Hepatitis B virus (HBV) is a major etiologic agent of chronic liver disease (CLD) and hepatocellular carcinoma (HCC). HBV encoded X antigen, HBx, and pathways implicated in the self-renewal of stem cells contribute to HCC, but it is not clear whether HBx expression promotes “stemness.” Thus, experiments were designed to test the hypothesis that HBx triggers malignant transformation by promoting properties that are characteristic of cancer stem cells (CSCs). To test this hypothesis, HepG2 cells were stably transduced with HBx and then assayed for phenotypic and molecular characteristics of “stemness.” The relationship between HBx and “stemness”-associated markers was also evaluated by immunohistochemical staining of liver and tumor tissue sections from HBV infected patients. The results showed that Oct-4, Nanog, Klf4, β-catenin and EpCAM were activated by HBx in vitro and in vivo. EpCAM was detected in the nuclei of human HCC cells from infected patients. HBx promotes “stemness” by activating β-catenin and epigenetic up-regulation of miR-181, both of which target EpCAM. HBx expression was also associated with depressed levels of E-cadherin. Moreover, HBx stimulated cell migration, growth in soft agar, and spheroid formation. This work is the first to propose that HBV promotes “stemness” in the pathogenesis of HCC. HBx associated up-regulated expression of multiple “stemness” markers support the hypothesis that HBx contributes to hepatocarcinogenesis, at least in part, by promoting changes in gene expression that are characteristics of CSCs.

Hepatitis B virus (HBV)-associated chronic liver diseases are treated with nucleoside analogs that target the virus polymerase. While these analogs are potent, drugs are needed to target other virus-encoded gene products to better block the virus replication cycle and chronic liver disease. This work further characterized GLS4 and compared it to the related BAY 41-4109, both of which trigger aberrant HBV core particle assembly, where the virus replication cycle occurs. This was done in HepAD38 cells, which replicate HBV to high levels. In vitro, GLS4 was significantly less toxic for primary human hepatocytes (P < 0.01 up to 100 M), inhibited virus accumulation in the supernantant of HepAD38 cells (P < 0.02 up to 100 nM), inhibited HBV replicative forms in the liver with a significantly lower 50% effective concentration (EC 50 ) (P < 0.02), and more strongly inhibited core gene expression (P < 0.001 at 100 to 200 nM) compared to BAY 41-4109. In vivo characterization was performed in nude mice inoculated with HepAD38 cells, which grew out as tumors, resulting in viremia. Treatment of mice with GLS4 and BAY 41-4109 showed strong and sustained suppression of virus DNA to about the same extents both during and after treatment. Both drugs reduced the levels of intracellular core antigen in the tumors. Alanine aminotransferase levels were normal. Tumor and total body weights were not affected by treatment. Thus, GLS4 was as potent as the prototype, BAY 41-4109, and was superior to lamivudine, in that there was little virus relapse after the end of treatment and no indication of toxicity.

Hepatitis B virus encoded X antigen (HBx) is a trans -regulatory protein that alters the activity of selected transcription factors and cytoplasmic signal transduction pathways. HBx transcriptionally up-regulates the expression of a unique gene, URG11, which in turn transcriptionally up-regulates β-catenin, thereby contributing importantly to hepatocarcinogenesis. HBx and URG11 also alter the expression of multiple microRNAs, and by miRNA array analysis, both were shown to promote the expression of miR-148a. Elevated miR-148a was also seen in HBx positive liver samples from infected patients. To study the function of miR-148a, anti-148a was introduced into HepG2 and Hep3B cells stably expressing HBx or stably over-expressing URG11. Anti-miR-148a suppressed cell proliferation, cell cycle progression, cell migration, anchorage independent growth in soft agar and subcutaneous tumor formation in SCID mice. Introduction of anti-miR-148a increased PTEN protein and mRNA expression, suggesting that PTEN was targeted by miR-148a. Anti-miR-148a failed to suppress PTEN expression when co-transfected with reporter gene mutants in the 3′UTR of PTEN mRNA. Introduction of anti-miR-148a also resulted in depressed Akt signaling by HBx and URG11, resulting in decreased expression of β-catenin. Thus, miR-148a may play a central role in HBx/URG11 mediated HCC, and may be an early diagnostic marker and/or therapeutic target associated with this tumor type.

