HBV-positive Cells Research Articles

Selective depletion of HBV-infected hepatocytes by class A capsid assembly modulators requires high levels of intrahepatic HBV core protein.

Capsid assembly mediated by hepatitis B virus (HBV) core protein (HBc) is an essential part of the HBV replication cycle, which is the target for different classes of capsid assembly modulators (CAMs). While both CAM-A ("aberrant") and CAM-E ("empty") disrupt nucleocapsid assembly and reduce extracellular HBV DNA, CAM-As can also reduce extracellular HBV surface antigen (HBsAg) by triggering apoptosis of HBV-infected cells in preclinical mouse models. However, there have not been substantial HBsAg declines in chronic hepatitis B (CHB) patients treated with CAM-As to date. To investigate this disconnect, we characterized the antiviral activity of tool CAM compounds in HBV-infected primary human hepatocytes (PHHs), as well as in HBV-infected human liver chimeric mice and mice transduced with adeno-associated virus-HBV. Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced a dose-dependent aggregation of HBc in the nucleus which is negatively regulated by the ubiquitin-binding protein p62. We confirmed that CAM-A, but not CAM-E, induced HBc-positive cell death in both mouse models via induction of apoptotic and inflammatory pathways and demonstrated that the degree of HBV-positive cell loss was positively correlated with intrahepatic HBc levels. Importantly, we determined that there is a significantly lower level of HBc per hepatocyte in CHB patient liver biopsies than in either of the HBV mouse models. Taken together, these data confirm that CAM-As have a unique secondary mechanism with the potential to kill HBc-positive hepatocytes. However, this secondary mechanism appears to require higher intrahepatic HBc levels than is typically observed in CHB patients, thereby limiting the therapeutic potential.

MicroRNA-642a-5p Targets DNA Damage-Inducible Transcript 4 to Suppress Hepatitis B Virus Hepatoma Carcinoma Cell

Background: Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors in clinical practice, with hepatitis B virus (HBV) being the most common risk factor for HCC. MicroRNAs (miRNAs) have emerged as a new marker for disease diagnosis and molecularly targeted therapies; however, the mechanism of miR-642a-5p in HBV-associated HCC remains unclear. Objectives: The aim of this study was to investigate the expression of miR-642a-5p, which targets DNA damage-inducible transcript 4 (DDIT4), in HBV-associated HCC, and its effect on the proliferation, migration, and invasion of HBV-positive HCC cells. Methods: miR-642a-5p in the serum of patients with HBV-associated liver cancer (LC), as well as miR-642a-5p and DDIT4 mRNA in LC tissues and cells, and HBV DNA in HBV-positive cells were detected. The targeting of DDIT4 by miR-642a-5p and the progression of cells were also examined. All cell experiments were repeated five times. Results: The results indicated that levels of miR-642a-5p were decreased, while levels of DDIT4 were increased in the serum, tissues, and cells of HBV-positive HCC patients. Overexpression of miR-642a-5p inhibited the progression of HBV-positive HCC cells, suppressed HBV DNA replication, cell proliferation, and invasion, and promoted apoptosis in HepG2.2.15 cells. Conclusions: In addition, miR-642a-5p directly targeted DDIT4, and knockdown of DDIT4 reversed the effects of miR-642a-5p upregulation, promoting the progression of HBV-positive HCC cells. In conclusion, miR-642a-5p is expressed at low levels in HBV-associated HCC and inhibits HBV DNA replication and tumor progression in HBV-positive HCC by targeting DDIT4. This study provides a foundation for molecular targeted therapy in HBV-positive HCC.

CARs derived from broadly neutralizing, human monoclonal antibodies identified by single B cell sorting target hepatitis B virus-positive cells.

To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ IgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2nd generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs.

