Background
Hepatitis B virus (HBV) infection is a global health problem and >350 million people are chronic carriers of the virus [
1,
2]. The infection is associated with a wide spectrum of clinical manifestations, ranging from acute or fulminant hepatitis to various forms of chronic infection, including asymptomatic carrier status, chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) [
3,
4].
Owing to the successful development of HBV vaccine, chronic HBV infection in children has been dramatically reduced worldwide [
5,
6]. However, adult HBV carriers still have a significant risk for HCC. In particular, HBV genotype C, which is found throughout Asia, has a higher risk for HCC than other genotypes [
7,
8]. Although the relationship between HBV infection and HCC development is clear, the mechanism by which HBV facilitates cancer development is not clear. Among the viral factors, HBV X protein (HBx) is closely related to HCC development [
9-
12].
One noteworthy universal epidemiological trait of HCC is the male predominance, with a male:female ratio range of 1.5–11:1 [
1,
13]. The male predominance is more pronounced in HBV-related than hepatitis-C-virus-related HCC [
14,
15]. Both higher androgen levels and more active androgen receptor gene alleles are correlated with an increased risk of HCC among male carriers of hepatitis B surface antigen [
16]. Recently, an intriguing interaction between HBx and the androgen axis in promoting male HCC was elucidated [
17,
18].
In addition to the androgen axis, the possible tumor-protective effect of the estrogen axis for HCC has been suggested. For example, longer exposure to estrogen, by taking oral contraceptives or postmenopausal hormone therapy, in female HBV carriers is associated with a lower risk of HCC [
19]. Moreover, in a diethylnitrosamine-induced HCC mouse model, estrogen-receptor-α-mediated inhibition of interleukin (IL)-6 secretion from Kupffer cells was critical in alleviating the carcinogenic process [
20,
21].
The role of HBV large surface proteins (LHBs) in the biology of the virus has yet to be clarified, but it is suggested that LHBs play a role in virus assembly [
22] and attachment to hepatocytes [
23]. Some studies have proposed that LHBs with mutations in the preS region contribute to hepatocarcinogenesis through induction of an endoplasmic reticulum stress pathway or by altering the transactivating capacity [
24-
26], which suggests there may be a disparity in the HCC-inducing capacity between wild-type (WT) LHBs and the variant with mutations associated with disease severity.
Recently, through a molecular epidemiological study, we discovered novel preS1 substitutions (W4P) related to HCC, in which tryptophan changed to proline at the fourth codon from the preS1 start [
27]. Notably, it occurred exclusively in male patients. Therefore, we hypothesize that the LHB variant with a W4P mutation may contribute to the male predominance in HCC. To address the hypothesis, we investigated whether an LHB variant with a W4P mutation from an HCC patient would exhibit gender disparity in tumorigenicity.
Discussion
South Korea is still a high-endemic area for HBV infection, and HCC is more prevalent by 4.2–5.9 times in men than in women [
28,
29]. This male-to-female ratio of HCC incidence is higher than the average of 2.9:1 worldwide [
30], which suggests that there are crucial viral factors in Korean chronic HBV patients favoring a male predominance. In an effort to determine HBV mutation types that contribute to gender disparity in HCC, we recently discovered the W4P/R mutation, which is found only in men, using a molecular epidemiological approach.
We selected W4P LHB and WT LHB from an HCC patient and a carrier with or without W4P mutation, respectively [
27]. Sequence comparison between W4P LHB and WT LHB showed that there were seven variations including W4P. However, in our careful inspection of the literature, we did not find any notable mutations, except for W4P, related to liver disease severity. Thus, although the acquisition of W4P mutation is likely to affect mostly the tumorigenic nature of variant LHB, the synergistic or additive effect of other variations cannot be excluded.
