Antiviral therapy for hepatitis B virus infection is beneficial for the prognosis hepatocellular carcinoma

Abstract

In this editorial, we comment on the article by Mu et al, published in the recent issue of the World Journal of Gastrointestinal Oncology. We pay special attention to the immune tolerance mechanism caused by hepatitis B virus (HBV) infection, the pathogenesis of hepatocellular carcinoma (HCC), and the role of antiviral therapy in treating HCC related to HBV infection. HBV infection leads to systemic innate immune tolerance by directly inhibiting pattern recognition receptor recognition and antiviral signaling pathways, as well as by inhibiting the immune functions of macrophages, natural killer cells and dendritic cells. In addition, HBV leads to an immunosuppressive cascade by expressing inhibitory molecules to induce exhaustion of HBV-specific cluster of differentiation 8 + T cells, ultimately leading to long-term viral infection. The loss of immune cell function caused by HBV infection ultimately leads to HCC. Long-term antiviral therapy can improve the prognosis of patients with HCC and prevent tumor recurrence and metastasis.

Key Words: Hepatitis B virus; Hepatocellular carcinoma; Hepatitis B virus-DNA; Immune tolerance; Antiviral therapy

Core Tip: Hepatocellular carcinoma (HCC) is the seventh most common cancer in the world and is usually associated with hepatitis B virus (HBV) infection. Radical resection and antiviral therapy are considered key clinical treatments for patients with HBV-related HCC. However, many patients have their HCC and HBV infection detected at the same time, so they receive remedial antiviral treatment beginning in the perioperative period, missing the opportunity for long-term preoperative antiviral therapy. Therefore, evaluating the clinical efficacy and relevant factors of perioperative remedial antiviral therapy will be valuable.

INTRODUCTION

Hepatocellular carcinoma (HCC) is currently one of the most common malignant tumors in the world, with its incidence rate ranking seventh in the world and its mortality ranking third in the world[1]. HCC is a complex and multifactorial disease, and its occurrence and development are influenced by both genetic and environmental factors. Multiple studies have shown that factors affecting HCC include hepatitis B virus (HBV) infection, hepatitis C virus (HCV) infection, alcohol consumption, and nonalcoholic fatty liver disease[2].

The immune tolerance caused by HBV can be divided into hepatic innate immune tolerance and adaptive immune tolerance. Hepatic intrinsic immunity is the frontline defense mechanism against HBV attacks, and pattern recognition receptors (PRRs) play important roles in immune responses. Numerous studies have shown that HBV infection interferes with PRR-mediated antiviral signaling[3-5]. HBV infection is one of the risk factors for HCC, accounting for 50%-80% of HCC cases worldwide. The tumor immune microenvironment of HCC is characterized by immunosuppression through a variety of mechanisms, including the recruitment of immunosuppressive cells, a reduction in antitumor effector cells, changes in cytokine levels, and increased expression of immune checkpoint proteins[6]. HBV infection leads to systemic innate immune tolerance by directly inhibiting PRR recognition and antiviral signaling pathways, as well as by inhibiting the immune functions of macrophages, natural killer (NK) cells and dendritic cells (DCs). In addition, HBV leads to an immunosuppressive cascade by expressing inhibitory molecules such as programmed death (PD)-1, programmed cell death 1 ligand 1 (PD-L1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), interleukin (IL)-10, T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) to induce exhaustion of HBV-specific cluster of differentiation (CD) 8 + T cells, ultimately leading to long-term viral infection. The loss of immune cell function caused by HBV infection ultimately leads to HCC. In this editorial, we comment on the article by Mu et al[7]. We elucidate the mechanisms of HBV infection and the pathogenesis of liver cancer and suggest that long-term antiviral therapy can improve the prognosis of patients with HCC and prevent tumor recurrence and metastasis.

