Hepatitis B virus X gene and hepatocarcinogenesis
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- Ng, S. & Lee, C. J Gastroenterol (2011) 46: 974. doi:10.1007/s00535-011-0415-9
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Chronic hepatitis B virus (HBV) infection has been identified as a major risk factor in hepatocellular carcinoma (HCC), which is one of the most common cancers worldwide. The pathogenesis of HBV-mediated hepatocarcinogenesis is, however, incompletely understood. Evidence suggests that the HBV X protein (HBx) plays a crucial role in HCC development. HBx is a multifunctional regulator that modulates transcription, signal transduction, cell cycle progression, apoptosis, protein degradation pathways, and genetic stability through interaction with host factors. This review describes the current state of knowledge of the molecular pathogenesis of HBV-induced HCC, with a focus on the role of HBx in hepatocarcinogenesis.
KeywordsHepatitis B virusHBxHepatocellualar carcinomaHepatocarcinogenesis
Hepatocellular carcinoma (HCC) is one of the most common cancers in the world with an annual incidence of more than 500,000 in the year 2000 [1, 2]. Its incidence is increasing in many countries. Significant differences in the geographic distribution of HCC incidence have led to the identification of chronic hepatitis B virus (HBV) infection as a major risk factor for HCC [1, 3]. In recent estimates, 53% of HCC cases worldwide are related to HBV . Studies have also estimated that hepatitis B surface antigen (HBsAg) carriers have a 25–37 times increased risk of developing HCC compared to non-infected people [4, 5]. Other risk factors for HCC include chronic hepatitis C virus infection, exposure to aflatoxin B1, and alcohol abuse. HCC patients have a dismal prognosis, with a 5-year survival rate of 6.5% and a median survival of less than 6 months [6, 7].
Although there is compelling evidence that HBV is a major etiologic factor in HCC, the association of chronic HBV infection with HCC remains obscure. Some of the mechanisms that have been suggested for HBV-mediated hepatocarcinogenesis include: persistent inflammation [8, 9] and viral integration resulting in chromosomal instability and insertional mutagenesis [10–17]; as well as the expression of certain viral proteins such as HBV X protein (HBx) and HBV surface antigens, which may exert their effects on cell cycle, cell growth, and apoptosis by interfering with cell signaling and transcription [14, 18–27].
This review will focus on the HBx gene, where much of the research on HBV-mediated hepatocarcinogenesis has been focused.
HBV, HBV X gene, and HBx
The HBx gene is maintained and transcribed in most integrated viral DNA and a few studies have now demonstrated both HBx RNA and protein expression in human HCC tumorous cells in the absence of HBV replication [30, 31]. Some studies have reported that HBx antibodies were more frequently encountered in patients with HCC than in those with chronic hepatitis B without cancer, suggesting a possible role of HBx in hepatocarcinogenesis [32, 33]. HBx appears to function as a regulatory protein for viral replication and is indispensable for the infectivity of woodchucks with woodchuck hepatitis virus (WHV), a member of the hepadnavirus family [34, 35]. Studies of HBx transgenic mice have also investigated the hepatocarcinogenic effect of HBx. Some studies reported that HBx transgenic mouse cells showed characteristics of malignant transformation [36, 37]. However, others demonstrated that although HBx transgenic mice exhibit no obvious pathology , these mice demonstrate an increased susceptibility to chemical carcinogens  or an acceleration of c-myc-induced HCC . These in vivo data indicate that HBx may at least function as a cofactor in hepatocarcinogenesis. Furthermore, HBV has been implicated in the process of hepatocarcinogenesis, as its DNA has been demonstrated to be integrated into the host genome in 85–90% of HBV-related HCC and this event precedes the development of HCC . It is, however, important to note that HBV integration, while commonly found in HBV-related HCC, may not always be required for HCC development. Nevertheless, HBV integration into the host genome appears to be an important step, and particularly that of HBx, as (1) a significant percentage of viral-host junctions are localized at the carboxyl-terminal part of the X gene conserving HBx function and (2) HBx is regularly detected in the tumors of HBV-associated HCC patients, unlike other HBV transcripts [41–44].
Effects of HBx
Summary list of studies examining role of HBx in hepatocarcinogenesis
1. Upregulation of interleukin (IL)-6
In vitro studies of transfected cell lines (Huh-7, HepG2 and X-expressing HepG2-4X) using calcium phosphate precipitation method
Lee et al. 
2. Induction of nitric oxide synthetase
In vitro studies of transfected cell lines: (i) Chang liver cell line, HepG2, and the 2.2.15 cell lines; (ii) HepG2 cell line transfected using Oligofectamine (Invitrogen, Life technologies, CA, USA)
3. Induction of Fas ligand
In vitro studies of transfected human hepatoma cell lines (Chang liver, SK-HEP-1, Hep
G2, Hep 3B, PLC/PRF/5, MOLT-4, Hep G2.2.15, SNU-182, SNU-354,
SNU-368, SNU-387, SNU-398, SNU-423, SNU-449 and SNU-475) with transient transfection of PLC/PRF/5 using Lipofectin (Invitrogen, Life technologies, CA, USA) and OPTI-MEM (Gibco/BRL) (Invitrogen, Life technologies, CA, USA)
Shin et al. 
