Potential conflict of interest: Nothing to report.
Hepatitis B virus X (HBx) protein is implicated in hepatitis B virus (HBV)-associated liver carcinogenesis. However, it remains unclear whether HBx-expressing hepatic progenitor cells (HPCs) are attributed to liver tumor formation. In this study, by using HBx transgenic mice and a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver injury model, the relationship between HBx expression and tumorigenicity of HPCs was analyzed. Compared with control mice, an elevated number of EpCAM+ cells with characteristics of HPCs was observed in HBx mice after 1 month and 4 months of DDC diet feeding. All HBx transgenic mice developed liver tumors characterized by histological features of both hepatocellular carcinoma (HCC) and cholangiocarcinoma after 7 months of DDC feeding. Notably, EpCAM+ HPCs isolated from premalignant HBx mice exposed to a DDC diet for 4 months formed subcutaneous mixed-lineage tumors (four out of six) in nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice, and none of the cells from wildtype (WT) induced tumor, indicating that HBx may induce malignant transformation of HPCs that contributes to tumorigenesis. We also found higher titers of circulating interleukin (IL)-6, activities of IL-6/STAT3, and Wnt/β-catenin signaling pathways in HBx transgenic mice, suggesting HBx may induce intrinsic changes in HPCs by way of the above signaling that enables HPCs with tumorigenicity potential. Finally, clinical evidence showed that high HBx expression in human HBV-related HCC was statistically associated with expansion of EpCAM+ or OV6+ tumor cells and aggressive clinicopathologic features. Conclusion: HBx induces intrinsic cellular transformation promoting the expansion and tumorigenicity of HPCs in DDC-treated mice, which may be a possible origin for liver cancer induced by chronic hepatitis infection. (HEPATOLOGY 2012)
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer-related mortality worldwide, causing ≈700,000 deaths yearly.1 Epidemiologic studies have provided overwhelming evidence that chronic infection with hepatitis B virus (HBV) is a major risk for HCC.2 However, the detailed mechanism about how HBV is involved in tumorigenesis of HCC is still not clear. Hepatitis B virus X (HBx), a small 17-kDa soluble protein, is known to be essential for HBV-induced carcinogenesis.3 It is one of four defined overlapping open reading frames (ORFs) in HBV genomic DNA and has been found in both nucleus and cytoplasm.4 To determine the role of HBx in the induction of HCC, an HBx transgenic mouse model was generated by introducing the HBx gene into the p21CIP1/WAF1 locus.5 About 60% of the HBx gene knockin transgenic mice developed HCC by 18 months of age,5 suggesting that HBx could be a promiscuous transactivator and activates transcription of viral and cellular genes during viral-induced HCC. Therefore, HBx transgenic mouse is an ideal model to study the detailed mechanisms by which chronic HBV infection promotes the occurrence of HCC.
It is widely accepted that cancer stem/progenitor cells are key players in tumorigenesis. Hepatic progenitor cells (HPCs) are considered to have bipotential ability to differentiate into hepatocyte or cholangiocyte.6 Activation of HPCs has been reported to be induced by 2-acetylaminofluorene (AAF) / partial hepatectomy (PH), 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), and choline-deficient or ethionine-supplemented diets.7-9 In addition, aberrant activation of Wnt/β-catenin,10-12 transforming growth factor beta (TGF-β), and interleukin (IL)-6 signaling,13 Bmi1,14 and Hippo-Salvador pathway15 contribute to the expansion and activation of HPCs, as well as transformation of HPCs. HPCs isolated from DDC-treated p53-null mice liver were able to induce tumors with characteristics of both HCC and cholangiocarcinoma in nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice.16 Clinical evidence has also shown that the degree of progenitor/stem cell activation is associated with the severity of inflammation and fibrosis in chronic hepatitis.17 The question is if activation of HPCs is involved in HBV-induced hepatocarcinoma. We hypothesized that HPCs may be affected and transformed by HBV and its associated proteins including HBx during chronic inflammation and HBx may be responsible for the development of HPC-derived liver tumors.
