Ni Tang

Epigenetic silencing of SFRP1 and SFRP5 by hepatitis B virus X protein enhances hepatoma cell tumorigenicity through Wnt signaling pathway

Secreted frizzled‐related proteins (SFRPs) are antagonists of the Wnt signaling pathway whose epigenetic downregulation have been shown to be involved in hepatocarcinogenesis. However, dysregulation of SFRPs induced by hepatitis B virus (HBV) X protein (HBx) has never been studied in HBV‐related hepatocellular carcinoma (HBV‐HCC). In this study, we sought to determine the clinical significance and underlying mechanism of HBx‐induced SFRPs dysregulation in hepatoma cells and HBV‐HCC patients. Our results showed that SFRP1 and SFRP5 expression were dramatically decreased by HBx in hepatoma cells. The repressed expression in hepatoma cells was partially rescued by a DNA methylation inhibitor and synergistically increased by a combination treatment with a histone deacetyltransferases inhibitor. In addition, we identified that SFRP1 and SFRP5 promoters were hypermethylated in both HBx‐expressing hepatoma cells and HBV‐HCC tissues. Downregulation of SFRP1 and SFRP5 in HBV‐HCC tissues was significantly correlated with overexpression of DNA methyltransferase 1 (DNMT1) and poor tumor differentiation. HBx facilitated the binding of DNMT1 and DNMT3A to SFRP1 and SFRP5 promoters, and resulted in epigenetic silencing of SFRP1 and SFRP5. Moreover, overexpression of SFRP1, SFRP5 or RNA interference mediated silencing of DNMT1 inactivated the Wnt signaling pathway and decreased the expression levels of Wnt target genes c‐Myc and CyclinD1, thus impeding HCC growth in vitro and in vivo, and regressing HBx‐induced epithelial–mesenchymal transition (EMT). Our findings strongly suggest that epigenetic silencing of SFRP1 and SFRP5 by HBx allows constitutive activation of Wnt signaling pathway and hence contributes to hepatocarcinogenesis.

Heparin sulphate D-glucosaminyl 3-O-sulfotransferase 3B1 plays a role in HBV replication.

Hepatitis B virus infection is a worldwide epidemic and is closely associated with the development of hepatocellular carcinoma. Nevertheless, the molecular mechanisms of HBV infection and carcinogenesis remain elusive. Using a hepatocyte model of HBV infection and comparing the gene expression profiling analysis we found that heparan sulfate D-glucosaminyl 3-O-sulfotransferase 3 B1 (HS3ST3B1,3-OST3-B) is down-regulated in the hepatocytes of chronic HBV infection model. HS3ST3B1 showed potent inhibitory effect on HBV replication. The inhibitory effect of HS3ST3B1 overexpression was lost upon gene silencing of HS3ST3B1 or when a catalytic inactive mutant of HS3ST3B1 was expressed. Our study revealed the anti-viral activity of HS3ST3B1 on HBV replication. It is conceivable that possible therapeutic applications of HBV infection could be devised by manipulating HS3ST3B1 activity.

HBx mutations promote hepatoma cell migration through the Wnt/β-catenin signaling pathway.

HBx mutations (T1753V, A1762T, G1764A, and T1768A) are frequently observed in hepatitis B virus (HBV)‐related hepatocellular carcinoma (HCC). Aberrant activation of the Wnt/β‐catenin signaling pathway is involved in the development of HCC. However, activation of the Wnt/β‐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/β‐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 β‐catenin. Moreover, the HBx Combo mutant increased and stabilized β‐catenin levels through inactivation of glycogen synthase kinase‐3β, resulting in upregulation of downstream target genes such as c‐Myc, CTGF, and WISP2. Enhanced activation of Wnt/β‐catenin was found in HCC tissues with HBx TA and Combo mutations. Knockdown of β‐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.

