBx) was higher in methylated groups of p16INK4A than in unmethylated groups, while hepatitis B surface antigen and hepatitis B core antigen, tissue HBV-DNA levels and HBV x gene mutations had no


relation to the methylation status of p16INK4A, suggesting that HBx may play an important role in the early stage of HBV-associated hepatocarcinogenesis via induction of hypermethylation of p16INK4A [85]. It was reported that DNMT1 and DNMT3A may play important roles in the process of HBx inducing hypermethylation of the p16INK4A in the early stages of HBV-associated HCC [86]. RASSF1A The Ras-Association Domain Family (RASSF) comprises ten members, termed RASSF1 to RASSF10 [87], RASSF1A is now described as a cell cycle-related tumor suppressor protein, which inhibits cyclin D1 and hence induces G1 phase arrest, though little is known about the underlying mechanisms. Since methylation of the RASSF1A promoter is described as an early and frequent event in tumorigenesis, RASSF1A could serve as a useful diagnostic marker in cancer screens [88]. Compared with common HCC arising in non-cirrhotic liver, frequent aberrant hypermethylation was found for the RASSF1A gene in fibrolamellar carcinoma [89]. Since RASSF1A promoter hypermethylation occurs frequently in HCC, researchers suggested that RASSF1A promoter hypermethylation may serve as a good prognostic factor [90]. RASSF1A methylation was reported to occur in more than 50% of HBV-infected livers, either neoplastic or non-neoplastic, and the methylation level is also considerably increased early in hepatocarcinogenesis. Probably because RASSF1A methylation might permit damaged hepatocytes to proceed further into the cell cycle by escaping G1 phase arrest [82]. Besides, a large-scale analysis of the genetic and epigenetic alterations in hepatocellular carcinoma demonstrated that frequency of RASSF1A hypermethylation was much higher than that in the p16INK4A genes in both HBV-positive HCC and neighboring tissues, indicating that deregulation of RASSF1A may precede the p16INK4A gene [91]. CDH1CDH1 gene encoded a protein named Cadherin-1 also known as epithelial cadherin (E-cadherin). Accumulating evidence shows that epithelial-mesenchymal transition (EMT) is a crucial event that mediates HCC invasion and metastasis which is often associated with poor clinical outcome [92,93]. Loss of E-cadherin expression is a hallmark of EMT which is associated with acquisition of metastatic capacity [94]. Numerous studies suggest that hypermethylation of CpG islands in E-cadherin gene (CDH1) is a major mechanism responsible for E-cadherin silencing in different tumors and cancer cell lines [95]. It was reported that the frequency of CDH1 promoter hypermethylation remained high in both nontumorous tissues and HCCs from HBsAg-positive patients, in contrast, it was lower in HCCs than in nontumorous tissues from HBsAg-negative patients, indicating that HBV infection may enhance or maintain GSTP1 and E-Cad promoter methylation and thereby affect hepatocarcinogenesis [96]. In another investment, the relationship between HBx, E-cadherin, was evaluated, decreased levels of E-cadherin, aswell as the hypermethylation of CDH1, were detected in HBx expressing HCC cells suggesting that HBx may downregulate E-cadherin expression by hypermethylation of CDH1 by HBx, may be important for the understanding of HBVrelated carcinogenesis [97]. GSTP1 The GSTP1 gene encodes a member of a family of enzymes called glutathione-S-transferase-pi (GST-), which could protect cells from oxidation damage and electrophilic carcinogens. GSTP1 has been suggested to play an important role in protecting cells against damage induced by carcinogens. This protection was believed to be via regulation of the conjugation of a wide range of xenobiotics for excretion of hydrophilic metabolites [98]. Significantly increased expression of GSTP1 was demonstrated in early hepatocarcinogenesis [99] and HCC specimens compared to their adjacent normal tissues or liver cirrhosis tissues [100,101]. GSTP1 CpG island hypermethylation changes have been detected in DNA from candidate prostate cancer precursor lesions, proliferative inflammatory atrophy. and prostatic intraepithelial neoplasia but not in normal prostate tissues or benign prostatic hyperplasia tissues. Moreover, GSTP1 CpG island hypermethylation has also been de-