In the present study, using HBx transgenic mice and human HBV-related HCC specimens, we demonstrated that expression of HBx promoted expansion and tumorigenicity of HPCs that contributed to HBx-mediated tumor formation in a DDC-induced mouse model. These studies shed novel light on the notorious role of HBx in the relationship between chronic hepatitis infection and liver cancer.
Liver specimens were obtained from patients with HCC after hepatectomy at the Eastern Hepatobiliary Surgery Hospital. All human sample collection procedures were approved by the China Ethical Review Committee.
HBx gene knock in transgenic mice (C57BL/6) were a kind gift from Xiao Yang (Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing, China) and maintained in the barrier facility under pathogen-free conditions. Littermates of HBx mice without insertion were used as controls and are henceforth referred to as wildtype (WT) mice. Male heterozygous transgenic HBx mice carrying a functional allele of p21CIP1/WAF1 and control mice at age 6-7 weeks were fed with normal chow diet containing 0.1% DDC (Sigma) for 1 to 7 months. All procedures regarding animals were conducted according to the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (publication 86-23 revised 1985).
The flow cytometry and labeling technique were carried out as described12 with MoFlo XDP (Beckman Coulter), using PE-conjugated EpCAM antibody (eBioscience), APC-conjugated CD45 antibody (Biolegend), and FITC-conjugated or PE-conjugated secondary antibody (Sigma).
Tumor Formation Assay.
One million EpCAM+ CD45− cells were suspended in a mixture of 50 μL phosphate-buffered saline (PBS) and 50 μL Matrigel (BD Bio-sciences) and injected subcutaneously into 6-week-old NOD/SCID male mice (Chinese Science Academy). Mice were killed at 8 weeks postinjection and tumors were harvested for further examination.
After acquiring all data for histological parameters and in vitro assays, Student's t test and χ2 test were applied to determine statistical significance. P < 0.05 was considered significant.
HBx Promotes the Expansion of HPCs in a DDC Model.
As we know, DDC is generally used to promote proliferation of murine HPCs.8 To investigate if DDC-induced proliferation of HPCs is involved in HBx-induced hepatocarcinogenesis, we fed the HBx transgenic and littermate control mice a DDC diet. After 1 month, hematoxylin and eosin (H&E) staining of liver sections displayed the HPC response (small round or oval cells with high nuclear-to-cytoplasmic ratio) in two groups (Fig. 1A). Because EpCAM and A6 were used for detection of HPCs in mice,18 we found that these cells were positive for both of EpCAM and A6 (Fig. 1B), suggesting that they may be HPCs. Ki67 is expressed most in active proliferative cells. By Ki67 histological staining we found that the number of progenitor cells entering the active cell cycle increased more than 1-fold in HBx mice compared to WT (Fig. 1C). These data thus suggested that HBx may promote expansion of HPCs in DDC-treated mice.
HBx Promotes Stem-Like Properties and Oncogene Expression in EpCAM+CD45− HPCs Isolated from Mice with DDC Diet Feeding.
It has been shown that the EpCAM+ cells isolated from DDC liver have adult progenitor potential that possess the capacity for unlimited proliferation and bidirectional differentiation.18 Flow cytometric analysis of nonparenchymal cells prepared from the liver of HBx mice fed with DDC for 1 month showed a higher percentage of EpCAM+CD45− HPCs (Fig. 1D). When cultured at low density, EpCAM+CD45− HPCs isolated from HBx mice were able to form more and larger colonies than EpCAM+CD45− cells from WT mice (Fig. 2A), suggesting that these cells may have a stronger stem-like property. To further determine the characteristics of HPCs, the gene expression profile of magnetic sorted primary EpCAM+CD45− cells (Supporting Figs. S1, S2) was examined by quantitative reverse-transcription polymerase chain reaction (RT-PCR). As shown in Fig. 2B, HPCs from HBx mice demonstrated stronger expression of stem/progenitor cell markers (EpCAM, CD133, CD90, ABCG2, AFP, and CK19) than those from WT controls. However, there were no significant differences in the expression of the oncogenes (K-ras, N-ras) between HPCs from HBx and control WT mice at 1 and 2 months DDC treatment (Fig. 2B; Fig. S3). To evaluate the self-renewal ability of HPCs, we performed a sphere formation assay. Compared with WT control, more spheres were observed in EpCAM+CD45− HPCs isolated from HBx mice (Fig. 2C).
