HBV-related HCC development in mice is STAT3 dependent and indicates an oncogenic effect of HBx.

Background & Aims: Although most hepatocellular carcinoma (HCC) cases are driven by hepatitis and cirrhosis, a subset of patients with chronic hepatitis B develop HCC in the absence of advanced liver disease, indicating the oncogenic potential of hepatitis B virus (HBV). We investigated the role of HBV transcripts and proteins on HCC development in the absence of inflammation in HBV-transgenic mice. Methods: HBV-transgenic mice replicating HBV and expressing all HBV proteins from a single integrated 1.3-fold HBV genome in the presence or absence of wild-type HBx (HBV1.3/HBVxfs) were analyzed. Flow cytometry, molecular, histological and in vitro analyses using human cell lines were performed. Hepatocyte-specific Stat3- and Socs3-knockout was analyzed in HBV1.3 mice. Results: Approximately 38% of HBV1.3 mice developed liver tumors. Protein expression patterns, histology, and mutational landscape analyses indicated that tumors resembled human HCC. HBV1.3 mice showed no signs of active hepatitis, except STAT3 activation, up to the time point of HCC development. HBV-RNAs covering HBx sequence, 3.5-kb HBV RNA and HBx-protein were detected in HCC tissue. Interestingly, HBVxfs mice expressing all HBV proteins except a C-terminally truncated HBx (without the ability to bind DNA damage binding protein 1) showed reduced signs of DNA damage response and had a significantly reduced HCC incidence. Importantly, intercrossing HBV1.3 mice with a hepatocyte-specific STAT3-knockout abrogated HCC development. Conclusions: Expression of HBV-proteins is sufficient to cause HCC in the absence of detectable inflammation. This indicates the oncogenic potential of HBV and in particular HBx. In our model, HBV-driven HCC was STAT3 dependent. Our study highlights the immediate oncogenic potential of HBV, challenging the idea of a benign highly replicative phase of HBV infection and indicating the necessity for an HBV 'cure'. Impact and implications: Although most HCC cases in patients with chronic HBV infection occur after a sequence of liver damage and fibrosis, a subset of patients develops HCC without any signs of advanced liver damage. We demonstrate that the expression of all viral transcripts in HBV-transgenic mice suffices to induce HCC development independent of inflammation and fibrosis. These data indicate the direct oncogenic effects of HBV and emphasize the idea of early antiviral treatment in the 'immune-tolerant' phase (HBeAg-positive chronic HBV infection).

Novel role of MHC class II transactivator in hepatitis B virus replication and viral counteraction.

BACKGROUND/AIMS: The major histocompatibility class II (MHC II) transactivator, known as CIITA, is induced by Interferon gamma (IFN-γ) and plays a well-established role in regulating the expression of class II MHC molecules in antigen-presenting cells. METHODS: Primary human hepatocytes (PHH) were isolated via therapeutic hepatectomy from two donors. The hepatocellular carcinoma (HCC) cell lines HepG2 and Huh7 were used for the mechanistic study, and HBV infection was performed in HepG2-NTCP cells. HBV DNA replication intermediates and secreted antigen levels were measured using Southern blotting and ELISA, respectively. RESULTS: We identified a non-canonical function of CIITA in the inhibition of hepatitis B virus (HBV) replication in both HCC cells and patient-derived PHH. Notably, in vivo experiments demonstrated that HBV DNA and secreted antigen levels were significantly decreased in mice injected with the CIITA construct. Mechanistically, CIITA inhibited HBV transcription and replication by suppressing the activity of HBV-specific enhancers/promoters. Indeed, CIITA exerts antiviral activity in hepatocytes through ERK1/2-mediated down-regulation of the expression of hepatocyte nuclear factor 1α (HNF1α) and HNF4α, which are essential factors for virus replication. In addition, silencing of CIITA significantly abolished the IFN-γ-mediated anti-HBV activity, suggesting that CIITA mediates the anti-HBV activity of IFN-γ to some extent. HBV X protein (HBx) counteracts the antiviral activity of CIITA via direct binding and impairing its function. CONCLUSION: Our findings reveal a novel antiviral mechanism of CIITA that involves the modulation of the ERK pathway to restrict HBV transcription. Additionally, our results suggest the possibility of a new immune avoidance mechanism involving HBx.