SUV39H1 is a novel biomarker targeting oxidative phosphorylation in hepatitis B virus-associated hepatocellular carcinoma

BackgroundAs a histone methyltransferase, suppressor of variegation 3–9 homolog 1 (SUV39H1) plays an important role in the occurrence and development of cancer. To explore the mechanism and biological function of SUV39H1 in hepatitis B virus-associated hepatocellular carcinoma (HBV-HCC) can gain an insight into the pathogenesis of HBV-HCC.MethodsThe effect of HBV infection on SUV39H1 in hepatoma cells was detected. CCK-8, colony growth assay and wound healing assay were used to assess the proliferation and migration of HBV-positive hepatoma cells. RNA sequencing (RNA-seq) was applied to find differential genes and enriched pathways. The serum SUV39H1 level in HBV-HCC patients was detected and its correlation with clinical indicators was analyzed.ResultsSUV39H1 was increased by HBV infection and promoted the proliferation and migration of hepatoma cells. SUV39H1 could upregulate the expression of mitochondrial oxidative phosphorylation (OXPHOS) pathway-related genes. OXPHOS pathway inhibitors could reduce the capacity of proliferation and migration of hepatoma cells after overexpressing SUV39H1. Serum SUV39H1 levels were higher in chronic hepatitis B (CHB) patients than in healthy controls and higher in HBV-HCC patients than in CHB patients. In the diagnosis of HCC, the predictive value of SUV39H1 combined with alpha-fetoprotein (AFP) was better than that of AFP alone.ConclusionSUV39H1 is regulated by HBV infection and promotes the proliferation and migration of hepatoma cells by targeting OXPHOS pathway. It indicates that SUV39H1 may be a new biomarker of the diagnosis of HCC.

Effect of lncRNA pvt1 on HBV replication in HepG2.2.15 cells, and its possible mechanism of action

Purpose: To investigate the influence of lncRNA pvt1 on hepatitis B virus replication in HepG2.2.15 cells and the underlying mechanism of action. 
 Methods: Concentrations of lncRNA pvt1 in HBV-positive liver cancer cell lines (HepAD38+, HepG2.2.15+, HepG2+, and HepaRG+), and 3 HBV-negative liver cancer cell lines were determined and compared. Differences in HBV DNA content, levels of HBsAg and HBeAg, and STAT3 axis signal pathway among groups were evaluated. 
 Results: LncRNA pvt1 levels in HBV-positive hepatoma cells were significantly higher than the corresponding expression levels in HBV-negative hepatoma cells. after si-pvt1 treatment in HepG2.2.15 cells, the 3.2K HBV DNA content increased significantly (p < 0.05), and the expression levels of HBsAg and HBeAg in HepG2.2.15 cells also significantly increased. In all groups, STAT3 protein level was comparable, but p-STAT3 protein expression in si-pvt1 group was significantly reduced, relative to those in Si NC and control groups (p < 0.05). 
 Conclusion: Levels of lncRNA pvt1 expression in HBV-positive and HBV-negative hepatoma cells differ significantly. Treatment with si-pvt1 decrease HBV DNA content in HepG2.2.15 cells through a mechanism that relates to the STAT3 axis signal pathway. This finding may be beneficial for follow-up treatment of HBV.

Phosphorylation of UHRF2 affects malignant phenotypes of HCC and HBV replication by blocking DHX9 ubiquitylation