Recent evidence shows that DDC not only causes liver damage along with the proliferation of HPCs, but also facilitates some oncogene-induced tumorigenesis in the adult liver.19 After consecutive administration of 0.1% DDC for 4 months, both HBx and WT mice showed an increase in liver size and toughness on the liver surface compared with livers from mice treated with DDC for 1 month (Fig. S4A,B). Histological and flow cytometric analysis revealed persistent HPC expansion in both groups, but more robust in HBx mice (Fig. 1C,D). HPCs from HBx mice also showed increased expression of stem/progenitor cell markers and stronger sphere formation ability compared with those from WT mice (Fig. 2B,C). Furthermore, the expression of the oncogenes (K-ras, N-ras) was up-regulated in HPCs from HBx mice (Fig. 2B), suggesting that HBx may also promote the HPCs to obtain transformed characteristics after long-term DDC treatment.
HBx Transgenic Mice Develop Live Tumors After Feeding a DDC Diet for 7 Months.
To further observe whether long-term exposure to a DDC diet could induce tumors, HBx and WT mice were continuously fed a DDC diet for 5-7 months. As the results in Fig. 3A show, all HBx mice (n = 15) developed liver tumors after 7 months, whereas none of WT mice (n = 15) had any liver tumors. Interestingly, after 5 months DDC treatment, although some HBx did not develop liver tumors, GSTpi and GPC3-positive dysplastic nodules were detectable in their liver tissues (Fig. S4C,D). Immunohistochemical staining of serial sections revealed that the GSTpi-positive cells coexpressed progenitor cells marker EpCAM, suggesting that these dysplastic cells had progenitor characteristics. Moreover, tumors developed from HBx mice exhibited phenotypes of mixed HCC and cholangiocarcinoma (CC) within the same liver. The HCC-like tumors exhibited features of hepatocellular morphologies, whereas CC-like tumors strongly resembled the “cholangiolocellular” subtype described in humans that exhibits poorly differentiated characteristics (Fig. 3B). Staining of tumor sections with AFP and CK19 confirmed that the tumors are composed of hepatocytes and cholangiocytes (Fig. 3C). Furthermore, we detected EpCAM+ tumor cells in both HCC and CC tumor tissues (Fig. 3D). The complete penetrance of both tumor types subsequent to HPC expansion suggested that tumors may derive from stem/progenitor cells and supported our hypothesis that HPCs are involved in HBx-induced tumorigenesis.
Only EpCAM+CD45− HPCs Isolated from Premalignant DDC-Treated HBx Transgenic Mice Formed Bilineage Tumors in NOD/SCID Mice.
As shown in Fig. 3A, consistent DDC treatment induced bilineage tumors in HBx-expressed livers. The results raised a question if tumors are derived from transformed HPCs. To identify if the bilineage tumor derived from HBx-induced HPCs, we isolated EpCAM+CD45− HPCs from HBx transgenic mice and WT control mice after 1, 2, 3, or 4 months of DCC treatment, respectively. One × 106 cells were then injected subcutaneously into NOD/SCID mice (n = 6). Eight weeks later, EpCAM+CD45− HPCs derived from all WT mice and 1, 2, or 3-month DDC-treated HBx mice did not produce any tumors, whereas EpCAM+CD45−HPCs from 4-month DDC-treated HBx mice formed tumor in four out of six mice (Fig. 4A,B). H&E staining and immunohistochemical analysis of AFP and CK19 revealed that these tumors contained mixed cell characteristics (Fig. 4C-E). EpCAM+ cells were also detected in these tumors (Fig. 4F). Therefore, these results demonstrate that chronic injury induced by DDC in the long term (at least 4 months) gradually enhanced the effect of HBx on HPCs and increased their tumorigenicity potential.