JNK/c-Jun pathway activation is essential for HBx-induced IL-35 elevation to promote persistent HBV infection.

BACKGROUND: Immunoregulation plays pivotal roles during chronic hepatitis B virus (HBV) infection. Studies have shown that Interleukin (IL)-35 is an important molecule associated with inadequate immune response against HBV. However, the mechanisms involved in the up-regulation of IL-35 expression during persistent HBV infection remain unknown. METHODS: In this study, we constructed a plasmid expressing the HBV X protein (pCMV-HBx) to evaluate the relationship between HBx and IL-35. Activation of the JNK/c-Jun pathway was analyzed and chromatin immunoprecipitation followed by sequencing and luciferase reporter assays were performed to determine whether c-Jun could regulate IL-35 transcription. RESULTS: HBx can significantly activate IL-35 promoter in both LO2 and HepG2 cells compared to the control plasmid (pCMV-Tag2) using the dual-luciferase assay. Whereas other viral proteins, such as S, preS1, the core protein, had no significant effect on IL-35 expression. Similarly, WB and qRT-PCR also showed that HBx can significantly promote IL-35 expression at protein and mRNA levels in the aforementioned cells. The relevant pathway mechanism showed that the expression of JNK and c-Jun genes was significantly higher in transfected cells carrying pCMV-HBx than in the pCMV-Tag2-transfected and -untransfected cells. WB analysis revealed that phosphorylated JNK and c-Jun were overexpressed after HBx action. Conversely, the addition of the JNK/c-Jun signaling pathway inhibitor could significantly suppress HBx-induced IL-35 expression in a dose-dependent manner. CONCLUSIONS: A novel molecular mechanism of HBV-induced IL-35 expression was revealed, which involves JNK/c-Jun signaling in up-regulating IL-35 expression via HBx, resulting in transactivation of the IL-35 subunit EBI3 and p35 promoter.

Poly(ADP-Ribose) Polymerase Inhibition as a Promising Approach for Hepatocellular Carcinoma Therapy.

Primary liver cancer is the sixth most common cancer in men and seventh in women, with hepatocellular carcinoma (HCC) being the most common form (75-85% of primary liver cancer cases) and the most frequent etiology being viral infections (HBV and HCV). In 2020, mortality represented 92% of the incidence-830,180 deaths for 905,677 new cases. Few treatment options exist for advanced or terminal-stage HCC, which will receive systemic therapy or palliative care. Although radiotherapy is used in the treatment of many cancers, it is currently not the treatment of choice for HCC, except in the palliative setting. However, as radiosensitizing drugs, such as inhibitors of DNA repair enzymes, could potentiate the effects of RT in HCC by exploiting the modulation of DNA repair processes found in this tumour type, RT and such drugs could provide a treatment option for HCC. In this review, we provide an overview of PARP1 involvement in DNA damage repair pathway and discuss its potential implication in HCC. In addition, the use of PARP inhibitors and PARP decoys is described for the treatment of HCC and, in particular, in HBV-related HCC.

E3 ubiquitin ligase TRIM21 restricts hepatitis B virus replication by targeting HBx for proteasomal degradation.

As a cytosol ubiquitin ligase and antibody receptor, Tripartite motif-containing 21 (TRIM21) has been reported to mediate the restriction of hepatitis B virus (HBV) through an HBx-antibody-dependent intracellular neutralization (ADIN) mechanism. However, whether TRIM21 limits HBV replication by targeting viral proteins remains unclarified. In this study, we demonstrate that TRIM21 inhibits HBV gene transcription and replication in HBV plasmid transfected and HBV-infected hepatoma cells. RING and PRY-SPRY domains are involved in this activity. TRIM21 interacts with HBx protein and targets HBx for ubiquitination and proteasomal degradation, leading to impaired HBx-mediated degradation of structural maintenance of chromosomes 6 (Smc6) and suppression of HBV replication. TRIM21 fails to restrict the replication of an HBx-deficient HBV. And knock-down of Smc6 largely impairs the anti-HBV activity of TRIM21 in HepG2 cells. In a hydrodynamic injection (HDI)-based HBV mouse model, we confirm an in vivo anti-HBV and anti-HBx therapeutic effect of TRIM21 by over-expression or knocking-out strategy. Our findings reveal a novel mechanism that TRIM21 restricts HBV replication through targeting HBx-Smc5/6 pathway, which may have an implication in the future TRIM21-based therapeutic application.