4. Inhibition of Fas-mediated apoptosis and upregulation of SAPK/JNK pathway
In vitro studies of cell lines: (i) transduction of HepG2 cells with recombinant retroviruses encoding HBxAg or CAT; (ii) Chang liver cells and DP-16-1 were transfected using Lipofectamine (Life Technologies), while primary human hepatocytes and mouse fibroblast cell lines were transfected using SuperFect (Qiagen, CA, USA)
5. Ras/Raf-1/MAP-signalling pathway
In vitro studies of transfected cell lines: (i) human HeLa cells transfected using calcium phosphate coprecipitation method; (ii) Chang cells transfected using calcium phosphate coprecipitation method; (iii) HepG2, HeLa, AR4-2J, DME-H21, CV-1, COS-7, F9, Ltk- cells, DME-H16 transfected using calcium phosphate coprecipitation, while Jurkat cells were transfected using DEAE dextran (GE Healthcare Biosciences, PA, USA); (iv) Chang cells were transfected using the polyethylenimine (PEI; Polysciences, PA, USA) method
6. Induction of JAK-STAT pathway
In vitro studies of transfected cell lines (Hepa 1–6) using Lipofectin
Lee and Yun 
7. VEGF, MMP2, MMP9, and MMP14
In vitro studies of transfected cell lines
Liu et al. 
8. Upregulation of HSP90alpha
In vitro studies of transfected cell lines (HepG2) by transfection with either an empty vector pcDNA3 or pcDNA3-X encoding the corresponding full-length HBx sequence (nt1374–1838)
Li et al. 
9. Induction of MTA-1
In vitro studies of transfected cell lines (HepG2, HEK 293 and NIH3T3 cells) using Oligofectamine (Invitrogen)
Bui-Nguyen et al. 
Nuclear factor of activated T cells (NF-AT)
1. Activation of tumor necrosis factor (TNF)-α
In vitro studies of transfected cell lines (CHL cells) using the DOSPER reagent (Boehringer Mannheim, Ingelheim, Germany)
Lara-Pezzi et al. 
Extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs)
1. Induction of AP-1
In vitro studies of cell lines (HepG2 cells) infected with adenovirus vectors
Benn et al. 
2. Shifting of TGF-beta signalling from tumor- suppressive pSMAD3C pathway to the oncogenic JNK-dependent pSMAD3L pathway
In vivo studies involving 90 patients with HBV-related chronic liver disease (chronic hepatitis: 70, cirrhosis: 10 and HCC: 10) who underwent liver biopsy
Murata et al. 
3. Over-expression of c-met
In vitro studies of transient transfected cell lines (Phoenix A cells) with infection of MHCC97-H cells with recombinant virus
Xie et al. 
4. Enhancement of androgen receptor-responsive gene
In vitro studies of transfected cell lines (HepG2 and Huh-7 cells) using Lipofectamine 2000 (Invitrogen, Life technologies, CA, USA)
Chiu et al. 
Cell cycle regulators and cell functions
1. Prolongs G1 → S transition via p53-independent pathway (p21waf1/cip1)
In vitro studies of transfected cell lines (stable transfectants of the HBV-X gene in hepatoma (Hep3B-6X, 8X) cell lines)
Park et al. 
2. Decreasing the levels of p15 and p16 and increasing the levels of the active G1-phase proteins p21, p27, cyclin D1, and cyclin E and the activities of CDK4
In vitro studies of transfected cell lines (primary rat hepatocytes) using Lipofectamine 2000 (Invitrogen)
Gearhart et al. 
3. Altered cellular function secondary to HBx binding to DDB1 and redirecting the ubiquitin ligase activity of the cullin 4-DNA-damage-binding protein 1 (CUL4-DDB1) E3 ligase
In vitro studies of transfected cell lines (HeLa cells)
Li et al. 
Early growth response factor
1. Induction of Fas ligand
In vitro studies of transfected cell lines (Chang cells, HepG2, HEK 293 and NIH-3T3 cells) transiently transfected using Polyfect® (Qiagen, CA, USA)
Yoo and Lee 
2. Stabilization of beta-catenin by suppression of glycogen synthase kinase 3 activity via the activation of Src kinase
In vitro studies of transfected cell lines (HepG2, Hep3B, Huh7, Fox Chase, PLC/PRF/5,23 SK-Hep1, and Chang liver) using calcium phosphate precipitation method
Cha et al. 
3. Activation of Erk which associates with GSK-3beta resulting in inactivation of GSK-3beta and upregulation of beta-catenin
In vitro studies of transfected cell lines (pCGN-GSK-3β and pGEX-GSK-3β KD) using Lipofectamine 2000 (Invitrogen) to perform the reporter assay, or the Nuclofector 1 System (Amaxa) (Lonza, Cologne, Germany) to perform the Western blot
Ding et al. 
Inflammatory markers (VEGF, COX)
1. Induction of hypoxia-inducible factor 1α (HIF-1α) and activation of CA9
In vitro studies of transfected cell lines: (i) HepG2, SK-HEP-1, SNU-368, and Hepa 1-6 cells; (ii) HEK 293 transfected using calcium phosphate precipitation method; (iii) Rat2 and HepG2 cells transfected using GenePorter II reagent (Genlantis, CA, USA)
2. Increased expression of cyclooxygenase-2 (COX-2) mRNA and protein
In vitro studies of transfected cell lines (Chang and Huh-7 cells) transfected using the polyethylenimine (PEI) method.
Lim et al. 
1. Upregulation of proteins such as MACF1, HMGB1, and Annexin A2 and downregulation of others such as Lamin A/C, resulting in decreased cell adhesion and increased cell migration
In vitro studies of transfected cell lines (HepG2) using Effectene Transfection Reagent (Qiagen, CA, USA) and Nucleofection Solution V (Amaxa) (Lonza, Cologne, Germany)
Feng et al. 