Hepatitis B virus (HBV) infection is one of main contributors to poor prognosis and rapid progression of hepatocellular cancer (HCC). We previously identified the important role of the phosphorylation of ubiquitin-like with PHD and ring finger domains (UHRF2) in HBV-associated HCC. In this study we identify upregulated UHRF2 protein levels in HBV-associated HCC cells and tissues. UHRF2 overexpression promotes the viability, proliferation, migration and invasiveness of HBV-positive HCC cell lines, and enhances HBV DNA replication. To obtain a comprehensive understanding of the interaction networks of UHRF2 and their underlying mechanism, this study suggests that UHRF2 facilitates the ubiquitin-proteasome-mediated proteolysis of DExD/H (Asp-Glu-Ala-His) -box helicase enzyme 9 (DHX9). However, phosphorylation of UHRF2 by HBx at S643 inhibits E3 ubiquitin ligase activity of UHRF2 and improves DHX9 protein stability. Furthermore, results suggest that HBx promotes phosphorylation of UHRF2 by the ETS1-CDK2 axis through the downregulation of miR-222-3p in HBV-associated HCC specimens and cells. Our findings suggest that HBx-induced phosphorylation of UHRF2 S643 acts as a “switch” in HBV-associated HCC oncogenesis, activating the positive feedback between phosphorylated UHRF2 and HBV, provide evidence that UHRF2 is a new regulator and a potential prognostic indicator of poor prognosis for HBV-associated HCC.

Activation of distinct antiviral T-cell immunity: A comparison of bi- and trispecific T-cell engager antibodies with a chimeric antigen receptor targeting HBV envelope proteins

Despite the availability of an effective prophylactic vaccine, 820,000 people die annually of hepatitis B virus (HBV)-related liver disease according to WHO. Since current antiviral therapies do not provide a curative treatment for the 296 million HBV carriers around the globe, novel strategies to cure HBV are urgently needed. A promising approach is the redirection of Tcells towards HBV-infected hepatocytes employing chimeric antigen receptors or T-cell engager antibodies. We recently described the effective redirection of T cells employing a second-generation chimeric antigen receptor directed against the envelope protein of hepatitis B virus on the surface of infected cells (S-CAR) as well as bispecific antibodies that engage CD3 or CD28 on T cells employing the identical HBV envelope protein (HBVenv) binder. In this study, we added a trispecific antibody comprising all three moieties to the tool-box. Cytotoxic and non-cytolytic antiviral activities of these bi- and trispecific T-cell engager antibodies were assessed in co-cultures of human PBMC with HBV-positive hepatoma cells, and compared to that of S-CAR-grafted T cells. Activation of T cells via the S-CAR or by either a combination of the CD3- and CD28-targeting bispecific antibodies or the trispecific antibody allowed for specific elimination of HBV-positive target cells. While S-CAR-grafted effector T cells displayed faster killing kinetics, combinatory treatment with the bispecific antibodies or single treatment with the trispecific antibody was associated with a more pronounced cytokine release. Clearance of viral antigens and elimination of the HBV persistence form, the covalently closed circular (ccc) DNA, through cytolytic as well as cytokine-mediated activity was observed in all three settings with the combination of bispecific antibodies showing the strongest non-cytolytic, cytokine-mediated antiviral effect. Taken together, we demonstrate that bi- and trispecific T-cell engager antibodies can serve as a potent, off-the-shelf alternative to S-CAR-grafted T cells to cure HBV.

Sex Differences in Genomic Features of Hepatitis B–Associated Hepatocellular Carcinoma With Distinct Antitumor Immunity

Aflatoxin exposure increases the risk for hepatocellular carcinoma (HCC) in hepatitis B virus (HBV)-infected individuals, particularly males. We investigated sex-based differences in the HCC genome and antitumor immunity. Whole-genome, whole-exome, and RNA sequencing were performed on 101 HCC patient samples (47 males, 54females) that resulted from HBV infection and aflatoxin exposure from Qidong. Androgen on the expression of aflatoxin metabolism-related genes and nonhomologous DNA end joining (NHEJ) factors were examined in HBV-positive HCC cell lines, and further tested in tumor-bearing syngeneic mice. Qidong HCC differed between males and females in genomic landscape and transcriptional dysfunction pathways. Compared with females, males expressed higher levels of aflatoxin metabolism-related genes, such as AHR and CYP1A1, and lower levels of NHEJ factors, such as XRCC4, LIG4, and MRE11, showed a signature of up-regulated type I interferon signaling/response and repressed antitumor immunity. Treatment with AFB1 in HBV-positive cells, the addition of 2 nmol/L testosterone to cultures significantly increased the expression of aflatoxin metabolism-related genes, but reduced NHEJ factors, resulting in more nuclear DNA leakage into cytosol to activate cGAS-STING. In syngeneic tumor-bearing mice that were administrated tamoxifen daily via oral gavage, favorable androgen signaling repressed NHEJ factor expression and activated cGAS-STING in tumors, increasing T-cell infiltration and improving anti-programmed cell death protein 1 treatment effect. Androgen signaling in the context of genotoxic stress repressed DNA damage repair. The alteration caused more nuclear DNA leakage into cytosol to activate the cGAS-STING pathway, which increased T-cell infiltration into tumor mass and improved anti-programmed cell death protein 1 immunotherapy in HCCs.