THE IMMUNE TOLERANCE MECHANISM OF HBV INFECTION

HBV has four open reading frames, namely, the S region, C region, P region, and X region, which encode hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), HBV polymerase, and hepatitis B X protein (HBx), respectively[8]. Because HBV does not cause the breakdown of infected cells through extensive proliferation, the immune system relies mainly on CD8 + T cells or NK cells to kill infected cells, release the virus, and then eliminate the virus with antibodies. Moreover, cytokines such as interferon (IFN)-α or tumor necrosis factor (TNF)-α can directly inhibit HBV replication or induce its degradation by activating apolipoprotein B mRNA editing enzyme catalytic subunit nucleic acid editing enzymes[9]. However, when the function of immune cells is disrupted or the release of cytokines is inhibited, immune tolerance can occur.

The immune tolerance caused by HBV can be divided into hepatic innate immune tolerance and adaptive immune tolerance. Hepatic intrinsic immunity is the first line of defense against HBV attacks, and PRRs play important roles in immune responses. Numerous studies have shown that HBV infection interferes with PRR-mediated antiviral signaling pathways[3-5]. HBeAg, HBsAg, and other viral particles inhibited the antiviral function induced by toll-like receptors (TLRs) in HBV-infected individuals[10]. The reduction in TLR3, TLR4, and TLR9 expression in hepatocytes is significantly reduced during the immune tolerance stage of HBV infection, and the TLR signaling pathway is disrupted. In addition, HBV polymerase inhibits TLR3-mediated IFN-α/β induction by blocking the activation of interferon regulatory factor 3 (IRF3). Research has indicated that HBV polymerase inhibits the phosphorylation, dimerization, and nuclear translocation of IRF3[11]. In addition, Lang et al[12] reported that HBeAg can inhibit the function of TLRs by inhibiting the activation of NF-κB[12]. HBsAg inhibits TLR2 on monocytes/macrophages by blocking the c-Jun N-terminal kinase-mitogen-activated protein kinase pathway and provides innate immune suppression[13]. HBV targets TLRs and downstream signaling pathways, leading to endogenous immune tolerance in the liver. Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic antiviral nucleic acid sensor that generates type 1 IFN, thereby inhibiting HBV replication[14]. HBx and HBV polymerase interfere with the RIG-I signaling pathway in human liver cells by disrupting the interaction between interferon beta promoter stimulator 1 and RIG-I and inhibiting the production of type I IFN[15].

Kupffer cells (KCs) and monocyte derived macrophages are two main types of macrophages in the liver. As an immunosuppressive organ, the liver exhibits an anti-inflammatory phenotype through the secretion of immune regulatory cytokines such as IL-10 under physiological conditions. However, during virus infection, the liver recruits Ly6c + monocytes to secrete pro-inflammatory factors IL-6 and IL-1β and TNF-α, controlling infection through inflammatory response[16]. Up-regulation of IL-10 during HBV infection can impair lymphocyte function and decelerate HBV clearance[17]. HBV infection promotes the production of IL-18 in KCs and affects the activity of NK cells. Research has shown that not only does the level of pro-inflammatory factors secreted by liver resident KCs decrease, but the level of pro-inflammatory factors secreted by M1 macrophages from periphery also decreases after HBV infected. In addition, HBV can enhance liver immune tolerance by stimulating monocyte differentiation into M2 macrophage, which is beneficial for infection maintenance[18]. Moreover, M2 macrophage may also be involved in the occurrence and development of HCC by releasing inhibitory regulatory factors.

NK cell number, IFN-γ production and cell lysis ability weakened in chronic hepatitis B (CHB) patients[19]. There are inhibitory receptors such as NK group 2A (NKG2A), Tim-3, and killer cell lectin like receptor G1 (KLRG1), and activate receptors such as NK group 2C, NK group 2D (NKG2D) and CD16 on NK cell surface[20,21]. HBeAg increase the expression of NKG2A and Tim-3 on the surface of peripheral blood NK cells of CHB patients[22,23]. The inhibitory KLRG1 + NK cells increased in the blood and liver of HBsAg positive CHB patients. In addition, peripheral blood mononuclear cells and NKG2D + NK cells in the liver of CHB patients decreased[24]. The phenotypic changes of NK cells are the basis of functional defects and the result of immune dysfunction. IFN-γ and TNF-α production decreased in conventional NK cells of CHB patients may related to the CD122 low expression on cell surface[25]. IL-15 mediated activation of the protein kinase B mammalian target of rapamycin pathway is impaired in NK cells of CHB patients[26]. The high level of inhibitory cytokine IL-10 in the liver of CHB patients has a significant inhibitory effect on the production of IFN-γ by NK cells[27].

