Molecular Mechanisms and Therapeutic Targets of Hepatitis B Virus Pre-S Mutant-Associated Hepatocellular Carcinoma Tumorigenesis

Abstract

Background: Despite significant progress in diagnosis and therapeutics, hepatocellular carcinoma (HCC) is still among the most commonly occurring and life-taking human cancers globally, raising an urgent need for discovering effective therapeutic targets.

Purpose: Chronic hepatitis B virus (HBV) infection is a major etiological factor associated with HCC development, progression, and prognosis. Pre-S mutants are naturally occurring mutated forms of HBV large surface proteins and predict a higher risk of HCC development and recurrence. Moreover, pre-S mutants function as important HBV oncoproteins which can promote HCC tumorigenesis through initiating a variety of oncogenic signaling pathways. Targeting pre-S mutant-induced oncogenic signaling pathways displays therapeutic potential in HCC.

Research Design: This review summarizes the underlying molecular mechanisms of pre-S mutant-associated HCC tumorigenesis and highlights their potential in serving as therapeutic targets for HCC.

Keywords

hepatocellular carcinoma pre-S mutant hepatitis B virus molecular mechanism therapeutic target

Background

Hepatocellular carcinoma (HCC) is the most predominant histologic subtype of primary liver cancer, which constitutes more than 90% of total liver malignancies.^1,2 Although many preventive and therapeutic strategies have been well established, HCC remains the sixth most frequent and third most deadly human cancer worldwide, leading to around 800,000 new cases and 800,000 new deaths annually.^3,4 Among the etiological agents of HCC development, chronic infection with hepatitis B virus (HBV) plays an essential role and accounts for up to 50% of all HCC cases worldwide.^5,6 Therefore, discovery of effective therapeutic targets for HBV-related HCC is urgently needed to ameliorate patient mortality and prognosis.

HBV surface gene comprises 3 continuous gene segments (pre-S1, pre-S2, and S) and encodes 3 different sizes of surface proteins (small, middle, and large) from the S gene segment, the pre-S2 and S gene segments, and all 3 gene segments, respectively, which collectively constitute the viral envelope proteins.^7,8 Among the 3 types of HBV surface proteins, small surface proteins are the most abundant type and harbor the major antigenic determinant that is exposed on the outside of viral envelope and recognized by neutralizing antibodies.^9,10 The pre-S2 domain of middle surface proteins is more immunogenic for B cells and important for facilitating the production of viral particles.^11,12 The pre-S1 domain of large surface proteins is indispensable for viral particle assembly and secretion and plays an essential role in mediating viral entry into hepatocytes.^13,14 In addition, because HBV polymerase lacks intrinsic proofreading activity, genomic mutations are frequently generated during the process of HBV DNA replication, responsible for the integration of viral genome into host cell genome and the production of mutated viral proteins, implicating in viral infection and liver disease progression.^15,16

Several naturally occurring deletion mutations occur in the pre-S1 gene segment (ranging from nucleotides (nt) 2854 to 3210) or pre-S2 gene segment (ranging from nt 3211 to 3221 and nt 1 to 154), leading to the production of mutated forms of large surface proteins which contain deleted pre-S1 domain (designated pre-S1 mutant) or deleted pre-S2 domain (designated pre-S2 mutant).^17,18 Although the frequency and pattern of pre-S gene mutations vary among different HBV genotypes,^19,20 the prevalence of pre-S mutants in HBV-related patients is gradually increased along the progression of liver diseases from chronic viral infection (approximately 20%) to liver cirrhosis (approximately 35%) and eventually HCC (up to 60%).^21-26 The presence of pre-S mutants in patients with chronic HBV infection and HBV-related HCC represents an independent factor for predicting a higher risk of HCC development and recurrence, respectively.^27-33 Moreover, pre-S mutants have been well demonstrated as important HBV oncoproteins that can activate multiple oncogenic signaling pathways to induce endoplasmic reticulum (ER) stress, oxidative stress, DNA damages, genomic and chromosomal instability, anchorage-independent cell growth, cell proliferation and survival, cell cycle progression, aerobic glycolysis, and lipid biosynthesis, which collectively contribute to HCC development.^34,35 Inhibition of the pre-S mutant-activated oncogenic signaling pathways exhibits potential therapeutic effects on HCC in transgenic mouse models.^36-39

This review summarizes the molecular evidence of pre-S mutant-induced HCC development and underscores the therapeutic potential of targeting pre-S mutant-activated oncogenic signaling pathways for HCC.

