Elsevier

Journal of Hepatology

Volume 47, Issue 3, September 2007, Pages 325-337
Journal of Hepatology

Modulation of MAPK pathways and cell cycle by replicating hepatitis B virus: Factors contributing to hepatocarcinogenesis

https://doi.org/10.1016/j.jhep.2007.03.025Get rights and content

Background/Aims

Chronic infection with the hepatitis B virus (HBV) is strongly associated with the development of hepatocellular carcinoma but the mechanism by which this occurs is unknown. Numerous studies have focused on the HBV X protein showing that it activates signal transduction pathways while few have investigated these changes in HBV-replicating hepatocytes.

Methods

We utilized the recombinant adenovirus system to deliver a replication competent HBV genome into Huh7 and primary marmoset hepatocytes (PMH) to examine the effects of active viral replication on the regulation of Ras-ERK signal transduction and related pathways.

Results

Huh7 cells and PMHs replicating HBV demonstrated significant upregulation in phosphorylated ERK, Akt, c-myc together with increased p53, cyclin B1 and p21cip1 expression and cell cycle progression to G2 phase in the absence of increased cell proliferation. Phosphorylation of the key cell survival kinase, Akt, was significantly increased, resulting in increased serine phosphorylation of the downstream target, GSK3-β.

Conclusions

These results demonstrated simultaneous activation of the MAP Kinase and Akt pathways in HBV-replicating hepatocytes that resulted in dysregulation in the control of cell cycle progression and which help explain the early pathogenic mechanisms that underlie malignant transformation associated with chronic hepatitis B infection.

Introduction

The mechanism by which HBV results in HCC development is not well understood and the risk of developing HCC remains significantly elevated despite suppression of viral replication by antiviral therapy and after clearance of serum HBeAg and HBV viremia [1], [2], [3].

The HBx protein of HBV has been proposed as an important oncogenic stimulus for the development of HCC in chronically infected individuals [4], [5], [6], [7]. However several studies have reported divergent effects of HBx on p53-, Fas- and TNF-α-[8], [9], [10], c-myc-, and Ras-mediated apoptosis [11], [12], [13] and transactivation of IL-8 [14], [15], [16]. Our understanding of how HBV causes HCC remains a challenge and the frequent discrepancies in experimental findings may reflect limitations in duplicating cellular changes that occur in natural infection. The HBV large (LHB) and middle (MHB) envelope proteins have also been shown to contribute to hepatocarcinogenesis of HBV [17], [18], further emphasizing the role of viral factors, other than HBx, and active HBV replication as mediators of HCC development.

To address the role of active HBV replication on hepatocarcinogenesis we used a recombinant adenovirus system to deliver a replication competent HBV genome rather than single viral genes into primary marmoset hepatocytes, and contrasted the changes to cell signaling pathways induced by HBV infection with those seen in the human hepatoma cell line, Huh7.

Section snippets

Plasmids

A 1.5× full length replication competent HBV (genotype A, subtype adw2) [17] was subcloned into pAdTrack (provided by B Vogelstein, Howard Hughes Medical Centre, Baltimore) which was pre-digested with HindIII and EcoRV (Promega). The plasmid pAdTrack-HBVwt was digested with PmeI and transformed into AdEasier-1 cells by electroporation (Bio-Rad Gene Pulser). Clones (AdEasy-HBV) were transformed into Top 10F′ cells (Invitrogen) and confirmed by DNA sequencing. An AdEasy-GFP control expressing

HBV replication in Huh7 cells and PMH

Although Huh7 cells were known to be permissive for HBV replication this was not known for PMH. Infection of both Huh7 cells and PMH with rAdHBV resulted in expression of GFP in >95% of cells (Fig. 1a, b, e and f). Intra- and extra-cellular HBV DNA replication intermediates (relaxed circular, double stranded linear and single stranded) were readily detectable 72 h PI for Huh7, increasing thereafter until day 6 (Fig. 1c), and 24 h PI for PMH cells (Fig. 1g). HBV cccDNA was detected by quantitative