A hepatitis B virus transgenic mouse model with a conditional, recombinant, episomal genome.

BACKGROUND & AIMS: Development of new and more effective therapies against hepatitis B virus (HBV) is limited by the lack of suitable small animal models. The HBV transgenic mouse model containing an integrated overlength 1.3-mer construct has yielded crucial insights, but this model unfortunately lacks covalently closed circular DNA (cccDNA), the episomal HBV transcriptional template, and cannot be cured given that HBV is integrated in every cell. METHODS: To solve these 2 problems, we generated a novel transgenic mouse (HBV1.1X), which generates an excisable circular HBV genome using Cre/LoxP technology. This model possesses a HBV1.1-mer cassette knocked into the ROSA26 locus and is designed for stable expression of viral proteins from birth, like the current HBV transgenic mouse model, before genomic excision with the introduction of Cre recombinase. RESULTS: We demonstrated induction of recombinant cccDNA (rcccDNA) formation via viral or transgenic Cre expression in HBV1.1X mice, and the ability to regulate HBsAg and HBc expression with Cre in mice. Tamoxifen-inducible Cre could markedly downregulate baseline HBsAg levels from the integrated HBV genome. To demonstrate clearance of HBV from HBV1.1X mice, we administered adenovirus expressing Cre, which permanently and significantly reduced HBsAg and core antigen levels in the murine liver via rcccDNA excision and a subsequent immune response. CONCLUSIONS: The HBV1.1X model is the first Cre-regulatable HBV transgenic mouse model and should be of value to mimic chronic HBV infection, with neonatal expression and tolerance of HBV antigens, and on-demand modulation of HBV expression. LAY SUMMARY: Hepatitis B virus (HBV) can only naturally infect humans and chimpanzees. Mouse models have been developed with the HBV genome integrated into mouse chromosomes, but this prevents mice from being cured. We developed a new transgenic mouse model that allows for HBV to be excised from mouse chromosomes to form a recombinant circular DNA molecule resembling the natural circular HBV genome. HBV expression could be reduced in these mice, enabling curative therapies to be tested in this new mouse model.

PARP inhibitors and radiation potentiate liver cell death in vitro. Do hepatocellular carcinomas have an achilles' heel?

BACKGROUND: A promising avenue for cancer treatment is exacerbating the deregulation of the DNA repair machinery that would normally protect the genome. To address the applicability of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) combined with radiotherapy for the treatment of hepatocellular carcinoma (HCC) two approaches were used: firstly, the in vitro sensitivity to the PARPi Veliparib and Talazoparib +/- radiation exposure was determined in liver cell lines and the impact of the HBV X protein (HBx) that deregulates cellular DNA damage repair via SMC5/6 degradation was investigated. Secondly, PARP expression profiles and DNA damage levels using the surrogate marker gammaH2AX were assessed in a panel of control liver vs HCC tissues. METHODS: Cell cytotoxicity was measured by clonogenic survival or relative cell growth and the DNA damage response using immunological-based techniques in Hep3B, PLC/PRF/5, HepG2- and HepaRG-derived models. Transcriptome changes due to HBx expression vs SMC6 loss were assessed by RNA sequencing in HepaRG-derived models. PARP and PARG transcripts (qPCR) and PARP1, H2AX and gammaH2AX protein levels (RPPA) were compared in control liver vs HBV-, HCV-, alcohol- and non-alcoholic steatohepatitis-associated HCC (tumor/peritumor) tissues. RESULTS: PARPi cytotoxicity was significantly enhanced when combined with X-rays (2Gy) with Talazoparib having a greater impact than Veliparib in most in vitro models. HBx expression significantly lowered survival, probably driven by SMC5/6 loss based on the transcriptome analysis and higher DNA damage levels. PARP1 and PARP2 transcript levels were significantly higher in tumor than peritumor and control tissues. The HBV/HCV/alcohol-associated tumor tissues studied had reduced H2AX but higher gammaH2AX protein levels compared to peritumor and control tissues providing evidence of increased DNA damage during liver disease progression. CONCLUSIONS: These proof-of-concept experiments support PARPi alone or combined with radiotherapy for HCC treatment, particularly for HBV-associated tumors, that warrant further investigation.