Human telomerase reverse transcriptase
1. Increased telomerase activity and hTERT expression
In vitro studies of transfected cell lines (HepG2, SMMC7721, HepG2.2.15, Bel7402, and COS-7) transiently transfected using Lipofectamine 2000 (Invitrogen)
Liu et al. 
1. Induction of Fas ligand by transactivation of NFκB
In vitro studies of transfected human hepatoma cell lines (Chang liver, SK-HEP-1, Hep G2, Hep 3B, PLC/PRF/5, MOLT-4, Hep G2.2.15, SNU-182, SNU-354,
SNU-368, SNU-387, SNU-398, SNU-423, SNU-449, and SNU-475) with transient transfection of PLC/PRF/5 using Lipofectin and OPTI-MEM (Gibco/BRL)
Shin et al. 
2. Induction of Fas ligand by enhancing transcriptional activity of Egr-2 and Egr-3
In vitro studies of transfected cell lines (HepG2, Chang, Chang X-34, NIH-3T3, and 293 cells) using Polyfect®
Yoo and Lee 
3. Induction of IL-18 which upregulates FasL
In vitro studies of transfected cell lines (Chang X-34 cells, SNU-354, SNU-368, SNU-387, SNU-398, and SNU-423) and in vivo mouse models (HBx homozygotes)
Lee et al. 
4. Induction of NF-kappaB activation by direct interaction with TNFR1 and thereby induced TNF-alpha production
In vitro studies of transfected cell lines using transient transfections using the Lipofectin reagent (Gibco) and in vivo transgenic mouse models
Kim et al. 
5. Upregulation of Bax and stimulation of TRAIL-induced apoptosis
In vitro studies of transfected cell lines (BEL7402) using Lipofectamine 2000 (Invitrogen) and in vivo mouse models (HBV complete genome (ayw) subtype transgenic BALB/c mice)
Liang et al. 
6. Inhibition of Bcl-xL
In vitro studies of transfected cell lines (Hep3B) using Lipofectamine 2000 (Invitrogen)
Miao et al. 
7. Activation of cyclin B1-CDK1 kinase
In vitro studies of transfected cell lines
Cheng et al. 
8. p53-induced apoptosis
In vitro studies of transfected cell lines (REF-52, NIH 3T3, BALByc3T3, and MEFs) that carry p53val135 temperature-sensitive allele
Chirillo et al. 
9. Stimulation of CD8+ T cells
In vivo mouse models (C57BL/6-based nude mice)
Chun et al. 
10. HBx-independent transactivation and stimulation of apoptosis
In vitro studies of transfected cell lines: (i) Chang liver cells transfected using calcium phosphate precipitation method; (ii) MHP53N, MHXP53N, and MMHD3 cells transfected using calcium phosphate precipitation method
11. Inhibition of Fas-mediated apoptosis and upregulation of SAPK/JNK pathway
In vitro studies of transfected cell lines (Chang liver cells, human primary hepatocytes, DP-16-1 cells, and mouse fibroblast cells) using Lipofectamine 2000 (Invitrogen) or SuperFect (Qiagen, Valencia, CA, USA)
Diao et al. 
12. Affects apoptosis via binding with UVDDB
In vitro studies of transfected cell lines: (i) Chang cells transfected using calcium phosphate precipitation method or Lipofectamine-Plus (Invitrogen, Life technologies, CA, USA); (ii) Chang cells transfected using calcium phosphate precipitation method
13. Can be pro- or anti-apoptosis depending on transcription of p21 which is affected by interaction of HBx and p53 in a concentration-dependent manner
In vitro studies of transfected cell lines (HepG2, Hep3B, and NIH3T3 cells) using calcium phosphate precipitation method
Ahn et al. 
1. Complexes with p53 in the cytoplasm, partially preventing its nuclear entry and ability to induce apoptosis
In vitro studies of transfected cell lines (GMO7532, HepG2, and normal human hepatocytes) using Lipofectin (GIBCO/BRL)
Elmore et al. 
2. HBx abrogates p53-induced apoptosis by direct interaction with p53
In vivo studies (liver biopsies from 10 HBV-infected patients with primary HCC)
Feitelson et al. 
3. Induction of the serine protease hepsin
In vitro studies of transfected cell lines/in vivo immunoprecipitation assays: HepG2cells co-transfected using calcium phosphate coprecipitation method
Zhang et al. 
4. Upregulation of survivin
In vitro studies of transfected cell lines/in vivo immunoprecipitation assays: H7402 and LO2 cells transfected using Lipofectamine (Gibco); and samples from 34 HCC tissues and adjacent tumor tissues, 30 cases of liver cirrhosis, and 6 normal livers without HBV infection obtained at autopsy
Zhang et al. 
5. Downregulation of ASPP1 and ASPP2 genes by DNA methylation
In vivo assays (HCC cell lines and tissues from HCC patients were used to examine the expression and methylation of ASPP1 and ASPP2)
Zhao et al. 
6. Downregulation of c-fos by c-myc
In vitro studies of transfected cell lines (Huh-7 cells) using Lipofectin (Invitrogen)
Kalra and Kumar 
1. Downregulation of mitochondrial enzymes involved in electron transport in oxidative phosphorylation (complexes I, III, IV, and V), increase in mitochondrial reactive oxygen species and lipid peroxide production
In vitro studies of transfected cell lines: (i) HepG2 cells; (ii) HepG2 and HuH7 cells; (iii) HepG2 and HuH7 cells transfected using calcium phosphate coprecipitation method
2. Regulation of HSP60 and HSP70
In vitro studies of transfected cell lines: (i) HEK293 and Huh-7 cells transfected using Effectene transfection reagent (Qiagen, Hilden, Germany); (ii) HepG2 and COS7 Lipofectamine 2000 (Invitrogen)
3. Regulation of VDAC3
In vitro studies with synthesis of HBx and HVDAC3 was carried out by using the TNT-coupled transcription-translation system (Promega, WI, USA); COS cells transiently cotransfected with pVDAC3 and pCXF using Superfect reagent (Qiagen)
Rahmani et al. 
