Hepatitis B virus integration and hepatocarcinogenesis

Authors

Ma L

Chen S

Wang H

Chen L

Doi

10.1016/j.livres.2025.09.002

PMID: 22634756 · DOI: 10.1016/j.livres.2025.09.002 · Journal: Liver Research (2025)

TL;DR

This review comprehensively summarizes the mechanisms by which hepatitis B virus (HBV) DNA integration into the host genome drives hepatocellular carcinoma (HCC) development. The authors describe three main oncogenic mechanisms: (1) insertional mutagenesis targeting recurrent hotspot genes such as TERT (23.7% of HCCs), KMT2D/MLL4 (11.8%), and CCNE1 (5%); (2) chromosomal instability induced by integrated viral DNA; and (3) the oncogenic activity of viral proteins HBx and PreS/S, which dysregulate transcription factors, tumor suppressor genes, noncoding RNAs, and cytoplasmic signaling pathways. The review also highlights the emerging role of extrachromosomal circular DNA (ecDNA) formed from HBV-oncogene integrations in amplifying oncogene expression.

Cohort & data

  • Study type: Narrative review (no original cohort).
  • Cancer type: HCC.
  • Scope: Synthesizes findings from multiple genomic, transcriptomic, and functional studies of HBV-related HCC, including genome-wide surveys of HBV integration sites (e.g., Sung et al., Nat Genet 2012; Chen et al., Nature 2024 — deep WGS of 494 HCCs), cell line experiments, and animal models.
  • Key referenced methods: whole-genome sequencing, RNA-seq, spatial transcriptomics, droplet digital PCR (ddPCR).

Key findings

  • HBV DNA integration occurs in approximately 90% of HBV-related HCCs and can be detected at early stages of infection both in vitro and in vivo.
  • Integration preferentially targets coding genes and regulatory regions, with recurrent hotspots in TERT promoter (23.7%), KMT2D (MLL4; 11.8%), and CCNE1 (5%).
  • HBV integration into TERT promoters induces telomerase expression and promotes resistance to cellular aging and death. HBV also inserts into TERT introns, upregulating expression through unclear mechanisms.
  • HBV-TERT integration can cyclize into extrachromosomal circular DNA (ecDNA), which was found in 27.3% of liver cancer samples and harbored 76 oncogenes including MYC.
  • HBV integration into KMT2D leads to epigenomic modification and downregulation of the TP53 tumor suppressor pathway, with >20-fold increase in KMT2D expression in HCC.
  • HBV integration into CCNE1 inhibits the RB1 pathway and drives abnormal cell cycle regulation; HBV-CCNE1 fusion transcripts increase cyclin E1 expression and are associated with reduced disease-free survival.
  • Chromosomal instability from HBV integration leads to copy number alterations; 10% of liver cancer samples showed loss of copy number at the caspase locus (CASP1, CASP4, CASP5, CASP12), inhibiting apoptosis.
  • The HBx-LINE1 chimeric transcript acts as a molecular sponge for miR-122, promotes EMT-like alterations, inhibits autophagy, and increases phosphorylated beta-catenin levels.
  • Nucleos(t)ide analogues reduce HBV DNA integration frequency (median 1.01 x 10^9 before treatment to 4.84 x 10^7 after 10 years) and decrease hepatocellular clonal expansion (from 2.41 x 10^5 to 2.55 x 10^4 after 10 years; n=28 patients).

