HBV Precore G1896A Mutation Promotes Malignancy of Hepatocellular Carcinoma by Activating Endoplasmic Reticulum Stress to Enhance Aerobic Glycolysis

Abstract

Hepatitis B virus (HBV) precore G1896A mutation is closely associated with poor prognosis of liver disease. We previously revealed that the G1896A mutation could enhance HBV replication and promote hepatocellular carcinoma (HCC) cell growth both in vitro and in vivo. However, the in-depth mechanisms by which this mutation promotes the malignancy of HCC still need to be explored. Here, we examined the activation of endoplasmic reticulum (ER) stress and glycolysis in HBV G1896A mutation–associated HCC. Bioinformatics, chromatin immunoprecipitation assay and dual-luciferase assay were performed to give insight into the underlying molecular interaction between ER stress and glycolysis. Here, we observed that HBV G1896A mutation also promoted HCC cell invasion and migration. Furthermore, HBV G1896A mutation induced ER stress, and specifically, PERK-ATF4 pathway was responsible for the HCC cell malignancy. Mechanistically, PERK-ATF4 signaling induced transcriptional activation of PFKFB3, a key gene in the process of glycolysis. Finally, in vitro rescue experiments and in vivo efficacy studies revealed that the ATF4-PFKFB3 axis is necessary for the HCC tumor growth and metastasis. These results highlight that the ER stress and glycolysis are involved in the HCC-promotion function of HBV G1896A mutation, providing new insights into HBV-related HCC.

Keywords

aerobic glycolysis / endoplasmic reticulum stress / HBV G1896A mutation / hepatocellular carcinoma

References

[1] P. Ganesan and L. M. Kulik, “Hepatocellular Carcinoma: New Developments,” Clinics in Liver Disease 27, no. 1 (2023): 85-102.

[2] C. H. Zhang, Y. Cheng, S. Zhang, J. Fan, and Q. Gao, “Changing Epidemiology of Hepatocellular Carcinoma in Asia,” Liver International 42, no. 9 (2022): 2029-2041.

[3] T. Garcia-Lezana, J. L. Lopez-Canovas, and A. Villanueva, “Signaling Pathways in Hepatocellular Carcinoma,” Advances in Cancer Research 149 (2021): 63-101.

[4] J. Wang, H. Huang, Y. Liu, et al., “HBV Genome and Life Cycle,” Advances in Experimental Medicine and Biology 1179 (2020): 17-37.

[5] D. Glebe, N. Goldmann, C. Lauber, and S. Seitz, “HBV Evolution and Genetic Variability: Impact on Prevention, Treatment and Development of Antivirals,” Antiviral Research 186 (2021): 104973.

[6] Y. M. Choi, S. Y. Lee, and B. J. Kim, “Naturally Occurring Hepatitis B Virus Reverse Transcriptase Mutations Related to Potential Antiviral Drug Resistance and Liver Disease Progression,” World Journal of Gastroenterology 24, no. 16 (2018): 1708-1724.

[7] C. L. Lin, Y. D. Chu, and C. T. Yeh, “Emergence of Oncogenic-Enhancing Hepatitis B Virus X Gene Mutants in Patients Receiving Suboptimal Entecavir Treatment,” Hepatology 69, no. 5 (2019): 2292-2296.

[8] W. Liu, S. Cai, R. Pu, et al., “HBV preS Mutations Promote Hepatocarcinogenesis by Inducing Endoplasmic Reticulum Stress and Upregulating Inflammatory Signaling,” Cancers (Basel) 14, no. 13 (2022): 3274.

[9] F. Wei, Q. Zheng, M. Li, and M. Wu, “The Association Between Hepatitis B Mutants and Hepatocellular Carcinoma: A Meta-Analysis,” Medicine 96, no. 19 (2017): e6835.

[10] B. Zhao, H. Qiao, Y. Zhao, et al., “HBV Precore G1896A Mutation Promotes Growth of Hepatocellular Carcinoma Cells by Activating ERK/MAPK Pathway,” Virologica Sinica 38, no. 5 (2023): 680-689.