(c) Modulation of repair
1. Modulation of transcriptional activation function of p53, increased sensitivity to UV damage and inhibition of DNA repair
In vitro studies of transfected cell lines (HepG2) using Superfect reagent (Qiagen)
Lee et al. 
2. Inhibition of transcription-coupled nucleotide excision repair via p53-dependent (transcription factor IIH (TFIIH), transcription NER factors, including XPB and XPD
In vitro studies of transfected cell lines: (i) TK6 and NH32 cells using Lipofectamine 2000 (Invitrogen); (ii) HepG2 and RKO-E6 cells using Lipofectin (Life Technology, Gaithersburg, MD, USA); (iii) HepG2 transiently transfected using Lipofectamine (Gibco/BRL, Burlington, Ontario)
3. Downregulates XPB, XPD
In vitro studies of transfected cell lines (HepG2) using Lipofectamine (Gibco/BRL, Burlington, Ontario)
Groisman et al. 
4. Binds to DDB1, compromising nucleotide excision repair
In vitro studies of transfected cell lines (HepG2) using Lipofectin (Life Technologies)
Becker et al. 
5. Inhibits ERCC3
In vitro studies of transfected cell lines (THLE-5b cells) using Lipofectin (Gibco/BRL)
Wang et al. 
6. Downregulation of hMYHalpha expression and accumulation of mutagenic DNA adduct 8-OHdG
In vitro studies of transfected cell lines (LO2) using the Effectene transfection reagent (Qiagen, Valencia, CA, USA)
Cheng et al. 
(d) Epigenetic changes
1. Upregulates DNA methyltransferases 1 and 3a, thereby downregulating expression of RAR-beta(2), abolished potential of retinoic acid (RA) to downregulate levels of G1-checkpoint regulators including p16, p21, and p27, resulting in activation of E2F1
In vitro studies of transfected cell lines (HepG2) with either pCMV-36HA1 or pCMV-36HA1-HBX3, followed by selection with G418 (Gibco) (Invitrogen, Life technologies, CA, USA) or transiently using WelFect-EX PLUS (WelGENE, Daego, South Korea)
Jung et al. 
2. Repression of E-cadherin transcription by activation of DNA methyltransferase 1
In vitro studies of transfected cell lines (HepG2) using the Fugenet 6 transfection kit (Roche, Bristol, UK) or the calcium phosphate precipitation method
Lee et al. 
3. HBx-induced hypermethylation of p16(INK4a) with positive correlation with DNA DNMT1 and DNMT3A at both mRNA and protein levels
In vivo studies (88 fresh tissue specimens of surgically resected HBV-associated HCC)
Zhu et al. 
4. Downregulation of p16(INK4a) leads to activation of G1-CDKs, phosphorylation of Rb, activation of E2F1, and finally evasion from G1 arrest induced by the premature senescence inducer, hydrogen peroxide
In vitro studies of transfected cell lines (HepG2) with pCMV-3 × HA1 or HBx, followed by selection with G418 (Life Technologies) or using WelFect-EX PLUS (WelGENE)
Kim et al. 
5. Upregulates 7 miRNAs and downregulates 11 miRNAs, including let-7a, which negatively regulates cellular proliferation and upregulation of STAT3
In vitro studies of transfected cell lines (HepG2 and SNU-182 cells)
Wang et al. 
(e) HBx mutations
1. Insert mutation at position 204: Insert 204AGGCCC and point mutations at 260 (G → A) and 264 (G/C/T → A)
In vivo studies (113 tumor tissue samples and 48 serum samples from patients with HCC)
Chen et al. 
2. Complex mutation involving T1766/A1768
In vivo studies (plasma samples of 852 HBsAg-positive subjects and 786 HBsAg-negative subjects)
Guo et al. 
3. Mutations at aa 127, 130, and 131 were frequently detected, but there was no distinct difference in point mutation profiles between tumor and non-tumor samples, whereas deletions in the HBx gene were more frequent in tumor-derived than in non-tumor-derived sequences
In vivo studies (frozen tumor and non-tumor samples from 8 patients with HBV-related HCC)
Iavarone et al. 
4. T1653, T1689, and/or T1762/A1764 mutations were associated with HCC development in Korean patients infected with HBV
In vivo studies (135 HCC patients infected with HBV genotype C2 and 135 HBeAg status-matched patients without HCC)
Kim et al. 
5. Compared to standard genotype B HBx, mutants I127T and I127T+K130 M+V131I showed higher transactivation and anti-proliferative activities, while mutants F132Y, K130 M+V131I, and K130 M+V131I+F132Y showed lower activities; compared to standard genotype C HBx, the I127T mutant showed higher transactivation activity, while four other types of mutants showed no differences, the F132Y and K130 M+ V131I mutants showed lower anti-proliferative activities, and the K130 M+V131I +F132Y mutant showed higher activity, while the I127T and I127T+K130 M+V131I mutants showed no differences
In vitro studies of transfected cell lines (Chang cells) using Lipofectamine 2000 (Invitrogen)
Lin et al. 
