The Effects and Underlying Mechanisms of Hepatitis B Virus X Gene Mutants on the Development of Hepatocellular Carcinoma.

Rui Pu,Guangwen Cao,Xi Chen,Shiliang Cai,Xiaomei Hou,Jianfeng Wu,Wenbin Liu,Jianhua Yin,Liping Chen,Xiaojie Tan,Fan Yang,Xinyu Zhou,Xin Wang

doi:10.3389/fonc.2022.836517

Abstract

We aimed to elucidate the mechanism by which hepatitis B virus X (HBx) mutations increase the occurrence of hepatocellular carcinoma (HCC) and identify novel putative therapeutic targets. Wild-type HBx (WT-HBx) and four HBx mutants (M1, A1762T/G1764A; M2, T1674G+T1753C+A1762T/G1764A; M3, C1653T+T1674G+A1762T/G1764A; and Ct-HBx, carboxylic acid-terminal truncated HBx) were delivered into Sleeping Beauty (SB) mouse models. The HCC incidence was higher in the M3-HBx- and Ct-HBx-injected SB mice. M3-HBx had a stronger capacity of upregulating inflammatory cytokines than other HBx variants. Ectopic expression of M3-HBx and Ct-HBx significantly increased proliferation and S phase proportion of HepG2 and HeLa cells, compared to WT-HBx. Plasminogen activator inhibitor-1 (PAI1) and cell division cycle 20 (CDC20) were identified as novel effectors by cDNA microarray analysis. M3-HBx and Ct-HBx significantly upregulated the expression of PAI1 and CDC20 in HepG2 and HeLa cells as well as the livers of SB mice. Silencing PAI1 attenuated the effects of M3-HBx and Ct-HBx on the growth of HepG2 and HeLa cells. PAI1, an important player bridging the HBx mutants and HCC, should be a promising candidate as a prognostic biomarker and therapeutic target in HBV-related HCC.

Highlights

Primary liver cancer (PLC) was the third leading cause of cancer death worldwide in 2020 and hepatocellular carcinoma (HCC) accounts for 75–85% of PLC cases [1]
In this cohort of hepatitis B virus (HBV)-infected patients, combo mutation A1762T/G1764A+C1653T+T1674G, A1762T/G1764A+T1674G+T1753C, and A1762T/ G1764A+C1653T+T1753C were all significantly associated with an increased risk of HCC, with an age- and genderadjusted Hazard ratio (HR) of 1.81 (1.42-2.32), 1.54 (1.11-2.14), and 1.83 (1.32-2.53), respectively (Figure 1)
The combo mutation A1762T/G1764A+C1653T+T1674G and A1762T/G1764A+T1674G+T1753C were selected for the following experiments because of higher frequencies in the HBV-infected population

Summary

Introduction

Primary liver cancer (PLC) was the third leading cause of cancer death worldwide in 2020 and hepatocellular carcinoma (HCC) accounts for 75–85% of PLC cases [1]. It has been demonstrated that the combination (combo) mutant, rather than HBx with single or double CP mutations, accelerates cell cycle progression and p21 degradation, possibly via increasing expression of S-phase kinase-associated protein 2 (SKP2) [10]. Gene expression profiling of human hepatocytes infected with HBV genotype F1b, the CP mutant, indicates more cancer-related signaling pathways compared with other genotypes [8]. HBV integration into the host genome often leads to the truncation of the HBV genome, at the C terminus of hepatitis B virus X (HBx), resulting in the generation of carboxylic acid terminal-truncated HBV X protein (Ct-HBx) [11]. The gene expression profiles of the HBV mutant-integrated liver of animal models with intact immune system, which represents the key molecular event in humans, are not reported. This study helps elucidate the mechanisms by which HBx mutants promote HBVinduced carcinogenesis and reveals effective therapeutic options for HBV-HCC

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