Increased IL-6/STAT3 Signaling Activity Is Found in DDC-Treated HBx Transgenic Mice.
Our results have shown that HBx induced expansion of HPCs with increased expression of stemness genes and oncogenes (Fig. 2B). Importantly, HPCs isolated from premalignant HBx mice induced a subcutaneous tumor xenograft (Fig. 4A,B). The question is, what is the mechanism underlying HBx-promoted expansion and transformation of HPCs? To answer the question we analyzed the liver injury, inflammatory response, and signaling pathways during the process of HPC's response to DCC. To determine if it was because of HBx exacerbated DDC-induced liver injury, we detected the serum alanine aminotransferase (ALT) level and found there was no difference between WT and HBx groups at any timepoint (Fig. 5A), concluding that the degree of liver damage is not associated with HPC proliferation. To determine whether inflammatory factors were related to expansion and transformation of HPCs, we examined the inflammation-related index in serum and liver at 1, 4, and 6 months after DDC treatment. F4/80 antibody staining displayed similar macrophage accumulation in livers of the two groups (Fig. 5B).
Interleukin-6 (IL-6) has been implicated in progenitor cells and inflammatory responses in the liver.20 As expected, the serum level of IL-6 and liver IL-6 messenger RNA (mRNA) expression were significantly higher in HBx mice than in WT (Fig. 5C). Increased IL-6 pathway activity in HPCs is critical for disturbed growth and tumorigenic differentiation of these liver precursors,13 acting through activation of STAT3 and transcription activity. Clearly, although DDC treatment increased the levels of P-STAT3 in both the WT and HBx liver tissues at 1 and 4 months, HBx mice exhibited higher activity of P-STAT3 (Fig. 5D). This was consistent with a recent report that HBx enhanced the synthesis and secretion of IL-6, which may be through an MyD88-dependent pathway to the activation of both nuclear factor kappa B (NF-κB) and ERK/p38 mitogen-activated protein (MAP) kinases in hepatic and hepatoma cells.21 In our results we also found that there was stronger activation of ERK and P38 in HBx murine livers compared with WT mice (Fig. 5D). In addition, tumors derived from liver of HBx mice fed with DDC for 7 months also showed higher activation of STAT3, ERK, and P38 compared with adjacent nontumor liver tissues (Fig. 5D). The results suggested that an increase of IL-6 production and its signaling activity may contribute to HBx-induced malignant transformation of HPCs.
Activation of Wnt/β-Catenin Signaling Pathways in HPCs and Tumors in DDC-Treated HBx Transgenic Mice.
The Wnt/β-catenin signaling pathway is known to be responsible for activation and transformation of stem/progenitor cells.10-12 To identify if this pathway is involved in expansion and tumorigenicity of HPCs, we detected the activity of Wnt/β-catenin signaling pathway in WT and HBx transgenic mice. As shown in Fig. 6A, mRNA levels of CyclinD1 and c-myc, well-known downstream targets of Wnt/β-catenin signaling, increased in HPCs isolated from HBx mice, and immunoblotting analysis of whole liver lysates showed similar results (Fig. 6B). Using immunohistochemical labeling, we observed stronger β-catenin staining in both cytoplasmic and nuclear of HPCs in HBx mice than those in WT mice (Fig. 6C). It is known that phosphorylation of GSK-3β is a major mechanism that leads to increased cellular expression of β-catenin.22 Therefore, we compared he kinase activity of GSK-3β between HBx mice and WT mice. Consistent with this notion, we found that phosphorylation of GSK-3β at the Ser9 residue in HBx mice was much stronger than that detected in WT mice (Fig. 6D). In addition, tumors isolated from liver of HBx mice fed with DDC for 7 months also showed higher cytoplasmic and nuclear β-catenin staining and phosphorylation of GSK-3β (Fig. 6B,E). These results suggest that higher activation of the Wnt/β-catenin pathway in HBx mice may be necessary for the expansion and transformation of HPCs.
HBx Expression Level Is Positively Associated with Percentage of HPCs in HCC Specimens.