Discussion

Dysregulation of the signal transduction pathways such as Ras-MAPK signaling, IRS1/IGF pathways, NFkB, cell cycle, Wnt/β-catenin and apoptotic pathways have all been implicated in the development of HBV-associated carcinogenesis [7], [25], [26], [27], [28], [29], [30]. However, it is difficult to derive a unifying theme to explain the precise mechanism(s) linking the initiating oncogenic stimulus with subsequent changes in signal transduction. We sought to examine how HBV infection, rather than

Acknowledgements

The AdEasy system was generously provided by B Vogelstein (Howard Hughes Medical Institute, Baltimore). We thank Dr Scott Bowden and Ms Kathy Jackson, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia, for kindly performing quantitative real time PCR analyses for HBV cccDNA. This work was supported by grants of the Deutsche Krebshilfe, “Dr. Mildred-Scheel-Stiftung fuer Krebsforschung”, Grant No. 10-2142-Bo1.

References (42)

  • M.F. Yuen et al.

    Prognostic determinants for chronic hepatitis B in Asians: therapeutic implications

    Gut

    (2005)
  • P. Lampertico et al.

    Five years of sequential LAM to LAM+ADV therapy suppresses HBV replication in most HBEAG-negative cirrhotics, preventing decompensation but not hepatocellular carcinoma

    Hepatology

    (2005)
  • C.-J. Chen et al.

    Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level

    JAMA

    (2006)
  • H.L. Pahl

    Activators and target genes of Rel/NF-kappaB transcription factors

    Oncogene

    (1999)
  • M.A. Feitelson

    Parallel epigenetic and genetic changes in the pathogenesis of hepatitis virus-associated hepatocellular carcinoma

    Cancer Lett

    (2005)
  • M.J. Bouchard et al.

    Activation of focal adhesion kinase by hepatitis B virus HBx protein: multiple functions in viral replication

    J Virol

    (2006)
  • M. Branda et al.

    Signal transduction cascades and hepatitis B and C related hepatocellular carcinoma

    Hepatology

    (2006)
  • L.W. Elmore et al.

    Hepatitis B virus X protein and p53 tumor suppressor interactions in the modulation of apoptosis

    Proc Natl Acad Sci USA

    (1997)
  • U.S. Park et al.

    Hepatitis B virus-X protein upregulates the expression of p21waf1/cip1 and prolongs G1  S transition via a p53-independent pathway in human hepatoma cells

    Oncogene

    (2000)
  • J. Pan et al.

    Hepatitis B virus X protein protects against anti-Fas-mediated apoptosis in human liver cells by inducing NF-kappa B

    J Gen Virol

    (2001)
  • F. Su et al.

    Hepatitis B virus HBx protein sensitizes cells to apoptotic killing by tumor necrosis factor alpha

    Proc Natl Acad Sci USA

    (1997)
  • Cited by (60)

    • Imidazole-pyridine hybrids as potent anti-cancer agents

      2023, European Journal of Pharmaceutical Sciences
      Citation Excerpt :

      In particular, since approximately 50% of all hepatocarcinomas are related to human hepatitis B virus (HBV) infections, with several HBV factors implicated with disease progression and prognosis (Chemin and Zoulim, 2009), three different LIHC cell lines were selected for these investigations: HepG2 (HBV-negative), HUH-7 (HBV-negative) and PLC/PRF/5 (derived from an HBV-infected liver). Importantly, HBV infections regulates liver cell tumorigenesis by affecting intracellular GSK-3β signaling pathways (Chin et al., 2007). According to previous results in lung and colon tumors, compounds 5a, 5d, 5e, and 5f showed the highest anti-cancer activity in all liver cancer cell lines (Fig. 5A).

    • DMAMCL exerts antitumor effects on hepatocellular carcinoma both in vitro and in vivo

      2020, Cancer Letters
      Citation Excerpt :

      It has also been found that the PI3K/Akt pathway plays an essential role in the development of HCC and is activated in 30%–50% of all HCC cases [41]. The infection of hepatocytes with HBV is associated with the activation of the PI3K/Akt pathway [42]. Currently, various inhibitors targeting the PI3K/Akt pathway are undergoing clinical assessment.

    View all citing articles on Scopus

    The authors who have taken part in this study declared that they have no relationship with the manufacturers of the product involved either in the past or present and did not receive funding from the manufacturers to carry out their research.

    View full text