Table of Contents Author Guidelines Submit a Manuscript
BioMed Research International
Volume 2016, Article ID 3956485, 13 pages
http://dx.doi.org/10.1155/2016/3956485
Review Article

Hepatoepigenetic Alterations in Viral and Nonviral-Induced Hepatocellular Carcinoma

1Division of Hepatology and Liver Research, Department of Medicine, Faculty of Health Sciences, University of Cape Town, Groote Schuur Hospital, Observatory 7925, Western Cape, South Africa
2MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK

Received 2 October 2016; Accepted 30 November 2016

Academic Editor: Jeroen T. Buijs

Copyright © 2016 Mankgopo M. Kgatle et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Hepatocellular carcinoma (HCC) is a major public health concern and one of the leading causes of tumour-related deaths worldwide. Extensive evidence endorses that HCC is a multifactorial disease characterised by hepatic cirrhosis mostly associated with chronic inflammation and hepatitis B/C viral infections. Interaction of viral products with the host cell machinery may lead to increased frequency of genetic and epigenetic aberrations that cause harmful alterations in gene transcription. This may provide a progressive selective advantage for neoplastic transformation of hepatocytes associated with phenotypic heterogeneity of intratumour HCC cells, thus posing even more challenges in HCC treatment development. Epigenetic aberrations involving DNA methylation, histone modifications, and noncoding miRNA dysregulation have been shown to be intimately linked with and play a critical role in tumour initiation, progression, and metastases. The current review focuses on the aberrant hepatoepigenetics events that play important roles in hepatocarcinogenesis and their utilities in the development of HCC therapy.

1. Introduction

HCC is the most common primary malignancy of hepatocytes that make up 70–80% of the liver mass, and it develops as a result of advanced hepatic disease and cirrhosis [1]. HCC is the third leading cause of cancer-related deaths worldwide, accounting for about 1 million deaths annually [2]. More than 80% of HCC cases occur in people who reside in sub-Saharan Africa, South East Asia, and eastern Mediterranean. HCC primarily occurs due to hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. Nonalcoholic steatohepatitis, aflatoxin exposure, haemochromatosis, obesity, severe alcohol intake, diabetes, and other metabolic factors are additional risk factors that can predispose to liver cancer. Patients with HCC generally present at an advanced stage due to compensated cirrhosis defined by the absence of pathognomonic symptoms, resulting in death within 6 to 20 months, suggesting an urgent need in treatment modalities that will dramatically decrease the mortality rate of HCC [24].

Liver diseases are characterised by chronic hepatic inflammation and damage, which appear to be important risk factors for hepatocarcinogenesis. Substantial evidence shows that alteration in the expression of NF-kB-induced proinflammatory cytokine TNF-α and interleukins, oncogenes, and tumour suppressor genes typically follows chronic hepatic inflammation associated with epigenetic aberrations [5]. Hepatocarcinogenesis has been described as CpG island methylator phenotype-positive (CIMP) multistep processes associated with the hallmark of successive accumulation of aberrant genetic and epigenetic alterations which co-operate to drive the malignant phenotype [6]. Deeper understanding of epigenetic aberrations, their interconnectivity, and clinical phenotypes in HCC patients may provide useful insights in the development of novel and more effective biomarkers for HCC treatment and better prognosis. In this review, we will highlight some of the hepatoepigenetic events that occur in response to nonviral and viral aetiologies, but mainly focusing on HBV and HCV infections.

2. Hepatoepigenetics

Hepatoepigenetics refers to activation or silencing in the expression of hepatic genes through chemical markers on DNA that do not involve mutations of the underlying sequence [710]. DNA methylation, histone modification, and noncoding miRNA are important epigenetic phenomena that collaboratively regulate gene expression and alter the normal function especially during pathological processes [710]. DNA methylation encompasses the attachment of a methyl group to the cytosine, guanine, or amino acids of histones wrapped with DNA, often leading to either normal or aberrant modification in gene function. DNA methylation often targets the CpG island promoter regions, which are a small (0.5–2 Kb) stretch of DNA with considerable quantity of CpG-rich regions as compared to the rest of the sequence [11, 12].

Addition or maintenance of methyl groups on the nucleotide sequence is usually catalysed by various DNA methyltransferases (DNMT) including DNMT1, DNMT3A, and DNMT3B. Aberrant DNA methylation is a common and well-described phenotype in HCC, and it can be defined as hypo- or hypermethylation depending on the targeted gene and alteration status [13]. Cancer-related hypermethylation denotes increased methylation in the CpG islands that is normally devoid methylation in normal cell and often results in the suppression of tumour suppressor genes [8, 14]. In contrast, hypomethylation signifies loss of DNA methylation and leads to activation of oncogenes in cancerous cells [7, 8]. Methyl groups can also be removed in the process known as DNA demethylation, a key regulator in tumour progression. Active DNA demethylation is governed by a group of ten-eleven translocation (Tet1, Tet2, and Tet3) enzymes that utilise oxygen to decarboxylate α-ketoglutaric acid and iron leading to oxidation of 5-hydroxymethylcytosine (5hmC) from 5-methylcytosine (5mC) [15, 16].

Histone modifications involving acetylation and methylation also contribute to hepatocarcinogenesis. Histone acetylation involves addition of an acetyl group from acetyl coenzyme via histone acetyltransferase [17]. Histone methylation uses histone methyltransferases (HMTs) to transfer 1 to 3 methyl groups from S-adenosyl-L-methionine to arginine (R) or lysine (K) of the histone proteins that package and order DNA into structural units called nucleosomes [1822]. Histone methylation regulates gene transcription and is implicated in carcinogenesis. Several histone variants associated with either activation or silencing in gene transcription are well characterised, including histone (H) 2A, H2B, H3K4, H3K9, H3K27, H3K36, H3K79, H4K5, H4K8, H4K12, H4K16, and H4K20. Methylation of H3K4, H3K36, and H3K79 is associated with transcriptional activation whereas methylation of H3K9 and H3K27 leads to transcriptional repression. Histone methylation can be either mono-, di-, or trimethylated [23, 24]. Altered histone methylation or acetylation targeting H3K4, H3K9, and H3K27 has been well-described in HCC and will be unpacked later in this review. Methylation of H3K4 is activated by SET-1 family enzymes SET1A, SET1B, MILL1, MILL2, MILL3, and MILL4. Set domain bifurcated 1 (SETDB1) regulates epigenetic repression of euchromatic genes via H3K9me3 by recruiting heterochromatin protein 1- (HP1-) related proteins to methylated histones [25, 26].

H3K27 methylation is catalysed by one of the two classes of polycomb-group proteins (PcGs) [27]. PRC2 represses the transcriptional activities of genes involved in cell cycle regulation, differentiation, and proliferation [27]. It is activated by its subunit, a famous enzyme enhancer of zester homolog-2 (EZH2), which requires its binding partners, suppressor of zeste 12 protein homolog (SUZ12), and embryonic ectoderm development (EED) for proper function [28]. EZH2 has been described as a useful marker for aggressive stages and its upregulation leads to HCC malignant progression signifying poor prognosis [29, 30]. PRC1, another PcG, is an E3 ubiquitin ligase that transfers monoubiquitin to the C-terminal tail of H2A at K118/119 [31, 32]. It is catalysed by RING class heterodimer, chromobox homolog 8 (CBX8), B-Lymphoma moloney murine leukemia virus insertion regional-1 (BMI1), and MEL18 paired with RING1A/B [30, 33]. Lysine specific demethylase 1 (LSD1) and Jumonji domain containing proteins (JMJD1A, JMJD2, JMJD3/UTX, and JARID1B) are two major histone demethylases (HDMTs) that have been identified to erase methylation from histone proteins in HCC [34].

Noncoding miRNA (miR) is another epigenetic mechanism that primarily regulates gene transcription at the posttranscriptional level and also contributes to hepatocarcinogenesis [35, 36]. miRs are a large class of 22 nucleotide long small RNAs processed from long endogenous transcripts that usually form local hairpin structure through Watson-Crick pairing (e.g., A-U and G-C) [37, 38]. Three key steps required for miR biogenesis [38] include nuclear processing of primary miR by DROSHA, nuclear export of precursor miR by Export in 5, and finally cytoplasmic processing of pre-miR by DICER [38, 39]. miRs do not encode for proteins, however, they can still alter gene transcription by base-pairing with complementary miR response element sequences located at the 3′-untranslated region of their target genes. miR-mediated gene regulation plays an important role in a variety of cellular processes such as cell differentiation and organ development, cell cycle progression, growth, proliferation, and apoptosis [40]. In HCC patients, miRs have been shown to alter gene transcription leading to tumour inhibition, progression, and poor prognosis [41]. This suggests that miRs may serve as transcription factors, oncogenes, or tumour suppressor genes contributing to hepatocarcinogenesis.

3. Nonviral-Induced HCC Hepatoepigenetic Aberrations

Substantive meta-analysis studies show that HCC tumours exhibit consistent CpG island promoter hypermethylation of classical tumour suppressor genes such as ras association domain family 1A (RASSF1A), p16ink4α/cyclin dependent kinase inhibitor 2A (CDKN2A), p15, suppressor of the cytokine signalling 1 (SOCS1), E-cadherin (CHD1), and glutathione-S-transferase Pi 1 (GSTP1) [42, 43] (Figure 1). CpG island promoter hypermethylation-mediated inactivation of these tumour suppressor genes especially RASSF1A, E-cadherin, GSTP1, and SOCS1 genes is associated with either increased risk of HCC and more aggressive clinical phenotype with high risk of metastasis [43, 44]. Promoter hypermethylation of RASSF1A was demonstrated to be a valuable diagnostic marker that can be used to complement the alpha fetoprotein (AFP) in screening for HCC [45]. As an important regulator of epithelial-mesenchymal transition (EMT), E-cadherin impedes cell adhesion within hepatic tissue resulting in loss of cell polarity and acquisition of mesenchymal phenotype. This promotes tumour cell infiltration, an essential feature associated with migration and metastasis into the surrounding or distant tissues and organs [46]. For example, aberrant expression of Notch1, which is an important silencer of E-cadherin through activation of Snail1, was associated with tumour-node-metastasis stages III-IV, tumour venous invasion, and poor prognosis in HCC patients [47]. Hypermethylation of GSTP1 gene also allows intrahepatic metastases of HCC by enhancing the expression of β-catenin molecules that mediate epithelial cell adhesion [48]. SOCS1 stimulates hepatocyte regeneration by negatively regulating the Janus kinase-Signal transducer and activator of transcription (JAK-STAT) signalling pathway. Aberrant epigenetically silenced SOCS1 leads to uncontrolled cell differentiation and proliferation associated with HCC aggressiveness [49, 50]. Ras association (ralgds/af-6) domain family member 1 (RASSF1A), insulin-like growth factor 2 (IGF-2), and adenomatous polyposis coli (APC) have also recently been shown to be hypermethylated in HCC patients in which they act as potential candidate epidrivers that predict poor clinical outcome [51].

Figure 1: Epigenetic alterations in hepatocarcinogenesis. DNA methylation, histone modification, and noncoding miRs cooperate in altering gene transcription and hepatic architecture leading to perturbed cellular processes associated with tumour initiation and metastases. Alteration in gene transcription is governed by several enzymes, including DNA methyltransferases (DNMTs), group of ten-eleven translocation (Tet1, Tet2, and Tet3) enzymes, histone methyltransferases (HMTs), and histone demethylases (HDMTs).