Based on our findings in the HBx transgenic mouse model, we asked whether HBx expression might also contribute to expansion of tumorigenic progenitor cells in humans. We therefore analyzed the expression levels of HBx in human HBV-related HCC tissues (n = 200). Patients were divided into high (n = 106) and low (n = 94) groups based on the average HBx level of all specimens (Fig. 7A). As shown in Table 1, patients with higher HBx expression were significantly associated with a high HBV DNA level, liver cirrhosis, multiple tumor number, absent tumor encapsulation, lower differentiation, portal vein thrombosis, vascular invasion, and high TNM stage of HCC, suggesting that overexpression of HBx promoted HCC development and progression. EpCAM+ or OV6+ cells have been reported to exhibit stronger cancer stem cell (CSC) characteristics than the corresponding EpCAM− or OV6− cells in HCC cell lines and HCC specimens.12, 23 The percentage of EpCAM+ and OV6+ cells were variable: some were semiquantitatively as low as 0% to <30% positive, others as high as ≥30% in HCC cells, by way of evaluating five medium-power fields of each tumor tissue by two independent observers. In addition, we also carried out immunohistochemical analysis to determine nuclear accumulation of β-catenin, a marker of Wnt/β-catenin signaling activation. Representative staining of each marker on serial sections is shown in Fig. 7B. Clearly, patients with higher HBx expression had much more EpCAM+ or OV6+ cells in their tumor tissues, accompanied by a higher frequency of nuclear β-catenin expression (Fig. 7C). These data suggest that overexpression of HBx may promote expansion of tumorigenic HPCs, and thus contribute to the development and progression of HCC.
Table 1. Correlation Between HBx mRNA Expression and Clinicopathological Characteristics in Human HBV-Related HCCs
High HBx Expression (No. of Cases)
Low HBx Expression (No. of Cases)
No. of tumor nodules
Maximal tumor size
Portal vein thrombosis
In this study we demonstrate for the first time that expression of HBx in liver contributed to expansion and transformation of HPCs during chronic liver injury in mice, providing novel evidence for the role of HPCs in HBV-related liver cancer.
Recent advances in the field of stem cells and cancer biology have shed light on CSCs, the origin of many hematological malignancies and solid tumors.24 There is a growing realization that some HCCs probably arise from transformed liver stem/progenitor cells.25-27 HPC-derived carcinomas, defined as having a progenitor cell phenotype, tend to have a more aggressive phenotype.28 The relationship between HPCs and hepatocarcinogenesis is further supported by the generation of tumors with bilineage phenotype from the progeny of a DDC-treated p53−/− liver-derived CD133+_HPCs.16 In our study we clearly showed that HBx promoted expansion and transformation of HPCs in DDC-treated mice, HBx mice developed liver tumor after long-term DDC treatment, and EpCAM+ HPCs from HBx transgenic mice induced bilineage tumor in NOD/SCID mice. These results not only provide new evidence that HPCs are involved in hepatocarcinogenesis, but also reveal for the first time that HBV-derived HCCs are possibly associated with overexpression of HBx that elicits and alternates intrinsic cellular signaling regulation and eventually endow HPCs with tumorigenicity potential. Given the close association between inflammation and carcinogenesis, it is reasonable to think that chronic and persistent liver injury induced by hepatitis viral infection might expand, activate, and transform the hepatic stem/progenitor cells, predisposing the patient to a high risk of cancer initiation.
A previous article reported that the HBx knockin transgenic mice developed HCC after the age of 18 months.5 Previous studies have shown that p21CIP1/WAF1 deficiency does not directly increase the susceptibility to HCC in mice,29 so the heterozygous HBx transgenic mice carrying a functional allele of p21CIP1/WAF1 in liver (Fig. S5) provided the ideal model to study the function of HBx in liver. DDC is used as an agent to stimulate proliferation of HPCs in mice. Compared with WT mice, short-term DDC-treated HBx knockin mice exhibited more EpCAM+ HPCs in the liver by histological analysis, immunofluorescent staining, and FCM analysis. Interestingly, although a long-term DDC diet also increased expansion of HPCs in WT mice, it failed to induce liver tumor formation. In contrast, all HBx mice developed liver tumors after 7 months of a DDC diet. This hepatotoxin promoted liver tumors histologically resembling both phenotypes of HCC and CC, and EpCAM+CD45− HPCs isolated from premalignant HBx mice exposed to a DDC diet for 4 months formed mixed-lineage tumors in NOD-SCID mice. Thus, our results strongly suggest that HBx expression induced malignant transformation of HPCs during DDC induced liver injury, and the bilineage tumors originated from transformed HPCs.