Both Pre-S1 and Pre-S2 Mutants Induce ER Stress-Dependent Signaling Pathways to Trigger Oxidative Stress, DNA Damages, Genomic Instability, Anchorage-Independent Cell Growth, Cell Proliferation, Aerobic Glycolysis, and Lipid Biosynthesis

As a major structural component of viral envelope, HBV large surface proteins are normally released from the host hepatocytes following viral particle assembly and budding. Unlike the large surface proteins with wild-type pre-S1 and pre-S2 domains, both pre-S1 and pre-S2 mutants are preferentially accumulated in the intracellular organelle ER and their retention in the ER induces ER stress and unfolded protein response (UPR) in human HCC cell lines and liver tissues of humanized mice and HCC patients.^17,40 Depending upon the induction of ER stress, pre-S mutants are capable of initiating multiple oncogenic signaling pathways to promote HCC tumorigenesis (Table 1). The study by Hsieh et al. found that pre-S mutants could increase the intracellular reactive oxygen species (ROS) levels to trigger oxidative stress and DNA damages, contributing to genomic instability in human HCC cell lines and liver tissues of transgenic mice and HCC patients.⁴¹ The study by Hung et al. revealed that pre-S mutants could upregulate cyclooxygenase-2 (COX-2) expression through activation of nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (p38 MAPK), leading to enhanced anchorage-independent cell growth in human HCC cell lines and liver tissues of transgenic mice and HCC patients.⁴² The study by Yang et al. unveiled that pre-S mutants could activate protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway through elevation of vascular endothelial growth factor (VEGF)-A expression, secretion, and autocrine binding to VEGF receptor-2 (VEGFR-2), resulting in increased cell proliferation in human HCC cell lines and liver tissues of transgenic mice and HCC patients.⁴³ The enhanced VEGF-A production by pre-S mutants could also promote endothelial cell proliferation in a paracrine fashion.⁴³ Moreover, the studies by Teng et al demonstrated that through the activation of mTOR, pre-S mutants could activate glucose metabolism-related Yin Yang 1 (YY1)/Myc/solute carrier family 2 member 1 (SLC2A1) signaling pathway to induce aerobic glycolysis and lipid metabolism-related sterol regulatory element-binding transcription factor 1 (SREBF1)/adenosine triphosphate citrate lyase (ACLY)/fatty acid desaturase 2 (FADS2) signaling pathway to enhance lipid biosynthesis in human HCC cell lines and transgenic mouse liver tissues, which combinedly promoted cell proliferation.^37,44

Table 1.

Oncogenic Signaling Pathways Activated by Pre-S1 Mutant And/or Pre-S2 Mutant in HCC.