Global DNA hypomethylation on pericentromeric satellite regions is a frequent and early event associated with chromosomal instability induced via heterochromatin decondensation and enhanced recombination enhancement in hepatocarcinogenesis [52]. DNA hypomethylation of chromosome 1 heterochromatin coincides with Q-arm copy gain in HCC. More than 90% of HCC tissues exhibit loss of heterozygosity (LOH) on different chromosomes in various tumour suppressor genes, and these genetic alterations further inactivate gene transcription to initiate tumour development [53, 54]. LOH located on chromosome 16 encoding axis inhibition protein 1 tumour suppressor gene was observed more frequently in poorly differentiated or metastatic tumours and therefore rather contributes to tumour aggressiveness than initiation [54, 55]. HCC-related DNA hypomethylation is commonly observed in promoter regions of transposable elements such as long interspersed nuclear element-1 (LINE-1) and monoacylglycerol acyltransferase-2 (MGAT3) [56]. Active demethylation mediated by Tet proteins was shown to play an important role in global DNA methylation and HCC. For instance, Tet 2 and Tet 3 proteins are abnormally regulated in HCC and result in the reduction of 5hmC, suggesting a new therapeutic modality for HCC [57].

Several histone posttranslational modifications also play an important role in altering the transcription of cellular genes including tumour suppressors that are commonly known to epigenetically promote malignant transformation [58, 59]. MacroH2A1 is a variant of histone H2A protein that correlates with suppressed gene transcription in the chromosomal region, and it has been found to be heavily upregulated in HCC [60]. The interaction of macroH2A1 and DNA hypermethylation was found to intercept HCC progression by attenuating chemotherapy-induced senescence in rodents and human livers [61]. This synergistic effect results in the suppression of a subset of tumour suppressor genes (CDKN2A, DLEC1, and RUNX2) and enhanced HCC cell growth in Hep G2 and Huh-7. Treatment with DNA hypomethylating agent guadecitabine reversed this effect leading to inhibition of HCC cell growth [62]. EZH2 is an important component of PRC2 that interacts with EED and SUZ12 to establish trimethylation of H3K27 (H3K27me3) leading to tumour initiation and progression [63, 64]. H3K27me3 is a well-established marker of transcriptionally silent chromatin implicated in hepatocarcinogenesis [59]. Gao and coauthors identified CDKN2A as a target of repression by H3K27me3 mediated by upregulation of EZH2 and SUZ12 enzymes in HCC human samples [64]. CDKN2A encodes proteins for p16ink4α and p14arf, two important tumour suppressor genes that prevent tumour formation by regulating cell growth, division, and apoptosis. The p1 protein attaches to cyclin dependent kinase- (CDK-) 4 or CDK-6 to inhibit cell cycle progression. The p1 protein protects p53 from degradation and promotes p21 activation leading to controlled cell division and enhanced apoptosis. Epigenetic repression of CDKN2A resulted in the obstruction of CDKN2A-TP53-P21 pathway leading to HCC initiation and aggressiveness. Importantly, reduced expression of PRC2 protein via H3K27me3 inhibitor restored CDKN2A-TP53-P21 pathway and effectively blocked the aggressive phenotype of HCC cells [63]. Epigenetic silencing by EZH2-mediated with H2K27me3 in HCC was also observed with other several tumour suppressor genes including deleted in lung cancer 1 (DLC1) and chromodomain helicase DNA binding proteins 5 (CHD5), and this correlates with metastasis and poor prognosis [58, 65]. Simultaneous activation of H3K27me3 and acetylation in association with aberrant expression of p53 gene was also reported in HCC with aggressive phenotype [66]. G9a, GLP, and suppressor of variegation 3-9 homolog 1 (SUV39H1) are another group of HMTs responsible for H3K9 methylation in association with p53. Increased expression of G9a-induced H3K9 methylation was also associated with poor prognosis in HCC patients [67].

Claudin 14 (CLDN14) was recently labelled a novel prognostic biomarker of HCC [68]. Integrative genome-wide analysis of H3K27me3 and gene expression profiling using chromatin immunoprecipitation with high-throughput sequencing and gene expression microarray showed that CLDN14 was another target for EZH2-mediated H3K27me3. Negative regulation of CLDN14 was associated with increased expression of EZH2 and H3K27me3. CLDN14 is a cell adhesion tight junction molecule that belongs to the claudin family proteins found in all epithelial and endothelial cells, and it plays an important role in selective paracellular permeability. Altered expression of CLDN14 was associated with activation of Wnt/β catenin signalling pathway leading to EMT of HCC cells. Low levels of CLDN14 expression consistently correlates with tumour aggressiveness and poor prognosis, suggesting that it is an effective prognostic marker and therapeutic target for HCC therapy. Silencing of CLDN14 protein expression with similar consequences was also observed in HCC tissues as a result of the CpG island promoter hypermethylation [69]. This suggested an intimate link of epigenetic mechanisms in synergistically dysregulating gene transcription [66].

LSD1 and JmjC domain containing H3K4 histone demethylase Jumonji AT-rich interactive domain 1B (JARID1B) are erasers for mono-, di-, and trimethylation of H3K4 (Figure 1). LSD1 is frequently overexpressed in HCC cells and promotes tumorigenesis by epigenetically dysregulating EMT and glycolytic and mitochondrial metabolism [70, 71]. JMJD1A is an important regulator of hypoxia-inducible transcription factor and also stimulates tumour growth. Upregulation of JMJD1 was significantly associated with HCC cell growth and recurrence, supporting the notion that histone demethylation plays an important role in hepatocarcinogenesis [34, 72]. JARID1B, also known as lysine demethylase 5B (KDM5B), acts as an oncogene and contributes to hepatocarcinogenesis by promoting cell migration and invasion. Most recently, it was demonstrated that knockdown of KDM5B blocks HCC cell proliferation and arrests cell cycle progression at G1/S-phase by upregulating p15 and p27 expression via H3K4 tri-methylation. Bone morphogenetic protein 7 (BMP7) is a secreted ligand for transforming growth factor-β that binds to and phosphorylates the SMAD family member proteins to activate gene transcription [7375]. BMP7 is also a KDM5B target protein, and its aberrant inactivation is associated with HCC invasiveness with phenotypic features of TGF-β-induced cell migration, invasion, and EMT [76]. KDM5B has been labelled a potential target for cancer treatment because it has also been shown to promote tumorigenesis by targeting and silencing tumour suppressors such as p21, breast cancer 1 (BRCA1), estrogen receptor (ER), forkhead box 1 (FOXA1), carbon tetrachloride (CCL4), and caveolin 1 in some human malignancies [77].

Regulation of noncoding RNAs by aberrant DNA methylation and histone modifications also provided some insights in the pathogenesis of HCC [78]. Bioinformatics studies revealed an array of several epigenetic-regulated miRs that emerged to be differentially pivotal in hepatocarcinogenesis. For instance, miR-101, miR-141, miR-133a, miR-145, miR-148a, miR-211, miR-377, and miR-431 reduce HCC progression by inducing apoptosis and suppressing cell proliferation, migration, and invasion. These miRs target various genes such as vascular endothelial growth factor C, hepatocyte nuclear factor-3β, fascin 1, sphingosine-1-phosphate receptor 1, secreted protein acidic and rich in cysteine, T-cell lymphoma invasion and metastases 1, and zinc finger E-box binding homeobox 1 [7984]. In particular, miR-145 was found to function as a tumour suppressor gene and inhibited HDAC2 oncogenic effects by restoring the expression of G1/S cell cycle proteins in HCC [85]. In contrast, miR-135a and miR-494 promote tumour growth and increase sorafenib treatment resistance by epigenetically regulating genes such as forkhead box O1 and phosphatase and tensin homolog (PTEN) [86, 87]. Additionally, upregulation of miR-210, miR-221, miR-224, and miR-519d was shown to promote hepatocarcinogenesis by targeting CDK1, CDKN1C/p57, CDKN1B/p27, CDKNA/p21, AKT serine/threonine kinase 3 (AKT3), TIMP metallopeptidase inhibitor 2 (TIMP2), and PTEN [8890]. Attenuated and noninfectious lentiviruses, adenoviruses, adenoassociated viruses, and herpes simplex viruses are commonly used as delivery vehicles for miR antagonists or mimics in various cancers [91]. However, there are limited data on miR-based therapeutics for HCC.

4. HBV-Induced HCC Hepatoepigenetic Alterations

Hepadnaviruses are the primary cause of hepatitis in both humans and animals. They are nonsegmented and very small genomes of 3.2 kb relaxed circular (rc) partially double-stranded DNA that, upon infection, are transported into the nucleus by nucleocapsid [92]. Inside the nucleus, covalently closed circular DNA (cccDNA) is converted from rcDNA and serves as a template for viral replication through RNA intermediates using reverse transcriptase. Orthohepadnavirus and Avihepadnavirus are two well-characterised hepadnavirus genera [93, 94].

During persistent infection, an Orthohepadnavirus HBV genome integrates in the host DNA leading to accumulation of profound genetic and epigenetic signatures that subsequently leads to HCC development [95]. Considerable data demonstrate that more than 90% of HCC cases exhibit integrated HBV genome within the host DNA [96]. HBV integration induces trans- and cis-activation in HBV and host genome, respectively. It has been shown that, following integration into the host DNA, HBV genome undergoes methylation induced as part of innate immune defense mechanisms to protect the host from increased viral replication [9597]. Vivekanandan and his coauthors demonstrated that long-term upregulation of DNMTs in the host may also become detrimental by methylating the surrounding hepatocyte CpG islands promoter regions. This may lead to activation of oncogenes or silencing in immunoregulatory and tumour suppressor genes that are critical in hepatocarcinogenesis [98]. Numerous studies showed that HBx gene, transcriptional activator, and oncogenic protein encoded by HBV manipulate DNMTs and induce promoter hypermethylation of wide array of cellular tumour suppressor genes. HBx-mediated methylation phenotype of these tumour suppressor genes was associated with tumour progression, aggressiveness and poor prognosis as a result of disrupted host cellular signalling pathways that regulate DNA repair, cell growth, proliferation, and apoptosis [99102].