6. Distal COOH-terminal region deletion resulting in loss of transcriptional activity, inhibitory effects on cell proliferation and transformation
In vivo studies: (i) 4 HBsAg-positive patients with HCC; (ii) tissue microarray specimens from 194 HBsAg-positive HCC patients and 20 frozen samples of tumor and matched non-tumor tissue and in vitro studies of transfected cell lines: (i) Chang, Huh7, and HepG2 cells using calcium phosphate precipitation method; (ii) HepG2 and MIHA cells transfected using lipofectamine (Life Technologies Bethesda Research Laboratories)
7. HBx-A31 mutation was associated with less effective transactivation of HBV enhancer I-X promoter complex, cell replication, and enhancing TNF-alpha induced increment of CPP32/caspase 3
In vivo studies (liver biopsy samples and sera from 100 patients with chronic hepatitis B and 77 patients with HBV-related HCC) and in vitro studies of transfected cell lines (HepG2) using calcium phosphate precipitation method
Yeh et al. 
8. Truncation mutation at 3′ end of HBx
In vivo studies (tumorous and non-tumorous tissues from 9 HBsAg-negative, HBV DNA-positive patients)
Poussin et al. 
9. Point mutations at X gene codons 130 (AAG → ATG) and 131 (GTC → ATC)
In vivo studies (frozen HCC tissues and adjacent nontumorous livers from 20 patients and sera of 26 patients who were chronically infected with HBV without HCC)
Hsia et al. 
10. Mutations at positions 1762 (A-to-T) and 1764 (G-to-A) within the core promoter
In vivo studies (sera from 40 HBsAg-positive HCC patients)
Takahashi et al. 
11. Deletion from 382 to 401 base pairs (HBxDelta127) promotes cell growth by activating SREBP-1c with involvement of 5-lipooxygenase and FAS
In vitro studies of transfected cell lines (HepG2 and H7402 cells) using Lipofectamine (Invitrogen)
12. HBx mutants have attenuated transactivation and proapoptotic functions but retain their ability to block p53-mediated apoptosis
In vitro studies of transfected cell lines (HHT4 cells) using Lipofectamine Plus Reagent (Invitrogen)
Jiang et al. 
Trans-activating mechanisms of HBx
The enigmatic HBx protein is a promiscuous transactivator that can activate a variety of viral and cellular promoters and enhancers . HBx protein does not bind directly to DNA. Instead, its transcriptional activity is mediated via a protein–protein interaction. The transactivation function of HBx may be exerted both in the cytoplasm, via signaling pathways, and in the nucleus, via DNA-binding proteins. HBx transcriptional activity was reported to be necessary for viral replication [46, 47]. Transcriptional factors directly interacting with HBx, as well as deregulated direct gene-targets of HBx from indirect protein-DNA binding, were recently identified .
HBx has been shown to upregulate the expression of a number of cellular and viral genes including the HBV enhancers, class II and III promoters, and proto-oncogenes such as c-jun, c-fos, and c-myc; as well, HBx has been shown to activate transcriptional factors such as nuclear factor κB (NFκB), activator protein-1 (AP-1), and activating transcription factor (ATF)/cAMP-responsive element binding transcription factor (CREB) in the nucleus [49–54]. Several studies have reported that the transactivation of NFκB by HBx indirectly leads to the upregulation of some host genes such as interleukin (IL)-6  and the induction of nitric oxide synthetase [56, 57] and Fas ligand [58, 59]. Resistance to Fas-mediated apoptosis in HepG2 cells was demonstrated to be responsible for the transactivation of the NFκB family by HBx via SAPK/Janus Kinase (JAK) pathway activation [19, 26], whereas the tumor necrosis factor (TNF)-α signaling pathway was shown to be regulated by HBx via NFAT activation in the cyclosporine A-dependent pathway . HBx also transactivates cellular promoters of genes associated with cell proliferation, such as IL-8, TNF, transforming growth factor (TGF)-β1, and early growth response factor (EGRF) . It has been observed that HBx shifts hepatocytic TGF-β signaling from the tumor-suppressive pSmad3C pathway to the oncogenic pSmad3L pathway in early carcinogenesis . HBx localized in the cytoplasm has been shown to activate signal transduction pathways such as RAS/RAF/ mitogen activated protein kinase (MAPK), JAK/STAT, and Src kinases [50, 63–65]. Stimulation of signaling cascades involving RAS/RAF/MAPK by HBx can activate transcription factors such as AP-1 and NFκB, resulting in the deregulation of cell cycle checkpoint controls [50, 66, 67]. HBx also upregulates the expression of p21waf1/cip1, a key regulatory protein in cell cycle progression, through an ets-binding element in a p53-independent manner . Gearhart and Bouchard demonstrated that HBx induces quiescent, normal hepatocytes to exit G0 but stall in the G1 phase of the cell cycle by decreasing the levels of p15 and p16, which would otherwise inhibit progression from G0 to G1, and increasing the levels of the active G1-phase proteins p21, p27, cyclin D1, and cyclin E and the activities of the G1-phase kinase, cyclin-dependent kinase 4 (CDK4) . HBx also inhibits the activation of CDK2, the late-G1/S-phase CDK . It was also demonstrated that HBx could activate Wnt/β-catenin signaling by stabilizing the cytoplasmic β-catenin through its interaction with Wnt-1  or activation of Erk . Another role that HBx plays is that of binding to DDB1 through an alpha-helical motif and redirecting the ubiquitin ligase activity of the cullin 4-DNA-damage-binding protein 1 (CUL4-DDB1) E3 ligase, which regulates diverse cellular functions . This mimics the DDB1-CUL4-associated factors (DCAFs) and is important for the assembly of cellular and virally hijacked CUL4-DDB1 E3 complexes .