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tected in urine, ejaculate, and plasma from men with prostate cancer [102]. In HCC aspect, GSTP1 is also commonly hypermethylated in hepatitis B virus (HBV)-associated with HCC tissues [103]. Since liver is the major organ for metabolism and detoxification, loss of GSTP1 in liver tissues may thus facilitate hepatocyte transformation by accumulation of tumor inducers or dysregulation of cell proliferation. Others Inactivation of p21WAF1/CIP1 cell cycle checkpoints has been reported to play roles in the multistep sequence, and there is increasing evidence for the role of aberrant DNA hypermethylation in the process. Previous investment reported that HBx suppressed ATRA-mediated induction of p16 INK4A and p21 WAF1/CIP1 in HepG2 cells via promoter hypermethylation, resulting in inactivation of this tumor suppressior protein, suggesting that HBx executes its potential by downregulating levels of p16 INK4A and p21 WAF1/CIP1 via DNA methylation. As cellular senescence is a tumor -suppression process, the present study provides a new strategy by which HBV promotes hepatocarcinogenesis [104]. The ankyrin-repeat– containing, SH3-domain–containing, and proline-rich-region– containing protein (ASPP) family of proteins regulates apoptosis through interaction with p53 and its family members. Report showed that ASPP1 and ASPP2 genes are frequently down-regulated by DNA methylation in HBV-positive HCC, which may play important roles in the development of HCC [105]. Moreover, data showed that HBx can interact directly with DNA methyltransferase3A (DNMT3A) and histone deacetylase-1 (HDAC1). Recruitment of DNMT3A enables HBx to the regulatory promoters of interleukin-4 receptor and metallothionein-1F and subsequently silenced their transcription via DNA methylation. The interaction of HBx and DNMT3A facilitates cellular epigenetic modification, providing an alternative mechanism within HBx-mediated transcriptional regulation [106]. Cyclooxygenase-2 (COX-2) is an inducible COX isoform required for the biosynthesis of prostaglandins and is involved in pathological processes such as inflammation, fibrogenesis, and carcinogenesis [107]. It was reported that HBx could mediate demethylation and recruitment of transcription factors binding to the promoter of COX-2 and eventually cause an upregulation of COX-2, which could enhance the inflammation and eventually develop HCC [108], DNMT3B was believed to take primary responsibility of demethylation event. HBV-MEDIATED ABERRANT EPIGENETIC ALTERATIONS OTHER THAN DNA METHYLATION Cancer cells maintain their telomere length mainly through activation of telomerase, an enzyme that synthesizes telomeric repeat DNA [109, 110]. Human telomerase reverse transcriptase (hTERT), is the key determinant of the enzymatic activity of human telomerase [111, 112]. hTERT mRNA was reported to increase with the progression of hepatocarcinogenesis [113], further experiment found that the HBV genome is integrated in the hTERT promoter region in hepatocellular carcinoma (HCC) cell line, in which the hTERT mRNA is transcribed from both the endogenous promoter and the HBV promoter [114]. Other data demonstrate that HBX upregulates the expression and activity of hTERT in hepatoma cells, suggesting that hTERT is associated with tumor development [115]. These findings showed that a structural alteration in the telomerase component genes resulted in the activation of telomerase in human cancer. Moreover, a research reported one case with the HBV genome integrated upstream of the hTERT gene [116]. Another group found that a primary HCC tumor and HCC cell lines had the HBV genome integrated in the hTERT gene locus, none of the integrations altered the hTERT coding sequence and all resulted in juxtaposition of viral enhancers near hTERT, with potential activation of hTERT expression. This work supports the hypothesis that the hTERT gene is a non-random integration site of, and a target for cisactivation by, the viral genome in a subset of HBV-positive HCCs [117]. IGFBP-3 is a member of the IGFBP family, which regulates insulin-like growth factor-1 (IGF-1) activity by binding to and se-