Pre-S Mutant	Deleted Region	Experimental Model	ER Stress Dependence	Signaling Pathway and Oncogenic Effect	Year	References
Both pre-S1 and pre-S2 mutants
Pre-S1 mutant	Pre-S1 domain (nt 3040 to 3111)	Human HCC cell lines, mouse liver tissues, and human liver tissues	ER stress-dependent	Accumulated in intracellular organelle ER to induce ER stress and UPR	2003; 2021	Wang et al⁹; Liang et al²⁴
Pre-S2 mutant	Pre-S2 domain (nt 2 to 55 or 23 to 55)		ER stress-dependent		2003; 2021	Wang et al⁹; Liang et al²⁴
Pre-S1 mutant	Pre-S1 domain (nt 3040 to 3111)	Human HCC cell lines, mouse liver tissues, and human liver tissues	ER stress-dependent	Increase intracellular ROS levels to trigger oxidative stress, DNA damages, and genomic instability	2004	Hsieh et al²⁵
Pre-S2 mutant	Pre-S2 domain (nt 4 to 57)		ER stress-dependent		2004	Hsieh et al²⁵
Pre-S1 mutant	Pre-S1 domain (nt 3040 to 3111)	Human HCC cell lines, mouse liver tissues, and human liver tissues	ER stress-dependent	Activate NF-κB/p38 MAPK/COX-2 signaling pathway to enhance anchorage-independent cell growth	2004	Hung et al²⁶
Pre-S2 mutant	Pre-S2 domain (nt 4 to 57)		ER stress-dependent		2004	Hung et al²⁶
Pre-S1 mutant	Pre-S1 domain (nt 3040 to 3111)	Human HCC cell lines, mouse liver tissues, and human liver tissues	ER stress-dependent	Activate VEGF-A/VEGFR-2/Akt/mTOR signaling pathway to increase cell proliferation	2009	Yang et al²⁷
Pre-S2 mutant	Pre-S2 domain (nt 4 to 57)		ER stress-dependent
Pre-S1 mutant	Pre-S1 domain (nt 3040 to 3111)	Human HCC cell lines and mouse liver tissues	ER stress-dependent	Activate YY1/Myc/SLC2A1 signaling pathway to induce aerobic glycolysis	2015	Teng et al²¹
Pre-S2 mutant	Pre-S2 domain (nt 4 to 57)	Human HCC cell lines and mouse liver tissues	ER stress-dependent		2015	Teng et al²¹
Pre-S1 mutant	Pre-S1 domain (nt 3040 to 3111)	Human HCC cell lines and mouse liver tissues	ER stress-dependent	Activate SREBF1/ACLY/FADS2 signaling pathway to enhance lipid biosynthesis	2015	Teng et al²⁸
Pre-S2 mutant	Pre-S2 domain (nt 4 to 57)	Human HCC cell lines and mouse liver tissues	ER stress-dependent		2015	Teng et al²⁸
Pre-S2 mutant alone
Pre-S2 mutant	Pre-S2 domain (nt 23 to 55)	Human HCC cell lines	ER stress-independent	Stimulate mitochondrial Ca²⁺ overload to cause mitochondrial dysfunction and energy metabolism impairment	2021	Liang et al²⁴
Pre-S2 mutant	Pre-S2 domain (nt 4 to 57)	Human HCC cell lines, mouse liver tissues, and human liver tissues	ER stress-independent	Enhance cyclin A expression and trigger μ-calpain-mediated cyclin A cleavage to increase ΔN-cyclin A levels to causes centrosome overduplication and chromosomal instability	2005; 2012; 2016	Wang et al²⁹; Wang et al³⁰; Yen et al³¹
Pre-S2 mutant	Pre-S2 domain (nt 4 to 57)	Human HCC cell lines and mouse liver tissues	ER stress-independent	Activate JAB1/p27/Cdk2/Rb signaling pathway to promote cell cycle progression	2007; 2013	Hsieh et al³²; Hsu et al³³
Pre-S2 mutant	Pre-S2 domain (nt 4 to 57)	Human HCC cell lines, mouse liver tissues, and human liver tissues	ER stress-independent	Interact with importin α1 to suppress nuclear transport of NBS1 to cause DNA double-strand break repair impairment and genomic instability	2015	Hsieh et al³⁴
Pre-S2 mutant	Pre-S2 domain (nt 4 to 57)	Human HCC cell lines and mouse liver tissues	ER stress-independent	Upregulate Bcl-2 expression to increased cell survival and drug resistance	2011	Hung et al³⁵

Abbreviations: HCC, hepatocellular carcinoma; ER, endoplasmic reticulum; nt, nucleotides; UPR, unfolded protein response; ROS, reactive oxygen species; NF-κB, nuclear factor-κB; p38 MAPK, p38 mitogen-activated protein kinase; COX-2, cyclooxygenase-2; VEGF vascular endothelial growth factor; VEGFR-2, VEGF receptor-2; Akt, protein kinase B; mTOR, mammalian target of rapamycin; YY1, Yin Yang 1; SLC2A1, solute carrier family 2 member 1; SREBF1, sterol regulatory element-binding transcription factor 1; ACLY, adenosine triphosphate citrate lyase; FADS2, fatty acid desaturase 2; Ca²⁺, calcium; ΔN-cyclin A, N-terminus truncated cyclin A; JAB1, Jun activation domain-binding protein 1; Cdk, cyclin-dependent kinase; Rb, retinoblastoma protein; NBS1, Nijmegen breakage syndrome 1; Bcl-2, B cell lymphoma-2.

