The truncated mutant HBsAg expression increases the tumorigenesis of hepatitis B virus by regulating TGF-β/Smad signaling pathway

Hepatitis B virus (HBV) chronic infection is the key cause of hepatocellular carcinoma (HCC) in Asians. Oral nucleos(t)ide analogs (NAs) have been proven to be effective in suppressing HBV replication, decreasing liver inflammation and fibrosis, and reducing the risk of HCC development [1]. Because all available NAs selectively target the reverse transcriptase (RT) domain of HBV DNA polymerase, mutations in HBV-RT region were naturally screened during the long term use of NAs [2]. The emergence of these mutants would not only inevitably lead to viral breakthrough and hepatic dysfunction, but also influence the biological characteristics and pathogenicity of HBV, which may increase risk of disease progression and HCC occurrence [3, 4].

In past decades, lamivudine and adefovir dipivoxil has been widely used in the treatment of patients with CHB. However, because of a low genetic barrier to the development of resistance, HBV rtA181T/V mutation is common in real-life medical practice [2]. As we known, HBV rtA181T mutant is an A → T mutation at position 181 of the P gene in the RT domain. In theory, its overlapping S gene should have two types of mutation at amino acid 172, which include rtA181T/sW172L (TGG CTC → TTA CTC) mutant coding substituted HBsAg protein and rtA181T/sW172*(TGG CTC → TGA CTC) mutant coding truncated HBsAg protein [5]. Our previously studies showed that the replication capacity of HBV rtA181T/sW172* mutant was higher than that of HBV rtA181T/sW172L mutant [6]. HBV rtA181T/sW172* mutant had a dominant secretion defect of HBsAg which may cause the accumulation of viral core particles in liver cells, and the latter may damage the liver microenvironment [6, 7]. Additionally, clinical study indicated that HBV carrying rtA181T/sW172* mutation may have an increased oncogenic potential [8]. However, it is still unknown about the molecular mechanism of tumorigenesis of HBV rtA181T/sW172* mutant expressing truncated HBsAg protein.

Thus, to better and fully understand the tumorigenicity of HBV rtA181T mutants, we designed this study to investigate the impact of different overlapping S gene mutation (sW172L and sW172*) on the tumorigenicity of HBV rtA181T mutant and reveal the possible mechanism.

Though HBV belongs to hepatotropic DNA virus family, its pathogenic mechanism is not directly damaging to liver cells. Instead, host cellular immunity and humoral immunity against HBV should be the main reason causing liver damage. It has been reported that the HBsAg produced by some HBV mutants may help virus particles escaping immune surveillance, thus hindering the body’s immune system killing HBV, leading to persistent infection of HBV and continuous liver injury, and finally resulting in the development of liver cirrhosis or even HCC [9, 10]. Additionally, HBsAg protein can interfere with some important signaling pathways in hepatocyte, which may induce malignant proliferation of hepatocytes and lead to the occurrence of hepatocellular carcinoma [4, 11]. In present study, because of the deletion of 55 amino acids at the carboxyl terminus of HBsAg, the spatial structure HBsAg expressed by HBV rtA181T/sW172* has been changed, and this may be the molecular basis for enhanced pathogenicity of HBV rtA181T/sW172* mutant.

Currently, both soft-agar colony forming assay and nude mouse tumorigenesis test have been recommended by WHO and US FDA for tumorigenicity evaluation. In present study, the results of above two experiments both suggested an increased tumorigenic ability of L02 cells expressing truncated mutant HBsAg as compared to that of L02 cells expressing substituted or wide-type HBsAg. And this finding is also consistent with the clinical increased risk of developing HCC in CHB patients with evidence of HBV rtA181T/sW172* mutant [8, 12]. In present study, as compared to L02 cell lines just expressing HBsAg proteins, the larger size and faster formation of cell mass and transplanted tumor in L02 cell lines expressing whole genome of HBV suggested that there was a synergistic effect of truncated mutant HBsAg in tumorigenicity with coding genes in other regions of HBV.

In our previous study, we found that the viral replication in pHBV4.1-HBs(sW172*) mutant increased and maintained significantly longer than that in pHBV4.1-HBs(sW172L) mutant, and truncated mutant HBsAg also could increase the activity of HBV core promoter which could further increase transcription and replication of HBV genome [6, 7]. As we known, active HBV replication is associated with the high risk of HCC development [13]. Thus the enhancement of HBV replication mediated by truncated mutant HBsAg may be involved in the pathogenicity enhancement of HBV.

