Does the hepatitis B antigen HBx promote the appearance of liver cancer stem cells?
ABSTRACT: Hepatitis B virus (HBV) is a major etiologic agent of chronic liver disease 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 (CSC). 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, Klf-4, ?-catenin, and epithelial cell adhesion molecule (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 ?-catenin and epigenetic upregulation 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 upregulated expression of multiple "stemness" markers supports the hypothesis that HBx contributes to hepatocarcinogenesis, at least in part, by promoting changes in gene expression that are characteristics of CSCs.
Project description:MYH9 has dual functions in tumors. However, its role in inducing tumor stemness in hepatocellular carcinoma (HCC) is not yet determined. Here, we found that MYH9 is an effective promoter of tumor stemness that facilitates hepatocellular carcinoma pathogenesis. Importantly, targeting MYH9 remarkably improved the survival of hepatocellular carcinoma-bearing mice and promoted sorafenib sensitivity of hepatocellular carcinoma cells in vivo. Mechanistic analysis suggested that MYH9 interacted with GSK3? and reduced its protein expression by ubiquitin-mediated degradation, which therefore dysregulated the ?-catenin destruction complex and induced the downstream tumor stemness phenotype, epithelial-mesenchymal transition, and c-Jun signaling in HCC. C-Jun transcriptionally stimulated MYH9 expression and formed an MYH9/GSK3?/?-catenin/c-Jun feedback loop. X protein is a hepatitis B virus (HBV)-encoded key oncogenic protein that promotes HCC pathogenesis. Interestingly, we observed that HBV X protein (HBX) interacted with MYH9 and induced its expression by modulating GSK3?/?-catenin/c-Jun signaling. Targeting MYH9 blocked HBX-induced GSK3? ubiquitination to activate the ?-catenin destruction complex and suppressed cancer stemness and EMT. Based on TCGA database analysis, MYH9 was found to be elevated and conferred poor prognosis for hepatocellular carcinoma patients. In clinical samples, high MYH9 expression levels predicted poor prognosis of hepatocellular carcinoma patients. These findings identify the suppression of MYH9 as an alternative approach for the effective eradication of CSC properties to inhibit cancer migration, invasion, growth, and sorafenib resistance in HCC patients. Our study demonstrated that MYH9 is a crucial therapeutic target in HCC.
Project description:HBx mutations (T1753V, A1762T, G1764A, and T1768A) are frequently observed in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Aberrant activation of the Wnt/?-catenin signaling pathway is involved in the development of HCC. However, activation of the Wnt/?-catenin signaling pathway by HBx mutants has not been studied in hepatoma cells or HBV-associated HCC samples. In this study, we examined the effects of HBx mutants on the migration and proliferation of HCC cells and evaluated the activation of Wnt/?-catenin signaling in HBx-transfected HCC cells and HBV-related HCC tissues. We found that HBx mutants (T, A, TA, and Combo) promoted the migration and proliferation of hepatoma cells. The HBx Combo mutant potentiated TOP-luc activity and increased nuclear translocation of ?-catenin. Moreover, the HBx Combo mutant increased and stabilized ?-catenin levels through inactivation of glycogen synthase kinase-3?, resulting in upregulation of downstream target genes such as c-Myc, CTGF, and WISP2. Enhanced activation of Wnt/?-catenin was found in HCC tissues with HBx TA and Combo mutations. Knockdown of ?-catenin effectively abrogated cell migration and proliferation stimulated by the HBx TA and Combo mutants. Our results indicate that HBx mutants, especially the Combo mutant, allow constitutive activation of the Wnt signaling pathway and may play a pivotal role in HBV-associated hepatocarcinogenesis.
Project description:Hepatitis B virus (HBV) is one of predisposing factors for hepatocellular carcinoma (HCC). The role of HBV x protein (HBx) in mediating the induction and maintenance of cancer stemness during HBV-related HCC attracts considerable attention, but the exact mechanism has not been clearly elucidated. Here, ABCG2-dependent stem-like side population cells, which are thought to be liver cancer stem cells (LCSCs), were present in HCC cells, and the fraction of this subset was increased in HBx-expressing HCC cells. In addition, glycolysis was upregulated in LCSCs and HBx-expressing HCC cells, and intervention of glycolysis attenuated cancer stem-like phenotypes. Mitochondria play an important role in the maintenance of energy homeostasis, BNIP3L-dependent mitophagy was also activated in LCSCs and HBx-expressing HCC cells, which triggered a metabolic shift toward glycolysis. In summary, we proposed a positive feedback loop, in which HBx induced BNIP3L-dependent mitophagy which upregulated glycolytic metabolism, increasing cancer stemness of HCC cells in vivo and in vitro. BNIP3L might be a potential therapeutic target for intervention of LCSCs-associated HCC. Anti-HBx, a monoclonal antibody targeting intracellular HBx, had the potential to delay the progression of HBV infection related-HCC.
