Hepatitis B virus surface proteins accelerate cholestatic injury and tumor progression in Abcb4-knockout mice

Abstract

Understanding of the pathophysiology of cholestasis associated carcinogenesis could challenge the development of new personalized therapeutic approaches and thus improve prognosis. Simultaneous damage might aggravate hepatic injury, induce chronic liver disease and even promote carcinogenesis. We aimed to study the effect of Hepatitis B virus surface protein (HBsAg) on cholestatic liver disease and associated carcinogenesis in a mouse model combining both impairments. Hybrids of Abcb4-/- and HBsAg transgenic mice were bred on fibrosis susceptible background BALB/c. Liver injury, serum bile acid concentration, hepatic fibrosis, and carcinogenesis were enhanced by the combination of simultaneous damage in line with activation of c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, and Signal transducer and activator of transcription 3 (STAT3). Activation of Protein Kinase RNA-like Endoplasmic Reticulum Kinase (PERK) and Eukaryotic translation initiation factor 2A (eIF2α) indicated unfolded protein response (UPR) in HBsAg-expressing mice and even in Abcb4-/- without HBsAg-expression.

Conclusion: Cholestasis-induced STAT3- and JNK-pathways may predispose HBsAg-associated tumorigenesis. Since STAT3- and JNK-activation are well characterized critical regulators for tumor promotion, the potentiation of their activation in hybrids suggests an additive mechanism enhancing tumor incidence.

Keywords: ER-stress; HBsAg; carcinogenesis; cholangitis; fibrosis.

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare that no conflicts of interest exist.

Figures

Figure 1. HBsAg expression elevates liver injury in Abcb4 knockout mice

A. Liver histology demonstrates accelerated portal inflammation and pronounced bile duct disease in Abcb4−/−/HBsAg+/− mice. Magnification x200, scale bars 100 μm. B. HBsAg expression increases serum ALT in Abcb4−/− mice. Open bars represent wild types, light grey bars Abcb4−/−, dark grey bars HBsAg+/−, and black bars Abcb4−/−/HBsAg+/−. C. Western blotting demonstrates reduced expression of HBsAg and ER stress in Abcb4−/−/HBsAg+/− mice. D. Immunohistochemistry visualizes reduced HBsAg expression in female Abcb4−/−/HBsAg+/− mice. Representative micrographs are shown.

Figure 2. Total serum BA levels raise while relative amount of protective BAs decrease in Abcb4−/−/HBsAg+/− mice

A. Total serum taurine conjugated Bas (T-BAs) increased in Abcb4−/−/HBsAg+/−-mice in comparison to controls. Serum T-BAs were measured by UHPLC-MS/MS at the day of sacrifice. Total T-BAs are depicted. Note, total T-BAs were increased in Abcb4−/−-mice in comparison to wt controls and in HBsAg+/− transgenic mice aged 52 weeks as well (#). B. Relative amount of protective TUDCA was decreased in Abcb4−/−/HBsAg+/−-mice. Results are presented as means ± SEM, n=4-10. *p<0.05 as indicated, #p=0.001 in comparison to wt control. Open bars represent wild types, light grey bars Abcb4−/−, dark bars HBsAg+/−, and black bars Abcb4−/−/HBsAg+/−. C. Scatter-plot demonstrates the correlation of ALT and total serum T-BA levels. All data of 52 week old animals are included; open dots wt, grey dots Abcb4−/−, dark grey dots HBsAg+/−, black dots Abcb4−/−/HBsAg+/−. D. Scatter-plot demonstrates the correlation of ALT and total serum T-BA levels in HBsAg+/−-mice. E and F. Absolute- (E) and relative (F) composition of the 8 most abundant bile acids in serum.

Figure 3. Hepatic fibrosis is elevated in Abcb4−/−/HBsAg+/− mice

A. Representative Sirius red staining demonstrates enhanced collagen accumulation in Abcb4−/−/HBsAg+/− mice. Magnification x100, scale bars 200 μm. B. Quantification of SiriusRed positive area was performed by computer-analysis (ImageJ). Open bars represent wild types, light grey bars Abcb4−/−, dark grey bars HBsAg+/−, and black bars Abcb4−/−/HBsAg+/−. C. HBsAg expression enhanced HSC activation and ductular reaction. Representative immunohistochemical stainings for quiescent HSC marker GFAP, HSC activation marker desmin, and transcription factor SOX9 in 12-16 week old mice. Magnification x100, scale bars 100μm. Results are presented as means ± SEM, n=6-10. *p<0.05 as indicated.

Figure 4. Induction of carcinogenic pathways enhanced tumor incidence in Abcb4−/−/HBsAg+/− mice

A. Macroscopic view of well vascularized primary tumors > 5mm in diameter which appeared more frequently in Abcb4−/−/HBsAg+/− mice. B. Tumor diameter is shown in Box and Whisker Plots. Enhanced incidence was calculated with Chi2 statistics. The upper and lower hinges of the box represent the 75th and 25th percentile, respectively. The line indicates the median value; error bars represent the minimum and maximum. C. Western blot analysis of phospho-SAPK/JNK (P-JNK), β-actin, phospho-c-Jun (P-cJun), c-Jun, phospho-STAT3 (P-STAT3), and STAT3 demonstrate enhanced induction of carcinogenic pathways in Abcb4−/−/HBsAg+/− mice.

Figure 5. Tumors in Abcb4−/−/HBsAg+/− mice show histological characteristics of HCC

A. A representative H&E staining demonstrates well defined tumor borders (dashed line). B. Tumor stroma is nearly free from Sirius red staining fibrillar collagen. Arrows point out fibrotic area in surrounding liver tissue. C. Ki67 staining (arrows) demonstrates pronounced proliferation in tumor stroma. D. Type IV collagen immunostaining depicts a lack of expression inside tumor stroma. E. Immunostaining for glutamine synthetase depicts expression in pericentral hepatocytes (arrows) and diffuse expression pattern in tumor stroma. F. Immunostaining for HBsAg demonstrates the loss of transgene expression in tumors. L liver, T tumor. Magnification x100 A-F, scale bars 100μm.