Hepatitis B Virus Utilizes a Retrograde Trafficking Route via the Trans-Golgi Network to Avoid Lysosomal Degradation

Hepatitis B virus (HBV) infection is difficult to cure owing to the persistence of covalently closed circular viral DNA (cccDNA). We performed single-cell transcriptome analysis of newly established HBV-positive and HBV-negative hepatocellular carcinoma cell lines and found that dedicator of cytokinesis 11 (DOCK11) was crucially involved in HBV persistence. However, the roles of DOCK11 in the HBV lifecycle have not been clarified. The cccDNA levels were measured by Southern blotting and real-time detection polymerase chain reaction in various hepatocytes including PXB cells by using an HBV-infected model. The retrograde trafficking route of HBV capsid was investigated by super-resolution microscopy, proximity ligation assay, and time-lapse analysis. The downstream molecules of DOCK11 and underlying mechanism were examined by liquid chromatography-tandem mass spectrometry, immunoblotting, and enzyme-linked immunosorbent assay. The cccDNA levels were strongly increased by DOCK11 overexpression and repressed by DOCK11 suppression. Interestingly, DOCK11 functionally associated with retrograde trafficking proteins in the trans-Golgi network (TGN), Arf-GAP with GTPase domain, ankyrin repeat, and pleckstrin homology domain-containing protein 2 (AGAP2), and ADP-ribosylation factor 1 (ARF1), together with HBV capsid, to open an alternative retrograde trafficking route for HBV from early endosomes (EEs) to the TGN and then to the endoplasmic reticulum (ER), thereby avoiding lysosomal degradation. Clinically, DOCK11 levels in liver biopsies from patients with chronic hepatitis B were significantly reduced by entecavir treatment, and this reduction correlated with HBV surface antigen levels. HBV uses a retrograde trafficking route via EEs-TGN-ER for infection that is facilitated by DOCK11 and serves to maintain cccDNA. Therefore, DOCK11 is a potential therapeutic target to prevent persistent HBV infection.

Chemical-induced degradation of PreS2 mutant surface antigen via the induction of microautophagy

Naturally evolved immune-escape PreS2 mutant is an oncogenic caveat of liver cirrhosis and hepatocellular carcinoma (HCC) during chronic hepatitis B virus (HBV) infection. PreS2 mutant is prevalent in above 50% of patients with HCC. In addition, intrahepatic expression of PreS2 mutant large surface antigen (PreS2-LHBS) induces endoplasmic reticulum stress, mitochondria dysfunction, cytokinesis failure, and subsequent chromosome hyperploidy. As PreS2-LHBS has no enzymatic activity, the development of PreS2-specific inhibitors can be challenging. In this study, we aim to identify inhibitors of PreS2-LHBS via the induction of protein-specific degradation. We set up a large-scale protein stability reporter platform and applied an FDA-approved drug library for the screening. We identified ABT199 as a negative modulator of PreS2-LHBS, which induced the degradation of PreS2-LHBS without affecting the general cell viability in both hepatoma and immortalized hepatocytes. Next, by affinity purification screening, we found that PreS2-LHBS interacted with HSC70, a microautophagy mediating chaperone. Simultaneously, inhibitions of lysosomal degradation or microautophagy restored the expression of PreS2-LHBS, suggesting microautophagy is involved in ABT199-induced PreS2-LHBS degradation. Notably, a 24-hr treatment of ABT199 was sufficient for the reduction of DNA damage and cytokinesis failure in PreS2-LHBS expressing hepatocytes. In addition, a persistent treatment of ABT199 for 3 weeks reversed chromosome hyperploidy in PreS2-LHBS cells and suppressed anchorage-independent growth of HBV-positive hepatoma cells. Together, this study identified ABT-199 as a negative modulator of PreS2-LHBS via mediating microautophagy. Our results indicate that long-term inhibition of PreS2-LHBS may serve as a novel strategy for the therapeutic prevention of HBV-mediated HCC.