Pre-S2 Mutant Additionally Initiates ER Stress-Dependent or -Independent Signaling Pathways to Promote Chromosomal Instability, DNA Repair Impairment, Cell Cycle Progression, and Cell Survival and Drug Resistance

Besides the both types of pre-S mutants-activated oncogenic signaling pathways, pre-S2 mutant can additionally induce several ER stress-dependent or -independent oncogenic signaling pathways (Table 1). The study by Liang et al. verified that pre-S2 mutant could stimulate ER stress-dependent mitochondrial calcium (Ca²⁺) overload, leading to mitochondrial ultrastructure aberration, mitochondrial membrane potential reduction, energy metabolism impairment, and mitochondrial dysfunction in human HCC cell lines.⁴⁰ The studies by Wang et al⁴⁵ and Wang et al⁴⁶ confirmed that pre-S2 mutant could trigger ER stress-dependent, Ca²⁺-dependent protease μ-calpain-mediated cyclin A cleavage and enhance ER stress-independent expression of cyclin A, which collectively lead to increased N-terminus truncated cyclin A (ΔN-cyclin A) levels in human HCC cell lines and liver tissues of transgenic mice and HCC patients. Distinct from the full-length cyclin A, which is mainly located in the nucleus of cells, ΔN-cyclin A is preferentially accumulated in the cytoplasm and causes centrosome overduplication, resulting in chromosomal instability.⁴⁶ Furthermore, the study by Yen et al. showed that pre-S2 mutant could stimulate ER stress-dependent Ca²⁺ efflux from the ER lumen, which induced stromal interaction molecule 1 (STIM1) oligomerization on the outer membrane of ER and recruitment of the STIM1-resident ER toward Ca²⁺ release-activated Ca²⁺ modulator 1 (Orai1)-localized plasma membrane, resulting in Ca²⁺ influx into the cytoplasm, which further facilitated cleavage of cyclin A by μ-calpain.⁴⁷ In addition, the studies by Hsieh et al⁴⁸ and Hsu et al⁴⁹ ascertained that pre-S2 mutant could induce ER stress-independent degradation of cyclin-dependent kinase (Cdk) inhibitor p27 through zinc-dependent binding with Jun activation domain-binding protein 1 (JAB1), which drived activation of Cdk2 and inactivation of retinoblastoma protein (Rb), leading to cell cycle progression in human HCC cell lines and transgenic mouse liver tissues. The study by Hsieh et al. further validated that pre-S2 mutant could interact with importin α1 in an ER stress-independent fashion to suppress transport of DNA repair protein Nijmegen breakage syndrome 1 (NBS1) into the cell nucleus, resulting in DNA double-strand break repair impairment and genomic instability in human HCC cell lines and liver tissues of transgenic mice and HCC patients.⁵⁰ Moreover, the study by Hung et al. identified that pre-S2 mutant could upregulate apoptosis inhibitor B cell lymphoma-2 (Bcl-2) expression without the mediation of ER stress, contributing to increased cell survival and drug resistance under the treatment of chemotherapy agent 5-fluorouracil in human HCC cell lines and transgenic mouse liver tissues.⁵¹

Pre-S Mutants Promote Hepatocyte Malignant Transformation and HCC Development and Inhibition of pre-S Mutant-Activated Oncogenic Signaling Pathways Exhibits Potential Therapeutic Effects on HCC

In addition to the ability of activating multiple oncogenic signaling pathways, pre-S mutants are able to trigger malignant transformation from hepatocytes into HCC development. The study by Wang et al. elucidated that pre-S2 mutant could drive cell proliferation, anchorage-independent cell growth, and multinucleation in human nonmalignant hepatocyte cell lines.⁴⁵ Furthermore, the studies by Teng et al,³⁶ Hung et al,⁴² Yang et al,⁴³ and Wu et al⁵² observed that liver-specific pre-S2 mutant-transgenic mice exhibited various liver pathologies, including chronic inflammation, hepatic steatosis, fibrosis/cirrhosis, hepatomegaly, and HCC tumor growth, along with ER stress and activation of multiple oncogenic signaling pathways during the pathological progression of liver diseases in transgenic mouse liver tissues. Moreover, targeting pre-S mutant-activated oncogenic signaling pathways can improve liver pathologies and prevent HCC tumor growth. The study by Teng et al. determined that suppression of mTOR/YY1/Myc/SLC2A1 signaling pathway activation by combination treatment with 2 natural plant products silymarin and resveratrol could reduce HCC tumor size in transgenic mouse liver tissues.³⁷ The study by Teng et al. also certified that inhibition of NF-κB and mTOR activation by treatment with a physically stable phytosomal formulation of natural plant product curcumin could ameliorate hepatic inflammation and lipid accumulation and prevent HCC tumor formation in transgenic mouse liver tissues.³⁸ Furthermore, the study by Hsieh et al. proved that disruption of the interaction between pre-S2 mutant and JAB1 by treatment with histone deacetylase inhibitor suberoylanilide hydroxamic acid, which increased thioredoxin-binding protein 2 expression for competitive interaction with JAB1, could decrease cell proliferation, multinucleation, and nucleus-to-cytoplasm ratio in transgenic mouse livers.³⁹

