Hepatitis B virus x protein in the pathogenesis of hepatitis B virus-induced hepatocellular carcinoma

Authors

  • Michael C Kew

    1. Department of Medicine, University of Cape Town and Groote Schuur Hospital, Cape Town, and Department of Medicine, University of the Witwatersrand, Johannesburg, South Africa
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  • Conflict of Interest
    The author does not any conflicts of interest to disclose.

Michael C Kew, Department of Medicine, K Floor, Old Main Hospital Building, Groote Schuur Hospital, Main Road, Observatory 7935, Cape Town, South Africa. Email: michael.kew@uct.ac.za

Abstract

Currently available evidence supports a role for the hepatitis B virus (HBV) x gene and protein in the pathogenesis of HBV-induced hepatocellular carcinoma (HCC). HBx gene is often included, and remains functionally active, in the HBV DNA that is frequently integrated into cellular DNA during hepatocellular carcinogenesis. HBx protein promotes cell cycle progression, inactivates negative growth regulators, and binds to and inhibits the expression of p53 tumour suppressor gene and other tumour suppressor genes and senescence-related factors. However, the molecular mechanisms responsible for HBx protein-induced HCC remain uncertain. Only some of the more fully documented or more recently recognised mechanisms are reviewed. During recent years evidence has accumulated that HBx protein modulates transcription of methyl transferases, causing regional hypermethylation of DNA that results in silencing of tumour suppressor genes, or global hypomethylation that results in chromosomal instability, thereby playing a role in hepatocarcinogenesis. HBx protein has both anti-apoptotic and pro-apoptotic actions, apparently contradictory effects that have yet to be explained. Particularly important among the anti-apoptotic properties is inhibition of p53. Recent experimental observations suggest that HBx protein may increase the expression of TERT and telomerase activity, prolonging the life-span of hepatocytes and contributing to malignant transformation. The protein also interferes with nucleotide excision repair through both p53-dependent and p53- independent mechanisms. Carboxy-terminal truncated HBx protein loses its inhibitory effects on cell proliferation and pro-apoptotic properties, and it may enhance the protein's ability to transform oncogenes. Dysregulation of IGF-II enhances proliferation and anti-apoptotic effects of oncogenes, resulting in uncontrolled cell growth.

Abbreviations:
AP-1

Activator protein-1

ASPP1 and ASPP2

ankyrin-repeat-containing, SH3-domain-containing, and proline- rich region-containing proteins 1 and 2

Bcl

B-cell lymphoma/leukaemia

COX-2

Cyclo-oxygenase 2

DNMT

DNA methyl transferase

ERCC1

excision repair cross-complementing complex 1

FAS

Fatty acid synthase

GSTP1

glutathione-S-transferase P1

HURP

hepatoma up-regulating protein

IGFBP-3

Insulin-like growth factor binding -3

IGF-II

Insulin-like growth factor II

MAPK

mitogen activated protein kinase

MAZ

myc-associated zinc-finger protein

PGE2

prostaglandin E2

RAR-β2

retinoic acid receptor-beta 2

RPA

replication protein A

TERT

Telomerase reverse transcriptase

TNF-α

Tumour necrosis factor-α

TRAIL

TNF-related apoptosis-inducing ligand

UV-DDB

ultraviolet-DNA-damaged binding protein

Introduction

Hepatocellular carcinoma (HCC) is one of the major malignant diseases in the world today. It ranks fifth in overall frequency and is either the most common tumour, or among the three most common tumours, in many of the most populous regions.1 More importantly, HCC ranks third in annual cancer mortality rates and has the highest annual fatality ratio of any tumour.1 Chronic hepatitis B virus (HBV) infection is the dominant global cause of HCC, accounting for 55% of cases worldwide and 80% or more of those in the eastern Pacific region and sub-Saharan Africa, the highest incidence regions of the tumour.1 The mechanisms whereby HBV causes malignant transformation remain uncertain. However, much of the evidence available supports a pathogenetic role forthe product of the HBV x gene, the HBx protein. HBV DNA is integrated into the chromosomal DNA of hepatocytes in the great majority of patients with HBV-induced HCC,2,3 and this is believed to play a pivotal role in the pathogenesis of the tumour. An exhausting number of putative mechanisms by which HBx protein might contribute to the development of HCC have been investigated—so much so that, as was aptly stated by Murakami4: “If there is a protein that is not among the list of HBx interactors, then this protein has probably not yet been tested”. Despite the plethora of experimental data published, the molecular genesis of HBx protein-induced HCC remains uncertain. It is certain, however, that the pathogenesis of the tumour will prove to be multifactorial, and that the mechanisms involved will include activation of oncogenes and silencing of tumour suppressor genes.

