HBV preS Mutations Promote Hepatocarcinogenesis by Inducing Endoplasmic Reticulum Stress and Upregulating Inflammatory Signaling

SIMPLE SUMMARY: Viral mutations at the preS region of hepatitis B virus (HBV) significantly increase the risk of developing hepatocellular carcinoma (HCC). Compared to HBV preS deletion, the oncogenic effect of preS combo mutation has rarely been investigated. With a cohort including 2114 subjects, we demonstrated that preS combo mutations G2950A/G2951A/A2962G/C2964A and C3116T/T31C significantly increased the risk of HCC in patients without antiviral treatment, whereas preS2 deletion significantly increased the risk of HCC in patients with antiviral treatment. The prevalence of C3116T/T31C (43.61%) was higher than preS2 deletion (7.16%). By using Sleeping Beauty mouse models and in vitro experiments, we found G2950A/G2951A/A2962G/C2964A, C3116T/T31C, and preS2 deletion promoted hepatocarcinogenesis by increasing levels of inflammatory cytokines, activating STAT3 pathway, enhancing endoplasmic reticulum stress, and altering gene expression profiles in inflammation- and metabolism-related pathways. These results suggest that preS combo mutations G2950A/G2951A/A2962G/C2964A and C3116T/T31C had similar oncogenic effects of preS2 deletion and should also be monitored. ABSTRACT: This study aimed to elucidate the effects and underlying mechanisms of hepatitis B virus (HBV) preS mutations on hepatocarcinogenesis. The effect of the preS mutations on hepatocellular carcinoma (HCC) occurrence was evaluated using a prospective cohort study with 2114 HBV-infected patients, of whom 612 received antiviral treatments. The oncogenic functions of HBV preS mutations were investigated using cancer cell lines and Sleeping Beauty (SB) mouse models. RNA-sequencing and microarray were applied to identify key molecules involved in the mutant-induced carcinogenesis. Combo mutations G2950A/G2951A/A2962G/C2964A and C3116T/T31C significantly increased HCC risk in patients without antiviral treatment, whereas the preS2 deletion significantly increased HCC risk in patients with antiviral treatment. In SB mice, the preS1/preS2/S mutants induced a higher rate of tumor and higher serum levels of inflammatory cytokines than did wild-type counterpart. The preS1/preS2/S mutants induced altered gene expression profiles in the inflammation- and metabolism-related pathways, activated pathways of endoplasmic reticulum (ER) stress, affected the response to hypoxia, and upregulated the protein level of STAT3. Inhibiting the STAT3 pathway attenuated the effects of the preS1/preS2/S mutants on cell proliferation. G2950A/G2951A/A2962G/C2964A, C3116T/T31C, and preS2 deletion promote hepatocarcinogenesis via inducing ER stress, metabolism alteration, and STAT3 pathways, which might be translated into HCC prophylaxis.