HBx may also contribute to tumorigenesis in HCC through modulation of the angiogenesis pathway. It was reported that HBx expression could induce upregulation of the transcription of the potent angiogenic factor, vascular endothelial growth factor (VEGF), as well as metalloproteinases (MMPs) such as MMP2, MMP9, and MMP14, thereby facilitating invasion and metastasis . HBx can also stabilize or even upregulate the expression of the hypoxia-inducible factor 1α (HIF-1α), which is the main transcriptional activator of carbonic anhydrase 9 (CA9) [73–75]. CA9 is involved in pH regulation, which helps tumor cells overcome intracellular acidosis and survive in hypoxic conditions, thereby aiding in the development of HCC by contributing to the survival of hepatocytes infected with HBV in the fibrotic liver parenchyma .
More recently, there have been several new findings. Firstly, several studies have uncovered various pathways which may be implicated in the increased motility of tumor cells, such as changes in the expression of proteins that may be involved in cell migration and decreased focal adhesion (e.g., upregulation of proteins such as MACF1, HMGB1, and Annexin A2 and downregulation of others such as Lamin A/C); activation of Rho GTPases and Rac1, with mutations at the sites of the proline-rich domain located in HBx; and upregulation of the expression of heat shock protein 90alpha (HSP90alpha) and c-met protein, as well as that of TANK-binding kinase-1 (TBK1), which induces the phosphorylation of NF-kappaB p65 at serine 536, at the transcriptional level [52, 76–79]. Secondly, the upregulation of inflammatory mediators by HBx is another pathway of interest. HBx has been shown to induce the expression of metastasis-associated protein 1 (MTA1) coregulator, which is a master chromatin modifier involved in carcinogenesis and a positive regulator of inducible nitric oxide synthase (iNOS) transcription [57, 80]. The HBx/MTA1 complex stimulates the production of NO, which has been implicated in hepatocarcinogenesis . HBx has also been demonstrated to increase the expression of cyclooxygenase-2 (COX-2) mRNA and protein . These findings provide further evidence that hepatic inflammation may be one of the pathways implicated in hepatocarcinogenesis. Thirdly, the predilection for males to develop HCC may be explained by the effect of HBx on the androgen receptor (AR) pathway, where in vitro studies have shown that HBx increased the anchorage-independent colony-formation potency of AR in a non-transformed mouse hepatocyte cell line, and acted as a positive transcriptional co-regulator to increase AR-mediated transcriptional activity via the c-SRC kinase signaling pathway in a concentration-dependent manner . Finally, HBx has also been shown to increase telomerase activity and human telomerase reverse transcriptase (hTERT) expression, which is associated with hepatocarcinogenesis .
Such modulation effects of HBx have wide-ranging effects on cell growth regulatory checkpoints and on hepatocyte proliferation and apoptosis, contributing to HCC tumorigenesis.
HBx and regulation of apoptosis
Apoptosis is necessary to eliminate redundant, damaged, and virally infected cells. Regulation of apoptosis involves the actions of three general classes of proteins: (i) effectors or initiators of apoptosis (for example, the caspases and upstream receptors, such as TNF receptors and Fas); (ii) inducers and suppressors of apoptosis, such as members of the B-cell lymphoma/leukemia-2-protein (Bcl-2) family of proteins; and (iii) intermediate proteins, which include transcription factors such as p53, Fos, Jun, and Myc .
HBx has been reported to either induce or block apoptosis. Initially, HBx was shown to abrogate p53-induced apoptosis by direct interaction with p53, thereby inactivating several critical p53-dependent activities, including p53-mediated apoptosis [84, 85], the transactivation properties of p53 [85–88], regulation of the cell cycle , tumor suppressor genes , and DNA repair genes [91, 92]. HBx can also exert antiapoptotic functions independently of p53 through the modulation of the serine protease hepsin activities  and the upregulation of survivin . Recently, HBx has also been shown to methylate the ankyrin-repeat-containing, SH3-domain-containing, and proline-rich-region-containing protein (ASPP) promoter, thereby downregulating the expression of ASPP2 and hence promoting tumor growth . However, it has also been shown that HBx can indeed induce apoptosis or sensitize cells to apoptotic death caused by proapoptotic stimuli. HBx has demonstrated proapoptotic effects in the livers of transgenic mice and in primary hepatocyte cultures [96, 97]. HBx protein may promote apoptosis via the p53 protein  or through regulation of the expression of Fas/FasL [58, 59, 97, 99], inactive procaspase-8, Bax/Bcl-2 [100, 101], the c-myc gene , UV-DDB1 [103, 104], the interaction of TNFR1 and NF-κB , and the sustained activation of cyclin B1-CDK1 kinase .
The apparently discrepant effects of HBx on apoptosis could reflect different concentration-dependent effects at various stages of HBV infection [18, 106]. It has been suggested that HBx inhibits apoptosis early during the infection of hepatocytes and later induces apoptosis to facilitate HBV replication and spread, allowing evasion of host cell-mediated immunity [18, 107]. Both the activation and inactivation of apoptosis by HBx could result in malignant cellular transformation, leading to HCC. An antiapoptotic effect may increase the risk of accumulated mutations, while enhanced compensatory hepatocyte proliferation induced by a pro-apoptotic effect may result in the selection of premalignant hepatocytes .
Another possible mechanism underlying HBx-related apoptotic cell death is that of the interaction of HBx with mitochondria, which causes an abnormal aggregation of mitochondrial structures in the cell. HBx exerts powerful effects on mitochondria, either directly through their channel-forming activity or indirectly through associations with endogenous channels [108–110]. Two mitochondrial proteins, heat-shock proteins 60 and 70 [111, 112], as well as the VDAC isoform, VDAC3 , have also been identified as cellular targets of HBx.