Rather than attempt a comprehensive, albeit tedious, review of the close to 450 scientific papers published during the past 12 years concerning postulated pathogenetic mechanisms of HBx protein-induced HCC, only the more fully documented or more recent information available in this regard will be reviewed.

Structure and functions of hepatitis B virus x protein

HBx gene is the smallest of the four partially overlapping open reading frames of HBV. It comprises 452 nucleotides that encode a 154-amino acid regulatory protein with a molecular mass of 17 kDa. HBx was the name assigned to the gene and protein because the deduced amino acid sequence did not show homology to any known protein.5 The protein is highly conserved among the different subtypes of the virus, and is common to all mammalian, but no avian, members of the Hepadnaviridae. It is present in the cytoplasm and, to a lesser extent, the nucleus of hepatocytes.

The functions of HBx protein are not fully understood, although it is known to play a regulatory role in HBV replication and is required for the establishment of viral infection.4 The protein is multi-functional. It functions by protein-protein interaction, and is a promiscuous transactivator of viral and cellular promoters and enhancers.6 HBx protein does not bind directly to DNA, but causes transcriptional activation by its interaction with nuclear transcription factors and modulation of cytoplasmic signal transduction pathways, including the Ras, Raf, c-jun, MAPK, NFκB, Jak-Stat, FAK, and protein kinase C pathways, as well as Src-dependent and phosphatiylinositol-3 kinase signalling cascades.4,6,7 Transcriptional activation is thought to be essential for replication of the virus.6 Activation of these signalling pathways may also contribute to HBx protein-mediated hepatocellular carcinogenesis through transactivation of cellular signalling cascades and oncogenes that stimulate proliferation of hepatocytes.8 HBx protein promotes cell cycle progression and inactivates negative growth regulators. In addition, it binds to and inactivates or down-regulates p53 tumour suppressor gene,9 as well as other tumour suppressors and senescence-related factors.8,9

Evidence for the hepatocarcinogenicity of hepatitis B virus x protein

Humans, woodchucks, and ground squirrels infected with their respective Orthohepadnaviruses, each of which encode the x protein, develop HCC, whereas birds infected with the other members of the Hepadnaviridae, the Avihepadnaviruses, which do not encode the x protein, do not develop the tumour.2

Although HBV DNA integration almost invariably results, to a greater or lesser degree, in loss and rearrangement of viral sequences, the HBx gene is the part of the HBV DNA that is most often included in integrants in the chromosomal DNA of patients with HCC.3 Moreover, integrated HBx gene, even when truncated, often encodes functionally active transactivator proteins and has been shown to express HBx protein.4

HBx protein acts as an oncogene in experimental hepatocellular carcinogenesis. It transforms rodent hepatocytes in vitro,10 and HBx encoding sequences persist in clonally expanding normal and malignant rodent hepatocytes.10 In addition, the protein transforms NIH3T3 cells in co-operation with ras,11 and in transgenic mice without cirrhosis it accelerates the development of HCC in the presence of myc.12,13 HBx protein also increases the susceptibility of these mice to develop HCC when exposed to the carcinogen, diethylnitrosamine.14 Moreover, RNA interference (RNAi) markedly reduces HBx mRNA and protein levels and the tumorigenicity of HCC cells that constitutively express HBx protein.15

Mechanisms of hepatitis B virus x protein-induced hepatocarcinogenesis

Epigenetic modification

Early research into the pathogenesis of HBx protein-induced HCC, as well as other aetiological forms of the tumour, was focused on changes at a genetic level. During recent years, epigenetic changes, resulting either in inactivation (silencing) of tumour suppressor genes or chromosomal instability, have been shown to play an increasingly important role in hepatocarcinogenesis. Epigenetic changes refer to changes in gene expression mediated by mechanisms other than alterations in the nucleotide sequence of that gene. They consist essentially of changes in methylation, either hypermethylation or hypomethylation. An important result of hypermethylation of DNA is inactivation of tumour suppressor genes.16,17 This involves hypermethylation of DNA of genes rich in CpG islands in the promoter regions of the tumour suppressor genes. Malignant transformation may also result from chromosomal instability caused by global hypomethylation of DNA.

