Hepatitis B surface antigen expression impairs endoplasmic reticulum stress-related autophagic flux by decreasing LAMP2

Abstract

Background & aims: Hepatitis B surface antigen (HBsAg) drives hepatocarcinogenesis. Factors and mechanisms involved in this progression remain poorly defined, hindering the development of effective therapeutic strategies. Therefore, the mechanisms involved in the HBsAg-induced transformation of normal liver into hepatocellular carcinoma (HCC) were investigated.

Methods: Hemizygous Tg(Alb1HBV)44Bri/J mice were examined for HBsAg-induced carcinogenic events. Gene set-enrichment analysis identified significant signatures in HBsAg-transgenic mice that correlated with endoplasmic reticulum (ER) stress, unfolded protein response, autophagy and proliferation. These events were investigated by western blotting, immunohistochemical and immunocytochemical staining in 2-, 8- and 12-month-old HBsAg-transgenic mice. The results were verified in HBsAg-overexpressing Hepa1-6 cells and validated in human HBV-related HCC samples.

Results: Increased BiP expression in HBsAg-transgenic mice indicated induction of the unfolded protein response. In addition, early-phase autophagy was enhanced (increased BECN1 and LC3B) and late-phase autophagy blocked (increased p62) in HBsAg-transgenic mice. Finally, HBsAg altered lysosomal acidification via ATF4- and ATF6-mediated downregulation of lysosome-associated membrane protein 2 (LAMP2) expression. In patients, HBV-related HCC and adjacent tissues showed increased BiP, p62 and downregulated LAMP2 compared to uninfected controls. In vitro, the use of ER stress inhibitors reversed the HBsAg-related suppression of LAMP2. Furthermore, HBsAg promoted hepatocellular proliferation as indicated by Ki67, cleaved caspase-3 and AFP staining in paraffin-embedded liver sections from HBsAg-transgenic mice. These results were further verified by colony formation assays in HBsAg-expressing Hepa1-6 cells. Interestingly, inhibition of ER stress in HBsAg-overexpressing Hepa1-6 cells suppressed HBsAg-mediated cell proliferation.

Conclusions: These data showed that HBsAg directly induces ER stress, impairs autophagy and promotes proliferation, thereby driving hepatocarcinogenesis. In addition, this study expanded the understanding of HBsAg-mediated intracellular events in carcinogenesis.

Impact and implications: Factors and mechanisms involved in hepatocarcinogenesis driven by hepatitis B surface antigen (HBsAg) are poorly defined, hindering the development of effective therapeutic strategies. This study showed that HBsAg-induced endoplasmic reticulum stress suppressed LAMP2, thereby mediating autophagic injury. The present data suggest that restoring LAMP2 function in chronic HBV infection may have both antiviral and anti-cancer effects. This study has provided insights into the role of HBsAg-mediated intracellular events in carcinogenesis and thereby has relevance for future drug development.

Keywords: HBV surface protein; cellular homeostasis; incomplete autophagy; liver cancer; lysosome; tumorigenesis.

Graphical abstract

Fig. 1

ER stress is induced in HBsAg-tg mice. (A) Clustering heat map of GSE84429 was acquired for hepatic ER stress signature comparing HBsAg-tg and WT mice. (B) Gene set enrichment analysis of GSE84429 for ER localization, ER stress and UPR distinguished HBsAg-tg and WT livers. Immunohistochemical staining show (C) HBsAg and (D) BiP expression in livers of WT and Alb-HBs mice at different ages (2-, 8-, 12-month-old, group sizes n = 3). Quantitative analysis based on integrated optical density of two randomly selected areas of each mouse (mean±SD). Scale bars: blue bar = 20 μm, black bar = 100 μm; p values (unpaired t test, unequal variances t-test) ∗∗∗∗p <0.0001. ER, endoplasmic reticulum; HBsAg, hepatitis B surface antigen; HBsAg-tg, HBsAg-transgenic; NES, normalised enrichment score; UPR, unfolded protein response; WT, wild-type.

Fig. 2

UPR is induced in HBsAg-transgenic mice. Western blot was performed to detect (A) HBsAg and (B) UPR components in primary murine hepatocytes isolated from WT and Alb-HBs mice (4-6-months-old). Western blot visualised (C) HBsAg and (D) UPR components in HBsAg-overexpressing Hepa1-6 cells (2 μg/well, 6 well plate, 24 h). Relative density is given as mean±SD of three independent experiments. (E) Gene expression of HSPA5, DDIT3, ATF4 and ATF6 was compared between HBV-associated HCC tissues and normal tissues (The Cancer Genome Atlas). p values (unpaired t-test, unequal variances t-test) ∗p <0.05, ∗∗p <0.01, ∗∗∗∗p <0.0001. HBsAg, hepatitis B surface antigen; HCC, hepatocellular carcinoma; L-HBsAg, large HBsAg; UPR, unfolded protein response; WT, wild-type.