Modulation of DNA repair by HBx
There is evidence that HBx interferes at multiple steps with DNA repair, to result in an accumulation of defects in the genome, which may favor cellular transformation [25, 114]. HBx was found to compromise nucleotide excision repair (NER) by binding to DDB1, a subunit of the UV-damaged DNA binding protein (UVDDB) that is bound to damaged DNA, and inactivating the UVDDB [24, 91, 115]. HBx binding to and inactivation of p53 can also compromise both NER and transcription-coupled repair by disrupting the normal interaction of p53 with transcription factor IIH (TFIIH) transcription NER factors, including Xeroderma Pigmentosum B (XPB) and XPD [92, 116], and by disrupting the normal interaction of p53 with the excision-repair cross-complementing 3 (ERCC3) that is involved in transcription-coupled repair . HBx also interferes with NER through p53-independent pathways by downregulating the expression of XPB and XPD . HBx also promotes the growth and malignant transformation of non-tumor hepatic LO2 cells in vitro, with the downregulation of hMYHalpha expression and accumulation of the mutagenic DNA adduct 8-OHdG .
HBx’s role in influencing epigenetics
HBx has the ability to deregulate cellular gene expression at the transcription level through its interaction with many cellular factors.
Jung et al.  also demonstrated that HBx induced promoter hypermethylation of retinoic acid receptor-beta (2) (RAR-beta(2)) via the upregulation of DNA methyltransferases 1 and 3a, thereby downregulating the expression of RAR-beta(2) in human HCC cells. HBx also abolished the potential of retinoic acid (RA) to downregulate levels of G1-checkpoint regulators including p16, p21, and p27, resulting in the activation of E2F1 in the presence of RA . Therefore, HBx-expressing cells were less susceptible to RA-induced cell growth inhibition, and it has been suggested that the epigenetic downregulation of RAR-beta2 by HBx can be an important step during hepatocarcinogenesis .
HBx has also been reported to influence Wnt activation via the repression of E-cadherin transcription through hypermethylation of the E-cadherin promoter by the activation of DNA methyltransferase 1 .
Thirdly, HBx-induced hypermethylation of p16(INK4a) has also been shown by 2 different groups. Zhu et al.  demonstrated that HBx was positively correlated with DNA methyltransferase (DNMT) 1 and DNMT3A at both the mRNA and protein levels, but HBx expression did not correlate with hypermethylation of the p16(INK4A) promoter or p16 protein expression in tumorous tissues. Kim et al.  provide evidence that downregulation of p16(INK4a) expression by HBx leads to the activation of G1-CDKs, phosphorylation of Rb, activation of E2F1, and finally evasion from G1 arrest induced by the premature senescence inducer, hydrogen peroxide.
Finally, Wang et al.  have also recently demonstrated HBx’s ability to deregulate cellular miRNA expression. HBx was shown to upregulate 7 miRNAs and downregulate 11 miRNAs. Particularly of note were the downregulation of Let-7a, which negatively regulates cellular proliferation, and the upregulation of STAT3 . These features allow for the deregulation of cellular proliferation, thereby contributing to hepatocarcinogenesis.
Mutations of HBx
Natural mutants of HBx have been described in the liver and serum of patients with HCC [125–132]. The frequency of such HBx gene mutation reported by several studies is very high, suggesting that mutant HBx may be important in tumorigenesis [125–128, 131, 132].
Some of the most frequently reported mutations are 3′ deletions of HBx [127, 131, 133, 134]. HBx mutations with 3′ end deletions are more frequently encountered in HCC tissue than in the non-tumorous tissue, as demonstrated in some reports examining small groups of patients [131, 134, 135], suggesting the possible role of these mutations in the tumorigenic properties of HBx. Poussin et al.  reported that the HBV X gene was truncated at its 3′ end in five of nine tumorous tissues but only one of eight non-tumorous tissues in a study of nine HCC patients. Tu et al.  demonstrated that the HBx sequences derived from six of seven HCC tissues contained a deletion in the distal COOH-terminal region, and that the mutant HBx had lost their transcriptional activity and inhibitory effects on cell proliferation and transformation. Furthermore, it was shown that COOH-terminally truncated HBx enhanced the transforming ability of ras and myc . A recent study by Ma et al.  also demonstrated that COOH-terminal truncated HBx detected in 111 of 194 HCC tissues contributed to hepatocarcinogenesis via the activation of cell proliferation and loss of proapoptotic ability. Indeed, the size of the deletion at the COOH terminus of HBx appears to affect HBx transcription activity, as several studies have reported that truncated HBx which exhibited the deletion within 14 amino acids retained its transactivation activity [131, 136]. A 14-amino acid deletion was sufficient to abrogate the inhibitory effects of HBx on cell growth and transformation, while an additional 2-amino acid deletion would affect HBx transcription activity . Thus, these last 14 amino acids at the COOH terminus of HBx are deemed unnecessary for transactivation activity [131, 136].
A study conducted by Kumar et al. to localize the regions of HBx important for transactivation demonstrated that deletions of residues 58–84, 85–119, and 120–140 that were separately introduced to HBx all resulted in a drastic loss of function. On the basis of their results, a mutant construct comprising only these regions (residues 58–140) was constructed, which proved to be an efficient transactivator, illustrating the importance of residues 58–140 for transcriptional activity . These regions have also been reported to be important in the interaction of HBx with TATA-binding protein , RPB5 subunit of RNA polymerase , and a serine protease .