Bufalin inhibits hepatitis B virus-associated hepatocellular carcinoma development through androgen receptor dephosphorylation and cell cycle-related kinase degradation.

PURPOSE: Hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), which has a male predominance, lacks effective therapeutic options. Previously, the cardiac glycoside analogue bufalin has been found to inhibit HBV infection and HCC development. As yet, however, its molecular role in HBV-associated HCC has remained obscure. METHODS: Colony formation and soft agar assays, xenograft and orthotopic mouse models and HBV X protein (HBx) transgenic mice with exposure to diethylnitrosamine were used to evaluate the effect of bufalin on HBV-associated HCC growth and tumorigenicity. HBx-induced oncogenic signaling regulated by bufalin was assessed using PCR array, chromatin immunoprecipitation, site-directed mutagenesis, luciferase reporter, transcription and protein expression assays. Synergistic HCC therapeutic effects were examined using combinations of bufalin and sorafenib. RESULTS: We found that bufalin exerted a more profound effect on inhibiting the proliferation of HBV-associated HCC cells than of non HBV-associated HCC cells. Bufalin significantly inhibited HBx-induced malignant transfromation in vitro and tumorigenicity in vivo. Androgen receptor (AR) signaling was found to be a target of bufalin resistance to HBV-associated hepatocarcinogenesis. We also found that bufalin induced both AR dephosphorylation and cell cycle-related kinase (CCRK) degradation to inhibit ß-catenin/TCF signaling, which subsequently led to cell cycle arrest via cyclin D1 down-regulation and p21 up-regulation, resulting in HCC regression. Furthermore, we found that bufalin reduced > 60% diethylnitrosamine-induced hepatocarcinogenesis in HBx transgenic mice, and improved the sensitivity of refractory HBV-associated HCC cells to sorafenib treatment. CONCLUSION: Our results indicate that bufalin acts as a potential anti-HCC therapeutic candidate to block HBx-induced AR/CCRK/ß-catenin signaling by targeting AR and CCRK, which may provide a novel strategy for the treatment of HBV-associated HCC.

Hepatitis B virus X protein-induced SH2 domain-containing 5 (SH2D5) expression promotes hepatoma cell growth via an SH2D5-transketolase interaction.

Hepatitis B virus X protein (HBx) critically contributes to the development of hepatocellular carcinoma (HCC). However, the mechanisms by which HBx promotes HCC remain unclear. In the present study, using a combination of gene expression profiling and immunohistochemistry, we found higher levels of SH2 domain-containing 5 (SH2D5) in liver tissue from HBV-associated HCC (HBV-HCC) patients than in adjacent nontumor tissues. Moreover, HBV infection elevated SH2D5 levels, and we observed that HBx plays an important role in SH2D5 induction. We also found that HBx triggers SH2D5 expression through the NF-κB and c-Jun kinase pathways. Employing SH2D5 overexpression or knockdown, we further demonstrate that SH2D5 promotes HCC cell proliferation both in vitro and in vivo While investigating the mechanism of SH2D5-mediated stimulation of HCC cell proliferation, we noted that HBV induces SH2D5 binding to transketolase (TKT), a pentose phosphate pathway enzyme, thereby promoting an interaction between and signal transducer and activator of transcription 3 (STAT3). Furthermore, HBx stimulated STAT3 phosphorylation at Tyr-705 and promoted the activity and downstream signaling pathway of STAT3 via the SH2D5-TKT interaction. Taken together, our results suggest that SH2D5 is an HBV-induced protein capable of binding to TKT, leading to induction of HCC cell proliferation.