Fig. 3

Autophagosomes are enriched in HBsAg-transgenic mice. (A) Gene set-enrichment analysis of GSE84429 for autophagosomes discriminates HBsAg-transgenic and WT mouse livers. (B) Immunohistochemistry detects LC3B in WT and Alb-HBs livers at different ages (2-, 8-, 12-month-old, group sizes n = 3). LC3B was quantified (mean±SD) by counting the positive hepatocytes from two randomly selected areas for each mouse. (C) Immunofluorescence staining shows HBsAg (red) and LC3B (green) in primary murine hepatocytes isolated from WT and Alb-HBs mice (4-6-months-old, representative of three independent experiments. (D) Immunofluorescence staining visualises autophagosomes in primary murine hepatocytes isolated from WT and Alb-HBs (4-6-months-old) mice. Hepatocytes were labelled with CYTO-ID® green detection reagent to detect autophagosomes. Hepatocytes treated with RAP (1 μM) in the presence of CQ (10 μM) for 24 h were used as a positive control; representative of three independent experiments. (E) Western blot detects LC3B-I, LCRB-II, p62 and ACTB in primary murine hepatocytes isolated from WT and Alb-HBs mice (4-6-months-old, left panel) and Hepa1-6 transfected with HBsAg-encoding plasmid (2 μg/well, 6 well plate, 24 h; right panel). Relative density is given as mean±SD of three independent experiments. Scale bars: blue bar = 20 μm, black bar = 100 μm; white bar, 10 μm; p values (unpaired t-test, unequal variances t-test); ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001. CQ, chloroquine; HBsAg, hepatitis B surface antigen; L-HBsAg, large HBsAg; NES, normalized enrichment score; RAP, rapamycin; WT, wild-type.

Fig. 4

Autophagy signalling is abnormal in HBsAg-tg mice. (A) Clustering heat map of GSE84429 was acquired for hepatic autophagy signature comparing HBsAg-tg and WT mice. (B) Gene set-enrichment analysis of GSE84429 for autophagy, autophagy positive regulation and autophagy negative regulation distinguished HBsAg-tg and WT livers. Immunohistochemical staining shows (C) BECN1 and (D) p62 expression in Alb-HBs and WT mouse livers at different ages (2-, 8-, 12-months-old, group sizes n = 3). BECN1 and p62 were quantified (mean±SD) by counting the positive hepatocytes from two randomly selected areas for each mouse (n = 3). Scale bars: blue bar = 20 μm, black bar = 100 μm; white bar = 10 μm; p values (unpaired t-test, unequal variances t-test), ∗∗∗p <0.001, ∗∗∗∗p <0.0001. HBsAg, hepatitis B surface antigen; HBsAg-tg, HBsAg-transgenic; NES, normalized enrichment score; WT, wild-type.

Fig. 5

Autophagic flux is impaired in HBsAg-transgenic mice. (A) Immunofluorescence staining shows HBsAg (red) and p62 (green) in primary murine hepatocytes isolated from WT and Alb-HBs (4-6-months-old), representative of three independent experiments. (B) Immunofluorescence staining shows lysosomal activity in primary murine hepatocytes isolated from WT and Alb-HBs mice (4-6-months-old). Primary murine hepatocytes were stained with 50 nM LysoTracker Red for 30 min. WT hepatocytes treated with CQ (10 mM) for 24 h were used as a positive control; representative of three independent experiments. Immunofluorescence staining shows lysosomal acidification in (C) primary murine hepatocytes isolated from WT and Alb-HBs mice (4-6-months-old) and (D) HBsAg-overexpressing Hepa1-6 cells. Cells were stained with AO (5 μM) for 15 min. Controls were treated with CQ (10 mM) for 24 h; representative of three independent experiments. The fluorescence intensity of AO-Red was analysed using an Olympus BX 51, upright epifluorescence microscope. (E) BECN1, MAP1LC3B2 and SQSTM1 gene expression was compared between HBV-associated HCC tissues and normal tissues (The Cancer Genome Atlas). Scale bars: white bar = 10 μm; p values (unpaired t-test, unequal variances t-test) ∗∗p <0.01, ∗∗∗∗p <0.0001. AO, acridine orange; CQ, chloroquine; HBsAg, hepatitis B surface antigen; HCC, hepatocellular carcinoma; WT, wild-type.