Mitosis of hepatitis B virus-infected cells in vitro results in uninfected daughter cells

Background & AimsThe chronicity of HBV (and resultant liver disease) is determined by intrahepatic persistence of the HBV covalently closed circular DNA (cccDNA), an episomal form that encodes all viral transcripts. Therefore, cccDNA is a key target for new treatments, with the ultimate therapeutic aim being its complete elimination. Although established cccDNA molecules are known to be stable in resting hepatocytes, we aimed to understand their fate in dividing cells using in vitro models.MethodsWe infected HepG2-NTCP and HepaRG-NTCP cells with HBV and induced mitosis by passaging cells. We measured cccDNA copy number (by precise PCR assays) and HBV-expressing cells (by immunofluorescence) with wild-type HBV. We used reporter viruses expressing luciferase or RFP to track number of HBV-expressing cells over time after mitosis induction using luciferase assays and live imaging, respectively.ResultsIn all cases, we observed dramatic reductions in cccDNA levels, HBV-positive cell numbers, and cccDNA-dependent protein expression after each round of cell mitosis. The rates of reduction were highly consistent with mathematical models of a complete cccDNA loss in (as opposed to dilution into) daughter cells.ConclusionsOur results are concordant with previous animal models of HBV infection and show that HBV persistence can be efficiently overcome by inducing cell mitosis. These results support therapeutic approaches that induce liver turnover (e.g. immune modulators) in addition to direct-acting antiviral therapies to achieve hepatitis B cure.Lay summaryChronic hepatitis B affects 300 million people (killing 884,000 per year) and is incurable. To cure it, we need to clear the HBV genome from the liver. In this study, we looked at how the virus behaves after a cell divides. We found that it completely clears the virus, making 2 new uninfected cells. Our work informs new approaches to develop cures for chronic hepatitis B infections.

Hepatitis B virus envelope proteins can serve as therapeutic targets embedded in the host cell plasma membrane.

Hepatitis B virus (HBV) infection is a major health threat causing 880,000 deaths each year. Available therapies control viral replication but do not cure HBV, leaving patients at risk to develop hepatocellular carcinoma. Here, we show that HBV envelope proteins (HBs)-besides their integration into endosomal membranes-become embedded in the plasma membrane where they can be targeted by redirected T-cells. HBs was detected on the surface of HBV-infected cells, in livers of mice replicating HBV and in HBV-induced hepatocellular carcinoma. Staining with HBs-specific recombinant antibody MoMab recognising a conformational epitope indicated that membrane-associated HBs remains correctly folded in HBV-replicating cells in cell culture and in livers of HBV-transgenic mice in vivo. MoMab coated onto superparamagnetic iron oxide nanoparticles allowed to detect membrane-associated HBs after HBV infection by electron microscopy in distinct stretches of the hepatocyte plasma membrane. Last but not least, we demonstrate that HBs located on the cell surface allow therapeutic targeting of HBV-positive cells by T-cells either engrafted with a chimeric antigen receptor or redirected by bispecific, T-cell engager antibodies. TAKE AWAYS: HBs become translocated to the plasma membrane. Novel, recombinant antibody confirmed proper conformation of HBs on the membrane. HBs provide an interesting target by T-cell-based, potentially curative therapies.