Hepatitis B Virus X Protein Increases 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine (8-Oxodg) Level via Repressing MTH1/ MTH2 Expression in Hepatocytes.

BACKGROUND/AIMS: Chronic hepatitis B virus (HBV) infection markedly increases the risk of development of hepatocellular carcinoma (HCC). Among the seven viral proteins that HBV encodes, HBV X protein (HBx) appears to have the most oncogenic potential. The mitochondria-associated HBx can induce oxidative stress in hepatocytes, leading to the production of abundant reactive oxygen species (ROS). High levels of ROS usually induce oxidative DNA damage and 8-hydroxy-2-deoxyguanosine (8-OHdG), also known as 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), which is one of the major products of DNA oxidation and an important biomarker for oxidative stress and carcinogenesis. Cells have evolved a mechanism to prevent oxidized nucleotides from their incorporation into DNA through nucleotide pool sanitization enzymes of MTH1 (NUDT1), MTH2 (NUDT15), MTH3 (NUDT18) and NUDT5. However, little is known as to whether HBx can regulate the expression of those enzymes and modulate the formation and accumulation of 8-oxodG in hepatocytes. METHODS: The level of 8-oxodG was assessed by ELISA in stable HBV-producing hepatoma cell lines, an HBV infectious mouse model, HBV and HBx transgenic mice and HBV-infected patients versus their respective controls. Expression of MTH1, MTH2, MTH3 and NUDT5 was determined by a real-time quantitative PCR and western blot analysis. Transcriptional regulation of MTH1 and MTH2 expression by HBx and the effect of HBx on MTH1 and MTH2 promoter hypermethylation were examined using a luciferase reporter assay and bisulfite sequencing analysis. RESULTS: In comparison with controls, significantly higher levels of 8-oxodG were detected in the genome and culture supernatant of stable HBV-producing HepG2.2.15 cells, in the sera and liver tissues of HBV infectious mice and HBV or HBx transgenic mice, and in the sera of HBV-infected patients. Expression of HBx in hepatocytes significantly increased 8-oxodG level and reduced the expression of MTH1 and MTH2 at both mRNA and protein levels. It was also demonstrated that HBx markedly attenuated the MTH1 or MTH2 promoter activities through hypermethylation. Furthermore, enhancement of 8-oxodG production by HBx was reversible by overexpression of MTH1 and MTH2. CONCLUSION: Our data show that HBx expression results in the accumulation of 8-oxodG in hepatocytes through inhibiting the expression of MTH1 and MTH2. This may implicate that HBx may act as a tumor promoter through facilitating the mutational potential of 8-oxodG thus connecting a possible link between HBV infection and liver carcinogenesis.

Hepatitis B virus X protein promotes epithelial-mesenchymal transition and metastasis in hepatocellular carcinoma cell line HCCLM3 by targeting HMGA2.

Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), and HBV X protein (HBx) serves an essential role in the development of HCC. However, its mechanism remains to be elucidated. The aim of the present study was to investigate the role and mechanism of the HBx protein in the epithelial-mesenchymal transition (EMT) and metastasis of HCC. The HCCLM3 cell line was transfected with a HBx-expressing vector. The effects of HBx overexpression on proliferation, migration, invasion and EMT capacities of the HCCLM3 cell line were evaluated using MTT, migration and invasion assays, and western blotting, respectively. Furthermore, the impact of High mobility group AT-hook 2 (HMGA2) knockdown on HBx-mediated metastasis was investigated in the HCC cell line HCCLM3. The results demonstrated that HBx significantly upregulated HMGA2 expression, and enhanced the proliferation, EMT, invasion and migration in HCC cells. Furthermore, HMGA2 knockdown almost abolished HBx-induced EMT and metastasis in HCC. The results of the present study suggest that HBx promotes the proliferation, EMT, invasion and migration of HCC cells by targeting HMGA2. HMGB2 may serve as a potential therapeutic target for HBV-associated HCC.

Reactive Oxygen Species-Mediated c-Jun NH2-Terminal Kinase Activation Contributes to Hepatitis B Virus X Protein-Induced Autophagy via Regulation of the Beclin-1/Bcl-2 Interaction.