Phenotypic assay of a hepatitis B virus strain carrying an rtS246T variant using a new strategy

Phenotypic assays of hepatitis B virus (HBV) play an important role in research related to the problem of drug resistance that emerges during long‐term nucleot(s)ide therapy in patients with chronic hepatitis B. Most of the phenotypic assay systems that are available currently rely on the transfection of recombinant replication‐competent HBV DNA into hepatoma cell lines. Cloning clinical HBV isolates using conventional digestion‐and‐ligation techniques to generate replication‐competent recombinants can be very difficult because of the sequence heterogeneity and unique structure of the HBV genome. In this study, a new strategy for constructing an HBV 1.1× recombinant was developed. The core of this strategy is the “fragment substitution reaction” (FSR). FSR allows PCR fragments to be cloned without digestion or ligation, providing a new tool for cloning fragments or genomes amplified from serum HBV DNA, and therefore making the assay of HBV phenotypes more convenient. Using this strategy, a phenotypic assay was performed on an HBV strain carrying an rtS246T variant isolated from a patient with chronic hepatitis B that was only responsive partially to entecavir therapy. The results indicated that this strain is sensitive to entecavir in vitro. J. Med. Virol. 84:34–43, 2011.

Chromatin remodeling gene ARID2 targets cyclin D1 and cyclin E1 to suppress hepatoma cell progression.

Exome and whole-genome sequencing studies have drawn attention to the role of somatic mutations in SWI/SNF chromatin remodeling complexes in the carcinogenesis of hepatocellular carcinoma (HCC). Here, we explored the molecular mechanisms underlying the biological roles of AT-rich interactive domain 2 (ARID2) in the pathogenesis of HCC. We found that ARID2 expression was significantly downregulated in HCC tissues compared with non-tumorous tissues. Restoration of ARID2 expression in hepatoma cells was sufficient to suppress cell proliferation and tumor growth in mice, whereas ARID2 knockdown contributed to the enhancement of cellular proliferation and tumorigenicity. Suppression of ARID2 expression accelerated G1/S transition associated with upregulation of cyclin D1, cyclin E1, CDK4, and phosphorylation of the retinoblastoma protein (Rb). Furthermore, we demonstrated that ARID2 physically interacts with E2F1 and decreases binding of E2F1/RNA Pol II to the promoters of CCND1 and CCNE1. Taken together, these results demonstrate that ARID2 suppresses tumor cell growth through repression of cyclin D1 and cyclin E1 expression, thereby retarding cell cycle progression and cell proliferation in hepatoma cells. These findings highlight the potential role of ARID2 as a tumor growth suppressor in HCC.

A novel baseline hepatitis B virus sequencing-based strategy for predicting adefovir antiviral response

Adefovir dipivoxil (ADV) is used as first-line monotherapy or rescue therapy in chronic hepatitis B (CHB) patients. In this study, we sought to identify nucleotide changes in the reverse transcriptase (RT) of hepatitis B virus (HBV) at baseline and explore their predictive value for ADV antiviral response. Ultra-deep pyrosequencing (UDPS) was utilized to determine HBV genetic variability within the RT region at baseline and during a 48-week ADV therapy. According to the viral load at the end of ADV treatment, all patients were classified into responders (HBV DNA level reduction of ⩾ 3 log 10 IU/mL) and suboptimal responders (HBV DNA level reduction of <3 log 10 IU/mL). Based on UDPS data at baseline, we identified 11 nucleotide substitutions whose combination frequency was significantly associated with the antiviral response among 36 CHB patients in the study group. However, the baseline distribution and frequency of rt181 and rt236 substitutions known to confer ADV resistance was a poor predictor for the antiviral response. Compared with baseline serum HBeAg, HBV-DNA and ALT levels, the baseline HBV sequence-based model showed higher predictive accuracy for ADV response. In an independent cohort of 31 validation patients with CHB, the sequence-based model provided greater predictive potency than the HBeAg/HBV-DNA/ALT and the HBeAg/HBV-DNA/ALT/sequence combinations. Taken together, we confirm the presence of ADV resistance variants in treatment-naïve patients and firstly unravel the predictive value of the baseline mutations in the HBV RT region for ADV antiviral response.

Pharmacological or transcriptional inhibition of both HDAC1 and 2 leads to cell cycle blockage and apoptosis via p21Waf1/Cip1 and p19INK4d upregulation in hepatocellular carcinoma

Histone deacetylases (HDACs) are commonly dysregulated in cancer and represent promising therapeutic targets. However, global HDAC inhibitors have shown limited efficacy in the treatment of solid tumours, including hepatocellular carcinoma (HCC). In this study, we investigated the therapeutic effect of selectively inhibiting HDAC1 and 2 in HCC.