Project description:Cancer stem cells (CSCs) are the key factor in determining cancer recurrence, metastasis, chemoresistance and patient prognosis in hepatocellular carcinoma (HCC). The role of miR-5188 in cancer stemness has never been documented. In this study, we investigated the clinical and biological roles of miR-5188 in HCC. Methods: MiRNA expression in HCC was analyzed by bioinformatics analysis and in situ hybridization. The biological effect of miR-5188 was demonstrated in both in vitro and in vivo studies through the ectopic expression of miR-5188. The target gene and molecular pathway of miR-5188 were characterized using bioinformatics tools, dual-luciferase reporter assays, gene knockdown, and rescue experiments. Results: MiR-5188 was shown to be upregulated and confer poor prognosis in HCC patient data from TCGA database. MiR-5188 was subsequently identified as a significant inducer of cancer stemness that promotes HCC pathogenesis. Specifically, the targeting of miR-5188 by its antagomir markedly prolonged the survival time of HCC-bearing mice and improved HCC cell chemosensitivity in vivo. Mechanistic analysis indicated that miR-5188 directly targets FOXO1, which interacts with ?-catenin in the cytoplasm to reduce the nuclear translocation of ?-catenin and promotes the activation of Wnt signaling and downstream tumor stemness, EMT, and c-Jun. Moreover, c-Jun transcriptionally activates miR-5188 expression, forming a positive feedback loop. Interestingly, the miR-5188-FOXO1/?-catenin-c-Jun feedback loop was induced by hepatitis X protein (HBX) through Wnt signaling and participated in the HBX-induced pathogenesis of HCC. Finally, analyses of transcriptomics data and our clinical data supported the significance of the abnormal expression of the miR-5188 pathway in HCC pathogenesis. Conclusions: These findings present the inhibition of miR-5188 as a novel strategy for the efficient elimination of CSCs to prevent tumor metastasis, recurrence and chemoresistance in patients with hepatocellular carcinoma. Our study highlights the importance of miR-5188 as a tumor stemness inducer that acts as a potential target for HCC treatment.
Project description:Although Hepatitis B virus (HBV) X gene mutations are frequently detected in HBV-related human hepatocellular carcinoma (HCC) patients, causative HBx mutations in the development of HCC have not yet been determined. We herein identified C1485T and C1653T mutations in the HBx gene as independent risk of HCC for HBV through the analysis using serum from chronic hepatitis B patients. We generated transgenic mice expressing wild-type (WT-HBxTg) and mutant (C1485T-HBxTg) HBx to assess the carcinogenic potential of mutated HBx. C1485T-HBxTg mice were more susceptible to diethylnitrosamine-induced hepatocarcinogenesis than WT-HBxTg mice and control non-Tg mice. The promotion of hepatocarcinogenesis in C1485T-HBxTg mice was accompanied by the activation of ?-catenin and Jun N-terminal kinase (JNK) signaling pathways as well as the production of reactive oxygen species, whereas the activation of nuclear factor-kappa B in the livers of C1485T-HBxTg mice was attenuated. These results demonstrate that the HBx C1485T mutation contributes to human and murine hepatocarcinogenesis.