DCs are specialized antigen-presenting cells, as well as participating in the production of cytokines which affect T cell polarization. The DC subpopulations mainly include myeloid DCs (mDCs) and plasma like DCs. The mDCs frequency reduced in CHB patients but it would be recovered after antiviral therapy[28]. The intrahepatic mDC was positively correlated with serum alanine aminotransferase level, and significantly negatively correlated with plasma HBV load[29]. The mDCs from HBV patients showed functional defects resulting in a reduction of IL-2, IFN-γ and TNF-α production by T cells due of IL-12 reduction[30]. NK cell and DC interactions caused by HBV may significantly impair the efficacy of antiviral immune response in CHB patients[31]. In conclusion, the persistence of HBV not only directly inhibits PRRs recognition and antiviral signal pathway, leading to endogenous immune tolerance of cells, but also inhibits the function of innate immune cells (including macrophages, NK cells and DCs), leading to hepatic innate immune tolerance.

The persistent existence of HBV will lead to CD8 + T cell frequency reduction and functional defects. In CHB infection, CD8 + T cells lose their proliferation capacity and antiviral function, which is characterized by excessive inhibitory signals, reduced production of cytokines, and T cells exhaustion[32]. PD-1 is a co-inhibitory receptor that has been shown to increase expression on antigen specific T cells during chronic viral infection and weaken T cell activation through co-inhibitory signaling. Blocking the PD-1/PD-L1 pathway can restore the function of exhausted T cells[33]. During the persistence of HBV, PD-1 was up-regulated on both peripheral blood monocytes and intrahepatic lymphocytes, especially on HBV specific CD8 + T cells. PD-1 interacted with PD-L1 on antigen presenting cells, leading to the functional inhibition and apoptosis of CD8 + T cells[34]. In CHB infection, the proportion of liver infiltrating T-reg cells increased. Multiple molecules are involved in T-reg-mediated immunosuppression, including CTLA-4, IL-10 and Tim-3. CTLA-4 and Tim-3 are up-regulated on HBV specific CD8 + T cells, which is closely related to the viral load, and plays an important role in T cell depletion in persistent HBV infection[35]. In addition, the immunosuppressive environment in the liver during HBV infection contributes to T cell tolerance. The level of IL-10 and transforming growth factor (TGF)-β is closely related to virus replication. The cell-intrinsic production of TGF-β mediates apoptosis of CD8 + T cells, thus blocking TGF-β may contribute to T cell reconstitution[36]. In summary, HBV forms an immunosuppressive cascade by inhibitory molecules such as PD-1, PD-L1, CTLA-4, IL-10, Tim-3 and immune cells CD8 + T cells, ultimately leading to long-term viral infection (shown in Figure 1).

Figure 1 Hepatitis B virus immune tolerance mechanism. In hepatocyte, hepatitis B virus (HBV) inhibits toll-like receptors mediated interferon (IFN)-α/β production by blocking the activation of interferon regulatory factor 3 HBV interferes with the retinoic acid-inducible gene-I (RIG-I) signaling pathway in hepatocyte and inhibits the production of IFN-α/β by disrupting the interaction between mitochondrial antiviral-signaling protein and RIG-I. HBV affects the activation of natural killer (NK) cells by inhibiting the production of interleukin (IL)-18 by macrophages. HBV promotes the generation of Tregs, which release IL-10 and transforming growth factor β to increase the expression of surface inhibitory receptors on NK cells and CD8 + T cells, thereby reducing IFN and tumor necrosis factor-α release. The expression of immune checkpoints on the surface of CD8 + T cells increases, leading to T cell exhaustion, resulting in immune tolerance of HBV. IL: Interleukin; HBV: Hepatitis B virus; NK: Natural killer; IFN: Interferon; NKG2A: Natural killer group 2A; NKG2D: Natural killer group 2D; Tim-3: T-cell immunoglobulin and mucin domain-containing protein 3; DC: Dendritic cell; CD: Cluster of differentiation; PD-1: Programmed death-1; CTLA4: Cytotoxic T lymphocyte-associated protein 4; TGF: Transforming growth factor; PD-L1: Programmed cell death 1 ligand 1; TNF: Tumor necrosis factor; TLR: Toll-like receptors; IRF-3: Interferon regulatory factor 3; IPS-1: Interferon-β promoter stimulator 1; RIG-I: Retinoic acid-inducible gene-I.