In this study, we demonstrated that the expression of LHBs enhanced cell proliferation and W4P LHB exerted a stronger effect. Expression of W4P LHB resulted in downregulation of the p53 pathway. It is likely that W4P LHB impairs the cell-cycle checkpoint at the G1/S phase by inhibiting the p53–p21 axis, which in turn may contribute to HCC by the accumulation of mutations due to the maintenance of genome stability. Cyclin A plays an important role in the S and G2/M phases of the cell cycle [
31]. It should be noted that W4P LHB upregulates expression of cyclin A in hepatocytes, because it is overexpressed in HCC tissue [
32,
33]. The enhanced colony-forming capability of W4P LHB provides further support for the potential role of the W4P mutation in HBV-related hepatocarcinogenesis. Similar patterns were observed in W4P-LHB-expressing Huh7 HCC cell lines. Taken together, our
in vitro data suggest that mutation in the LHB region, such as W4P, during the natural course of chronic hepatitis B, may contribute to HCC generation. This strongly supports the previous epidemiological finding showing a higher prevalence of W4P mutation in patients with severe forms of liver disease than in those with milder forms of liver disease [
27].
HBx contributes to gender disparity in HBV infection or HCC generation mainly due to signaling via the androgen axis [
18,
34,
35]. However, although it is important for the initial replication in the host, it is not essential for HBV life cycle or chronic infection, so its function has been lost occasionally in chronic patients during the course of chronic hepatitis B via deletion events [
34,
36]. Therefore, another viral factor providing a likely explanation of gender disparity in HCC generation, mainly in the final stage of chronic hepatitis B, should be considered. Our
in vivo mouse data clearly proved that W4P LHB, but not WT LHB, had higher potential for tumor formation in male than in female mice, suggesting mutations in LHB, such as W4P, occurring in a later stage of chronic hepatitis B, could contribute to the gender disparity in HCC generation.
Our
in vivo data also showed that the gender disparity in W4P-LHB-induced tumorigenicity was closely related to the difference in IL-6 production between the genders. IL-6 is known to play a crucial role in the fibrosis and HCC related to liver regeneration [
20,
21,
37]. Moreover, HCC patients with W4P variant displayed a higher IL-6 serum level than HCC patients with WT. The NIH3T3 cell line constitutively expressing W4P LHB induced preferential tumor formation in male nude mice over females, as well as having oncogenic potential at a high level, with 95% tumor generation incidence within 4 weeks. Our
in vivo system showed a significant positive correlation between tumor size and IL-6 secretion, supporting previous reports regarding the positive role of IL-6 in tumorigenesis [
20,
21]. Estrogen was also shown to evoke biological defense against hepatocarcinogenesis by functioning as a negative regulator of IL-6 production in liver Kupffer cells [
37]. In our study, we showed that estrogen suppressed production of IL-6 and subsequently reduced W4P-induced tumor growth in male mice, which suggests that a high level of estrogen in female mice suppresses tumorigenesis by W4P variant HBV through regulating IL-6 signaling. Thus, a likely explanation of the preponderance of tumor generation in male mice observed in our W4P-injected mouse model is that female hormonal factors play a suppressive role in W4P-induced tumorigenicity by inhibiting IL-6 production. Several studies have shown the relation between menopause and HCC risk in women. For example, a 1-year delay in the onset of menopause reduced the risk of HCC by 21%, and hormone replacement therapy was associated with a low risk of HCC [
38,
39]. Our data provide some insights and explanations for the gender disparity in HCC and how estrogen suppresses, at least in part, the tumorigenicity induced by HBV mutations.
Methods
Generation of stable cell lines
Sequences encoding WT and variant LHBs were amplified from a patient with HBV carrier status and an HCC patient, which were proven without and with W4P/R mutation by a real-time polymerase chain reaction method as described previously [
27]. Amplified products were cloned into the pIRES2 vector. NIH3T3 murine cell lines and Huh7 human HCC cell lines constitutively expressing the WT LHB and W4P variant were established by transfection with pIRES2-WT or pIRES2-W4P, followed by the selection with 500 μg/ml neomycin.
Analysis of cell proliferation and cell cycle
To analyze the proliferation of cells, 104 cells were seeded on 100-mm tissue culture dishes. The number of viable cells was determined at each time point by counting after trypan blue staining. To analyze the cell cycle, the DNA content of cells was assessed via propidium iodide staining, followed by flow cytometry. To analyze the role of JAK2 and stat3, cells were transfected with siRNAs targeting them (Bioneer, Daejeon, Korea) and subjected to conventional 3-(4, 5)-dimethylthiazol (−z-y1)-diphenyltetrazolium bromide (MTT) cell viability assay.