questering IGF-1 away from its receptor in the extracellular milieu [118, 119]. IGF-1 binding to its cellular receptor activatesmitogenic and anti-apoptotic processes in cells, therefore, IGFBP-3 is a multifunctional protein that has both IGF-dependent as well as IGFindependent effects on cell proliferation and survival. It was reported that de-differentiated hepatocyte cell lines exhibited selective HBx-induced expression of proapoptotic genes IGFBP-3 [120]. In contrast, other research showed that we verified that HBx transcriptionally represses IGFBP-3 by promoting HBx/histone deacetylase 1 complex formation, suggesting that HBx may induce IGFBP-3 repression and the modification of chromatin structure [121]. To date, HBV-derived histone modification is still rare compared to the methylational alteration. Interestingly, however, histone modification on HBV genome integrated to host was reported widely. Covalently closed circular DNA (cccDNA) is the main replicative intermediate of HBV cccDNA of HBV plays an important role in regulating HBV replication. It was reported that phosphorylation and methylation occur in the remodeling of HBV minichromosomes during HBV replication. The modifications of cccDNA-bound H3 histones were associated with the level of HBV replication, suggesting that alterations in the extent of minichromosome remodeling might be a potential target to inhibit HBV replication in the development of effective novel antiviral agents [122]. HBV-derived noncoding RNA dysregulation was reported abundantly in HCC, especially on microRNA-induced RNA interference [123,124]. To date, miR-152 [125], miR-602 [126] and miR-143 [127] were regarded as key responsors of HBV which could regulate important cellular genes including DNMT1, RASSF1A and FNDC3B affecting pathways related to cell death, DNA damage, recombination, and signal transduction, eventually induced oncogenesis. Moreover, long noncoding RNA high expression in HBV-related HCC is significantly associated with recurrence and is an independent prognostic factor for survival, which was associated with enhancer of zeste homolog 2 (EZH2) and that this association was required for the repression of EZH2 target Table 1.

genes [128]. These findings expand the knowledge on epigenetic mechanism of HBV-related oncogenesis in HCC. HCV-DERIVED EPIGENETIC ALTERATION RELATED TO HCC TUMORIGENESIS HCV infection is the second leading cause of chronic hepatitis, liver cirrhosis and HCC worldwide. HCV is a positive-stranded, enveloped, RNA virus belonging to the flaviviridae family, it is the only member of the genus hepacivirus [129]. There is suggestive experimental evidence that HCV infection itself can promote the development of HCC [130]. Different HCV proteins, especially core [131], NS3 [132] and NS5A [133, 134], have been reported to be involved in the process of hepatocarcinogenesis. HCV core protein has been proposed to be involved in apoptosis, signal transduction, reactive oxygen species (ROS) formation, lipid metabolism, transcriptional activation, transformation and immune modulation.DNA methyltransferases (DNMTs) play a key role in DNA methylation, it was confirmed that HCV core protein could upregulate both mRNA and protein expression levels of DNMT1 and DNMT3b, which may probably affect epigenetic alteration in HCVinfected hepatocytes [135]. To date, sorts of epigenetic alteration related to HCC tumorigenesis induced by HCV infection were uncovered. Interestingly, other than HBV, HCV caused epigenetic alteration mainly occurred on DNA repair-related genes (Table 1). HCV-MEDIATED ABERRANT DNA METHYLATION In fact, the difference between HCV and HBV etiologies was emphasized in two recent methylation studies. In the first, researchers examined methylation of 19 epigenetic markers in 77 paired HCC and matching noncancerous liver tissues along with 22 normal liver tissues [136]. The authors found that 7/19 epigenetic markers (COX2, MINT1, CACNA1G, RASSF2, MINT2, Reprimo, and DCC) were hypermethylated in HCV tissues in comparison to both HBV and normal liver tissues. The authors concluded that HCV infection may accelerate the methylation process. In another research, investigators examined 24 different gene promoter regions using MSP in

HBV/HCV-induced gene alterations in epigenetic manner.