Retinoic acid receptor β2 (RARβ2) is one of the isoforms encoded by RARβ, which is a nuclear receptor gene that was first identified in HCC where it flanks a HBV integration site. It binds to and inactivates the E2F1 transcription factor, which is essential for cell cycle progression [103105]. HBx protein was shown to induce the hypermethylation of RARβ2 promoter region by upregulating DNMT1 and DNMT3A activities leading to repression in the expression of RARβ2 protein [104, 106]. This cis-activation with RARβ2 gene was associated with activation of E2F1 transcription factor, which abolishes the ability of retinoic acid to regulate expression of G1 checkpoint regulators such as p16, p21, and p27 proteins. Under the influence of HBx transcriptional activities in the cytoplasm, RAR may also promote hepatocarcinogenesis by interacting with other proteins such as AFP. It was recently shown that interaction of HBx-induced AFP with RAR resulted in perturbed RAR signalling pathway, leading to repression of growth arrest and DNA damage 45α (GADD45α) protein expression [107]. GADD45α is an 18.4 kDa RNA-binding acidic protein that is expressed in response to DNA damage for repairing and induction of apoptosis by inhibiting G2/M transition of cell cycle. Downregulation of GADD45α in HCC was associated with uncontrolled HBV-infected hepatocytes growth and hepatocarcinogenesis, suggesting its effects in allowing the cells to evade senescence and apoptosis [104, 107]. Although the role of GADD45α in promoting instability through DNA demethylation has been reported previously, it has not been explored in HBV-induced HCC and therefore warrants investigation [108, 109]. Caveolin- 1, encoded by caveolin-1 gene, is an integral membrane protein abundantly expressed in adipose, fibrous, and endothelial tissue [110]. HCC cells expressing high levels of Caveolin-1 protein are associated with uncontrolled cell growth, motility, in vivo tumour aggressiveness, and metastasis [111]. Conversely, HBx-induced methylation of caveolin-1 gene promoter region suppresses its transcriptional activities leading to reduced tumour aggressiveness and metastasis, indicating a good prognostic marker of the disease [110].

Deleted in Lung and Esophageal Cancer 1 (DLEC1) is a functional tumour suppressor gene silenced by promoter hypermethylation in several human malignancies including HBV-induced HCC through activation of DNMT1 and HATs [112114]. Insulin-like growth factor binding 3 (IGJBP-3) is another potential tumour suppressor gene which was shown to be methylated and histone deacetylated in The Chang Liver, HuH-7, and HEP-G2 cells expressing HBx-construct [115]. Downregulation of IGJBP-3 gene expression following hypermethylation of CpG island within IGJBP-3 promoter regions was observed in HBV-induced HCC tissues, and the expression was restored by azacytidine-2′-deoxycytidine [116].

In mouse and cell line models, repression of p16ink4α gene via H3K27me3 was found to be an early aberrant epigenetic event associated with HCC initiation and progression [117]. Several studies showed similar effects through HBx-induced hypermethylation within the promoter region of p16ink4α gene via DNMT1/3A, and this was associated with advanced HBV infection [118120]. These data may suggest synergistically relationship between H3K27me3 and hypermethylation in promoting HBV-related hepatocarcinogenesis via alteration of p16ink4α gene. Cooperation in the activity of epigenetic mechanisms has also been observed with deleted in liver cancer 1 (DLC1) gene. EZH2 and other demethylating agents also play an important role in HBV-related hepatocarcinogenesis. Synchronous epigenetic regulation targeting classic tumour suppressor genes known to be altered in HBV-related HCC was also demonstrated with aberrant acetylation and trimethylation of H3K27 catalysed by CBX8, BMI1, EZH2, and SUZ12 enzymes [66]. It was recently shown that epithelial cell adhesion molecule (EpCAM) was upregulated via active DNA demethylation catalysed by EZH2 and Tet2 in conjunction with nuclear factor kappa B (NF-Kβ) and RelA [121]. EZH2 promotes HCC motility and metastasis by epigenetically silencing the expression of multiple tumour suppressor miRs including miR-99a, miR-101, miR-125b, miR-139-5p, and let-7c [122]. Downregulation of miR-99a in HCC is associated with suppressed tumour growth [123]. Downregulation of miR-125 promotes proliferation and migration of HCC by upregulating SUV39H1 [124]. MiR-101 is negatively regulated by c-Myc-EZH2 complex, and its epigenetic silencing is associated with HCC poorer prognosis [65, 125]. Serum miR-150 was found to be a promising novel noninvasive diagnostic and prognostic biomarker for HBV-related HCC [126]. SETDB1 is an oncogene that marks the transcriptional repression of euchromatic gene via H3K9me3. In the absence of HBx, SETDB1 represses HBV-cccDNA transcription by regulating chromatin organization via methylation of H3K9me3 [127]. SETB1 is habitually upregulated in HBV-related HCC through multiple complementary acting mechanisms that occur at certain chromosomal (gain of SETB1 copy number at chromosome 1q21), transcriptional (hyperactivation of SP1 transcription factor), and posttranscriptional levels (methylation of p53 and loss of miR-29). Anomalous regulation of SETB1 correlates with tumour growth, aggressiveness, and poorer prognosis in HBV-related HCC patients [128130].

5. Hepatoepigenetic Alterations Elicited by HCV-Induced HCC

HCC and liver failure are the life-threatening conditions associated with untreated chronic HCV infection. Development of HCV-induced HCC is a multistep process that involves chronic liver inflammation and repetitive-cycles of hepatic fibrosis, which may occur over years leading to hepatic failure or cirrhosis and/or malignant transformation [131]. The recent development of directly acting antiviral agents (DAAs) has been an important revolution and exciting progressive era in the field of HCV therapy. Excellent clinical outcomes from increased SVR rates to curing HCV were observed in more than 90% of HCV-patients including those previously regarded as difficult-to-treat. This raised hope not only for the HCV eradication in the near future but also for the dramatic decline in the risk of developing HCV-induced HCC or recurrence of disease in previously HCC-treated patients [132, 133]. Unfortunately, recent data shows that DAAs may promote tumour development in patients with hepatic cirrhosis and recurrence of HCC in patients who had previously been cured, suggesting the need for effective HCV-related HCC treatment with better response and a higher safety profile [134].

HCV is an icosahedral blood-borne RNA virus of 9.6 kb genome and a member of the Flaviviridae family [135]. HCV genome encodes for a large polyprotein processed by viral and cellular proteinases to produce structural and nonstructural (NS) proteins [136, 137]. Amongst NS proteins are NS3, NS4A, NS4B, NS5A, and NS5B. The NS3-5B coding region serves as the HCV replicase, and it is therefore required for proper RNA replication in cell culture and chimpanzees [135, 138]. In particular, NS3, NS5A, and NS5A play an important role in HCV pathogenesis and potentiate oncogenic transformation. Unlike HBV, HCV genome is unable to integrate into the human genome. However, it is able to cause epigenetic changes that favour its own replication through NS3, NS5A, and NS5B oncogenic events that are associated with the development of liver cancer [139]. Apolipoprotein E, which is required for the replication and infectivity of HCV, is known to be hypermethylated in chronic HCV infection. This is associated with increased viral replication and an increased risk of developing malignancy [140, 141].

Tumour suppressor genes such as E-cadherin and p16 are implicated in HCV core protein transcriptional activities and epigenetically dysregulated via DNMTs [142]. For instance, HCV core protein stimulates immortalization of hepatocytes by silencing p16 expression and E-cadherin (CDH1) via upregulation of DNMT1 and DNMT3B [142, 143]. Upregulation of DNMT1 and DNMT3B was associated with suppressed CDH1 mRNA in Huh-7 cells expressing HCV core protein of genotype 1b but not in genotypes 2a, 3a, 4h, and 5a. However, CDH1 protein expression level was downregulated in cells expressing HCV core of genotypes 1b, 2a, and 3a [144, 145]. Downregulation of secreted frizzled-related protein 1 (SFPR1) gene via HCV core protein-mediated DNMT was associated with aberrant activation of Wnt-signalling pathway leading to HCC aggressiveness by EMT [146]. Silent SFPR1 was induced by the HCV core protein-induced methylation of SFRP1 promoter region via DNMT1, and this enhanced cell proliferation, migration, and invasiveness. Inhibition of HCV core-activated ECM by either knockdown of DNMT1 and HDAC1 or restoration of SFPR1 disrupted Wnt/β-catenin-c-Myc-cyclin D1 pathways leading to abolished tumour growth and aggressiveness [146].

The epigenetic alterations in HCV-induced HCC may also implicate CDKN2A gene. Several histone posttranslational modifications also play an important role in altering the transcription of cellular genes, including classic tumour suppressors that are commonly known to epigenetically promote malignant transformation. EZH2 is an important component of PRC2 that interacts with EED and SUZ12 to establish trimethylation of H3K27 (H3K27me3) leading to HCV-related HCC tumour initiation and progression. H3K27me3 is a well-established marker of transcriptionally silent chromatin implicated in hepatocarcinogenesis [59]. Cyclin dependent kinase inhibitor 2A (CDKN2A) was identified as a target of repression by H3K27me3 mediated by upregulation in EZH2 and SUZ12 enzymes in HCC human samples [64]. CDKN2A encodes for p1 and p1, two important tumour suppressor genes that prevent tumour formation by regulating cell growth, division, and apoptosis. The p1 protein attaches to cyclin dependent kinase (CDK) 4 or CDK6 to and inhibit cell cycle progression. The p1 protein protects p53 from degradation and promotes p21 activation leading to controlled cell division and enhanced apoptosis. Epigenetic repression of CDKN2A resulted in the obstruction in CDKN2A-TP53-P21 pathway leading to HCC initiation and aggressiveness. Importantly, reduced expression of PRC2 protein via H3K27me3 inhibitor restored CDKN2A-TP53-P21 pathway and effectively blocked the aggressive phenotype of HCC cells [64]. Epigenetic silencing by EZH2-mediated H2K27me3 in HCC was also observed with other several tumour suppressor genes including DLC1 and chromodomain helicase DNA binding proteins 5 (CHD5) [58]. Reduced levels of CHD5 in HCC cells coincide with metastasis and poor prognosis [65].

6. Epigenetic Drug Therapy and Immunotherapy of HCC

The primary objective of epigenetic therapy is to pharmacologically reverse the aberrant epigenetic alterations and to restore altered gene expression by specifically targeting abnormal cells while leaving normal cells unaffected. There are four epigenetic reversals currently approved for cancer treatment by the US Food and Drug Administration (FDA). Decitabine, azacytidine, vorinostat, romidepsin, and belinostat inhibitors are well-known epigenetic inhibitors that were developed to treat several haematopoietic cancers and explored in HCC [147, 148]. Decitabine (5-aza-2′-deoxycytidine) and azacytidine (5-azacytidine) are potent epigenetic reversals that incorporate into DNA and inhibit DNMTs activities leading to DNA hypermethylation and reactivation of gene transcription. Although these DNA methylation inhibitors were both approved to treat acute leukemia and myelodysplastic syndrome, azacytidine is more potent than decitabine and provides better clinical outcome [149, 150]. However, recent phase I/II studies showed that lower-dose decitabine based therapy exhibited antitumour activities and tolerance in patients with advanced HCC [151]. Decitabine and azacytidine also differ in the manner in which they mediate apoptosis and senescence in solid tumours [152]. In squamous cell carcinoma, senescence is mediated by decitabine-induced overexpression of p16ink4a [153]. Silencing of p53 and upregulation of GADD45 genes through azacytidine is associated with caspase activation and enhanced apoptosis in HCC and colon tumour cells [152, 154].