DNA hypermethylation is catalysed by a family of DNMTs, including DNMT1, DNMT3A, and DNMT3B. This family catalyses the addition of a methyl group to the 5′CpG dinucleotide of the cytosine ring to form methylcytosine, using S-adenosylmethionine as the methyl donor.16,17 The normal function of DNMT1 is to re-establish the DNA methylation pattern during replication, whereas DNMT3A and DNMT3B function as de novo DNMTs. They co-operate to maintain regional hypermethylation of tumour suppressor genes.16,17

Recent studies of virus-associated tumours have shown that aberrant hypermethylation of CpG islands in tumour suppressor genes, resulting from up-regulation of the activity of DNMTs, represses their transcription and silences them, thereby playing a role in the pathogenesis of tumours.16 The importance of this mechanism in hepatocellular carcinogenesis was shown by the observation that methylation of at least one tumour suppressor gene was present in 82% of the tumours studied,18 although the prevalence specifically in HBx protein-induced HCCs has not been published.

HBx protein can modulate the transcription of DNMT1 and DNMT3. By virtue of this ability the protein acts as a potent epigenetic deregulating agent; and epigenetic changes in a number of tumour suppressor genes have been described in patients with HBx protein-induced HCC.

By inducing DNMT1 transcription, HBx protein represses the expression of the tumour suppressor gene, E-cadherin.19,20 As a consequence of such repression, the CpG island1 of the E-cadherin promoter is inactivated. Lysine-30 in place of methionine-30 in the transactivating domain of HBx protein is essential to the repression of E-cadherin by the protein.19 This result might be achieved by way of up-regulation of AP-1, because the DNMT1 promoter contains the binding motifs of the AP-1 complex.20

The p16INK4A gene encodes a negative regulator of cell cycle progression, and is one of the most frequently altered tumour suppressor genes in human cancers, including HCC.21 Hypermethylation of the p16INK4A promoter correlates closely with higher HBx protein levels in precancerous liver tissue and HCCs.21,22 HBx protein induces transcriptional activation of DNMT1, resulting in DNA hypermethylation of p16INK4A promoter and repressing its expression.23 A similar pathway can also inactivate E-cadherin expression.18,19 Activation of DNMT1 expression in this context may be mediated by the pRb-E2F pathway.24

Genomic hypomethylation and regional hypermethylation of the tumour promoter, IGFBP-3, correlate significantly with HBx protein expression in patients with HBx protein-induced HCC.25 Shortly after transfection with HBx, hypermethylation of the IGFBP-3 promoter region is observed, mediated by DNMT1 and DNMT3A1 and 3A2. Induction of DNMT1 may be explained by activation of the Ras signalling pathway. Down-regulation of the DNMT3B signalling pathway results in gradual hypomethylation of IGFBP-3.25 In another study, transcription of IGFBP-3 was activated by HBx.26 It was suggested that this discrepant result might reflect the operation of different subtypes of HBx protein.

The tumour suppressor gene, p53, plays a role in regulating expression levels of the DNMTs.23 The ASPP1 and ASPP2 family of proteins regulate apoptosis through interaction with p53.27,28 They function as tumour suppressor genes and specifically enhance the binding of p53 to promoters of proapoptotic genes and their transactivation.27 The expression of ASPP2 and, less often, ASSP1 is down-regulated in HCC cells by epigenetic silencing induced by hypermethylation of their promoters.27 Knockdown of ASPP2 and ASPP1 promotes the growth of HCC in nude mice and soft agar and decreases the sensitivity of the malignant cells to apoptotic stimuli, although ASPP2 is more effective than ASPP1 in this regard.27 In addition, ASPP2 is methylated as a result of HBx protein expression.28 ASPP2 may thus play a more important role than ASPP1 in hepatocarcinogenesis in general, and HBx protein-induced HCC in particular.