Infected Virus

Reported gene

Epigenetic event

Contribution in HCC development

Reference

HBV

p16INK4A

DNA hypermethylation

Cell cycle dysregulation

[79-86]

GSTP1


Loss the protection from oxidation damage and electrophilic carcinogens

[87-92]

CDH1


Cell adhension and metastasis

[93-98]

RASSF1A



[82,99-103]



[104-106]

hTERT

Cis-activation

Dysregulation of telomerase enzymatic activity

[112,115]

COX-2

DNA demethylation

Inflammation

[122]

SOCS-1


Loss suppression of JAK/STAT protumor signaling

[134-140]

Gadd45


Response to genotoxic stress

[141-145]

MGMT


Dysfunction of DNA repair

[92,146-148]

APC


Cell cycle dysregulation/Dysfunction of DNA repair

[155-157]

STAT1

DNA hypomethylation

Upregulation of JAK/STAT protumor signaling

[149-154]

PP2Ac

Histone modification

Inflammation

[161]

P21

HCV

WAF1/CIP1

5


conjunction with DNA sequencing in 28 HBV/HCC tissues and corresponding non-tumorous tissues and found that 15 of the 24 were more frequently methylated among HCC versus HCV tissues. These evidences suggested that monitoring important epigenetic markers may be of clinical utility but that different markers would be needed for HCV and HBV. Despite the information mentioned above, several genes were conformed to be selectively modulated by HCV infection. SOCS-1 Suppressor of cytokine signaling (SOCS) 1 is a negative regulator of the JAK/STAT pathway17 and is therefore an attractive candidate. Expression of SOCS1 leads to reduced JAK /STAT phosphorylation, reduced STAT dimerization and imports to the nucleus and reduced transcription of target genes [137]. Since JAK/STAT pathway was regarded as a protumor signaling [138], SOCS-1 could be treated as a tumor suppressor gene [139]. Aberrant methylation of SOCS-1 promoter sequence has been reported in several kinds of human cancer. SOCS-1 CpG island hypermethylation is an early event in human carcinogenesis [140]. It was reported that methylation of the SOCS-1 gene was detected in HCVinduced chronic hepatitis and liver cirrhosis, whereas the methylation frequency increased with fibrosis stage with the highest proportion in liver cirrhosis [141]. In HCC, methylation of SOCS-1 was more frequently seen in hepatitis C virus-positive HCC than hepatitis C virus/hepatitis B virus-negative HCC [142]. In another research, SOCS-1 methylation was found in 163 (57%) of 284 HCCs. and positively associated with HCV infection status, occurred at a 4.34 times higher prevalence in HCV-positive cases than in HCVnegative cases. Interestingly, the very data suggested that methylation of SOCS-1 was inversely associated with HBV infection [143]. Gadd45 Growth arrest and DNA damage-inducible gene 45 (Gadd45) which is a Member of the Gadd45 family plays central roles in the cellular response to genotoxic stress and has been implicated in several human cancers, including hepatocellular carcinomas [144, 145]. Gadd45 expression was significantly decreased in tumors compared with normal tissue and its association with histological grading suggests a critical role for Gadd45 in cancer [146]. Hypermethylation of Gadd45 was reported in other cancer, suggesting GADD45 is a novel tumor suppressor expression of which blocks proliferation, survival, and tumorigenesis [147]. Experiment had confirmed that HCV could down regulate Gadd45 expression and eventually lead to defective cell cycle arrest which could cause HCC tumorigenesis, indicating that HCV proteins have a direct effect on liver tumorigenesis by promoting the hypermethylation of the Gadd45 promoter in the absence of chronic inflammation, affecting DNA repair and cell cycle regulation [148]. MGMTO6-methylguanine DNA methyltransferase (MGMT) is an important DNA repair gene with the highest activity in the liver [149]. Several chemotherapeutic drugs such as N-nitroso compounds could alkylate DNA. Among several alkylation lesions introduced in the DNA, the most mutagenic is the alkylation at the O6-position of the DNA base guanine. The contribution of MGMT, which repairs these adducts, to cancer prevention has been discussed recently [150, 151]. It was also reportedthat consistent DNA hypomethylation of MGMT, the gene encoding an enzyme suggesting that the loss of methyl-cytosine at the MGMT gene promoter may be an early and transient biomarker of hepatocarcinogenesis. It is perhaps surprising to find a consistent loss of methyl cytosine in the MGMT promoter in both cirrhotic and HCC tissues. No association between methylation of any gene and HCV RNA and HBSAg status was found, with the exception of MGMT, which exhibited a significantly higher level of methylation in the HCV RNA negative group than in the HCV RNA positive group [103]. STAT1 The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway was identified as one of the principal signaling cascades mediating cytokine receptor-derived signals in mammals [152]. Originally implicated in the regulation of survival, proliferation and differentiation of hematopoietic cells, the