Autophagy is closely associated with the regulation of hepatitis B virus (HBV) replication. HBV X protein (HBx), a multifunctional regulator in HBV-associated biological processes, has been demonstrated to be crucial for autophagy induction by HBV. However, the molecular mechanisms of autophagy induction by HBx, especially the signaling pathways involved, remain elusive. In the present investigation, we demonstrated that HBx induced autophagosome formation independently of the class I phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling pathway. In contrast, the class III PI3K(VPS34)/beclin-1 pathway was revealed to be critical for HBx-induced autophagosome formation. Further study showed that HBx did not affect the level of VPS34 and beclin-1 expression but inhibited beclin-1/Bcl-2 association, and c-Jun NH2-terminal kinase (JNK) signaling was found to be important for this process. Moreover, it was found that HBx treatment led to the generation of reactive oxygen species (ROS), and inhibition of ROS activity abrogated both JNK activation and autophagosome formation. Of importance, ROS-JNK signaling was also revealed to play an important role in HBV-induced autophagosome formation and subsequent HBV replication. These data may provide deeper insight into the mechanisms of autophagy induction by HBx and help in the design of new therapeutic strategies against HBV infection.IMPORTANCE HBx plays a key role in diverse HBV-associated biological processes, including autophagy induction. However, the molecular mechanisms of autophagy induction by HBx, especially the signaling pathways involved, remain elusive. In the present investigation, we found that HBx induced autophagy independently of the class I PI3K/AKT/mTOR signaling pathway, while the class III PI3K(VPS34)/beclin-1 pathway was revealed to be crucial for this process. Further data showed that ROS-JNK activation by HBx resulted in the release of beclin-1 from its association with Bcl-2 to form a complex with VPS34, thus enhancing autophagosome formation. Of importance, ROS-JNK signaling was also demonstrated to be critical for HBV replication via regulation of autophagy induction. These data help to elucidate the molecular mechanisms of autophagy induction by HBx/HBV and might be useful for designing novel therapeutic approaches to HBV infection.

Apoptosis of Hepatitis B Virus-expressing Liver Tumor Cells Induced by a High Concentration of Nucleos(t)ide Analogue.

BACKGROUND/AIM: We investigated the expression of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and HBV X protein (HBx) in human hepatocellular carcinoma (HCC) and evaluated the effect of high-concentration nucleos(t)ide analogs (NUCs) on liver tumor cell lines. MATERIALS AND METHODS: This study consisted of three parts: part I used human blood and non-tumor liver tissues; part II used human HCC and adjacent liver tissues; and part III used an HBV-expressing liver tumor cell line. RESULTS: There were close correlations among blood and liver HBV DNA and liver cccDNA. HBV cccDNA and HBx were highly up-regulated in HCC compared to adjacent liver tissues despite NUC therapy. HBV cccDNA and HBx were highly up-regulated in the cccDNA-expressing HepG2.2.15 cell line. Their expression was down-regulated and apoptosis was induced by a very high concentration of NUCs in dose- and time-dependent manner. CONCLUSION: Very high concentrations of NUCs may have a novel potential to kill replicating HBV-expressing liver tumor cells.

High-dose tenofovir is not effective in suppressing hepatitis B virus replication in patients with hepatocellular carcinoma progression: a preliminary result.

BACKGROUNDS/AIMS: Nucleos(t)ide analogues (NUCs) effectively suppress hepatitis B virus (HBV) replication, but hepatocellular carcinoma (HCC) recurrence often leads to HBV replication despite NUC therapy. The aim of this study was to determine whether high-dose tenofovir (TNF) therapy can suppresses HCC recurrence-associated HBV replication. METHODS: We performed a single-arm prospective study to assess the clinical feasibility of high-dose TNF (hdTNF). We recruited 10 patients during September 2015 and followed up for 3 months or early drop-out. RESULTS: All 10 patients had HCC of advanced stages due to HCC recurrence and gradual progression. The average age of patients was 51.2±4.7 years and 9 were male. Three patients did not tolerate the increased TNF dosage and were dropped out early. The other 7 patients were relatively tolerable to the increased dosage of TNF 5 tablets per day. One patient had mild gastrointestinal symptoms and another patient complained of insomnia. Increased HBV replication and HCC progression was observed despite hdTNF for 4-8 weeks. All 7 patients showed tumor progression during the 3 month follow-up. In these patients, blood HBV DNA before hdTNF was 50-200 copies/ml; and 4-8 weeks after hdTNF, the HBV replication status was not improved with blood HBV DNA of 50-300 copies/ml. This clinical study was terminated early after these negative results were confirmed. CONCLUSIONS: The results of this study indicated that high dose of TNF up to 5-fold the recommended dosage is not tolerated by a considerable proportion of patients and also ineffective in suppressing HCC progression-associated HBV replication.