Retinoids inhibit carcinogenesis by blocking the promotion of initiated or transformed cells. HBx protein-induced hypermethylation of RAR-β2 via up-regulation of DNMT1 and DNMT3A results in down-regulation of its expression in HCC cells.29 Hypermethylation also abolishes the potential of retinoic acid to down-regulate levels of G1-check point regulators, including p16, p21, and p27. As a result, HBx protein-expressing cells are less susceptible to retinoic acid-induced cell growth inhibition. Because RAR-β2 is a major executor of the anti-tumour potential of retinoic acid, its epigenetic down-regulation by HBx protein is likely to be an important event in hepatocellular carcinogenesis.29

HBx protein represses the expression of, and silences, the tumour suppressor gene, GSTP1, as a result of hypermethylation of its promoter region by DNMT1.30 This is an early event in the pathogenesis of HCC, and also that of several other tumours.

Apoptosis

Maintenance of normal tissue homeostasis depends upon a balance between cell growth and programmed cell death (apoptosis). Apoptosis is responsible for the removal of redundant, damaged, or virally-infected cells. Regulation of apoptosis involves the actions of three categories of proteins: (i) effectors or initiators of apoptosis, such as the caspases and “death receptors”, including TNF-α, TRAIL and Fas; (ii) inducers and suppressors of apoptosis, for example, members of the Bcl-2 proteins; and (iii) intermediate proteins, including transcription factors such as p53, Fos, Jun, and myc.31–33

The function of the p53 tumour suppressor gene is central to the maintenance of genomic integrity by controlling the cell's response to stresses, including DNA damage and nucleotide deprivation.31,32 Cells respond to DNA damage by increasing the intracellular p53 concentration. This induces cell cycle arrest, allowing for repair of damaged DNA or p53-dependent apoptosis, or elimination of potentially mutated cells.

One of the more fully documented mechanisms by which HBx protein contributes to the development of HCC is its effects on apoptosis. These are complex, with HBx protein having both anti-apoptotic effects, which occur in association with the p53 gene, and pro-apoptotic effects. These apparently contradictory effects have not been explained, but one possible explanation is that high levels of HBx protein promote apoptosis, whereas low levels inhibit apoptosis.34 Another, less plausible, explanation is that anti-apoptotic effects occurring early in tumorigenesis allow the development and persistence of initiated hepatocytes, whereas pro-apoptotic effects later in the course allow accumulation of transforming mutations and progression of malignant transformation.

Anti-apoptotic effects of HBx protein

A number of ways in which HBx protein may induce anti-apoptotic effects have been described. The most important of these is the ability of HBx protein to inhibit p53-mediated apoptosis. HBx has been shown both in vivo and in vitro to form a complex with p53 in the cytoplasm, sequestering the protein and preventing its entry into the nucleus, blocking its transcriptional transactivating properties, and disrupting protein-to-protein interactions between p53 and other factors in its apoptotic pathway.35–39 One consequence of this effect is the failure of p53, in the presence of HBx protein, to up-regulate genes, such as p21CIPWAF1, Bax, or Fas. These genes are known to be involved in the apoptotic pathway and are normally up-regulated during p53-mediated apoptosis.35 Another consequence is that, because binding of p53 protein to the XPB and XPD constituents of the TFIIH transcription-nucleotide excision repair complex may be involved in inducing programmed cell death, and because HBx protein binds to the carboxy (COOH)-terminal of p53 protein and inhibits its binding to XPB and XPD, p53 induced apoptosis may be disrupted by HBx protein.36,39 This sequence may provide a selective advantage for hepatocytes containing HBx protein in the early stages of hepatocarcinogenesis.