Discussion

To date, the functions of the oncogenic signaling pathways activated by pre-S mutants are mainly to regulate the intrinsic malignancy of HCC cells. Although the pre-S mutant-enhanced VEGF-A production can upregulate endothelial cell proliferation, the role of pre-S mutants in regulating the communication between tumor cells and stromal cells in the tumor microenvironment of HCC is still largely unclarified. Recently, the studies by Teng et al^53,54 and Jeng et al⁵⁵ unraveled that pre-S2 mutant-positive HCC patients had increased expression of pro-tumor immune checkpoint molecule programmed death ligand 1 (PD-L1) and elevated number and activity of pro-tumor immune cell regulatory T cells (Tregs) in tumor tissues, both of which independently predicted a higher risk of HCC recurrence. Moreover, the study by Jeng et al. showed that the plasma levels of the pro-tumor chemokine monocyte chemoattractant protein-1 (MCP-1) were increased in pre-S2 mutant-positive HCC patients and independently associated with a higher risk of HCC recurrence.⁵⁶ These findings suggest that pre-S2 mutant may play a potential role in facilitating tumor immune evasion although the exact regulation and underlying mechanisms remain to be elucidated. In addition, besides pre-S mutants, C-terminal truncated HBV X protein (HBx) is another naturally occurring mutated viral protein that plays important role in HCC tumorigenesis.⁵⁷ It has been shown that C-terminal truncated HBx promotes hepatocarcinogenesis through enhancing cell proliferation, migration, and invasion abilities as well as reprogramming glucose metabolism.^58-60 The study by Wu et al. found that co-expression of pre-S2 mutant and wild-type HBx exhibited enhanced effects on activation of oncogenic signaling pathways and promotion of HCC tumor growth in human HCC cell lines and liver tissues of transgenic mice and HCC patients.⁵² The combined oncogenic effect of pre-S mutants and C-terminal truncated HBx and its implication for HBV-induced hepatocarcinogenesis are worthwhile for further investigation. Also, several naturally occurring point mutations in the precore/core region of HBV genome lead to expression of mutated core proteins, which have been shown to induce ER stress, ROS production, and inflammatory cytokine expression, contributing to HCC progression.^61,62 Furthermore, atezolizumab (anti-PD-L1 antibody) plus bevacizumab (anti-VEGF antibody) has been approved by the US Food and Drug Administration (FDA) as a first-line therapy for advanced HCC.⁶³ Considering the increased expression of VEGF-A and PD-L1 in pre-S mutant-positive HCC patients, combination of atezolizumab and bevacizumab may hold great promise in the treatment of this patient population. In contrast to targeting viral protein-disturbed host signaling pathways, several therapeutic strategies have been developed for targeting specific viral protein without interfering with the host signaling pathways, such as ubiquitination-targeted drugs and cell-penetrating whole molecule antibodies.^64,65 The study by Yang et al. identified that degradation of intracellular pre-S2 mutant through induction of microautophagy by selective Bcl-2 inhibitor ABT199 could diminish pre-S2 mutant-stimulated DNA damages and chromosomal instability.⁶⁶ Additionally, the study by Khodadad et al characterized the structure of pre-S1 and pre-S2 domains of large surface proteins,⁶⁷ supporting the promise in discovering structure-based therapeutics for specifically targeting pre-S mutants.

Conclusions

This review summarizes the molecular mechanisms explaining the oncogenic functions of pre-S mutants in promoting HBV-related HCC development and proposes targeting the pre-S mutant-activated oncogenic signaling pathways as a promising therapeutic strategy for HCC chemoprevention.

Footnotes

Author Contributions

Conceptualization, Long-Bin Jeng, Wen-Ling Chan, and Chiao-Fang Teng; Funding acquisition, Chiao-Fang Teng; Visualization, Long-Bin Jeng, Wen-Ling Chan, and Chiao-Fang Teng; Writing—original draft, Chiao-Fang Teng; Writing—review and editing, Chiao-Fang Teng.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the China Medical University, Taichung, Taiwan (grant number CMU112-ASIA-06).

Ethical Statement

ORCID iD

Chiao-Fang Teng

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