Sirma et al.  reported that mutations in the HBx gene evolving in HCC abolished both HBx-induced growth arrest and apoptosis. Furthermore, using a panel of engineered mutants, it was demonstrated that mutations introduced into the transactivation relevant domains of HBx abolished its growth-suppressive effect . Such abrogation of the anti-proliferative and apoptotic effects of natural mutants of HBx renders the hepatocytes susceptible to uncontrolled growth and contributes to HBV-mediated hepatocarcinogenesis.
Certain point mutations may also be involved in modifying the functions of HBx. HBx with mutations in amino acids 130 and 131 has been frequently reported in HCC [140, 141], but its functional consequences are still unclear. Tu et al.  demonstrated that this linked-point mutation (amino acids 130 and 131), together with an additional mutation in amino acid 129, completely abrogated the growth-suppressive and inhibition of focus formation effects of HBx in vitro. Mutations at amino acids 61, 69, and 137 of HBx have also been shown to result in a dramatic loss of transactivation capability . In a recent study of 113 Chinese Hong Kong patients with HCC, a special pattern of insertion at nucleotide 204 and point mutations at nucleotides 260 and 264 was frequently detected in the tumor tissues, and it appears to be associated with the nuclear localization of HBx protein, suggesting a carcinogenetic potential of this mutation pattern .
In another study of six HCC patients by Iavarone et al.  combining laser capture microdissection and polymerase chain reaction (PCR)-derived techniques, it was demonstrated that mutations at amino acids 127, 130, and 131 were frequently detected but there was no distinct difference in point mutation profiles between tumorous and non-tumorous samples derived from the same patient. An in vitro study by Tu et al.  showed that these mutations did not change HBx influence on cell viability and proliferation. In contrast, deletions in the HBx gene that were detected in five of six patients in the study by Iavarone et al.  were more frequent in tumor-derived sequences (6/18) than in non-tumor derived sequences (1/20) .
A cross-sectional case-control study of 135 Korean HCC patients infected with HBV, carried out by Kim et al.  to examine the relationship between HCC and mutations in the enhancer II/core promoter and precore regions of HBV, has led to the identification of the T1653, T1689, and/or T1762/A1764 mutations that were found to be associated with the development of HCC. In another study, by Guo et al. , double mutant T1762/A1764 was also detected and, unexpectedly, the adjacent T1766/A1768 mutation was found to significantly increase the risk of HCC in HBV-infected patients. The prevalence of triple mutations in the HBV basal core promoter was significantly higher in patients with HCC than in those with chronic hepatitis .
Recently, Wang et al. [142, 143] identified a natural mutant of the hepatitis B virus X gene (HBx) with a deletion from 382 to 401 base pairs (HBxDelta127), which was shown to promote hepatoma cell growth in a human hepatoma HepG2 (or H7402) cell line by activating the transcription factor sterol regulatory element-binding protein 1c (SREBP-1c) with the involvement of 5-lipooxygenase and FAS. Jiang et al.  have also demonstrated that certain tumor-derived HBx variants encoded by HBV exhibit attenuated transactivation and proapoptotic functions but retain their ability to block p53-mediated apoptosis. Furthermore, certain HBx variants cooperate with p53-249(ser) to further contribute to hepatocarcinogenesis .
Belloni et al.  reported that the HBx regulatory protein is recruited onto the cccDNA minichromosome, and the kinetics of HBx recruitment on the cccDNA parallel the HBV replication. In an HBV mutant that does not express HBx, its replication is impaired and exogenously expressed HBx transcomplements the replication defects . Hence, it is possible that mutations in HBx may contribute greatly to hepatocarcinogenesis. Therefore, the true impact of structural deletions and point mutations on the regulation of the biological function of HBx should be further evaluated.
Interestingly, from a potential therapeutic perspective, the knockdown of HBx by RNA interference results in decreased HBx expression and cessation of cell growth in certain cell lines, as well as the sensitization of the cells to chemotherapeutic agents (i.e., 5-fluorouracil and cisplatin) [146, 147]. The reduced tumorigencity resulting from the knockdown of HBx, coupled with findings that HBV-DNA integration has been demonstrated in HCC cells and that the HBx coding region has also often been included in the HBV-DNA integrant, provide further support to the notion that HBx is involved in hepatocarcinogenesis [146, 148]. Xian et al.  also reported that p53 can reduce the levels of HBx protein and shorten its half life, but that this process may be MDM2-dependent. These findings may provide further research opportunities for therapeutic advances in the treatment of HCC.
At present, HBV-associated hepatocarcinogenesis can be viewed as a multi-factorial process that includes both direct and indirect mechanisms that may act cooperatively. However given the avalanche of studies implicating many different molecules and pathways in the development of HCC, the exact mechanism/s through which HBx induces hepatocarcinogenesis remain/s quite controversial. As such, more integrated studies are needed to comprehensively understand the role of HBx in HCC. Furthermore, most current studies have primarily examined the effects of the in vitro over-expression of the HBx gene in cells, a finding which is consistent with the observation that the HBx protein was found to be over-expressed in the tumors of HCC compared to the adjacent non-tumorous liver tissues . Nonetheless, the extrapolation of the in vivo effects of HBx remains to be determined. With the advent of new technologies, further exploration of the molecular mechanisms underlying HBV-associated HCC development will enhance our understanding in this arena. Biologically elucidating the role of HBx in hepatocarcinogenesis may ultimately lead to novel therapeutic strategies for managing HBV-associated HCC patients, as well as contribute to the clinical therapy of HCC.