One hundred cells of each cell line were seeded in a six-well culture plate. After incubation for 14 days, colonies were fixed and stained with 0.5% methylene blue in ethanol for 10 min at room temperature. The number of cell colonies in each dish was counted under a microscope.
Immunoblot analysis
Anti-preS1 (Aprogen, Daejeon, Korea), anti-cyclin A, anti-cyclin D1, anti-cdk2, anti-cdk4, anti-PCNA (Abcam, Cambridge, UK), anti-p53 and anti-β-actin (Santa Cruz Biotechnology Santa Cruz, CA, USA) antibodies were used for immunoblotting.
ELISA
The amounts of secreted IL-6 and TNF-α were determined by mIL-6 and TNF-α ELISA kits (eBioscience, San Diego, CA, USA). The cells were incubated for 48 h and the supernatants were subjected to ELISA. To investigate the effect of estradiol on secretion of IL-6, cell lines were treated with 20 nM β-estradiol for 48 h. To examine the secretion of cytokines by macrophages, J774A.1 cells were incubated in the presence or absence of W4P-LHB-expressing tumor homogenate (10 mg) for 48 h with or without 20 mM estrogen.
In vivo tumorigenicity study
About 107 NIH3T3, WT-LHB-NIH3T3 and W4P-LHB-NIH3T3 cells were injected subcutaneously into the right hind legs of 8-week-old nude mice. Tumor formation was monitored over a 4-week period. Tumor volumes were calculated using the following equation: length × (width)2 × 0.52. The resected tumor masses were fixed and processed in an alcohol–xylene series followed by paraffin embedding. For histological examination, sections were stained with hematoxylin and eosin or subjected to immunohistochemistry with anti-preS1 monoclonal antibodies. To examine the effect of estrogen on tumor growth, male mice were injected with β-estradiol (0.5 mg/kg) or PBS. One week later, mice were injected with W4P-LHB-NIH3T3 cells subcutaneously together with β-estradiol (0.5 mg/kg) or PBS injection. Mice were injected with β-estradiol (0.5 mg/kg) or PBS twice weekly. Serum IL-6 and TNF-α levels were determined using a Bio-Plex cytokine panel (BioRad, CA). All of the animal experiments were conducted following NIH guidelines for housing and care of laboratory animals and in accordance with the protocol approved by Institutional Animal Care and Use Committee (IAUAC) of Seoul National University College of Medicine (protocol number SNU-111025).
Patients
Sera from 22 male HCC and liver cirrhosis patients with WT and W4P LHBs were subjected to multiplex cytokine measurement. Patients information was previously described [
27]. The Ethical Committee of Seoul National University Hospital (IRB No. C-1007-021-322) approved this research protocol and waived the need for written informed consent because routine diagnostic data were analyzed anonymously (Additional file
5: Table S1).
Statistical analysis
All ELISA and cell proliferation assays in this study were repeated at least three times, and the results were expressed as percentages, means ± SD, or as medians (range). Differences between categorical variables were analyzed using Fisher’s exact test or χ2 test. For continuous variables, Student’s t test was used when the data showed a normal distribution, or the Mann–Whitney U test was used when the data were not normally distributed. A value of P < 0.05 (two-tailed) was considered to be statistically significant.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SAL: design, acquisition of data, analysis and interpretation of data, writing of the manuscript; HK: design, acquisition of data, analysis and interpretation of data, writing of the manuscript; YSW: acquisition, analysis and interpretation of data; SHS: analysis and interpretation of data, writing of the manuscript; YRN: analysis and interpretation of data, writing of the manuscript; HBS: acquisition, analysis and interpretation of data; KSI: design, analysis and interpretation of data, writing of the manuscript; BJK: obtaining funding, study supervision, analysis and interpretation of data, writing of the manuscript. All authors read and approved the final manuscript.