Interaction of the hepatitis B virus X protein with the lysine methyltransferase SET and MYND domain-containing 3 induces activator protein 1 activation.

Hepatitis B virus (HBV) is a widespread human pathogen that often causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The detailed mechanisms underlying HBV pathogenesis remain poorly understood. The HBV X protein (HBx) is a multifunctional regulator that modulates viral replication and host cell functions, such as cell cycle progression, apoptosis and protein degradation through interaction with a variety of host factors. Recently, the nonstructural protein 5A (NS5A) of hepatitis C virus has been reported to interact with methyltransferase SET and MYND domain-containing 3 (SMYD3), which is implicated in chromatin modification and development of cancer. Because HBx shares fundamental regulatory functions concerning viral replication and pathogenesis with NS5A, it was decided to examine whether HBx interacts with SMYD3. In the present study, it was demonstrated by co-immunoprecipitation analysis that HBx interacts with both ectopically and endogenously expressed SMYD3 in Huh-7.5 cells. Deletion mutation analysis revealed that the C-terminal region of HBx (amino acids [aa] 131-154) and an internal region of SMYD3 (aa 269-288) are responsible for their interaction. Immunofluorescence and proximity ligation assays showed that HBx and SMYD3 co-localize predominantly in the cytoplasm. Luciferase reporter assay demonstrated that the interaction between HBx and SMYD3 activates activator protein 1 (AP-1) signaling, but not that of nuclear factor-kappa B (NF-κB). On the other hand, neither overexpression nor knockdown of SMYD3 altered production of HBV transcripts and HBV surface antigen (HBsAg). In conclusion, a novel HBx-interacting protein, SMYD3, was identified, leading to proposal of a novel mechanism of AP-1 activation in HBV-infected cells.

Sleeping Beauty transposon system for genetic etiological research and gene therapy of cancers.

Carcinogenesis is etiologically associated with somatic mutations of critical genes. Recently, a number of somatic mutations and key molecules have been found to be involved in functional networks affecting cancer progression. Suitable animal models are required to validate cancer-promoting or -inhibiting capacities of these mutants and molecules. Sleeping Beauty transposon system consists of a transposon that carries gene(s) of interest and a transposase that recognizes, excises, and reinserts genes in given location of the genome. It can create both gain-of-function and loss-of-function mutations, thus being frequently chosen to investigate the etiological mechanisms and gene therapy for cancers in animal models. In this review, we summarized current advances of Sleeping Beauty transposon system in revealing molecular mechanism of cancers and improving gene therapy. Understanding molecular mechanisms by which driver mutations contribute to carcinogenesis and metastasis may pave the way for the development of innovative prophylactic and therapeutic strategies against malignant diseases.

Hepatitis B virus X protein accelerates the development of hepatoma.