Project description:Background: Hepatitis B-X Protein (HBx) encoded in Hepatitis B virus (HBV) is known to play a critical role in development and progression of HBV induced hepatocellular carcinoma (HCC). HBx interacts with and activates various cells in HCC microenvironment to promote tumor initiation, progression and invasion. In this study, we investigated how surrounding stromal cells interact with HBx-infected hepatoma cells by a series of in vitro co-culture studies. Methods: Huh7 hepatoma cells were cultured and transfected with the mammalian expression vector pGFP-HBx. Co-culture assays were performed between HBx-transfected Huh7 cells and conditioned media (CM) from stromal cells [endothelial cell lines (HUVECs) and hepatic stellate cell lines (LX2 cells)]. The effect of these interactions was studied by a series of functional assays like chemotaxis, invasion, and wound healing scratch assays. Also, quantitative real time (RT)-PCRs of the mesenchymal genes was performed in the hepatoma cells with and without the co-cultures. Hep3B cells with an integrated HBV genome were taken as positive controls. Results: HBx-transfected Huh7 cells cultured in presence of CM from HUVECs illustrated enhanced migration and tube formation as compared to HBx-transfected cells cultured alone or co-cultured with LX2 cells. HBx-transfected hepatoma cells incubated with CM from HUVECs also expressed mesenchymal genes including Thy1, CDH2, TGF?R1, VIM, and CD133. ELISAs revealed increased levels of TGF-? in CM from HUVECs. In comparison to unstimulated HBx-transfected Huh7 cells, TGF-? stimulated cells displayed increased invasive properties and mesenchymal gene expression. RT-PCR and flow cytometry analysis further demonstrated that incubation with either CM from HUVECs or TGF-? significantly increased the expression of a stemness marker, CD133 in HBx-infected hepatoma cells. Gene inhibition experiments with CD133 siRNA showed a downregulation of mesenchymal gene expression and properties in TGF-? induced HBx-infected hepatoma cells as compared to that observed in control siRNA treated cells, indicating CD133 as one of the key molecules affecting epithelial to mesenchymal transition (EMT) in HBx-infected cells. Conclusion: The study indicates that secretory factors like TGF-? from neighboring endothelial cells may enhance expression of CD133 and impart an aggressive EMT phenotype to HBx-infected hepatoma cells in HBV induced HCC.
Project description:AIM:To investigate the hepatitis B virus (HBV)x gene (HBx) state in the tissues of HBV-related hepatocellular carcinoma (HCC) in Chinese patients and whether there were particular HBx mutations. METHODS:HBx gene was amplified and direct sequencing was used in genomic DNA samples from 20 HCC and corresponding non-cancerous liver tissues from HBsAg-positive patients. HBV DNA integration and HBx deleted mutation were validated in 45 HCC patients at different stages by Southern blot analysis and polymerase chain reaction methods. RESULTS:The frequencies of HBx point mutations were significantly lower in HCC than their corresponding non-cancerous liver tissues (11/19 vs 18/19, P=0.019). In contrast, deletions in HBx gene were significantly higher in HCC than their non-cancerous liver tissues (16/19 vs 4/19, P<0.001). The deletion of HBx COOH-terminal was detected in 14 HCC tissues. A specific integration of HBx at 17p13 locus was also found in 8 of 16 HCC, and all of them also exhibited full-length HBx deletions. Integrated or integrated coexistence with replicated pattern was obtained in 45.5% (20/45)-56.8% (25/45) tumors and 40.9% (18/45)-52.3% (23/45) non-tumor tissues. CONCLUSION:HBx deletion, especially the COOH-terminal deletion of HBx is a frequent event in HBV-associated HCC tissues in China. HBV integration had also taken place in partial HCC tissues. This supporting the hypothesis that deletion and probably integrated forms of the HBx gene may be implicated in liver carcinogenesis.
Project description:BACKGROUND: The biological pathways and functional properties by which misexpressed microRNAs (miRNAs) contribute to liver carcinogenesis have been intensively investigated. However, little is known about the upstream mechanisms that deregulate miRNA expressions in this process. In hepatocellular carcinoma (HCC), hepatitis B virus (HBV) X protein (HBx), a transcriptional trans-activator, is frequently expressed in truncated form without carboxyl-terminus but its role in miRNA expression and HCC development is unclear. METHODS: Human non-tumorigenic hepatocytes were infected with lentivirus-expressing full-length and carboxyl-terminal truncated HBx (Ct-HBx) for cell growth assay and miRNA profiling. Chromatin immunoprecipitation microarray was performed to identify the miRNA promoters directly associated with HBx. Direct transcriptional control was verified by luciferase reporter assay. The differential miRNA expressions were further validated in a cohort of HBV-associated HCC tissues using real-time PCR. RESULTS: Hepatocytes expressing Ct-HBx grew significantly faster than the full-length HBx counterparts. Ct-HBx decreased while full-length HBx increased the expression of a set of miRNAs with growth-suppressive functions. Interestingly, Ct-HBx bound to and inhibited the transcriptional activity of some of these miRNA promoters. Notably, some of the examined repressed-miRNAs (miR-26a, -29c, -146a and -190) were also significantly down-regulated in a subset of HCC tissues with carboxyl-terminal HBx truncation compared to their matching non-tumor tissues, highlighting the clinical relevance of our data. CONCLUSION: Our results suggest that Ct-HBx directly regulates miRNA transcription and in turn promotes hepatocellular proliferation, thus revealing a viral contribution of miRNA deregulation during hepatocarcinogenesis.