HBx protein does not prevent p53-induced apoptosis by directly inactivating the tumour suppressor gene. Rather, it achieves protection against apoptotic death through an HBx-P3K-Akt-Bad pathway and by inactivating caspase 3 activity,40 or in association with H-ras oncogene through activation of the phosphatidyl inositol-3 kinase and AKt pathway.11

COX-2 is highly expressed in HCC. It mediates HBx protein actions in opposing p53.41 Enforced expression of HBx sequestrates p53 in the cytoplasm and abolishes p53-induced apoptosis by activation of the COX-2/PGE2 pathway. The anti-apoptotic Mcl-1 protein is suppressed by p53 but reactivated by HBx protein.40

Another possible explanation for the anti-apoptotic effect of HBx protein involves the accumulation of the anti-apoptotic protein, survivin.42 Hep3B cells expressing HBx protein have increased levels of HURP RNA and protein and show resistance to cisplatinin-induced apoptosis.42 Knock-down of HURP in these cells reverses this effect. The anti-apoptotic effect of HBx protein was shown to require activation of the p38/MAPK pathway. In addition, the expression of survivin was up-regulated by HBx protein in an HURP-dependent manner.41 These results indicate that HBx protein activates the expression of HURP via the p38/MAP pathway, culminating in the accumulation of survivin.

Pro-apoptotic effects of HBx protein

In addition to its anti-apoptotic effects, HBx protein may also induce apoptosis. HBx induces apoptosis or sensitizes cells to apoptotic death induced by death-receptor activating stimuli in HBx transgenic livers, as well as in primary hepatocytes in culture, and in mice with a p53 null mutation.43,44 The formation of transformed foci of NIH3T3 cells and Chang hepatocytes transfected with a variety of oncogenes is completely inhibited by HBx protein and is reversed by the apoptosis inhibitor, Bcl-2; this confirms that HBx protein induces apoptosis.45 Bcl-xL is a member of the anti-apoptotic Bcl-2 family. Expression of HBx protein reduces Bcl-xL mRNA and protein levels.44 It also sensitises HepG2 cells to apoptotic killing, and renders cells that were resistant to apoptotic killing by high doses of TNF-α sensitive to very low levels of the cytokine.46 In addition, HBx protein induces apoptosis by activating cyclin B1.47 HBx protein also sensitizes hepatocytes to p53-mediated apoptosis by stabilizing p53 through induction of E2F1and p53 activation via ATR.48

HBx protein increases the sensitivity of PLC/PRF/5 cells to TNF-α mediated apoptosis and reduces the expression of C-myc.49 TRAIL preferentially induces apoptosis of tumour cells and virus-infected cells but not normal cells. HBx protein sensitizes hepatocytes to TRAIL-induced apoptosis.50

The induction of apoptosis by full-length HBx protein but not by the COOH-truncated HBx protein51 strongly suggests that the COOH-terminal region is required for pro-apoptosis. If this observation is confirmed, this would suggest that the pro-apoptotic effect of HBx protein would seldom occur in practice because of the high frequency of COOH-terminal truncation in the integrated HBx gene.

Telomerase activity

Telomeres, which cap the ends of eukaryotic chromosomes, are composed of structural proteins and telomeric DNA, the latter comprised of conserved homogeneous repeats (TTAGGG)n that extend up to 10–15 kb in length.52–54 The structural proteins stabilize the telomeres and regulate their length.55 With each cell division the telomeres normally reduce in length with the loss of 30–150 base-pairs of telomeric DNA. The cells cease to divide when telomeres have been shortened to a certain length. At this point chromosomal instability occurs, and cell death or malignant transformation will ensue if cells continue to divide with short telomeres.52 Telomere length can be restored or maintained by telomerase, a ribonuclear enzyme. Telomerase consists of two proteins, telomerase reverse transcriptase (TERT) and telomerase-associated protein (TEP1), and an RNA template (TERC).52–55 TERT is considered the limiting factor of telomerase activity. Telomerase synthezises the telomere sequence de novo and adds it to the end of the telomere. Its activation is thus an important event in preventing immortalization and malignant transformation of cells, and it might therefore be a critical event in cancer development.56

There is no significant telomerase activity in normal liver tissue.52–55 In contrast, 80% of HCCs show high levels of telomerase activity.57 Telomerase activation is an important event in immortalization and malignant transformation of cells. The activation has been linked to re-expression of TERT. There is some uncertainty as to the timing of TERT expression in hepatocarcinogenesis, but it appears to be essential for growth of HCC cells.58

Telomere shortening appears to have a dual role in HCC development and progression. On the one hand, it induces chromosomal instability and the initiation of cancer; on the other hand, tumour progression requires stabilization of telomeres.58

The information available on the role played by HBx protein in HCC occurring in the presence of high telomerase activity is limited to 3 reports. Two studies showed that HBx protein increases TERT expression and telomerase activity in HBx-transfected cells, HBx protein-positive HCC tissues, or cultured HCC cells.59,60 In each of these, c-myc appeared to play a role in the process of telomerase activation: it induced telomerase activity and prolonged the life-span of normal cells.