The chronic infection of hepatitis B virus (HBV) is closely related to the occurrence and development of hepatocellular carcinoma (HCC). Accumulated evidence has shown that HBV X protein (HBx protein) is a multifunctional regulator with a crucial role in hepatocarcinogenesis. However, information on the mechanism by which HBV induces HCC is lacking. This review focuses on the pathological functions of HBx in HBV-induced hepatocarcinogenesis. As a transactivator, HBx can modulate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and transcription factor AP-2. Moreover, HBx can affect regulatory non-coding RNAs (ncRNAs) including microRNAs and long ncRNAs (lncRNAs), such as miRNA-205 and highly upregulated in liver cancer (HULC), respectively. HBx is also involved in epigenetic modification, including methylation and acetylation. HBx interacts with various signal-transduction pathways, such as protein kinase B/Akt, Wnt/ß-catenin, signal transducer and activator of transcription, and NF-κB pathways. Moreover, HBx affects cellular fate by shifting the balance toward cell survival. HBx may lead to the loss of apoptotic functions or directly contributes to oncogenesis by achieving transforming functions, which induce hepatocarcinogenesis. Additionally, HBx can modulate apoptosis and immune response by direct or indirect interaction with host factors. We conclude that HBx hastens the development of hepatoma.

15d-PGJ2 decreases PGE2 synthesis in HBx-positive liver cells by interfering EGR1 binding to mPGES-1 promoter.

Microsomal prostaglandin E synthase 1 (mPGES-1) is the terminal regulator of PGE2 synthesis. The expression of mPGES-1 is increased by stimulating inflammatory factors in various human cancers. However, whether hepatitis B virus (HBV) infection affects mPGES-1 and its molecular mechanism in liver cells has not been studied. In this study, we observed that mPGES-1 expression was positively correlated with HBV X protein (HBx) in hepatocellular carcinoma cancerous tissue, and HBx enhanced the mPGES-1 promoter activity in HL7702 liver cells. Mechanistic investigations revealed that HBx can increase the early growth response 1 (EGR1) binding to the transcription site of mPGES-1 promoter. The overexpression and knockdown of EGR1 did not affect cyclooxygenase-2 (COX-2) transcription and expression in HL7702-HBx cells. We also investigated the unique function of 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2), a kind of PGE2 inhibitor, in the regulation of mPGES-1 expression in HBx-positive liver cells. In the presence of 15d-PGJ2, the expression of COX-2 was unaffected, but that of the EGR1-mPGES-1-PGE2 axis was inhibited. Moreover, the capacity of EGR1 binding to the mPGES-1 promoter decreased, and the change in HL7702-HBx cells was more significant. The results indicated that EGR1 is a specific transcription factor in the up-regulation of mPGES-1 expression by HBx, and targeting EGR1 may contribute to inhibiting the change from inflammation to HBV-induced cancer.

Hepatitis B virus X induces cell proliferation in the hepatocarcinogenesis via up-regulation of cytoplasmic p21 expression.

BACKGROUND: Hepatitis B virus X protein (HBx) has been shown to induce hepatocarcinogenesis by disrupting the functions of intracellular molecules. Cyclin-dependent kinase inhibitor p21 (Cip1/WAF1), known as a tumour-suppressor gene, has been reported to have paradoxical function, that is, acting as an oncogene, particularly when expressed in the cytoplasm. The effects of HBx on the expression and function of p21 also remain controversial. AIMS: We attempted to investigate the role of HBx in the hepatocarcinogenic process, focusing on the association with this paradoxical function of p21. The results obtained were further verified with experiments using the antihepatocarcinogenic action of interferon (IFN)-ß. METHODS: HBx transgenic mice (Xg) and HBx-transfected hepatoma cell lines were used. Intracellular localization of p21 was determined by Western blot analysis and immunofluorescence. RESULTS: Xg and HBx-transfected cells exhibited increased expression of p21. Up-regulation of p21 was positively correlated with the expression of cyclin D1 and inactive phosphorylation of retinoblastoma protein (pRb). These HBx-induced cell proliferative responses were cancelled by knockdown of p21, which resulted in growth reduction in HBx-expressing cells, suggesting the oncogenic properties of HBx-induced p21. HBx induced accumulation of p21 in the cytoplasm, and activation of PKCα was involved. Finally, IFN-ß-treated Xg liver, as well as hepatoma cells, showed a shift of cytoplasmic p21 to the nucleus, accompanied by the abrogation of HBx-induced oncogenic modulation. CONCLUSIONS: Our results suggest that HBx induces hepatocarcinogenesis via PKCα-mediated overexpression of cytoplasmic p21 and IFN-ß suppressed these molecular events by shifting p21 to the nucleus.