Reticulon 3-mediated Chk2/p53 activation suppresses hepatocellular carcinogenesis and is blocked by hepatitis B virus

ObjectiveDysfunction of endoplasmic reticulum (ER) proteins is closely related to homeostasis disturbance and malignant transformation of hepatocellular carcinoma (HCC). Reticulons (RTN) are a family of ER-resident proteins critical for maintaining...

T-cell engager antibodies enable T cells to control HBV infection and to target HBsAg-positive hepatoma in mice

Current antiviral therapies control but rarely eliminate HBV, leaving chronic HBV carriers at risk of developing hepatocellular carcinoma (HCC). Lacking or dysfunctional virus-specific adaptive immunity prevents control of HBV and allows the virus to persist. Restoring antiviral T-cell immunity could lead to HBV elimination and cure of chronically infected patients. We constructed bispecific T-cell engager antibodies that are designed to induce antiviral immunity through simultaneous binding of HBV envelope proteins (HBVenv) on infected hepatocytes and CD3 or CD28 on T cells. T-cell engager antibodies were employed in co-cultures with healthy donor lymphocytes and HBV-infected target cells. Activation of the T-cell response was determined by detection of pro-inflammatory cytokines, effector function (by cytotoxicity) and antiviral effects. To study invivo efficacy, immune-deficient mice were transplanted with HBVenv-positive and -negative hepatoma cells. The 2T-cell engager antibodies synergistically activated T cells to become polyfunctional effectors that in turn elicited potent antiviral effects by killing infected cells and in addition controlled HBV via non-cytolytic, cytokine-mediated antiviral mechanisms. Invivo in mice, the antibodies attracted T cells specifically to the tumors expressing HBVenv resulting in T-cell activation, tumor infiltration and reduction of tumor burden. This study demonstrates that the administration of HBVenv-targeting T-cell engager antibodies facilitates a robust T-cell redirection towards HBV-positive target cells and provides a feasible and promising approach for the treatment of chronic viral hepatitis and HBV-associated HCC. T-cell engager antibodies are an interesting, novel therapeutic tool to restore immunity in patients with chronic hepatitis B. As bispecific antibodies, they bind envelope proteins on the surface of the hepatitis B virus (HBV) and CD3 or CD28 on T cells. This way, they induce a potent antiviral and cytotoxic T-cell response that leads to the elimination of HBV-positive cells. These bispecific T-cell engager antibodies are exciting therapeutic candidates for chronic hepatitis B and HBV-associated hepatocellular carcinoma.

HBx promotes hepatocarcinogenesis by enhancing phosphorylation and blocking ubiquitinylation of UHRF2.

The major cause of Hepatocellular carcinoma (HCC) is acute or chronic infection caused by hepatotropic viruses and HBV infection is the main cause. UHRF2, a ubiquitin-protein ligase E3, is associated with cancer development. This study aimed to investigate the connection and mechanism between UHRF2 and HBV-associated HCC. The expression of UHRF2 in human HBV-positive HCC tissues and paracancerous tissues was detected by western blot and tissue microarray. The effects of UHRF2 on invasion, migration and proliferation were detected in HBV-positive hepatoma cell lines. Furthermore, western blot, immunofluorescence, Co-immunoprecipitation and ubiquitination assays were used to explore the relationship and mechanism between UHRF2 and HBV-associated HCC. HBV-positive HCC tissues had higher UHRF2 expression levels than adjacent non-tumor tissues. The HBV-positive HCC patients with a low UHRF2 level in cancer tissues had longer overall and recurrence-free survival compared with those with a high UHRF2 level. UHRF2 induced invasion, migration and proliferation in human HBV-positive HCC cell lines HepG2.2.15 and Hep AD38(-). HBx, an encoding protein of HBV, maintained the stability of UHRF2 by blocking the ubiquitination of UHRF2. HBx up-regulated CDK2 expression through ETS1. UHRF2 bound to CDK2 directly and enhanced UHRF2 phosphorylation at serine 643. These results suggest that HBx-ETS1-CDK2-UHRF2 pathway plays an important role in the pathogenesis of HBV-associated HCC and represents new therapeutic targets for human HCC. ChiCTR2000041416.