Rongrui et al.

JAK-STAT pathway has later on also been linked to developmental processes, growth control and maintenance of homeostasis in a variety of other cells and tissues [153, 154]. Among the members of the STAT family, accumulating evidence now indicates an important role for STAT1 in various forms of cell death. STAT1dependent regulation of cell death is largely dependent on a transcriptional mechanism such as the activation of death-promoting genes. The importance of STAT1 in IFN- and IFN-/ signaling was clearly established by studies using the mutant cell lines [155] and the generation of STAT1-deficient mice [156]. It was reported that HCV interferes with interferon- signaling via hypomethylation of STAT1, and increased STAT1-PIAS1 association, resulting in reduced transcriptional activation of interferon--stimulated genes, indicating that hypomethylation of STAT1 could be an important epigenetic mechanism in HCV-derived HCC [157]. Others The adenomatous polyposis coli (APC) tumor suppressor gene encodes a large protein with multiple cellular functions and interactions, including signal transduction in the Wnt-signaling pathway [158]. Preliminary reports suggest that the APC promoter is methylated in up to 81% of patients with viral hepatitis-induced HCC [159]. Moreover, a high-throughput assessment of CpG site methylation for distinguishing between HCV-cirrhosis and HCVassociated hepatocellular carcinoma was executed. The data showed that when comparing paired HCC tissues to their corresponding pre-neoplastic nontumorous tissues, APC was significantly hypermethylated [160]. In the very assessment, in contrast, NOTCH4, EMR3, HDAC9, DCL1, HLA-DOA, HLA-DPA1, and ERN1 were found to be hypomethylated in HCC. HCV-MEDIATED ABERRANT EPIGENETIC ALTERATIONS OTHER THAN DNA METHYLATION It was suggested that the induction of HCV proteins or the infection of HCC cells with HCVcc resulted in an inhibition of histone H4 methylation/acetylation and histone H2AX phosphorylation, in a significantly changed expression of genes important for hepatocarcinogenesis, and inhibited DNA damage repair, indicating that HCV-induced overexpression of PP2Ac contributes to hepatocarcinogenesis through dysregulation of epigenetic histone modifications [161]. Another research found that HCV could increase histone deacetylation activity through affecting hepcidin expression, which was a key negative regulator of iron availability [162], highlighting an HCV-induced oxidative stress which suppresses hepcidin expression through increased histone deacetylase activity. CONCLUSIONS AND FUTURE DIRECTIONS Although HBV and HCV are the major risk factors leading to the development of HCC, the precise pathogenetic mechanisms linking viral infection and HCC remain uncertain. Aberrant epigenetic events play an important role in the onset and progression of hepatocellular carcinoma, and have been associated with all HCC subtypes. At the cellular level, aberrant epigenetic events influence critical cellular events, which are further modulated by risk factor exposures and thus define the severity/ subtype of HCC. Methylation of DNA has been shown to be the most consistent molecular change in many neoplasms. Current techniques are focused on identifying the presence of disease, and to a much lesser extent, on early detection of malignancies [163, 164]. Thus, an epigenetic alteration might function as a marker for HBV/HCVinfected patients [165]. In fact, many of the epigenetic events described in this review have been detected in premalignant tissue, as well as in cancers. In addition, several of the frequently methylated genes can be detected in the serum of patients prior to or concurrent with the diagnosis of cancer. Although negative evidence was given that plasma samples might have limited usage for HCC diagnosis [166], the potential advantage of diagnosis is tempting. In fact, epigenetic machineries are tightly associated with each other and exert their functions in a hierarchical fashion. It was be-