Project description:CYP2E1, one of the cytochrome P450 mixed-function oxidases located predominantly in liver, plays a key role in metabolism of xenobiotics including ethanol and procarcinogens. Recently, down-expression of CYP2E1 was found in hepatocellular carcinoma (HCC) with the majority to be chronic hepatitis B virus (HBV) carriers. In this study, we tested a hypothesis that HBx may inhibit CYP2E1 gene expression via hepatocyte nuclear factor 4? (HNF4?). By enforced HBx gene expression in cultured HepG2 cells, we determined the effect of HBx on CYP2E1 mRNA and protein expression. With a bioinformatics analysis, we found a consensus HNF-4? binding sequence located on -318 to -294 bp upstream of human CYP2E1 promoter. Using reporter gene assay and site-directed mutagenesis, we have shown that mutation of this site dramatically decreased CYP2E1 promoter activity. By silencing endogenous HNF-4?, we have further validated knockdown of HNF-4? significantly decreased CYP2E1 expression. Ectopic overexpression of HBx in HepG2 cells inhibits HNF-4? expression, and HNF-4? levels were inversely correlated with viral proteins both in HBV-infected HepG2215 cells and as well as HBV positive HCC liver tissues. Moreover, the HBx-induced CYP2E1 reduction could be rescued by ectopic supplement of HNF4? protein expression. Furthermore, human hepatoma cells C34, which do not express CYP2E1, shows enhanced cell growth rate compared to E47, which constitutively expresses CYP2E1. In addition, the significantly altered liver proteins in CYP2E1 knockout mice were detected with proteomics analysis. Together, HBx inhibits human CYP2E1 gene expression via downregulating HNF4? which contributes to promotion of human hepatoma cell growth. The elucidation of a HBx-HNF4?-CYP2E1 pathway provides novel insight into the molecular mechanism underlining chronic HBV infection associated hepatocarcinogenesis.
Project description:Chronic hepatitis B virus (HBV) infection is a major risk factor for developing hepatocellular carcinoma (HCC), and HBV X protein (HBx) acts as cofactor in hepatocarcinogenesis. In liver tumors from animals modeling HBx- and HBV-mediated hepatocarcinogenesis, downregulation of chromatin regulating proteins SUZ12 and ZNF198 induces expression of several genes, including epithelial cell adhesion molecule (EpCAM). EpCAM upregulation occurs in HBV-mediated HCCs and hepatic cancer stem cells, by a mechanism not understood. Herein we demonstrate HBx induces EpCAM expression via active DNA demethylation. In hepatocytes, EpCAM is silenced by polycomb repressive complex 2 (PRC2) and ZNF198/LSD1/Co-REST/HDAC1 chromatin-modifying complexes. Cells with stable knockdown of SUZ12, an essential PRC2 subunit, upon HBx expression demethylate a CpG dinucleotide located adjacent to NF-?B/RelA half-site. This NF-?B/RelA site is in a CpG island downstream from EpCAM transcriptional start site (TSS). Chromatin immunoprecipitation (ChIP) assays demonstrate HBx-dependent RelA occupancy of NF-?B half-site, whereas RelA knockdown suppresses CpG demethylation and EpCAM expression. Tumor necrosis factor-? activates RelA, propagating demethylation to nearby CpG sites, shown by sodium bisulfite sequencing. RelA-dependent demethylation occurring upon HBx expression requires methyltrasferase EZH2, TET2 a key factor in cytosine demethylation and inactive DNMT3L, shown by knockdown assays and sodium bisulfite sequencing. Co-immunoprecipitations and sequential ChIP assays demonstrate that RelA in the presence of HBx forms a complex with EZH2, TET2 and DNMT3L, although the role of DNMT3L remains to be understood. Interestingly, the human EpCAM gene also has a CpG island downstream from its TSS, and a NF-?B-binding site flanked by CpGs. HepG2 cells derived from human HCC exhibit demethylation of these NF-?B-flanking CpG sites, and HBV replication propagates demethylation to nearby CpG sites. DLK1, another PRC2 target gene, also upregulated in HBV-mediated HCCs, is demethylated in liver tumors at CpG dinucleotides flanking the NF-?B-binding sequence, supporting that this active DNA demethylation mechanism functions during oncogenic transformation.