THE PATHOGENESIS OF HCC

HCC is currently one of the most common malignant tumors worldwide[1]. The occurrence and development of HCC are influenced by both genetic and environmental factors. Multiple studies have shown that factors affect HCC, including HBV infection, HCV infection, alcohol consumption, and nonalcoholic fatty liver disease[2]. At present, the occurrence and development of HCC is a multigene, multistep and multistage process. With the development of molecular biology, research on the pathogenesis of HCC is gradually becoming systematic, which may provide new approaches for the treatment of HCC.

The occurrence of HCC is related to the unbalance activation of oncogenes and antioncogenes. Under normal circumstances, oncogenes maintain low expression levels and play important physiological functions. But under certain conditions, such as viral infection, chemical carcinogens or radiation effects, oncogenes can be abnormally activated and induce cancerous transformation. Common oncogenes include the RAS family, MYC family, SRC family, SIS family and MYB family[37]. Antioncogenes play a crucial negative regulatory role in controlling cell growth, proliferation, and differentiation. They interact with oncogenes to maintain a normal physiological activity of cells. P53 is an important antioncogene that can induce apoptosis of cancer cells. However, when P53 mutates, it loses control of cell proliferation and leads cell canceration[38]. P53 can inhibit tumor progression by controlling the composition of microRNAs carried by exosomes and secreting cytokines. On the contrary, P53 mutants can promote tumor progression by regulating the content of exosomes, leading M2 macrophages polarization and form immunosuppressive microenvironment[39]. P21 gene is closely linking tumor inhibition with cell cycle control processes[40]. P16 is a fundamental gene in the cell cycle, whose expression products are directly involved in the negative regulation of cell proliferation[41]. The bnormal methylation of P53, P21 and P16 in serum DNA play an important role in early detection of HCC[42]. These findings indicate that the inactivation of P16 is a major event in the development of liver cancer. In addition, there are a large number of antioncogenes, such as PTEN, Rb, which are closely related to the occurrence of HCC.

The occurrence of HCC is also related to abnormal activation of signaling pathways. The key proteins in Wingless (Wnt)/β-catenin signaling pathway undergo mutations will lead to abnormal cell proliferation and promote HCC occurrence. Frizzled family, casein kinase 1, desheveled, glycogen synthase kinase 3, APC, AXIN, β-catenin and transcription factor T-cell factor (TCF)/lymphoid enhancer factor family compose the main Wnt signaling pathway[43,44]. When there is no Wnt signal, β-catenin is effectively phosphorylated and polyubiquitinated in the AXIN complex. AXIN gene mutations can prevent β-catenin degradation, which accumulates in the cytoplasm and enters the nucleus to bind to TCF family proteins, enhances the transcription of downstream genes such as c-MYC, survivin and Cyclin-D1, promotes cell proliferation, and inhibits cell apoptosis. Abnormal activation of the Wnt pathway can cause unlimited proliferation of tumor cells and invasion and metastasis of tumors[45]. The expression of β-catenin and its downstream target genes such as c-MYC and Cyclin-D1 is reduced when Wnt-10B gene knockout. Therefore, the abnormal expression of Wnt is involved in HCC occurrence[46]. PARP6 can affect wnt/β-catenin by inhibiting the expression of XRCC6 to inhibit HCC progression[47]. Curcumin inhibit the proliferation of liver cancer cells, induce cell cycle arrest and apoptosis by reducing the expression of β-catenin and inducing inactivation of wnt/β-catenin signaling pathway[48]. Based on these themes, future experiments will focus on developing new therapies for wnt/β-catenin in HCC treatment[49].