A third study showed the opposite effect to that reported in the other studies, that is, a down-regulation of telomerase expression by transcriptional repression of its promoter.61 The authors suggested that there may be different isoforms of HBx protein that might produce opposing results. They attributed their finding to HBx protein acting as a transcriptional co-repressor of MAZ's activity on the telomerase promoter.61,62 MAZ is a transcription factor that binds to GC-rich regions to regulate the expression of target genes.62 The telomerase promoter possesses four MAZ sites, and MAZ suppresses the telomerase promoter. Thus, HBx protein may cause down-regulation of telomerase expression by acting as a transcriptional co-represser of MAZ on the telomerase promoter.61–63

Telomerase activity, telomere length and human telomerase reverse transcriptase expression were shown not to differ between patients with HBV-induced and hepatitis C virus (HCV)-induced HCC.64 However, no attempt was made to distinquish between HBV-induced HCC and that caused specifically by HBx protein.

Further information is needed on the role played by telomerase in HBx protein-induced HCC.

Nucleotide excision repair

The maintenance of genomic integrity through the recognition and repair of damaged or altered DNA is essential for the propagation of cellular life. The nucleotide excision repair (NER) pathway is responsible for the repair of a number of DNA lesions,65,66 particularly those resulting in helix distortions, for example those caused by carcinogenic adducts. Repair is achieved by the concerted actions of the products of as many as 30 genes, including the series XPA to XPG.65,66 NER excision repair is initiated by the binding of XPA to damaged DNA in a process that is enhanced by the interaction of XPA with the single-stranded DNA-binding protein, RPA. The transcription factor, TFIIH, is subsequently recruited to the site of damage. The TFIIH complex includes XPB and XPD helicases, which are responsible for open complex formation around the DNA lesion. P53 interacts with XPB and XPD to inhibit their translocation from sites of damaged DNA. This is thought to stabilize the formation of repair complexes.67 In addition to this function, p53 plays a role in a number of mechanisms involved in the regulation of DNA repair, including the regulation of cell cycle checkpoint mechanisms required for DNA repair.32,67 The ERCC-1 complex and XPG unit are positioned at the repair site through association with XPA and RPA, where they incise the DNA 5′ and 3′ to the lesion.66,67 A single-stranded piece of DNA of approximately 29 nucleotides in length, including the damaged site, is then removed. DNA polymerase γ or ε, DNA ligase, and accessory proteins then effect the repair synthesis.

Currently available evidence indicates that hepatocytes do not readily undergo apoptosis in response to DNA damage.68 Moreover, the efficiency of DNA repair in these cells is relatively high, suggesting an emphasis on repair mechanisms rather than self-destruction.

HBx protein may interfere with NER through both p53-independent and p53-dependent mechanisms. HBx protein inhibits cell cycle checkpoint mechanisms required for DNA repair and binds to damaged DNA, interfering with NER and facilitating the accumulation of host DNA mutations.32,69–71 The protein also binds to several cellular proteins known to be involved in DNA repair pathways. Cells containing these proteins show decreased repair capacity, which further contributes to the accumulation of host DNA mutations.32 The repair factors that are disrupted include the human homologue of the UV-DDB.70–72 In addition, HBx protein has been shown to bind to and repress XPB and XPD components of TFIIH that are essential for NER in both p53-proficient and p53-deficient hepatocytes,72,73 and to stimulate TFIIH-mediated DNA helicase activity.74 This effect on TFIIH-mediated DNA helicase activity differs from that of the HBx protein in association with p53 protein (see next paragraph) because it occurs during the assembly of the repair complex. The XPB and XPD proteins of the TFIIH complex are also involved in transcription-coupled repair,73 and p53 plays a part in the regulation of this process.75 Several transcription factor responsive elements are present in both XPB and XPD promoters, including Sp1. Inhibition of transcription of XPB and XPD is Sp1 dependent.75 Sp1 transcription factor has been shown to be a specific target for HBx protein resulting in impairment of its DNA binding properties.76