Immunotherapy involves the use of substances either produced by the body or in the laboratory to boost the host’s innate and adaptive immune responses to fight infectious diseases and cancer. Although this method has shown to elicit impressive response rates in eradicating the transformed hepatocytes and controlling tumour growth/remission, the emergence of resistance and drug toxicity has been problematic [155, 156]. Combination therapy with decitabine and suberoylanilide hydroxamic acid (SAHA) significantly reduces cell growth and proliferation in a xenograft hepatoma model [157]. On the other hand, azacytidine induces apoptosis by inhibiting protein biosynthesis when used in combination with tumour necrosis factor related apoptosis inducing ligand (TRAIL) [158]. Guadecitabine is a hypomethylating agent that has been formulated as a dinucleotide antimetabolite of a decitabine that is linked to a guanosine through a phosphodiester bond. Preclinical studies showed that the combination of guadecitabine and cytotoxic agent oxaliplatin yielded a synergistic response by inhibiting cell growth and proliferation through disruption of Wnt/EGF/IGF signalling pathways in HCC models, suggesting that the combination of epigenetic inhibitors and immunotherapies may deliver a better response to HCC [159]. Combination of azacytidine and alendronate also elicited cytotoxic effects in Huh-7 HCC cells [160]. Recently, it was demonstrated that azacytidine promotes not only passive demethylation pathway of DNA but also TET2/3-mediated generation of 5hmC via active demethylation pathway in [57]. Combination of vitamin C and azacytidine enhances Tet activities in HCC cells leading to induction of active demethylation that suppresses the expression of Snail, a key regulator of EMT and cell cycle process [57].

Vorinostat (also known as SAHA) and romidepsin are two HDAC inhibitors (HDACIs) that were developed to treat a heterogeneous group of lymphoproliferative cutaneous T-cell lymphomas [161163]. These HDACIs alter gene transcription by interfering with class 1 and II HDACs, leading to cell cycle arrest and apoptosis in a wide variety of transformed cells [13]. Vorinostat or SAHA perturbs the ERK/NF-κB signalling pathway, which is an important inducer for cell proliferation/and invasiveness and suppressor for apoptosis [164]. Sorafenib is a tyrosine kinase inhibitor targeted therapy for metastatic HCC, thyroid and renal cell cancers. A recent study showed that combination of SAHA and sorafenib has potential as a novel strategy for treating metastatic HCC (Table 1). This study demonstrated that prolonged treatment with sorafenib boosted therapeutic efficacy of SAHA against HCC in both cell line and animal models through inhibition of ERK/NF-κB signalling pathway [165]. Belinostat is another HDACI that was approved by FDA in 2014 for the treatment of patients with relapsed or refractory peripheral T-cell lymphoma [166, 167]. Phase I and II-based study presented in 2012 Gastrointestinal Cancers Symposium reported disease stabilization and favourable safety profile in advanced HCC patients treated with belinostat [168]. Bortezomib is a potent proteasome inhibitor approved for the treatment of patients with mantle cell lymphoma [169]. Combination of belinostat and bortezomib exhibited significant antiproliferative and proapoptotic activities in HCC cell lines [170]. Thus the combination of epigenetic drugs and immunotherapy may provide highly promising, feasible, and intriguing therapeutic benefit to HCC patients (Table 1) [155, 156, 171].

Table 1: Strategies of HCC treatment by combining epigenetic drugs and immunotherapy.

miRs are also important targets for epigenetic therapy. Substantiating data show that inhibition of miRs that act as oncogenes may eradicate tumour growth. For instance, inhibition of miR-221 blocks hepatocarcinogenesis, suggesting the potential of miRs as potential therapeutic targets for treating HCC [88]. It is evident that miRs play an important role in hepatoepigenetics and related tumorigenesis enlightening their utility to serve as potential biomarkers for HCC prediction, diagnosis, prognosis, and therapeutic targets.

7. Conclusion

Understanding the manner in which epigenetic mechanisms alter gene transcription and genome architecture is currently a major challenge but one which should yield better cancer therapeutic approaches [172]. Hepatoepigenetics is becoming increasingly visible with a growing understanding of the roles of specific epigenetic aberrations in the liver and their utilities in the development of cancer therapy. Substantiating data shows that DNA methylation, histone modifications, and noncoding miRs synergistically cooperate in aberrantly altering gene transcription and critical cellular processes that lead to hepatocarcinogenesis and metastasis. Epigenetics alterations are pharmaceutically reversible with various inhibitors, offering an opportunity for therapeutic intervention. Combination of epigenetic inhibitors and immunotherapies emerge as a better therapeutic approach for HCC and may help in eradicating cancer cells especially those that are refractory to standard treatment.

Competing Interests

The authors declare no conflict of interests.

Acknowledgments

This work was supported by grants from Oppenheimer Memorial Trust, National Research Foundation, and University of Cape Town, Research Office.