In present study, we also investigated the molecular mechanism of tumorigenesis of truncated mutant HBsAg using L02 cells with stably transfected pHBV4.1-HBs(sW172*), and L02 cells with stably transfected pHBV-HBs(sW172L) and pHBV4.1-HBs(wt) were used as control. The key molecules involving in TGF-β/Smad pathway were significantly changed, with low expression of anti-tumor factor TGFBI and high expression of CyclinD1(oncogene) in downstream of TGFBI. And the trends of TGFBI, CREB, Smad2, Smad3 and CyclinD1 expression in L02 cells with truncated, substitute and wide-type HBsAg stable expression were all consistent with corresponding finding of soft-agar colony forming assay and nude mouse tumorigenesis experiment. And the difference in viral replication capacity of pHBV4.1-HBs(wt), pHBV4.1-HBs(sW172L) and pHBV4.1-HBs(sW172*) should be an important cause of different expression of Smad3/2 and CREB in present study.

It is well-known that CyclinD1 is an important regulator of host cell cycle, and it could promote cells from G1 phase to S phase that would shorten the period of cell cycle and accelerate cell proliferation. Conversely, the decreasing of CyclinD1would result in a reduction of cell proliferation but an increasing of cell apoptosis. Evidences have shown that excessive promotion of cell proliferation play important roles in cell malignant transformation. In fact, we also found that the proliferation of L02 cells was significantly increased in the presence of truncated mutant HBsAg (data unshown). TGFBI(transforming growth factor, β-induced), also known as βig-H3, is a protein induced by TGFβ and found in a wide variety of tissues. It conduces to cell adhesion by interactions with integrins as same as many parts of the extracellular matrix (ECM). There are quite a few causative evidences revealing either the negative or positive role of TGFBI playing in tumorigenesis. TGFBI functions as a tumor suppressor has been found in many tumors, as in human lung carcinoma, ovarian carcinoma and breast carcinoma [14, 15]. In present study, after restoration of TGFBI expression in L02 cells with stably transfected pHBV4.1-HBs(sW172*), the tumor volume was significantly reduced in nude mice; and the expression of CyclinD1 decreased in L02 cells accompanied by a decreasing of cell proliferation and increasing of cell apoptosis(data unshown). Thus, we speculated that the pathogenic mechanism of truncated mutant HBsAg inducing tumorigenesis may be associated with TGF-β/Smad pathway via regulating cell cycle. In fact, we also have other evidences in support of this speculation. For example, Porf. Locarnini et al. has been reported that the presence of HBV rtV191I/sW182* mutant may induce cell canceration via inhibiting TGFBI expression and increasing CyclinD1expression [16]. And Porf. Lee et al.found that the percentage of S-phase cells increased in the presence of HBV rtV191I/sW182* as compared to that in the presence of wide-type HBV, and the possible mechanism may be associated with the down-regulation of p53 and p21 and up-regulation of Cyclin A and CDK4 gene expressions [17], which also could promote cells from G1 phase to S phase thus accelerating cell proliferation.

In present study, there was no significant pathogenicity enhancement of sW172L mutation as compared to sW172*. The main reason may be associated with spatial structure difference between substituted mutant HBsAg and truncated mutant HBsAg. As we known, truncated mutant HBsAg lost the C-terminal membrane translocation signal because of the C-terminal large deletions, and this change of spatial structure would impair the secretion of truncated mutant HBsAg and result in accumulation of truncated mutant HBsAg in cytoplasm. As a product of pre-S2 /S gene with at least 50 amino acids deletion in C-terminal, truncated mutant HBsAg is also reported to be a trans activator that could induce activation of a large number of oncogene via combining with PKC and regulating transcription factors such as AP-2. Additionally, truncated mutant HBsAg is also could directly regulate the activation of some gene promoters [18]. Thus, the transcriptional activation of truncated mutant HBsAg may be related to the increased possibility of tumorigenesis in HBV-rtA181T/sW172* mutant.

In present study, we also investigated the molecular mechanism of tumorigenesis of truncated mutant HBsAg using L02 cells with stably transfected pHBV4.1-HBs(sW172*), and L02 cells with stably transfected pHBV4.1-HBs (sW172L) and pHBV4.1-HBs(wt) were used as control. Though the changes of key molecules in LEF/Wnt pathway and proteinase C (PKC)-dependent c-Raf-1/Erk2 pathway were not significant than key molecules in TGF-β/Smad pathway, we could not completely deny the possible role of LEF/Wnt and PKC- dependent c-Raf-1/Erk2 pathways in the tumorigenic mechanism of HBV-sW172* truncated mutant, and further research is still needed to clarify this issue.

The emergence of sW172* mutant coding truncated mutant HBsAg may increase possibility of HBV tumorigenesis, and its mechanism may be associated with down-regulated expression of TGFBI in TGF-β/Smad signaling pathway. However, the exact biological characteristics of HBV mutants coding truncated mutant HBsAg is still unclear and the exact mechanism of truncated mutant HBsAg increasing the tumorigenesis of HBV is still need more studies to reveal.