In addition, HBx may compromise the role that p53 plays in NER by binding to the COOH-terminus of the tumour suppressor gene, inducing its sequestration from the nucleus to the cytoplasm, and inhibiting its effects on cell cycle arrest and DNA repair.32 HBx protein also represses 53-mediated sequence-specific transcriptional activity, disrupts protein-to-protein interactions between p53 and other factors in its apoptotic pathway,4 promotes cell susceptibility to carcinogen-induced mutations of p53,77 binds to UV-DDB,78 and prevents binding of p53 to the transcription-repair factors, XPB and XPD. These are essential DNA helicases in the TFIIH nucleotide excision-basal transcription complex.32,69,70 Both in vitro and in vivo studies have shown that HBx protein can disrupt p53 binding directly to TFIIH, which is necessary for NER,32,78 and also inhibits TFIIH-mediated helicase activity during assembly of the repair complex.19,79

The binding domain of p53 for interaction with HBx protein has been mapped to residues between 293 and 393.32 This region also binds XPB and XPD. It is therefore possible that HBx may interfere in the NER pathway by masking the p53-COOH-terminal domain and by blocking p53 from binding to XPB and XPD.32 p53 associates with XPB, XPD and p62 of the TFIIH complex that is involved in nucleotide excision repair, as well as in the transcription coupled repair mechanism.66,67,82

Truncation of the carboxy terminal region of hepatitis B virus x protein

The COOH-terminal region of HBx protein is crucial to the protein's transactivational capability and for controlling cell proliferation and viability.80 This region is also required for the pro-apoptotic effect of the protein.81 HBV DNA sequences from HCC patients, particularly those isolated from integrated viral DNA, invariably, or very often, have a deletion in the distal COOH-terminal region of the x gene;82–86 this suggests that the deletion may be implicated in the pathogenesis of HBx protein-induced HCC. Truncation of the COOH-terminal end of HBx protein has also been documented in patients with chronic hepatitis B,87 suggesting that truncation may be selected during the development of the tumour and may play a role in the early stage of hepatocarcinogenesis. The deletion occurs during, and possibly also after, HBV DNA integration, although the explanation for its high frequency remains to be determined. COOH-terminal truncated HBx protein loses its transcriptional activity and inhibitory effects on cell proliferation and transformation,82 as well as its pro-apoptotic effect.83 It may also enhance the protein's ability to transform ras and myc.83

A number of mechanisms that could contribute to the carcinogenic potential of COOH-truncated HBx protein have been described. Wnt-5a is up-regulated in many human tumours.88,89 The expression of Wnt-5a differs significantly and in opposite directions when Huh7 HCC cells are transfected with mutant HBx gene, with either a HBx3′ minus 30 (30 amino acid deletion) or a HBx3′ minus 40 deletion in the COOH-terminal region. The HBx3′ minus 40 mutant promotes cellular proliferation, tumorigenicity, invasive growth, and metastasis by promoting cell cycle progression from G0/G1 to S phase, whereas the HBx3′ minus 30 deletion repressed cell proliferation by arresting cells in G1 phase.90 Wnt-5a is up-regulated by the minus 30 mutant but down-regulated by the minus 40 mutant. Wnt-5a antagonises the canonical Wnt/β-catenin pathway by promoting the degradation of β-catenin, possibly suppressing tumour formation.88,89

Fatty acid synthase (FAS) plays a crucial role in cancer cell survival and proliferation. The COOH-terminal truncated HBx protein increases the transcriptional activity of FAS in HCC cell lines.89 5-lipoxygenase is responsible for the up-regulation of FAS, and its expression results in an increase in the release of leukotriene, a metabolite of 5-lipoxygenase. FAS up-regulates the expression of 5-lipoxygenase in a feedback manner. Thus, the COOH-deleted HBx protein could promote cell proliferation through a positive feedback loop involving 5-lipoxygenase and FAS.91