References

  1. S. S. Thorgeirsson and J. W. Grisham, “Molecular pathogenesis of human hepatocellular carcinoma,” Nature Genetics, vol. 31, no. 4, pp. 339–346, 2002. View at Publisher · View at Google Scholar · View at Scopus
  2. R. L. Siegel, K. D. Miller, and A. Jemal, “Cancer statistics, 2015,” CA: A Cancer Journal for Clinicians, vol. 65, no. 1, pp. 5–29, 2015. View at Publisher · View at Google Scholar · View at Scopus
  3. H. B. El-Serag, “Epidemiology of viral hepatitis and hepatocellular carcinoma,” Gastroenterology, vol. 142, pp. 1264–1273. e1, 2012. View at Google Scholar
  4. S. Mittal and H. B. El-Serag, “Epidemiology of hepatocellular carcinoma: consider the population,” Journal of Clinical Gastroenterology, vol. 47, supplement, pp. S2–S6, 2013. View at Google Scholar · View at Scopus
  5. Y. Kondo, L. Shen, S. Suzuki et al., “Alterations of DNA methylation and histone modifications contribute to gene silencing in hepatocellular carcinomas,” Hepatology Research, vol. 37, no. 11, pp. 974–983, 2007. View at Publisher · View at Google Scholar · View at Scopus
  6. B. Li, W. Liu, L. Wang et al., “CpG island methylator phenotype associated with tumor recurrence in tumor-node-metastasis stage I hepatocellular carcinoma,” Annals of Surgical Oncology, vol. 17, no. 7, pp. 1917–1926, 2010. View at Publisher · View at Google Scholar · View at Scopus
  7. S. B. Baylin and J. E. Ohm, “Epigenetic gene silencing in cancer—a mechanism for early oncogenic pathway addiction?” Nature Reviews Cancer, vol. 6, no. 2, pp. 107–116, 2006. View at Publisher · View at Google Scholar · View at Scopus
  8. P. A. Jones and S. B. Baylin, “The epigenomics of cancer,” Cell, vol. 128, no. 4, pp. 683–692, 2007. View at Publisher · View at Google Scholar · View at Scopus
  9. P. A. Jones and S. B. Baylin, “The fundamental role of epigenetic events in cancer,” Nature Reviews Genetics, vol. 3, no. 6, pp. 415–428, 2002. View at Google Scholar · View at Scopus
  10. P. A. Jones and P. W. Laird, “Cancer epigenetics comes of age,” Nature Genetics, vol. 21, no. 2, pp. 163–167, 1999. View at Publisher · View at Google Scholar · View at Scopus
  11. A. P. Bird, “CpG-rich islands and the function of DNA methylation,” Nature, vol. 321, no. 6067, pp. 209–213, 1986. View at Publisher · View at Google Scholar · View at Scopus
  12. P. A. Jones and D. Takai, “The role of DNA methylation in mammalian epigenetics,” Science, vol. 293, no. 5532, pp. 1068–1070, 2001. View at Publisher · View at Google Scholar · View at Scopus
  13. J. J. Issa, “Epigenetics in cancer: what's the future?” Oncology, vol. 25, p. 220, 2011. View at Google Scholar
  14. C. Ozen, G. Yildiz, A. T. Dagcan et al., “Genetics and epigenetics of liver cancer,” New Biotechnology, vol. 30, no. 4, pp. 381–384, 2013. View at Publisher · View at Google Scholar · View at Scopus
  15. S. Ito, A. C. Dalessio, O. V. Taranova, K. Hong, L. C. Sowers, and Y. Zhang, “Role of tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification,” Nature, vol. 466, no. 7310, pp. 1129–1133, 2010. View at Publisher · View at Google Scholar · View at Scopus
  16. R. M. Kohli and Y. Zhang, “TET enzymes, TDG and the dynamics of DNA demethylation,” Nature, vol. 502, no. 7472, pp. 472–479, 2013. View at Publisher · View at Google Scholar · View at Scopus
  17. T. Jenuwein and C. D. Allis, “Translating the histone code,” Science, vol. 293, no. 5532, pp. 1074–1080, 2001. View at Publisher · View at Google Scholar · View at Scopus
  18. J. Govin, C. Caron, C. Lestrat, S. Rousseaux, and S. Khochbin, “The role of histones in chromatin remodelling during mammalian spermiogenesis,” European Journal of Biochemistry, vol. 271, no. 17, pp. 3459–3469, 2004. View at Publisher · View at Google Scholar · View at Scopus
  19. J. Nakayama, J. C. Rice, B. D. Strahl, C. D. Allis, and S. I. S. Grewal, “Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly,” Science, vol. 292, no. 5514, pp. 110–113, 2001. View at Publisher · View at Google Scholar · View at Scopus
  20. S. Rea, F. Eisenhaber, D. O'Carroll et al., “Regulation of chromatin structure by site-specific histone H3 methyltransferases,” Nature, vol. 406, pp. 593–599, 2000. View at Publisher · View at Google Scholar · View at Scopus
  21. R. D. Kornberg and Y. Lorch, “Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome,” Cell, vol. 98, no. 3, pp. 285–294, 1999. View at Publisher · View at Google Scholar · View at Scopus
  22. A. Kirmizis, S. M. Bartley, A. Kuzmichev et al., “Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27,” Genes and Development, vol. 18, no. 13, pp. 1592–1605, 2004. View at Publisher · View at Google Scholar · View at Scopus
  23. D. Bonenfant, M. Coulot, H. Towbin, P. Schindler, and J. van Oostrum, “Characterization of histone H2A and H2B variants and their post-translational modifications by mass spectrometry,” Molecular and Cellular Proteomics, vol. 5, no. 3, pp. 541–552, 2006. View at Publisher · View at Google Scholar · View at Scopus
  24. C. L. Peterson and M.-A. Laniel, “Histones and histone modifications,” Current Biology, vol. 14, no. 14, pp. R546–R551, 2004. View at Publisher · View at Google Scholar · View at Scopus
  25. X. Li and X. Zhao, “Epigenetic regulation of mammalian stem cells,” Stem Cells and Development, vol. 17, no. 6, pp. 1043–1052, 2008. View at Publisher · View at Google Scholar · View at Scopus
  26. X. Li and X. Zhao, “Comprehensive review: stem cells and development,” Stem Cells and Development, vol. 17, no. 6, pp. 1043–1052, 2008. View at Publisher · View at Google Scholar
  27. M. G. Lee, R. Villa, P. Trojer et al., “Demethylation of H3K27 regulates polycomb recruitment and H2A ubiquitination,” Science, vol. 318, no. 5849, pp. 447–450, 2007. View at Publisher · View at Google Scholar · View at Scopus
  28. R. Cao, L. Wang, H. Wang et al., “Role of histone H3 lysine 27 methylation in polycomb-group silencing,” Science, vol. 298, no. 5595, pp. 1039–1043, 2002. View at Publisher · View at Google Scholar · View at Scopus
  29. D. P. F. Tsang and A. S. L. Cheng, “Epigenetic regulation of signaling pathways in cancer: role of the histone methyltransferase EZH2,” Journal of Gastroenterology and Hepatology, vol. 26, no. 1, pp. 19–27, 2011. View at Publisher · View at Google Scholar · View at Scopus
  30. M. Sasaki, H. Ikeda, K. Itatsu et al., “The overexpression of polycomb group proteins Bmi1 and EZH2 is associated with the progression and aggressive biological behavior of hepatocellular carcinoma,” Laboratory Investigation, vol. 88, no. 8, pp. 873–882, 2008. View at Publisher · View at Google Scholar · View at Scopus
  31. A. M. Farcas, N. P. Blackledge, I. Sudbery et al., “KDM2B links the polycomb repressive complex 1 (PRC1) to recognition of CpG islands,” eLife, vol. 2012, no. 1, Article ID e00205, 2012. View at Publisher · View at Google Scholar · View at Scopus
  32. L. Tavares, E. Dimitrova, D. Oxley et al., “RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3,” Cell, vol. 148, pp. 664–678, 2012. View at Google Scholar
  33. J. K. Stock, S. Giadrossi, M. Casanova et al., “Ring1-mediated ubiquitination of H2A restrains poised RNA polymerase II at bivalent genes in mouse ES cells,” Nature Cell Biology, vol. 9, no. 12, pp. 1428–1435, 2007. View at Publisher · View at Google Scholar · View at Scopus
  34. S.-J. Park, J.-G. Kim, T. G. Son et al., “The histone demethylase JMJD1A regulates adrenomedullin-mediated cell proliferation in hepatocellular carcinoma under hypoxia,” Biochemical and Biophysical Research Communications, vol. 434, no. 4, pp. 722–727, 2013. View at Publisher · View at Google Scholar · View at Scopus
  35. C. M. Croce, “Causes and consequences of microRNA dysregulation in cancer,” Nature Reviews Genetics, vol. 10, no. 10, pp. 704–714, 2009. View at Publisher · View at Google Scholar · View at Scopus
  36. M. Esteller, “Non-coding RNAs in human disease,” Nature Reviews Genetics, vol. 12, no. 12, pp. 861–874, 2011. View at Publisher · View at Google Scholar · View at Scopus
  37. E. C. Lai, “Predicting and validating microRNA targets,” Genome Biology, vol. 5, no. 9, article no. 115, 2004. View at Publisher · View at Google Scholar · View at Scopus
  38. T. Du and P. D. Zamore, “microPrimer: the biogenesis and function of microRNA,” Development, vol. 132, no. 21, pp. 4645–4652, 2005. View at Publisher · View at Google Scholar · View at Scopus
  39. E. Berezikov and R. H. A. Plasterk, “Camels and zebrafish, viruses and cancer: a microRNA update,” Human Molecular Genetics, vol. 14, no. 2, pp. R183–R190, 2005. View at Publisher · View at Google Scholar · View at Scopus
  40. N. Valeri, I. Vannini, F. Fanini, F. Calore, B. Adair, and M. Fabbri, “Epigenetics, miRNAs, and human cancer: a new chapter in human gene regulation,” Mammalian Genome, vol. 20, no. 9-10, pp. 573–580, 2009. View at Publisher · View at Google Scholar · View at Scopus
  41. Y. Ladeiro, G. Couchy, C. Balabaud et al., “MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations,” Hepatology, vol. 47, no. 6, pp. 1955–1963, 2008. View at Publisher · View at Google Scholar · View at Scopus
  42. O. C. Araújo, A. S. Rosa, A. Fernandes et al., “RASSF1A and DOK1 promoter methylation levels in hepatocellular carcinoma, cirrhotic and non-cirrhotic liver, and correlation with liver cancer in brazilian patients,” PLOS ONE, vol. 11, no. 4, Article ID e0153796, 2016. View at Publisher · View at Google Scholar
  43. J. Chen, J. Zhao, R. Ma, H. Lin, X. Liang, and X. Cai, “Prognostic significance of E-cadherin expression in hepatocellular carcinoma: a meta-analysis,” PLoS ONE, vol. 9, no. 8, Article ID e103952, 2014. View at Publisher · View at Google Scholar · View at Scopus
  44. L. Hu, G. Chen, H. Yu, and X. Qiu, “Clinicopathological significance of RASSF1A reduced expression and hypermethylation in hepatocellular carcinoma,” Hepatology International, vol. 4, no. 1, pp. 423–432, 2010. View at Publisher · View at Google Scholar · View at Scopus
  45. Z.-H. Zhao, Y.-C. Fan, Y. Yang, and K. Wang, “Association between Ras association domain family 1A promoter methylation and hepatocellular carcinoma: a meta-analysis,” World Journal of Gastroenterology, vol. 19, no. 41, pp. 7189–7196, 2013. View at Publisher · View at Google Scholar · View at Scopus
  46. Y.-M. Li, S.-C. Xu, J. Li et al., “Epithelial-mesenchymal transition markers expressed in circulating tumor cells in hepatocellular carcinoma patients with different stages of disease,” Cell Death & Disease, vol. 4, no. 10, article e831, 2013. View at Publisher · View at Google Scholar · View at Scopus
  47. X. Q. Wang, W. Zhang, E. L. H. Lui et al., “Notch1-Snail1-E-cadherin pathway in metastatic hepatocellular carcinoma,” International Journal of Cancer, vol. 131, no. 3, pp. E163–E172, 2012. View at Publisher · View at Google Scholar · View at Scopus
  48. C.-N. Guan, X.-M. Chen, H.-Q. Lou, X.-H. Liao, B.-Y. Chen, and P.-W. Zhang, “Clinical significance of axin and β-catenin protein expression in primary hepatocellular carcinomas,” Asian Pacific Journal of Cancer Prevention, vol. 13, no. 2, pp. 677–681, 2012. View at Publisher · View at Google Scholar · View at Scopus