lieved that HBV/HCV infection-induced epigenetic alteration may be a ligase reaction. DNMTs were believed to be the original procedure in many epigenetic events. Although DNMTs were reported to be deregulated in sorts of cancer included HCC [167, 168], rare evidence shows a direct interaction between HBV and DNMTs. Briefly, RASSF1 was thought to be the initial mediator degerulated in HBV infection. p16INK4A, p21 WAF1/CIP1 may induce a cell cycle dysregulation subsequently to RASSF1 [91]. The other genes such as GSTP1, CHD1, were thought to be more subsequential events. The exact procedure of HCV-induced epigenetic events was more obscure since HCV itself could upregulate both mRNA and protein expression levels of DNMT1 and DNMT3b [135]. DNMTdependent fashion could be thought of the primary manner in HCVinduced epigenetic alterations. Still, more investigations are required to elucidate the exact mechanism. The current benefit of epigenetic studies that are involved in HCC malignancies is that we have gained a better understanding of the processes that drive liver carcinogenesis. It was known that HBV and HCV are the major risk factors leading to the development of HCC. Uncovering the epigenetic alterations of HBV/HCVinduced HCC carcinogenesis could highlight a new strategy for deciphering the mechanism of HCC tumorigenesis and development. CONFLICT OF INTEREST The authors have no conflict of interest to declare. ACKNOWLEDGEMENTS This work was supported by program for changjiang Scholars and Innovative Research Team in University (PCSIRT:1171). ABBREVIATIONS APC = Adenomatous polyposis coli ASPP = Ankyrin-repeat–containing, SH3-domain– containing, and proline-rich-region–containing protein cccDNA = Covalently closed circular DNA DMR = Differentially methylated region DNMT = DNA-methyltransferase eHCC = Early hepatocellular carcinoma EMT = Epithelial-mesenchymal transition EZH = Enhancer of zeste homolog Gadd = Growth arrest and DNA damage-inducible gene GST- = Glutathione-S-transferase-pi HBV = Hepatitis B virus HBx = Hepatitis B virus X protein HCC = Hepatocellular carcinoma HCV = Hepatitis C virus HDAC = Histone deacetylases HGDN = High-grade dysplastic nodules hTERT = Human telomerase reverse transcriptase ICR = Imprinting control region IGF = Insulin-like growth factor JAK = Janus kinase LGDN = Low-grade dysplastic nodules LOI = Loss of imprinting MGMT = O6-methylguanine DNA methyltransferase miRNAs = Micrornas ncRNAs = Noncoding rnas pHCC = Progressed hccs


RASSF ROS SAM SNF SOCS STAT

= = = = = =

7

Ras-association domain family Reactive oxygen species S-adenosylmethionine Sucrose non-fermenting Suppressor of cytokine signaling Signal transducer and activator of transcription

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Received: April 20, 2013

Accepted: July 18, 2013

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