[11] H. Lee, H. Kim, S. A. Lee, et al., “Upregulation of Endoplasmic Reticulum Stress and Reactive Oxygen Species by Naturally Occurring Mutations in Hepatitis B Virus Core Antigen,” Journal of General Virology 96, no. Pt 7 (2015): 1850-1854.

[12] G. Li, D. Yang, X. Liu, et al., “Precore Mutation Enhances Viral Replication to Facilitate Persistent Infection Especially in HBeAg-Negative Patients,” Virologica Sinica 39, no. 2 (2024): 319-330.

[13] S. Wu, S. Ye, X. Lin, et al., “Small Hepatitis B Virus Surface Antigen Promotes Malignant Progression of Hepatocellular Carcinoma via Endoplasmic Reticulum Stress-Induced FGF19/JAK2/STAT3 Signaling,” Cancer Letters 499 (2021): 175-187.

[14] I. L. Lemmer, N. Willemsen, N. Hilal, and A. Bartelt, “A Guide to Understanding Endoplasmic Reticulum Stress in Metabolic Disorders,” Molecular Metabolism 47 (2021): 101169.

[15] C. Salvagno, J. K. Mandula, P. C. Rodriguez, and J. R. Cubillos-Ruiz, “Decoding Endoplasmic Reticulum Stress Signals in Cancer Cells and Antitumor Immunity,” Trends in Cancer 8, no. 11 (2022): 930-943.

[16] A. Fernandes-da-Silva, C. S. Miranda, D. A. Santana-Oliveira, et al., “Endoplasmic Reticulum Stress as the Basis of Obesity and Metabolic Diseases: Focus on Adipose Tissue, Liver, and Pancreas,” European Journal of Nutrition 60, no. 6 (2021): 2949-2960.

[17] C. Luna-Marco, A. Ubink, M. Kopsida, and F. Heindryckx, “Endoplasmic Reticulum Stress and Metabolism in Hepatocellular Carcinoma,” American Journal of Pathology 193, no. 10 (2023): 1377-1388.

[18] S. W. Xia, Z. M. Wang, S. M. Sun, et al., “Endoplasmic Reticulum Stress and Protein Degradation in Chronic Liver Disease,” Pharmacological Research 161 (2020): 105218.

[19] Y. M. Choi, SY. Lee, and B. J. Kim, “Naturally Occurring Hepatitis B Virus Mutations Leading to Endoplasmic Reticulum Stress and Their Contribution to the Progression of Hepatocellular Carcinoma,” International Journal of Molecular Sciences 20, no. 3 (2019): 597.

[20] Y. Guo, J. Shao, R. Zhang, et al., “Large HBV Surface Protein-Induced Unfolded Protein Response Dynamically Regulates p27 Degradation in Hepatocellular Carcinoma Progression,” International Journal of Molecular Sciences 24, no. 18 (2023): 13825.

[21] F. Yang, L. Hilakivi-Clarke, A. Shaha, et al., “Metabolic Reprogramming and Its Clinical Implication for Liver Cancer,” Hepatology 78, no. 5 (2023): 1602-1624.

[22] G. Jiang, J. Hong, L. Sun, et al., “Glycolysis Regulation in Tumor-Associated Macrophages: Its Role in Tumor Development and Cancer Treatment,” International Journal of Cancer 154, no. 3 (2024): 412-424.

[23] L. Chen, X. Lin, Y. Lei, et al., “Aerobic Glycolysis Enhances HBx-Initiated Hepatocellular Carcinogenesis via NF-kappaBp65/HK2 Signalling,” Journal of Experimental & Clinical Cancer Research 41, no. 1 (2022): 329.

[24] J. Zhong, Q. Kang, Y. Cao, et al., “BMP4 Augments the Survival of Hepatocellular Carcinoma (HCC) Cells Under Hypoxia and Hypoglycemia Conditions by Promoting the Glycolysis Pathway,” American Journal of Cancer Research 11, no. 3 (2021): 793-811.

[25] J. Li, Z. Q. Hu, S. Y. Yu, et al., “CircRPN2 Inhibits Aerobic Glycolysis and Metastasis in Hepatocellular Carcinoma,” Cancer Research 82, no. 6 (2022): 1055-1069.