  49. R. C. Zhao, J. Zhou, J. Y. He, Y. G. Wei, Y. Qin, and B. Li, “Aberrant promoter methylation of SOCS-1 gene may contribute to the pathogenesis of hepatocellular carcinoma: a meta-analysis,” Journal of BUON, vol. 21, pp. 142–151, 2016. View at Google Scholar
  50. Z. Qu, Y. Jiang, H. Li, D.-C. Yu, and Y.-T. Ding, “Detecting abnormal methylation of tumor suppressor genes GSTP1, P16, RIZ1, and RASSF1A in hepatocellular carcinoma and its clinical significance,” Oncology Letters, vol. 10, no. 4, pp. 2553–2558, 2015. View at Publisher · View at Google Scholar · View at Scopus
  51. A. Villanueva, A. Portela, S. Sayols et al., “DNA methylation-based prognosis and epidrivers in hepatocellular carcinoma,” Hepatology, vol. 61, no. 6, pp. 1945–1956, 2015. View at Publisher · View at Google Scholar · View at Scopus
  52. Y. Saito, Y. Kanai, M. Sakamoto, H. Saito, H. Ishii, and S. Hirohashi, “Overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, associated with DNA hypomethylation on pericentromeric satellite regions during human hepatocarcinogenesis,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 15, pp. 10060–10065, 2002. View at Publisher · View at Google Scholar · View at Scopus
  53. T. Oda, H. Tsuda, M. Sakamoto, and S. Hirohashi, “Different mutations of the p53 gene in nodule-in-nodule hepatocellular carcinoma as a evidence for multistage progression,” Cancer Letters, vol. 83, no. 1-2, pp. 197–200, 1994. View at Publisher · View at Google Scholar · View at Scopus
  54. P. Laurent-Puig and J. Zucman-Rossi, “Genetics of hepatocellular tumors,” Oncogene, vol. 25, no. 27, pp. 3778–3786, 2006. View at Publisher · View at Google Scholar · View at Scopus
  55. S. M. Mazzoni and E. R. Fearon, “AXIN1 and AXIN2 variants in gastrointestinal cancers,” Cancer Letters, vol. 355, no. 1, pp. 1–8, 2014. View at Publisher · View at Google Scholar · View at Scopus
  56. M. Klasić, J. Krištić, P. Korać et al., “DNA hypomethylation upregulates expression of the MGAT3 gene in HepG2 cells and leads to changes in N-glycosylation of secreted glycoproteins,” Scientific Reports, vol. 6, Article ID 24363, 2016. View at Publisher · View at Google Scholar
  57. S. O. Sajadian, S. Ehnert, H. Vakilian et al., “Induction of active demethylation and 5hmC formation by 5-azacytidine is TET2 dependent and suggests new treatment strategies against hepatocellular carcinoma,” Clinical Epigenetics, vol. 7, no. 1, article 98, 2015. View at Publisher · View at Google Scholar · View at Scopus
  58. S. L.-K. Au, C. C.-L. Wong, J. M.-F. Lee, C.-M. Wong, and I. O.-L. Ng, “EZH2-mediated H3K27me3 is involved in epigenetic repression of deleted in liver cancer 1 in human cancers,” PLoS ONE, vol. 8, no. 6, Article ID e68226, 2013. View at Publisher · View at Google Scholar · View at Scopus
  59. M.-Y. Cai, Z.-T. Tong, F. Zheng et al., “EZH2 protein: a promising immunomarker for the detection of hepatocellular carcinomas in liver needle biopsies,” Gut, vol. 60, no. 7, pp. 967–976, 2011. View at Publisher · View at Google Scholar · View at Scopus
  60. F. Rappa, A. Greco, C. Podrini et al., “Correction: immunopositivity for histone MacroH2A1 isoforms marks steatosis-associated hepatocellular carcinoma,” PLOS ONE, vol. 8, no. 3, 2013. View at Publisher · View at Google Scholar · View at Scopus
  61. M. Borghesan, C. Fusilli, F. Rappa et al., “DNA Hypomethylation and histone variant macroH2A1 synergistically attenuate chemotherapy-induced senescence to promote hepatocellular carcinoma progression,” Cancer Research, vol. 76, no. 3, pp. 594–606, 2016. View at Publisher · View at Google Scholar · View at Scopus
  62. S. Jueliger, J. Lyons, S. Cannito et al., “Efficacy and epigenetic interactions of novel DNA hypomethylating agent guadecitabine (SGI-110) in preclinical models of hepatocellular carcinoma,” Epigenetics, vol. 11, no. 10, pp. 709–720, 2016. View at Publisher · View at Google Scholar
  63. S.-B. Gao, B. Xu, L.-H. Ding et al., “The functional and mechanistic relatedness of EZH2 and menin in hepatocellular carcinoma,” Journal of Hepatology, vol. 61, no. 4, pp. 832–839, 2014. View at Publisher · View at Google Scholar · View at Scopus
  64. S.-B. Gao, Q.-F. Zheng, B. Xu et al., “EZH2 represses target genes through H3K27-dependent and H3K27-independent mechanisms in hepatocellular carcinoma,” Molecular Cancer Research, vol. 12, no. 10, pp. 1388–1397, 2014. View at Publisher · View at Google Scholar · View at Scopus
  65. C.-R. Xie, Z. Li, H.-G. Sun et al., “Mutual regulation between CHD5 and EZH2 in hepatocellular carcinoma,” Oncotarget, vol. 6, no. 38, pp. 40940–40952, 2015. View at Publisher · View at Google Scholar · View at Scopus
  66. A. Hayashi, N. Yamauchi, J. Shibahara et al., “Concurrent activation of acetylation and tri-methylation of H3K27 in a subset of hepatocellular carcinoma with aggressive behavior,” PLoS ONE, vol. 9, no. 3, Article ID e91330, 2014. View at Publisher · View at Google Scholar · View at Scopus
  67. K. Bai, Y. Cao, C. Huang, J. Chen, X. Zhang, and Y. Jiang, “Association of histone methyltransferase G9a and overall survival after liver resection of patients with hepatocellular carcinoma with a median observation of 40 months,” Medicine, vol. 95, no. 2, Article ID e2493, 2016. View at Publisher · View at Google Scholar · View at Scopus
  68. C. Li, M. Cai, L. Jiang et al., “CLDN14 is epigenetically silenced by EZH2-mediated H3K27ME3 and is a novel prognostic biomarker in hepatocellular carcinoma,” Carcinogenesis, vol. 37, no. 6, pp. 557–566, 2016. View at Publisher · View at Google Scholar
  69. L. Jiang, Y.-D. Yang, L. Fu et al., “CLDN3 inhibits cancer aggressiveness via Wnt-EMT signaling and is a potential prognostic biomarker for hepatocellular carcinoma,” Oncotarget, vol. 5, no. 17, pp. 7663–7676, 2014. View at Publisher · View at Google Scholar · View at Scopus
  70. S. Hino, K. Kohrogi, and M. Nakao, “Histone demethylase LSD1 controls the phenotypic plasticity of cancer cells,” Cancer Science, vol. 107, no. 9, pp. 1187–1192, 2016. View at Publisher · View at Google Scholar
  71. A. Sakamoto, S. Hino, K. Nagaoka et al., “Lysine demethylase LSD1 coordinates glycolytic and mitochondrial metabolism in hepatocellular carcinoma cells,” Cancer Research, vol. 75, no. 7, pp. 1445–1456, 2015. View at Publisher · View at Google Scholar · View at Scopus
  72. D. Yamada, S. Kobayashi, H. Yamamoto et al., “Role of the hypoxia-related gene, JMJD1A, in hepatocellular carcinoma: clinical impact on recurrence after hepatic resection,” Annals of Surgical Oncology, vol. 19, no. 3, pp. S355–S364, 2012. View at Publisher · View at Google Scholar · View at Scopus
  73. J. T. Buijs, G. Van Der Horst, C. Van Den Hoogen et al., “The BMP2/7 heterodimer inhibits the human breast cancer stem cell subpopulation and bone metastases formation,” Oncogene, vol. 31, no. 17, pp. 2164–2174, 2012. View at Publisher · View at Google Scholar · View at Scopus
  74. M. Khalaf, J. Morera, A. Bourret et al., “BMP system expression in GCs from polycystic ovary syndrome women and the in vitro effects of BMP4, BMP6, and BMP7 on GC steroidogenesis,” European Journal of Endocrinology, vol. 168, no. 3, pp. 437–444, 2013. View at Publisher · View at Google Scholar · View at Scopus
  75. H. P. H. Naber, TGF-Beta and BMP in Breast Cancer Cell Invasion, Signal Transduction and Ageing Section, Department of Molecular Cell Biology, Faculty of Medicine, Leiden University Medical Center (LUMC), Leiden University, 2012.
  76. X. Ji, S. Jin, X. Qu et al., “Lysine-specific demethylase 5C promotes hepatocellular carcinoma cell invasion through inhibition BMP7 expression,” BMC Cancer, vol. 15, no. 1, article no. 801, 2015. View at Publisher · View at Google Scholar · View at Scopus
  77. M. R. Zou, J. Cao, Z. Liu, S. J. Huh, K. Polyak, and Q. Yan, “Histone demethylase Jumonji AT-rich Interactive Domain 1B (JARID1B) controls mammary gland development by regulating key developmental and lineage specification genes,” Journal of Biological Chemistry, vol. 289, no. 25, pp. 17620–17633, 2014. View at Publisher · View at Google Scholar · View at Scopus
  78. X.-X. He, S.-Z. Kuang, J.-Z. Liao et al., “The regulation of microRNA expression by DNA methylation in hepatocellular carcinoma,” Molecular BioSystems, vol. 11, no. 2, pp. 532–539, 2015. View at Publisher · View at Google Scholar · View at Scopus
  79. Z. Liu, J. Wang, Y. Mao, B. Zou, and X. Fan, “MicroRNA-101 suppresses migration and invasion via targeting vascular endothelial growth factor-C in hepatocellular carcinoma cells,” Oncology Letters, vol. 11, no. 1, pp. 433–438, 2016. View at Publisher · View at Google Scholar · View at Scopus
  80. L. Lin, H. Liang, Y. Wang et al., “MicroRNA-141 inhibits cell proliferation and invasion and promotes apoptosis by targeting hepatocyte nuclear factor-3β in hepatocellular carcinoma cells,” BMC Cancer, vol. 14, no. 1, article no. 879, 2014. View at Publisher · View at Google Scholar · View at Scopus
  81. S.-L. Zhang and L. Liu, “microRNA-148a inhibits hepatocellular carcinoma cell invasion by targeting sphingosine-1-phosphate receptor 1,” Experimental and Therapeutic Medicine, vol. 9, no. 2, pp. 579–584, 2015. View at Publisher · View at Google Scholar · View at Scopus
  82. B. Deng, L. Qu, J. Li et al., “MiRNA-211 suppresses cell proliferation, migration and invasion by targeting SPARC in human hepatocellular carcinoma,” Scientific Reports, vol. 6, article 26679, 2016. View at Publisher · View at Google Scholar
  83. G. Chen, L. Lu, C. Liu, L. Shan, and D. Yuan, “MicroRNA-377 suppresses cell proliferation and invasion by inhibiting TIAM1 expression in hepatocellular carcinoma,” PLoS ONE, vol. 10, no. 3, Article ID e0117714, 2015. View at Publisher · View at Google Scholar · View at Scopus
  84. K. Sun, T. Zeng, D. Huang et al., “MicroRNA-431 inhibits migration and invasion of hepatocellular carcinoma cells by targeting the ZEB1-mediated epithelial-mensenchymal transition,” FEBS Open Bio, vol. 5, pp. 900–907, 2015. View at Publisher · View at Google Scholar · View at Scopus
  85. J. H. Noh, Y. G. Chang, M. G. Kim et al., “MiR-145 functions as a tumor suppressor by directly targeting histone deacetylase 2 in liver cancer,” Cancer Letters, vol. 335, no. 2, pp. 455–462, 2013. View at Publisher · View at Google Scholar · View at Scopus
  86. Y. Zeng, X. Liang, G. Zhang et al., “miRNA-135a promotes hepatocellular carcinoma cell migration and invasion by targeting forkhead box O1,” Cancer Cell International, vol. 16, no. 1, 2016. View at Publisher · View at Google Scholar
  87. K. Liu, S. Liu, W. Zhang et al., “miR-494 promotes cell proliferation, migration and invasion, and increased sorafenib resistance in hepatocellular carcinoma by targeting PTEN,” Oncology Reports, vol. 34, no. 2, pp. 1003–1010, 2015. View at Publisher · View at Google Scholar · View at Scopus
  88. F. Fornari, L. Gramantieri, M. Ferracin et al., “MiR-221 controls CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma,” Oncogene, vol. 27, no. 43, pp. 5651–5661, 2008. View at Publisher · View at Google Scholar · View at Scopus
  89. Y. Wang, H. C. Toh, P. Chow et al., “MicroRNA-224 is up-regulated in hepatocellular carcinoma through epigenetic mechanisms,” FASEB Journal, vol. 26, no. 7, pp. 3032–3041, 2012. View at Publisher · View at Google Scholar · View at Scopus