The COOH-truncated HBx protein can effectively transform an immortalized tumour cell line (MIHA cells) and increase the tumorigenicity of Hep3B and MIHA cells.81

Insulin-like growth factor II

IGF-II is normally expressed in a number of foetal tissues, including the liver, where it is believed to play a role in the proliferation and differentiation of foetal cells.92 The key molecules in the IGF-II pathway are the ligands IGF-I and II, IGF binding proteins (IGFBP I—6), membrane associated receptors (IGF-IR and mannose-phosphate receptor) and insulin receptor substrates. The pattern of IGF-II transcripts is both tissue-specific and developmental stage-dependent. After birth, the expression level decreases rapidly and remains at a very low level in adult tissues. Transcription of the growth factor is regulated by four promoters (P1-P4), which act in a tissue-specific and dose-dependent manner. Dysregulation of the evolutionary highly conserved IGF pathway is involved in the proliferation and anti-apoptotic pathways of cancer cells, and is associated with uncontrolled tumour growth and chemo-resistance. Raised levels of IGF-II play a role in the malignant transformation of hepatocytes.

IGF-II and HBx protein show simultaneous positive staining in liver tissue of the great majority of patients with HCC.93 Moreover, all of the positively stained tissues show DNA aneuploidy. Up-regulation of IGF-II mRNA and IGF-II transcripts has been demonstrated in HBV-induced HCC in culture, and a role for HBx protein in this process is indicated by the observation that IGF-II expression was closely related to the expression of this protein by means of a transactivation mechanism.78,94,95 The presence of IGF-II receptors was also demonstrated in HCC cells in culture.94 In most cells, the IGF-II was localised to the cytoplasm. Expression of IGF-II correlated with the expression of HBx protein. These observations suggest that IGF-II may play a role in the pathogenesis of HBx protein-induced HCC. However, in another study, although all patients with HCC in whom liver tissue stained positive for HBx protein also expressed IGF-II, those patients staining negative for HBx protein also expressed IGF-II.96

HBx protein increases the endogenous IGF-II expression from promoters 3 and 4. As mentioned earlier Sp-1 is a ubiquitous transcription factor involved in the regulation of a large number of genes.94 Analysis of the fourth promoter (P4) shows that HBx protein positively regulates transcription and that two Sp1 binding sites on the promoter are responsible. Sp-1 is phosphorylated and its DNA binding activity up-regulated by HBx protein, and the protein positively regulates Sp1-mediated transcriptional activity of IGF-II.94 In a subsequent study from the same laboratory it was shown that protein kinase C (PKC) and p44/p42 MAPK signalling mediated the HBX protein-induced transcriptional activation of IGF-II gene.78

Dietary exposure to the fungal toxin, aflatoxin B1, contributes importantly to hepatocarcinogenesis in sub-Saharan Africa, China, and Taiwan. Its hepatocarcinogenic effects are synergistic with those of HBV. Exposure to aflatoxin B1 induces a “hot spot” mutation in the p53, a G to T (arginine to serine) transversion in codon 249 (p53ser249 mutation), which inactivates the tumour suppressor gene. This mutation is found in 50 to 60% of patients with HCC in regions of high exposure to AFB1.96,97

Transfection of hepatocytes having the p53ser249 mutation impaired the induction of apoptosis by the HBx protein.98 The p53ser249 mutation also markedly increases IGF-II transcription, mainly from P4.98 p53ser249 enhances the formation of transcriptional complexes through enhanced DNA-Sp1 interaction. This might be attributed to the fact that the p53ser249 mutation stimulates phosphorylation of transcription factor Sp-1 but wild-type p53 mutation does not.98 The blocking of apoptosis through enhanced production of IGF-II might provide a favourable opportunity for selection of transformed hepatocytes.

Overall perspectives and future directions

Papers continue to be published describing putative mechanisms by which HBx protein may contribute to the pathogenesis of HBV-induced HCC. The number and diversity of the proposed mechanisms are overwhelming and, although there is little doubt that the protein will ultimately be proven to play a pivotal role in HBV-induced hepatocarcinogenesis, it is not possible at this time to predict the unified series of mechanisms that will be responsible. Future research should be planned with this goal in mind.

Ancillary