The infection efficiency and replication ability of circularized HBV DNA optimized the linear HBV DNA in vitro and in vivo.

Studies on molecular mechanisms of the persist infection of hepatitis B virus have been hampered by a lack of a robust animal model. We successfully established a simple, versatile, and reproducible HBV persist infection model in vitro and in vivo with the circularized HBV DNA. The cells and mice were transfected or injected with circularized HBV DNA and pAAV/HBV1.2, respectively. At the indicated time, the cells, supernatants, serum samples, and liver tissues were collected for virological and serological detection. Both in vitro and in vivo, the circularized HBV DNA and pAAV/HBV1.2 could replicate and transcribe efficiently, but the infection effect of the former was superior to the latter (p < 0.05). The injection of circularized HBV genome DNA into the mice robustly supported HBV infection and approximately 80% of HBV infected mice established persistent infection for at least 10 weeks. This study demonstrated that the infection efficiency and replication ability of the circularized structure of HBV DNA overmatched that of the expression plasmid containing the linear structure of HBV DNA in vitro and in vivo. Meanwhile, this research results could provide useful tools and methodology for further study of pathogenic mechanisms and potential antiviral treatments of human chronic HBV infection in vitro and in vivo.

Validation of a multi-omics strategy for prioritizing personalized candidate driver genes

Significant heterogeneity between different tumors prevents the discovery of cancer driver genes, especially in a patient-specific manner. We previously prioritized five personalized candidate mutation-driver genes in a hyper-mutated hepatocellular carcinoma patient using a multi-omics strategy. However, the roles of the prioritized driver genes and patient-specific mutations in hepatocarcinogenesis are unclear. We investigated the impact of the tumor-mutated allele on structure-function relationship of the encoded protein and assessed both loss- and gain-of-function of these genes and mutations on hepatoma cell behaviors in vitro. The prioritized mutation-driver genes act as tumor suppressor genes and inhibit cell proliferation and migration. In addition, the loss-of-function effect of the patient-specific mutations promoted cell proliferation and migration. Of note, the HNF1A S247T mutation significantly reduced the HNF1A transcriptional activity for hepatocyte nuclear factor 4 alpha (HNF4A) but did not disrupt nuclear localization of HNF1A. The results provide evidence for supporting the validity of our proposed multi-omics strategy, which supplies a new avenue for prioritizing mutation-drivers towards personalized cancer therapy.

Cisplatin Enhances Hepatitis B Virus Replication and PGC-1α Expression through Endoplasmic Reticulum Stress

Chronic hepatitis B infection remains a serious public health issue worldwide. Hepatitis B virus (HBV) reactivation is commonly reported in patients receiving anticancer therapy, immunosuppressive therapy, or organ and tissue transplantation. However, the precise mechanisms underlying chemotherapeutic agent-related HBV reactivation remain unclear. Here, we report that peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) plays a central role in cisplatin-induced HBV transcription and replication. First, cisplatin treatment upregulated the expression levels of PGC-1α and hepatocyte nuclear factor 4 alpha (HNF-4α) in both HBV-replicating cells and an HBV-transgenic mouse model. PGC-1α coactivates with HNF-4α, which interacts with a core promoter and enhancer II region of HBV genome, thereby promoting HBV production. In contrast, knockdown of PGC-1α and HNF-4α by RNA interference in hepatoma cells reversed HBV activation in response to cisplatin. Additionally, PGC-1α upregulation depended on cisplatin-mediated endoplasmic reticulum (ER) stress. We further observed that the recruitment of cyclic AMP-responsive element-binding protein plays a crucial role for PGC-1α transcriptional activation in cisplatin-treated cells. Finally, pharmacologic inhibition of ER stress impaired PGC-1α upregulation and HBV production induced by cisplatin treatment. These findings demonstrate novel molecular mechanisms indicating that ER stress-PGC1α signaling pathway plays a critical role in cisplatin-evoked HBV reactivation.