  90. Y. Wang, A. T. C. Lee, J. Z. I. Ma et al., “Profiling microRNA expression in hepatocellular carcinoma reveals microRNA-224 up-regulation and apoptosis inhibitor-5 as a microRNA-224-specific target,” Journal of Biological Chemistry, vol. 283, no. 19, pp. 13205–13215, 2008. View at Publisher · View at Google Scholar · View at Scopus
  91. D. M. Pereira, P. M. Rodrigues, P. M. Borralho, and C. M. P. Rodrigues, “Delivering the promise of miRNA cancer therapeutics,” Drug Discovery Today, vol. 18, no. 5-6, pp. 282–289, 2013. View at Publisher · View at Google Scholar · View at Scopus
  92. J. S. Tuttleman, C. Pourcel, and J. Summers, “Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells,” Cell, vol. 47, no. 3, pp. 451–460, 1986. View at Publisher · View at Google Scholar · View at Scopus
  93. M. S. Chapman and L. Liljas, “Structural folds of viral proteins,” Advances in Protein Chemistry, vol. 64, pp. 125–196, 2003. View at Publisher · View at Google Scholar · View at Scopus
  94. N. Paran, A. Cooper, and Y. Shaul, “Interaction of hepatitis B virus with cells,” Reviews in Medical Virology, vol. 13, no. 3, pp. 137–143, 2003. View at Publisher · View at Google Scholar · View at Scopus
  95. W.-K. Sung, H. Zheng, S. Li et al., “Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma,” Nature Genetics, vol. 44, no. 7, pp. 765–769, 2012. View at Publisher · View at Google Scholar · View at Scopus
  96. M. A. Feitelson and J. Lee, “Hepatitis B virus integration, fragile sites, and hepatocarcinogenesis,” Cancer Letters, vol. 252, no. 2, pp. 157–170, 2007. View at Publisher · View at Google Scholar · View at Scopus
  97. Z. Jiang, S. Jhunjhunwala, J. Liu et al., “The effects of hepatitis B virus integration into the genomes of hepatocellular carcinoma patients,” Genome Research, vol. 22, no. 4, pp. 593–601, 2012. View at Publisher · View at Google Scholar · View at Scopus
  98. P. Vivekanandan, H. D.-J. Daniel, R. Kannangai, F. Martinez-Murillo, and M. Torbenson, “Hepatitis B virus replication induces methylation of both host and viral DNA,” Journal of Virology, vol. 84, no. 9, pp. 4321–4329, 2010. View at Publisher · View at Google Scholar · View at Scopus
  99. S. Lee, H. J. Lee, J.-H. Kim, H.-S. Lee, J. J. Jang, and G. H. Kang, “Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis,” The American Journal of Pathology, vol. 163, no. 4, pp. 1371–1378, 2003. View at Publisher · View at Google Scholar · View at Scopus
  100. M.-P. Lambert, A. Paliwal, T. Vaissière et al., “Aberrant DNA methylation distinguishes hepatocellular carcinoma associated with HBV and HCV infection and alcohol intake,” Journal of Hepatology, vol. 54, no. 4, pp. 705–715, 2011. View at Publisher · View at Google Scholar · View at Scopus
  101. F. H. T. Duong, V. Christen, S. Lin, and M. H. Heim, “Hepatitis C virus-induced up-regulation of protein phosphatase 2A inhibits histone modification and DNA damage repair,” Hepatology, vol. 51, no. 3, pp. 741–751, 2010. View at Publisher · View at Google Scholar · View at Scopus
  102. Y.-J. Kim, J. K. Jung, S. Y. Lee, and K. L. Jang, “Hepatitis B virus X protein overcomes stress-induced premature senescence by repressing p16INK4aexpression via DNA methylation,” Cancer Letters, vol. 288, no. 2, pp. 226–235, 2010. View at Publisher · View at Google Scholar · View at Scopus
  103. Y. Edamoto, A. Hara, W. Biernat et al., “Alterations of RB1, p53 and Wnt pathways in hepatocellular carcinomas associated with hepatitis C, hepatitis B and alcoholic liver cirrhosis,” International Journal of Cancer, vol. 106, no. 3, pp. 334–341, 2003. View at Publisher · View at Google Scholar · View at Scopus
  104. J. K. Jung, S.-H. Park, and K. L. Jang, “Hepatitis B virus X protein overcomes the growth-inhibitory potential of retinoic acid by downregulating retinoic acid receptor-β2 expression via DNA methylation,” Journal of General Virology, vol. 91, no. 2, pp. 493–500, 2010. View at Publisher · View at Google Scholar · View at Scopus
  105. J. K. Jung, P. Arora, J. S. Pagano, and L. J. Kyung, “Expression of DNA methyltransferase 1 is activated by hepatitis B virus X protein via a regulatory circuit involving the p16INK4a-cyclin D1-CDK 4/6-pRb-E2F1 pathway,” Cancer Research, vol. 67, no. 12, pp. 5771–5778, 2007. View at Publisher · View at Google Scholar · View at Scopus
  106. D.-L. Zheng, L. Zhang, N. Cheng et al., “Epigenetic modification induced by hepatitis B virus X protein via interaction with de novo DNA methyltransferase DNMT3A,” Journal of Hepatology, vol. 50, no. 2, pp. 377–387, 2009. View at Publisher · View at Google Scholar · View at Scopus
  107. C. Zhang, X. Chen, H. Liu et al., “Alpha fetoprotein mediates HBx induced carcinogenesis in the hepatocyte cytoplasm,” International Journal of Cancer, vol. 137, no. 8, pp. 1818–1829, 2015. View at Publisher · View at Google Scholar · View at Scopus
  108. G. Barreto, A. Schäfer, J. Marhold et al., “Gadd45a promotes epigenetic gene activation by repair-mediated DNA demethylation,” Nature, vol. 445, no. 7128, pp. 671–675, 2007. View at Publisher · View at Google Scholar · View at Scopus
  109. W. Guo, Z. Dong, Y. Guo, Z. Chen, G. Kuang, and Z. Yang, “Methylation-mediated repression of GADD45A and GADD45G expression in gastric cardia adenocarcinoma,” International Journal of Cancer, vol. 133, no. 9, pp. 2043–2053, 2013. View at Publisher · View at Google Scholar · View at Scopus
  110. J. Yan, Q. Lu, J. Dong, X. Li, K. Ma, and L. Cai, “Hepatitis B virus X protein suppresses caveolin-1 expression in hepatocellular carcinoma by regulating DNA methylation,” BMC Cancer, vol. 12, article no. 353, 2012. View at Publisher · View at Google Scholar · View at Scopus
  111. E. Y. Ting Tse, F. C. Fat Ko, E. K. Kwan Tung et al., “Caveolin-1 overexpression is associated with hepatocellular carcinoma tumourigenesis and metastasis,” Journal of Pathology, vol. 226, no. 4, pp. 645–653, 2012. View at Publisher · View at Google Scholar · View at Scopus
  112. J. Kwong, L. S.-N. Chow, A. Y.-H. Wong et al., “Epigenetic inactivation of the deleted in lung and esophageal cancer 1 gene in nasopharyngeal carcinoma,” Genes, Chromosomes and Cancer, vol. 46, no. 2, pp. 171–180, 2007. View at Publisher · View at Google Scholar · View at Scopus
  113. J. Kwong, J.-Y. Lee, K.-K. Wong et al., “Candidate tumor-suppressor gene DLEC1 is frequently downregulated by promoter hypermethylation and histone hypoacetylation in human epithelial ovarian cancer,” Neoplasia, vol. 8, no. 4, pp. 268–278, 2006. View at Publisher · View at Google Scholar · View at Scopus
  114. G.-H. Qiu, M. Salto-Tellez, J. A. Ross et al., “The tumor suppressor gene DLEC1 is frequently silenced by DNA methylation in hepatocellular carcinoma and induces G1 arrest in cell cycle,” Journal of Hepatology, vol. 48, no. 3, pp. 433–441, 2008. View at Publisher · View at Google Scholar · View at Scopus
  115. I. Y. Park, B. H. Sohn, E. Yu et al., “Aberrant epigenetic modifications in hepatocarcinogenesis induced by hepatitis B virus X protein,” Gastroenterology, vol. 132, no. 4, pp. 1476–1494, 2007. View at Publisher · View at Google Scholar · View at Scopus
  116. T. Hanafusa, Y. Yumoto, K. Nouso et al., “Reduced expression of insulin-like growth factor binding protein-3 and its promoter hypermethylation in human hepatocellular carcinoma,” Cancer Letters, vol. 176, no. 2, pp. 149–158, 2002. View at Publisher · View at Google Scholar · View at Scopus
  117. J.-Y. Yao, L. Zhang, X. Zhang et al., “H3K27 trimethylation is an early epigenetic event of p16INK4a silencing for regaining tumorigenesis in fusion reprogrammed hepatoma cells,” Journal of Biological Chemistry, vol. 285, no. 24, pp. 18828–18837, 2010. View at Publisher · View at Google Scholar · View at Scopus
  118. Y.-H. Shim, G.-S. Yoon, H.-J. Choi, Y. H. Chung, and E. Yu, “p16 hypermethylation in the early stage of hepatitis B virus-associated hepatocarcinogenesis,” Cancer Letters, vol. 190, no. 2, pp. 213–219, 2003. View at Publisher · View at Google Scholar · View at Scopus
  119. Y.-Z. Zhu, R. Zhu, J. Fan et al., “Hepatitis B virus X protein induces hypermethylation of p16INK4A promoter via DNA methyltransferases in the early stage of HBV-associated hepatocarcinogenesis,” Journal of Viral Hepatitis, vol. 17, no. 2, pp. 98–107, 2010. View at Publisher · View at Google Scholar · View at Scopus
  120. R. Zhu, B.-Z. Li, H. Li et al., “Association of p16INK4A hypermethylation with hepatitis B virus X protein expression in the early stage of HBV-associated hepatocarcinogenesis,” Pathology International, vol. 57, no. 6, pp. 328–336, 2007. View at Publisher · View at Google Scholar · View at Scopus
  121. H. Fan, H. Zhang, P. E. Pascuzzi, and O. Andrisani, “Hepatitis B virus X protein induces EpCAM expression via active DNA demethylation directed by RelA in complex with EZH2 and TET2,” Oncogene, vol. 35, no. 6, pp. 715–726, 2016. View at Publisher · View at Google Scholar · View at Scopus
  122. S. L.-K. Au, C. C.-L. Wong, J. M.-F. Lee et al., “Enhancer of zeste homolog 2 epigenetically silences multiple tumor suppressor microRNAs to promote liver cancer metastasis,” Hepatology, vol. 56, no. 2, pp. 622–631, 2012. View at Publisher · View at Google Scholar · View at Scopus
  123. D. Li, X. Liu, L. Lin et al., “MicroRNA-99a inhibits hepatocellular carcinoma growth and correlates with prognosis of patients with hepatocellular carcinoma,” Journal of Biological Chemistry, vol. 286, no. 42, pp. 36677–36685, 2011. View at Publisher · View at Google Scholar · View at Scopus
  124. D. Ngo-Yin Fan, F. Ho-Ching Tsang, A. Hoi-Kam Tam et al., “Histone lysine methyltransferase, suppressor of variegation 3-9 homolog 1, promotes hepatocellular carcinoma progression and is negatively regulated by microRNA-125b,” Hepatology, vol. 57, no. 2, pp. 637–647, 2013. View at Publisher · View at Google Scholar · View at Scopus
  125. L. Wang, X. Zhang, L.-T. Jia et al., “C-Myc-mediated epigenetic silencing of microRNA-101 contributes to dysregulation of multiple pathways in hepatocellular carcinoma,” Hepatology, vol. 59, no. 5, pp. 1850–1863, 2014. View at Publisher · View at Google Scholar · View at Scopus
  126. F. Yu, Z. Lu, B. Chen, P. Dong, and J. Zheng, “microRNA-150: a promising novel biomarker for hepatitis B virus-related hepatocellular carcinoma,” Diagnostic Pathology, vol. 10, article no. 129, 2015. View at Publisher · View at Google Scholar · View at Scopus
  127. L. Rivière, L. Gerossier, A. Ducroux et al., “HBx relieves chromatin-mediated transcriptional repression of hepatitis B viral cccDNA involving SETDB1 histone methyltransferase,” Journal of Hepatology, vol. 63, no. 5, pp. 1093–1102, 2015. View at Publisher · View at Google Scholar · View at Scopus
  128. C. Cicchini, C. Battistelli, and M. Tripodi, “SETDB1 is a new promising target in HCC therapy,” Chinese Clinical Oncology, vol. 5, pp. 504–504, 2016. View at Publisher · View at Google Scholar
  129. Q. Fei, K. Shang, J. Zhang et al., “Histone methyltransferase SETDB1 regulates liver cancer cell growth through methylation of p53,” Nature Communications, vol. 6, article no. 8651, 2015. View at Publisher · View at Google Scholar