Genes & alterations

  • TERT: Most frequent HBV integration hotspot (23.7% of HCCs); promoter and intronic insertions induce telomerase expression. HBV-TERT integrations cyclize into ecDNA for amplified expression.
  • KMT2D (MLL4): Second most frequent integration target (11.8%); intronic and exonic insertions cause epigenomic modification and downregulate the p53 pathway. >20-fold expression increase in HCC.
  • CCNE1: Recurrent integration target (5%); HBV-CCNE1 fusions increase cyclin E1 expression and inhibit the RB pathway, disrupting G1/S cell cycle regulation.
  • TP53: HBx binds p53 directly, inhibiting nucleotide excision repair and transcription-coupled repair. HBx also indirectly reduces p53 expression via ASPP1/ASPP2 downregulation and Pin1 interaction.
  • RB1: HBx suppresses CDK inhibitors through promoter methylation, leading to RB inactivation.
  • CTNNB1: HBx destabilizes beta-catenin regulation through GSK3B degradation; the Wnt/beta-catenin pathway is activated by both HBx and MHBst proteins.
  • GSK3B: Degraded by HBx-mediated ubiquitination, leading to dysregulated CTNNB1 stability.
  • MTOR: Activated by HBx via asparagine synthase (ASNS) promoter binding, increasing asparagine levels and promoting tumor cell proliferation.
  • DNMT3A: Relocated by HBx to tumor suppressor gene promoters (e.g., p16/INK4A), causing regional hypermethylation.
  • MYC: Identified among 76 oncogenes harbored on ecDNA in HBV-related HCC.
  • CASP1: Loss of copy number at the caspase locus (CASP1, CASP4, CASP5, CASP12) observed in 10% of liver cancer samples, inhibiting apoptosis.

Clinical implications

  • Biomarker — virus-host chimera DNA (vh-DNA): Cell-free vh-DNA detectable in blood via ddPCR serves as a circulating biomarker for HCC. In tumors >1.5 cm, junctional vh-DNA abundance correlates with tumor size. Postoperative vh-DNA levels predict early recurrence risk and can distinguish de novo tumors from true recurrence.
  • Antiviral treatment reduces HCC risk: Nucleos(t)ide analogues significantly decrease HBV DNA integration frequency and hepatocellular clonal expansion over 10 years of treatment. Spatial transcriptomics data show transcriptionally active HBV integration is nearly undetectable in HBsAg-deficient patients.
  • Precision medicine potential: Heterogeneity of HBV integration sites may lead to distinct prognostic outcomes and differential responses to immune checkpoint inhibitors or targeted therapies, suggesting integration-site-based patient stratification.
  • Therapeutic vaccine development: HBV-host chimeric antigens from integration sites may enable development of therapeutic vaccines or antigen-specific T-cell therapies for HBV-induced HCC.

Limitations & open questions

  • This is a narrative review; no original data or meta-analysis was performed.
  • The precise mechanism by which HBV DNA integrates into ecDNA remains unknown, and implications for viral elimination are unclear.
  • How HBV selects specific target genes for integration is not well understood.
  • HBV-LINE1 fusion transcripts appear restricted to HBV genotype C in Asian populations and have not been identified in European HCC patients, limiting generalizability.
  • The specific regulatory mechanism by which HBV intronic insertion into TERT leads to gene upregulation is unclear.
  • How to develop effective precision therapies based on individual HBV integration site profiles remains an open challenge.

Citations from this paper used in the wiki

  • “Approximately 90% of HBV-related HCCs [show] HBV DNA integration into host chromosomes […] and this integration can be observed at the early stages of HBV infection in vitro and in vivo.” (Section 3.1)
  • “TERT gene (23.7%) […] MLL4 gene (11.8%) […] CCNE1 gene (5%)” (Table 1)
  • “We reported ecDNA in 27.3% of liver cancer samples and identified a total of 76 oncogenes in ecDNA, including proto-oncogenes, such as MYC.” (Section 4.1)
  • “Nucleos(t)ide analogues reduced HBV DNA integration (median integration frequency was 1.01 x 10^9 before treatment and 5.74 x 10^8 after 1 year of treatment). The median integration frequency after 10 years of treatment was 4.84 x 10^7.” (Section 3.1)
  • “10% of liver cancer samples exhibited a loss of copy number at the caspase site.” (Section 4.2)
  • “In HCC tumors >1.5 cm, the signature junctional vh-DNA fragment can be detected in blood, and its abundance strongly correlates with tumor size.” (Section 5)

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