The Hedgehog (Hh) signaling pathway controls cell growth and proliferation, and the occurrence of tumors is a result of uncontrolled cell growth and proliferation[50]. In the presence of Hh ligands, when Hh ligands bind to patched (Ptch)-1, Ptch-1 is internalized, which relieves the inhibition of smoothened (Smo). Smo dissociates SUFU from GLI and then forms GLI activators, which promotes the expression of target genes. In the absence of Hh ligands, Ptch-1 inhibits the expression of Smo, and Gli binds to SUFU to form GLIR, inhibiting the expression of target genes. Zhou et al[51] discovered that the molecular mechanism of chemotherapy resistance in HCC has been revealed by targeting the Hh signaling pathway, providing new insights for alleviating resistance in refractory HCC. He et al[52] demonstrated that circznf609 activates the Hh pathway by regulating the expression of GLI2, thereby enhancing the proliferation and metastasis of liver cancer cells. In summary, numerous studies have shown that abnormal expression of the Hh pathway promotes the occurrence of HCC.

The NOTCH signaling pathway is also closely related to the occurrence of HCC. NOTCH signaling affects multiple processes of normal cellular morphogenesis, including apoptosis, proliferation, and formation of cell boundaries[53,54]. In the pathogenesis of HCC, the NOTCH signaling pathway can play a pathogenic role by regulating other HCC related signaling pathways. NOTCH signaling can promote the progression of HCC by upregulating the Wnt signaling pathway[55]. NOTCH1 and Akt may play a crucial role in the resistance of HCC cells to sorafenib. Valproic acid may overcome resistance and enhance the sensitivity of HCC cells to sorafenib by inhibiting the NOTCH/Akt signaling pathway[54]. Xie et al[56] described a significant correlation between high expression of TSPAN5 and some clinical pathological features, including invasion length, vascular invasion, clinical staging and low overall survival rate in HCC patients. In clinical HCC samples, the expression of TSPAN5 is closely related to many key molecules involved in NOTCH signaling, highlighting its role in regulating NOTCH signaling and demonstrating the important role of the NOTCH signaling pathway in the pathogenesis of HCC. In addition, the abnormal activation of epidermal growth factor (EGF)/EGF receptor signaling pathway, MAPK signaling pathway, vascular endothelial growth factor (VEGF)/VEGF receptor signaling pathway also promotes tumor cell proliferation. There are complex intersections and cascades between various signaling pathways, and these reaction processes are crucial for the treatment of HCC (shown in Figure 2).

Figure 2 The pathogenesis of liver cancer. The occurrence of hepatocellular carcinoma is related to the abnormal activation of multiple signaling pathways. When there is a wnt signal, abnormalities in the AXIN gene can inhibit β-catenin degradation, accumulating in the cytoplasm and entering the nucleus to bind with T-cell factor family proteins, enhancing downstream gene transcription. In the presence of hedgehog (Hh) ligands, the binding of Hh ligand to patched (Ptch)-1 can lead to the endocytosis of Ptch-1, thereby relieving the inhibition of smoothened (Smo). The accumulation and activation of Smo lead to the dissociation of suppressor of fused and GLI, forming GLI activators that promote the expression of target genes. Notch protein is cleaved by furin like proteins and expressed on the cell surface in the form of heterodimers. After binding with ligands, it is cleaved by proteases, and then cleaved by γ-secretase complex in the transmembrane region. At this time, the activated molecule notch intracellular domain enters the nucleus to regulate the transcription of target genes. Hh: Hedgehog; Smo: Smoothened; TCF/LEF: Transcription factor T-cell factor/lymphoid enhancer factor.