  130. T. Longerich, “Dysregulation of the epigenetic regulator SETDB1 in liver carcinogenesis—more than one way to skin a cat,” Chinese Clinical Oncology, vol. 5, pp. 318–318, 2016. View at Publisher · View at Google Scholar
  131. A. S. Lok, L. B. Seeff, T. R. Morgan et al., “Incidence of hepatocellular carcinoma and associated risk factors in hepatitis C-related advanced liver disease,” Gastroenterology, vol. 136, no. 1, pp. 138–148, 2009. View at Publisher · View at Google Scholar · View at Scopus
  132. F. Conti, F. Buonfiglioli, A. Scuteri et al., “Early occurrence and recurrence of hepatocellular carcinoma in HCV-related cirrhosis treated with direct-acting antivirals,” Journal of Hepatology, vol. 65, no. 4, pp. 727–733, 2016. View at Publisher · View at Google Scholar
  133. J. M. Llovet and A. Villanueva, “Liver cancer: effect of HCV clearance with direct-acting antiviral agents on HCC,” Nature Reviews Gastroenterology & Hepatology, vol. 13, no. 10, pp. 561–562, 2016. View at Publisher · View at Google Scholar
  134. M. Reig, Z. Mariño, C. Perelló et al., “Unexpected high rate of early tumor recurrence in patients with HCV-related HCC undergoing interferon-free therapy,” Journal of Hepatology, vol. 65, no. 4, pp. 719–726, 2016. View at Publisher · View at Google Scholar
  135. B. D. Lindenbach and C. Rice, “Flaviviridae: the viruses and their replication,” in Fields Virology, vol. 1, pp. 991–1041, Lippincott Williams & Wilkins, 2001. View at Google Scholar
  136. T. Heintges and J. R. Wands, “Hepatitis C virus: epidemiology and transmission,” Hepatology, vol. 26, no. 3, pp. 521–526, 1997. View at Publisher · View at Google Scholar · View at Scopus
  137. D. Moradpour, F. Penin, and C. M. Rice, “Replication of hepatitis C virus,” Nature Reviews Microbiology, vol. 5, no. 6, pp. 453–463, 2007. View at Publisher · View at Google Scholar · View at Scopus
  138. K. J. Blight, J. A. McKeating, J. Marcotrigiano, and C. M. Rice, “Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture,” Journal of Virology, vol. 77, no. 5, pp. 3181–3190, 2003. View at Publisher · View at Google Scholar · View at Scopus
  139. L. Rongrui, H. Na, L. Zongfang, J. Fanpu, and J. Shiwen, “Epigenetic mechanism involved in the HBV/HCV-related hepatocellular carcinoma tumorigenesis,” Current Pharmaceutical Design, vol. 20, no. 11, pp. 1715–1725, 2014. View at Publisher · View at Google Scholar · View at Scopus
  140. Y.-B. Deng, G. Nagae, Y. Midorikawa et al., “Identification of genes preferentially methylated in hepatitis C virus-related hepatocellular carcinoma,” Cancer Science, vol. 101, no. 6, pp. 1501–1510, 2010. View at Publisher · View at Google Scholar · View at Scopus
  141. T. Hishiki, Y. Shimizu, R. Tobita et al., “Infectivity of hepatitis C virus is influenced by association with apolipoprotein E isoforms,” Journal of Virology, vol. 84, no. 22, pp. 12048–12057, 2010. View at Publisher · View at Google Scholar · View at Scopus
  142. P. Arora, E.-O. Kim, J. K. Jung, and K. L. Jang, “Hepatitis C virus core protein downregulates E-cadherin expression via activation of DNA methyltransferase 1 and 3b,” Cancer Letters, vol. 261, no. 2, pp. 244–252, 2008. View at Publisher · View at Google Scholar · View at Scopus
  143. S.-H. Park, J. S. Lim, S.-Y. Lim, I. Tiwari, and K. L. Jang, “Hepatitis C virus Core protein stimulates cell growth by down-regulating p16 expression via DNA methylation,” Cancer Letters, vol. 310, no. 1, pp. 61–68, 2011. View at Publisher · View at Google Scholar · View at Scopus
  144. G. Benegiamo, M. Vinciguerra, G. Mazzoccoli, A. Piepoli, A. Andriulli, and V. Pazienza, “DNA methyltransferases 1 and 3b expression in Huh-7 cells expressing HCV core protein of different genotypes,” Digestive Diseases and Sciences, vol. 57, no. 6, pp. 1598–1603, 2012. View at Publisher · View at Google Scholar · View at Scopus
  145. M. Ripoli, R. Barbano, T. Balsamo et al., “Hypermethylated levels of E-cadherin promoter in Huh-7 cells expressing the HCV core protein,” Virus Research, vol. 160, no. 1-2, pp. 74–81, 2011. View at Publisher · View at Google Scholar · View at Scopus
  146. H. Quan, F. Zhou, D. Nie et al., “Hepatitis C virus core protein epigenetically silences SFRP1 and enhances HCC aggressiveness by inducing epithelial-mesenchymal transition,” Oncogene, vol. 33, no. 22, pp. 2826–2835, 2014. View at Publisher · View at Google Scholar · View at Scopus
  147. K. C. Lakshmaiah, L. A. Jacob, S. Aparna, D. Lokanatha, and S. C. Saldanha, “Epigenetic therapy of cancer with histone deacetylase inhibitors,” Journal of Cancer Research and Therapeutics, vol. 10, no. 3, pp. 469–478, 2014. View at Publisher · View at Google Scholar · View at Scopus
  148. K. B. Glaser, “HDAC inhibitors: clinical update and mechanism-based potential,” Biochemical Pharmacology, vol. 74, no. 5, pp. 659–671, 2007. View at Publisher · View at Google Scholar · View at Scopus
  149. K. Tohyama, “Editorial [hot topic: new treatment strategy of the myelodysplastic syndromes],” Current Pharmaceutical Design, vol. 18, no. 22, pp. 3147–3148, 2012. View at Publisher · View at Google Scholar · View at Scopus
  150. E. A. Griffiths and S. D. Gore, “Epigenetic therapies in MDS and AML,” in Epigenetic Alterations in Oncogenesis, pp. 253–283, Springer, Berlin, Germany, 2013. View at Google Scholar
  151. Q. Mei, M. Chen, X. Lu et al., “An open-label, single-arm, phase I/II study of lower-dose decitabine based therapy in patients with advanced hepatocellular carcinoma,” Oncotarget, vol. 6, no. 18, pp. 16698–16711, 2015. View at Publisher · View at Google Scholar · View at Scopus
  152. S. Venturelli, A. Berger, T. Weiland et al., “Differential induction of apoptosis and senescence by the DNA methyltransferase inhibitors 5-azacytidine and 5-aza-2′-deoxycytidine in solid tumor cells,” Molecular Cancer Therapeutics, vol. 12, no. 10, pp. 2226–2236, 2013. View at Publisher · View at Google Scholar · View at Scopus
  153. S. Timmermann, P. W. Hinds, and K. Münger, “Re-expression of endogenous p16(ink4a) in oral squamous cell carcinoma lines by 5-aza-2'-deoxycytidine treatment induces a senescence-like state,” Oncogene, vol. 17, no. 26, pp. 3445–3453, 1998. View at Google Scholar · View at Scopus
  154. S.-I. Suh, H.-Y. Pyun, J.-W. Cho et al., “5-Aza-2′-deoxycytidine leads to down-regulation of aberrant p16INK4A RNA transcripts and restores the functional retinoblastoma protein pathway in hepatocellular carcinoma cell lines,” Cancer Letters, vol. 160, no. 1, pp. 81–88, 2000. View at Publisher · View at Google Scholar · View at Scopus
  155. K. B. Chiappinelli, C. A. Zahnow, N. Ahuja, and S. B. Baylin, “Combining epigenetic and immunotherapy to combat cancer,” Cancer Research, vol. 76, no. 7, pp. 1683–1689, 2016. View at Publisher · View at Google Scholar
  156. A. E. Dear, “Epigenetic modulators and the new immunotherapies,” The New England Journal of Medicine, vol. 374, no. 7, pp. 684–686, 2016. View at Publisher · View at Google Scholar · View at Scopus
  157. S. Venturelli, S. Armeanu, A. Pathil et al., “Epigenetic combination therapy as a tumor-selective treatment approach for hepatocellular carcinoma,” Cancer, vol. 109, no. 10, pp. 2132–2141, 2007. View at Publisher · View at Google Scholar · View at Scopus
  158. S. Venturelli, A. Berger, T. Weiland et al., “Dual antitumour effect of 5-azacytidine by inducing a breakdown of resistance-mediating factors and epigenetic modulation,” Gut, vol. 60, no. 2, pp. 156–165, 2011. View at Publisher · View at Google Scholar · View at Scopus
  159. Y. Kuang, A. El-Khoueiry, P. Taverna, M. Ljungman, and N. Neamati, “Guadecitabine (SGI-110) priming sensitizes hepatocellular carcinoma cells to oxaliplatin,” Molecular Oncology, vol. 9, no. 9, pp. 1799–1814, 2015. View at Publisher · View at Google Scholar · View at Scopus
  160. A. Ilyas, Z. Hashim, and S. Zarina, “Effects of 5′-azacytidine and alendronate on a hepatocellular carcinoma cell line: a proteomics perspective,” Molecular and Cellular Biochemistry, vol. 405, no. 1-2, pp. 53–61, 2015. View at Publisher · View at Google Scholar · View at Scopus
  161. V. M. Richon, J. Garcia-Vargas, and J. S. Hardwick, “Development of vorinostat: current applications and future perspectives for cancer therapy,” Cancer Letters, vol. 280, no. 2, pp. 201–210, 2009. View at Publisher · View at Google Scholar · View at Scopus
  162. L. Barbarotta and K. Hurley, “Romidepsin for the treatment of peripheral T-cell lymphoma,” Journal of the Advanced Practitioner in Oncology, vol. 6, no. 1, pp. 22–36, 2015. View at Google Scholar
  163. S. P. Iyer and F. F. Foss, “Romidepsin for the treatment of peripheral T-cell lymphoma,” Oncologist, vol. 20, no. 9, pp. 1084–1091, 2015. View at Publisher · View at Google Scholar · View at Scopus
  164. I.-T. Chiang, Y.-C. Liu, W.-H. Wang et al., “Sorafenib inhibits TPA-induced MMP-9 and VEGF expression via suppression of ERK/NF-kappaB pathway in hepatocellular carcinoma cells,” In Vivo, vol. 26, no. 4, pp. 671–681, 2012. View at Google Scholar · View at Scopus
  165. F.-T. Hsu, Y. -C. Liu, I.-T. Chiang et al., “Sorafenib increases efficacy of vorinostat against human hepatocellular carcinoma through transduction inhibition of vorinostat-induced ERK/NF-κB signaling,” International Journal of Oncology, vol. 45, no. 1, pp. 177–188, 2014. View at Publisher · View at Google Scholar · View at Scopus
  166. Food and Drug Administration, “Food and Drug Administration. FDA approves Beleodaq to treat rare, aggressive form of non-Hodgkin lymphoma. July 3, 2014,” FDA Approves Beleodaq to Treat Rare, Aggressive Form of Non-Hodgkin Lymphoma, July 2014.
  167. M. Mottamal, S. Zheng, T. L. Huang, and G. Wang, “Histone deacetylase inhibitors in clinical studies as templates for new anticancer agents,” Molecules, vol. 20, no. 3, pp. 3898–3941, 2015. View at Publisher · View at Google Scholar · View at Scopus
  168. S. L. Chan, H. C. Chung, L. Wang et al., “Efficacy of belinostat in advanced hepatocellular carcinoma (HCC): phase I and II multicentered study of the mayo phase 2 consortium (P2C) and the cancer therapeutics research group (CTRG),” in Proceedings of the ASCO Annual Meeting Proceedings, p. 259, 2012.
  169. A. Milano, R. V. Iaffaioli, and F. Caponigro, “The proteasome: a worthwhile target for the treatment of solid tumours?” European Journal of Cancer, vol. 43, no. 7, pp. 1125–1133, 2007. View at Publisher · View at Google Scholar · View at Scopus
  170. J. L. Spratlin, T. M. Pitts, G. N. Kulikowski et al., “Synergistic activity of histone deacetylase and proteasome inhibition against pancreatic and hepatocellular cancer cell lines,” Anticancer Research, vol. 31, no. 4, pp. 1093–1103, 2011. View at Google Scholar · View at Scopus
  171. K. Weintraub, “Take two: combining immunotherapy with epigenetic drugs to tackle cancer,” Nature Medicine, vol. 22, no. 1, pp. 8–10, 2016. View at Publisher · View at Google Scholar · View at Scopus
  172. J. S. You and P. A. Jones, “Cancer genetics and epigenetics: two sides of the same coin?” Cancer Cell, vol. 22, no. 1, pp. 9–20, 2012. View at Publisher · View at Google Scholar · View at Scopus