[26] X. T. Liu, Y. Huang, D. Liu, et al., “Targeting the SphK1/S1P/PFKFB3 Axis Suppresses Hepatocellular Carcinoma Progression by Disrupting Glycolytic Energy Supply That Drives Tumor Angiogenesis,” Journal of Translational Medicine 22, no. 1 (2024): 43.

[27] R. Zhou, W. Ni, C. Qin, et al., “A Functional Loop Between YTH Domain Family Protein YTHDF3 Mediated M(6)A Modification and Phosphofructokinase PFKL in Glycolysis of Hepatocellular Carcinoma,” Journal of Experimental & Clinical Cancer Research 41, no. 1 (2022): 334.

[28] J. Tao, L. Yin, A. Wu, et al., “PDIA2 Bridges Endoplasmic Reticulum Stress and Metabolic Reprogramming During Malignant Transformation of Chronic Colitis,” Frontiers in Oncology 12 (2022): 836087.

[29] R. L. Wiseman, JS. Mesgarzadeh, and L. M. Hendershot, “Reshaping Endoplasmic Reticulum Quality Control Through the Unfolded Protein Response,” Molecular Cell 82, no. 8 (2022): 1477-1491.

[30] LWS. Finley, “What Is Cancer Metabolism?,” Cell 186, no. 8 (2023): 1670-1688.

[31] S. McGuirk, Y. Audet-Delage, and J. St-Pierre, “Metabolic Fitness and Plasticity in Cancer Progression,” Trends in Cancer 6, no. 1 (2020): 49-61.

[32] R. Gao, R. K. R. Kalathur, M. Coto-Llerena, et al., “YAP/TAZ and ATF4 Drive Resistance to Sorafenib in Hepatocellular Carcinoma by Preventing Ferroptosis,” EMBO Molecular Medicine 13, no. 12 (2021): e14351.

[33] J. K. C. Kress, C. Jessen, A. Hufnagel, et al., “The Integrated Stress Response Effector ATF4 Is an Obligatory Metabolic Activator of NRF2,” Cell Reports 42, no. 7 (2023): 112724.

[34] H. Akrami, M. R. Monjezi, S. Ilbeigi, F. Amiri, and M. R. Fattahi, “The Association Between Hepatitis B Virus Mutations and the Risk of Liver Disease and Hepatocellular Carcinoma,” Current Molecular Medicine 22, no. 6 (2022): 514-523.

[35] M. Levrero and J. Zucman-Rossi, “Mechanisms of HBV-Induced Hepatocellular Carcinoma,” Journal of Hepatology 64, supplement, no. S1 (2016): S84-S101.

[36] E. Bobrovnikova-Marjon, C. Grigoriadou, D. Pytel, et al., “PERK Promotes Cancer Cell Proliferation and Tumor Growth by Limiting Oxidative DNA Damage,” Oncogene 29, no. 27 (2010): 3881-3895.

[37] H. K. Cho, K. J. Cheong, H. Y. Kim, and J. Cheong, “Endoplasmic Reticulum Stress Induced by Hepatitis B Virus X Protein Enhances Cyclo-oxygenase 2 Expression via Activating Transcription Factor 4,” Biochemical Journal 435, no. 2 (2011): 431-439.

[38] N. Bhat and A. Mani, “Dysregulation of Lipid and Glucose Metabolism in Nonalcoholic Fatty Liver Disease,” Nutrients 15, no. 10 (2023): 2323.

[39] Y. Zhang, Z. Zhai, J. Duan, et al., “Lactate: The Mediator of Metabolism and Immunosuppression,” Frontiers in Endocrinology 13 (2022): 901495.

[40] J. Zhang, J. Yang, C. Lin, et al., “Endoplasmic Reticulum Stress-Dependent Expression of ERO1L Promotes Aerobic Glycolysis in Pancreatic Cancer,” Theranostics 10, no. 18 (2020): 8400-8414.

[41] J. DeCaprio and T. O. Kohl, “Chromatin Immunoprecipitation,” Cold Spring Harbor Protocols 2020, no. 8 (2020): 098665.

RIGHTS & PERMISSIONS

2025 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.