RELATION BETWEEN HBV AND HCC

Most HCCs arise from underlying chronic inflammation, including HBV and HCV infections, alcoholic steatohepatitis, nonalcoholic steatohepatitis, and exposure to toxic substances such as aflatoxins. HBV infection is one of the risk factors for HCC, accounting for 50%-80% of HCC cases worldwide. The tumor immune microenvironment of HCC is characterized by immunosuppression through a variety of mechanisms, including the recruitment of immunosuppressive cells, a reduction in antitumor effector cells, changes in cytokine levels and increased expression of immune checkpoint proteins[8]. The tumor microenvironment can induce macrophage differentiation. Macrophages can differentiate into M1-type and M2-type macrophages in different microenvironments. Many activated macrophages infiltrate tumor tissue; these activated macrophages are named tumor-associated macrophages (TAMs), which have multiple M2 phenotypes and can promote tumor growth, invasion, and metastasis by secreting a variety of active substances. TAMs are among the most infiltrative inflammatory cells in the tumor microenvironment and can inhibit the antitumor immune effect of HCC. Some studies have shown that immunosuppressive cytokines, such as IL-4, IL-13, C-C motif chemokine ligand 2, chemokine (C-X-C Motif) ligand 12, and colony stimulating factor-1, can promote the differentiation of TAMs, leading to innate or adaptive immunity defects[57]. TAMs can block antitumor immune responses and accelerate tumor progression by increasing the expression of PDGF, EGF and IGF[58].

NK cells, as an important part of immune system, play an important role at different stages of HCC development. Their function is primarily regulated by interactions with other immune cells, which are mediated by different types of cytokines, ligands, and their receptors. Tregs can impair the function of NK cells by releasing a variety of cytokines (IL-8, IL-10, and TGF-β1). Recent studies have shown that in HBV transgenic mice, NK cell-derived IFN-γ induces HCC through the epithelial cell adhesion molecule-epithelial mesenchymal transition axis and promotes HBsAg-positive hepatocyte injury through activated NK cells, thereby stimulating HCC development[59]. Studies have shown reduced cytotoxicity and production of IFN-γ and TNF-α in NK cells in HBV-induced HCC patients compared to healthy individuals[60].

TAMs can inhibit anti-tumor responses by damaging effector T cells, reducing cytotoxicity in NK cells, reducing tumor-infiltrating lymphocytes, and amplifying immune checkpoint signaling in HCCs[61]. Immune checkpoints mainly include PD-1/PD-L1, CTLA-4 and Tim-3, which play a key role in the process of tumor immune evasion. PD-1 is expressed on activated B cells, T cells, DCs, and NK cells and inhibits the activation of these immune cells by binding to PD-L1 to generate inhibitory signals, protecting tumor cells from attack[62]. Interestingly, in HCC patients, immune checkpoint-related molecules are often overexpressed due to long-term chronic inflammation, leading to apoptosis of CD8 + T cells, which are less active against tumors[63].

CONCLUSION

Elevated levels of HBV-DNA during the perioperative period are associated with postoperative recurrence of liver cancer. Therefore, antiviral therapy is necessary to suppress serum HBV DNA levels. Currently, antiviral drugs include nucleotide analogs (NAs), viral DNA polymerase inhibitors, and polyethylene glycol IFN (PEG-IFN-α). The NAs used for antiviral therapy include tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, entecavir, etc., which can effectively inhibit HBV replication and reduce the viral load to undetectable levels after 48 weeks of treatment[64]. A recent meta-analysis revealed that antiviral therapy with NA can reduce HCC-related mortality and postoperative recurrence of HCC and improve the overall survival rate of patients with HBV-related HCC[65]. However, NA therapy rarely reduces HBsAg levels, and virus replication typically rebounds after the termination of NA therapy[66]. Therefore, to maintain beneficial therapeutic effects, lifelong NA treatment is necessary. IFN-α has dual antiviral and anti-proliferative properties. Some meta-analyses have indicated that IFN-α effectively clears HBeAg and continuously decreases serum HBV-DNA levels[67]. A recent study using urokinase-type plasminogen activator/severe combined immunodeficiency liver-humanized mice revealed that PEG-IFN-α treatment resulted in a decrease in HBx, restoration of structure maintenance of chromosome (SMC) 5/6, and decreased cccDNA transcriptional activity. However, the antiviral effect did not persist after treatment, and SMC5/6 degraded again[68]. A recent report involving RNA sequencing of liver biopsies from patients with CHB after PEG-IFN-α treatment revealed that hepatic tumor protein p53 binding protein 2 levels were significantly higher in the HBsAg loss group than in the HBsAg persistence group, indicating an increased probability of serum HBsAg loss in PEG-IFN-α-treated CHB patients[69]. In short, the rational and long-term use of antiviral drugs that target different HBV components and activate antiviral immune responses can improve patient prognosis and reduce the recurrence and metastasis of HCC.