Hepatitis B virus core protein promotes the expression of neuraminidase 1 to facilitate hepatocarcinogenesis

AbstractNeuraminidase 1 (NEU1) has been reported to be associated with hepatocellular carcinoma (HCC). However, the function and associated molecular mechanisms of NEU1 in hepatitis B virus (HBV)-related HCC have not been well investigated. In the present study, the expression of NEU1 mediated by HBV and HBV core protein (HBc) was measured in hepatoma cells. The expression of NEU1 protein was detected via immunohistochemical analysis in HBV-associated HCC tissues. The role of NEU1 in the activation of signaling pathways and epithelial-mesenchymal transition (EMT) and the proliferation and migration of hepatoma cells mediated by HBc was assessed. We found that NEU1 was upregulated in HBV-positive hepatoma cells and HBV-related HCC tissues. HBV promoted NEU1 expression at the mRNA and protein level via HBc in hepatoma cells. Mechanistically, HBc was able to enhance the activity of the NEU1 promoter through NF-κB binding sites. In addition, through the increase in NEU1 expression, HBc contributed to activation of downstream signaling pathways and EMT in hepatoma cells. Moreover, NEU1 facilitated the proliferation and migration of hepatoma cells mediated by HBc. Taken together, our findings provide novel insight into the molecular mechanism underlying the oncogenesis mediated by HBc and demonstrate that NEU1 plays a vital role in HBc-mediated functional abnormality in HCC. Thus, NEU1 may serve as a potential therapeutic target in HBV-associated HCC.

Hepatitis B Virus Core Protein Mediates the Upregulation of C5α Receptor 1 via NF-κB Pathway to Facilitate the Growth and Migration of Hepatoma Cells.

PurposeC5α receptor 1 (C5AR1) is associated with the development of various human cancers. However, whether it is involved in the development of hepatitis B virus (HBV)–related hepatocellular carcinoma (HCC) is poorly understood. We explored the expression, biological role, and associated mechanisms of C5AR1 in HBV-related hepatoma cells.Materials and MethodsThe expression of C5AR1 mediated by HBV and HBV core protein (HBc) was detected in hepatoma cells. The function of nuclear factor κB (NF-κB) pathway in HBc-induced C5AR1 expression was assessed. The roles of C5AR1 in the activation of intracellular signal pathways, the upregulation of inflammatory cytokines, and the growth and migration of hepatoma cells mediated by HBc, were investigated. The effect of C5α in the development of HCC mediated by C5AR1 was also measured.ResultsC5AR1 expression was increased in HBV-positive hepatoma cells. Dependent on HBc, HBV enhanced the expression of C5AR1 at the mRNA and protein levels. Besides, HBc could promote C5AR1 expression via the NF-κB pathway. Based on the C5AR1, HBc facilitated the activation of JNK and ERK pathways and the expression and secretion of interleukin-6 in hepatoma cells. Furthermore, C5AR1 was responsible for enhancing the growth and migration of hepatoma cells mediated by HBc. Except these, C5α could promote the malignant development of HBc-positive HCC via C5AR1.ConclusionWe provide new insight into the mechanisms of hepatocarcinogenesis mediated by HBc. C5AR1 has a significant role in the functional abnormality of hepatoma cells mediated by HBc, and might be utilized as a potential therapeutic target for HBV-related HCC.