How does HBx affect the function of HPCs and what is the mechanism of HBx inducing transformation of HPCs? We know that IL-6 is a multifunctional cytokine involved in hepatic response to infections or systemic inflammation. An increase of IL-6 in serum is often seen in chronic liver inflammation, including alcoholic hepatitis, HBV, and HCV infections.30, 31 In addition, a high serum IL-6 level also serves as a symbol for future HCC development in a prospective clinical study.32 In our data, IL-6 and STAT3 activity were increased after DDC treatment in HBx mice, suggesting that HBx may regulate HPCs through the IL-6/STAT3 pathway, which not only results in enhanced HPC proliferation, but also contributes to the development of liver cancers by transformation of HPCs.13
The Wnt/β-catenin pathway is widely associated with tumor and stem/progenitor cells and elicits different impacts on developmental stages. Aberrant activation of Wnt/β-catenin is primarily involved in the pathogenesis of hepatic tumors, especially HCC.33 Enhanced self-renewal capacity by way of Wnt/β-catenin and Bmi-1 signaling drives hepatic tumor formation.14 We and other groups have reported that β-catenin can also regulate the proliferative response of hepatic progenitor cells in rodent models and expansion of cancer stem cells in HCC.11, 12, 23 Consistent with in vitro results that HBx is involved in activating Wnt/β-catenin signaling in hepatoma cells,34 we found here that HBx transgenic mice had much stronger β-catenin staining in HPCs in DDC-induced injury models, suggesting that Wnt/β-catenin signaling likely regulates the expansion and transformation of HPC compartments in the HBx transgenic mice model. HBx protein may function as a transactivator to activate many signaling pathways in HPCs and induce a variety of cellular genes including pro-oncogenes. Thus, other molecular mechanisms underlying the effects of HBx on HPCs are still under investigation.
In human HCC specimens, biomarker of HPCs, such as EpCAM,23 CD133,35, 36 OV6,12 are also used to identify CSC-like cancer cells, which have tumor-initiating ability and stem/progenitor cell characteristics. In order to find a relationship between HBx and HPCs in HCC, we analyzed a large series of HBV-related human HCC specimens. The fact that patients with higher HBx expression levels also had a higher percentage of EpCAM or OV6-positive tumor cells supports our hypothesis that HPCs are involved in HBx-mediated hepatocarcinogenesis. During the course of the present study, Arzumanyan et al.37 also reported that stable transduction of HBx into HepG2 cells promoted “stemness” of tumor cells. According to the aggressive clinicopathologic features observed in patients with higher HBx expression, there may exist a possible mechanism that HBx may promote HCC progression through its regulation on CSC cells.
Although HPCs are useful for cell and gene therapy to treat metabolic liver diseases, it is clearly shown here that their aberrant activation and transformation also played an important role in liver tumorigenesis. Our present study highlights that HBx contributes to activation and transformation of HPCs and further initiates liver tumorigenesis during chronic liver injury. Therefore, inhibition of HBx expression by antiviral treatment undoubtedly will decrease the incidence of HCC. Future studies will focus on other HBV-related molecules and the detailed mechanism involved in CSC/HPC-mediated liver tumor to clarify the mechanisms of viral hepatitis related to liver cancer.
We thank Dong-Ping Hu, Lin-Na Guo, Dan Cao, Shan-Hua Tang, Dan-Dan Huang, and Shan-Na Huang for technical assistances. We also thank Gen-Sheng Feng for helpful suggestions. We thank for Mark A. Feitelson and Valentina Factor for sharing the HBx antibody and A6 antibody for these studies.