The hepatitis B virus (HBV) encoded X protein (HBx) contributes centrally to the pathogenesis of hepatocellular carcinoma (HCC). Aberrant activation of the Hedgehog (Hh) pathway has been linked to many tumor types including HCC. Thus, experiments were designed to test the hypothesis that HBx promotes HCC via activation of Hh signaling. HBx expression correlated with an up-regulation of Hh markers in human liver cancer cell lines, in liver samples from HBV infected patients with HCC, and in the livers of HBx transgenic mice (HBxTg) that develop hepatitis, steatosis, and dysplasia, culminating in the appearance of HCC. The findings in human samples provide clinical validation for the in vitro results and those in the HBxTg. Blockade of Hh signaling inhibited HBx stimulation of cell migration, anchorage independent growth, tumor development in HBxTg and xenograft growth in nude mice. Results suggest that the ability of HBx to promote cancer is at least partially dependent upon the activation of the Hh pathway. This study provides biological evidence for the role of Hh signaling in the pathogenesis of HBV mediated HCC and suggests cause and effect for the first time. The observation that inhibition of Hh signaling partially blocked the ability of HBx to promote growth and migration in vitro and tumorigenesis in two animal models implies that Hh signaling may represent an “oncogene addiction” pathway for HBV associated HCC. This work could be central to designing specific treatments that target early development and progression of HBx mediated HCC.

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Hepatitis B virus (HBV) carriers are at high risk for the development of hepatocellular carcinoma (HCC), but there are no reliable markers that will identify such high-risk carriers. The objective of this work is to identify serologic markers that may indicate the early presence of HCC. Since HBV-encoded X antigen (HBxAg) likely contributes to HCC by upor down-regulation of host gene expression, X positive and negative HepG2 cells were made and subjected to cDNA subtraction. When specific ELISAs were constructed measuring differentially expressed antigens and corresponding antibodies, antibodies to several differentially expressed genes were detected. In cross-sectional and longitudinal studies, antibodies were predominantly present in patients with HBV-associated cirrhosis and HCC, but not in most carriers with hepatic inflammation alone or without active liver disease. Antibodies were also present in patients with hepatitis C virus (HCV)-related HCC, but rarely detected in sera from uninfected individuals, those with tumors other than HCC, or those with drug-induced hepatitis. Statistical analysis showed that HCC patients with four or more antibodies detectable before the appearance of HCC had decreased survival, suggesting that these markers may reflect stepwise hepatocarcinogenesis. Hence, these antibodies may serve as preneoplastic markers for HCC in HBV carriers with chronic liver disease, and may be identified by a simple blood test.

Woodchuck hepatitis virus (WHV)-infected woodchucks have been used for preclinical development of drugs against hepatitis B virus (HBV).However, there is no simple in vivo model to evaluate small amounts of compounds against HBV. To develop such a model, HepAD38 cells, in which HBV replication is regulated by tetracycline (tet), were grown as subcutaneous tumours in nude mice. Mice developing viraemia were then left untreated or given tet in the drinking water. In some of the mice given tet, it was removed and the mice were injected intraperitoneally with phosphate buffer saline (PBS), lamivudine (3TC), clevudine (CLV) or tenofovir dipivoxil fumarate (TDF). Virus DNA titres were measured by realtime PCR during and after drug treatment. In water-fed and PBS-injected mice, virus titres reached ~10 9 copies/ml serum within 35 days of HepAD38 injection, whereas in tet-treated mice, virus titres remained at 10 4 -10 5 copies/ml. HBV DNA levels were suppressed by 3TC, TDF and CLV, with the latter two drugs showing more sustained virus suppression compared with 3TC. Combination therapy with CLV plus TDF was much more effective than either drug alone in suppressing virus titre for at least 3 weeks after the end of treatment. There was no demonstrable toxicity to HepAD38 cells in drug-treated mice. Hence, a robust tet-controlled system for HBV replication in vivo was demonstrated, validated with monotherapies against HBV and shown to be useful in assessing combination therapy. This system will be useful for preclinical assessment of small amounts of single or multiple compounds against HBV in vivo.