RSK2-inactivating mutations potentiate MAPK signaling and support cholesterol metabolism in hepatocellular carcinoma

Mutational profiling of patient tumors has suggested that hepatocellular carcinoma (HCC) development is mainly driven by loss-of-function mutations in tumor suppressor genes. p90 ribosomal S6 kinase 2 (RSK2) functions as a direct downstream kinase of ERK1/2 and elevated RSK2 expression has been reported to support oncogenic functions in some cancers. We investigated if RSK2 was also dysregulated by inactivating mutations in cancers including HCC. We performed exome sequencing and targeted DNA sequencing on HBV-associated HCCs to examine recurrent RSK2 mutations. The functional significance and mechanistic consequences of RSK2 mutations were examined in natural RSK2-null HCC cells, and RSK2-knockout HCC cells. The potential downstream pathways underlying RSK2 mutations were investigated by RNA sequencing, qRT-PCR and mass spectrometry. We detected recurrent somatic RSK2 mutations at a rate of 6.3% in our HCC cohorts and revealed that, among many cancer types, HCC was the cancer most commonly harboring RSK2 mutations. The RSK2 mutations were inactivating and associated with a more aggressive tumor phenotype. We found that, functionally, restoring RSK2 expression in natural RSK2-null HBV-positive Hep3B cells suppressed proliferation and migration in vitro and tumorigenicity in vivo. Mechanistically, RSK2-inactivating mutations attenuated a SOS1/2-dependent negative feedback loop, leading to the activation of MAPK signaling. Of note, this RSK2 mutation-mediated MAPK upregulation rendered HCC cells more sensitive to sorafenib, a first-line multi-kinase inhibitor for advanced HCC. Furthermore, such activation of MAPK signaling enhanced cholesterol biosynthesis-related gene expression in HCC cells. Our findings reveal the mechanistic and functional significance of RSK2-inactivating mutations in HCC. These inactivating mutations may serve as an alternative route to activate MAPK signaling and cholesterol metabolism in HCC. In this study, we identified and functionally characterized RSK2-inactivating mutations in human hepatocellular carcinoma and demonstrated their association with aggressive tumor behavior. Mutations in RSK2 drive signaling pathways with known oncogenic potential, leading to enhanced cholesterol biosynthesis and potentially sensitizing tumors to sorafenib treatment.

Combinatorial Treatment of Birinapant and Zosuquidar Enhances Effective Control of HBV Replication In Vivo.

Chronic hepatitis B virus (HBV) infection remains a global health threat and affects hundreds of millions worldwide. Small molecule compounds that mimic natural antagonists of inhibitor of apoptosis (IAP) proteins, known as Smac-mimetics (second mitochondria-derived activator of caspases-mimetics), can promote the death of HBV-replicating liver cells and promote clearance of infection in preclinical models of HBV infection. The Smac-mimetic birinapant is a substrate of the multidrug resistance protein 1 (MDR1) efflux pump, and therefore inhibitors of MDR1 increase intracellular concentration of birinapant in MDR1 expressing cells. Liver cells are known to express MDR1 and other drug pump proteins. In this study, we investigated whether combining the clinical drugs, birinapant and the MDR1 inhibitor zosuquidar, increases the efficacy of birinapant in killing HBV expressing liver cells. We showed that this combination treatment is well tolerated and, compared to birinapant single agent, was more efficient at inducing death of HBV-positive liver cells and improving HBV-DNA and HBV surface antigen (HBsAg) control kinetics in an immunocompetent mouse model of HBV infection. Thus, this study identifies a novel and safe combinatorial treatment strategy to potentiate substantial reduction of HBV replication using an IAP antagonist.

SAT460 - HBV specific ImmTAV induce a broad immune response enabling the elimination of HBV positive cells

SAT460 - HBV specific ImmTAV induce a broad immune response enabling the elimination of HBV positive cells