Fig. 6

HBsAg suppresses LAMP2 expression. (A) Clustering heat map of GSE84429 for lysosome signature was acquired comparing HBsAg-tg and WT mouse livers. (B) Gene set-enrichment analysis of GSE84429 for lysosome signature distinguished HBsAg-tg and WT mouse livers. (C) Western blot shows LAMP1/2 proteins in primary murine hepatocytes isolated from WT and Alb-HBs mice (4-6-months-old). (D) Immunofluorescence staining shows HBsAg (red) and LAMP2 (green) expression in primary murine hepatocytes isolated from WT and Alb-HBs mice (4-6-months-old), representative of three independent experiments. (E) Western blot shows LAMP2 expression in Hepa1-6 cells transfected with HBsAg-encoding plasmid and controls (2 μg/well, 6 well plate, 24 h). Western blot shows ER stress and autophagy components in primary murine hepatocytes isolated from WT and Alb-HBs mice (4–6-months-old) treated with (F) TUDCA (1 mM, 48 h) and (G) GSK2606414 (GSK, 1 mM, 48 h). (H) Dual luciferase reporter assay for the LAMP2 promoter was performed 48 h after the transfection with 20 ng/rxn and 80 ng/rxn of pcDNA3.1-ATF4, pcDNA3.1-ATF6 and pcDNA3.1-PafA in HEK293T cells. For western blots, relative densities are given as mean±SD of three independent experiments. Scale bars: white bar = 10 μm; p values (unpaired t-test, unequal variances t-test) ∗p <0.1, ∗∗p <0.01, ∗∗∗p <0.001; n.s., not significant. HBsAg, hepatitis B surface antigen; L-HBsAg, large HBsAg; NES, normalized enrichment score; rxn, reaction; TUDCA, tauroursodeoxycholic acid; WT, wild-type.

Fig. 7

HBsAg-induced ER stress drives proliferative activity. (A) Gene set-enrichment analysis of GSE84429 for proliferation distinguished HBsAg-tg and WT mouse livers. Immunohistochemical staining shows (B) Ki67, (C) cleaved caspase-3 and (D) AFP positive hepatocytes in WT and Alb-HBs mouse livers at different ages (2-, 8-, 12-months-old, group sizes n = 3), quantified in two randomly selected areas for each mouse. (E) Colony formation assays were performed with Hepa1-6 cells transfected with HBsAg plasmid for 24 h (2 μg/well, 6-well plate). The number of colonies was counted (mean±SD of three independent experiments). (F) CCK-8 viability analysis was performed. Hepa1-6 cells, transfected with HBsAg-encoding plasmid for 24 h, were trypsinized, seeded (5 × 10⁵/well) and cultured in 6-well culture plates. TUDCA or GSK were added for 24 h. Hepa1-6 cells were trypsinized again, seeded (5 × 10³/well) and cultured in 96-well culture plate. Cell viability was assessed at 24 h, 48 h and 72 h. Scale bars: blue bar = 20 μm, black bar = 100 μm; p values (unpaired t-test, unequal variances t-test) ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001; n.s., not significant. ER, endoplasmic reticulum; HBsAg, hepatitis B surface antigen; NES, normalized enrichment score; TUDCA, tauroursodeoxycholic acid; WT, wild-type.

Fig. 8

HBsAg accumulation induces ER stress-associated autophagy impairment by suppressing LAMP2 in patients with HCC and HBV infection. (A) Expression of HBsAg, BiP, p62 and LAMP2 was verified by immunohistochemical staining in paraffin-embedded HCC specimens with (n = 5) or without (n = 5) HBV infection. Tumour and AT were analysed. Quantitative analysis of HBsAg, BiP, p62 and LAMP2 based on integrated optical density of five randomly selected areas for each patient. (B) Spearman's rank correlation was applied to the integrated optical density values of HBsAg and LAMP2. Scale bar, 100 μm. p values (unpaired t-test, unequal variances t-test) ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001; ∗∗∗∗p <0.0001; n.s., not significant. AT, adjacent tissue; ER, endoplasmic reticulum; HBsAg, hepatitis B surface antigen; HCC, hepatocellular carcinoma.