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

Yaojie Liang; Xufeng Luo; Stefan Schefczyk; Lorraine T Muungani; Hui Deng; Baoju Wang; Hideo A Baba; Mengji Lu; Heiner Wedemeyer; Hartmut H Schmidt; Ruth Broering

doi:10.1016/j.jhepr.2024.101012

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

JHEP Rep. 2024 Jan 28;6(4):101012. doi: 10.1016/j.jhepr.2024.101012. eCollection 2024 Apr.

Authors

Yaojie Liang¹, Xufeng Luo^{1

2}, Stefan Schefczyk¹, Lorraine T Muungani¹, Hui Deng³, Baoju Wang³, Hideo A Baba⁴, Mengji Lu⁵, Heiner Wedemeyer^{1

6}, Hartmut H Schmidt¹, Ruth Broering¹

Affiliations

¹ Department of Gastroenterology, Hepatology and Transplant Medicine, Medical Faculty, University of Duisburg-Essen, Essen, Germany.
² Institute for Lymphoma Research, Henan Cancer Hospital, The Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou, China.
³ Department of Infectious Diseases, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
⁴ Institute of Pathology, Medical Faculty, University of Duisburg-Essen, Essen, Germany.
⁵ Institute for Virology, Medical Faculty, University of Duisburg-Essen, Essen, Germany.
⁶ Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.

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.