Viral hijacking of the TENT4-ZCCHC14 complex protects viral RNAs via mixed tailing.

TENT4 enzymes generate 'mixed tails' of diverse nucleotides at 3' ends of RNAs via nontemplated nucleotide addition to protect messenger RNAs from deadenylation. Here we discover extensive mixed tailing in transcripts of hepatitis B virus (HBV) and human cytomegalovirus (HCMV), generated via a similar mechanism exploiting the TENT4-ZCCHC14 complex. TAIL-seq on HBV and HCMV RNAs revealed that TENT4A and TENT4B are responsible for mixed tailing and protection of viral poly(A) tails. We find that the HBV post-transcriptional regulatory element (PRE), specifically the CNGGN-type pentaloop, is critical for TENT4-dependent regulation. HCMV uses a similar pentaloop, an interesting example of convergent evolution. This pentaloop is recognized by the sterile alpha motif domain-containing ZCCHC14 protein, which in turn recruits TENT4. Overall, our study reveals the mechanism of action of PRE, which has been widely used to enhance gene expression, and identifies the TENT4-ZCCHC14 complex as a potential target for antiviral therapeutics.

SERPINA3 is a key modulator of HNRNP-K transcriptional activity against oxidative stress in HCC.

Most studies about serpin peptidase inhibitor, clade A member 3 (SERPINA3) has been limited to its inhibitory functions and mechanisms. Herein, we report a novel role of SERPINA3 in transcriptional regulation of HCC progression-related genes. Among 19 selected genes through HCC cell isolation system based on telomere length, microarray analyses, and cell-based studies, SERPINA3 was the strongest determinant of increases in telomere length, HCC cell proliferation, survival, migration, and invasion. We also found that SERPINA3 strongly interacted with heterogeneous nuclear ribonucleoprotein K (HNRNP-K) under H2O2 exposure, and the oxidation-elicited SERPINA3-HNRNP-K complex enhanced the promoter activities and transcript levels of a telomere-relating gene (POT1) and HCC-promoting genes (UHRF1 and HIST2H2BE). Intriguingly, the inhibition of SERPINA3 oxidation rendered the transcriptional activity of the SERPINA3-HNRNP-K complex suppressed. Moreover, the co-immunoprecipitated HNRNP-K with SERPINA3 quantitatively correlated with not only the level of SERPINA3 oxidation but also the level of POT1, UHRF1, and HIST2H2BE transcripts and telomere length in HCC tissues. Therefore, the upregulated transcriptional activity of HNRNP-K mediated by SERPINA3 promotes HCC cell survival and proliferation and could be an indicator of poor prognosis for HCC patients.

Cetylpyridinium chloride interaction with the hepatitis B virus core protein inhibits capsid assembly.

Hepatitis B virus (HBV) infection is a major risk factor for chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC) worldwide. While multiple hepatitis B drugs have been developed, build up of drug resistance during treatment or weak efficacies observed in some cases have limited their application. Therefore, there is an urgent need to develop substitutional pharmacological agents for HBV-infected individuals. Here, we identified cetylpyridinium chloride (CPC) as a novel inhibitor of HBV. Using computational docking of CPC to core protein, microscale thermophoresis analysis of CPC binding to viral nucleocapsids, and in vitro nucleocapsid formation assays, we found that CPC interacts with dimeric viral nucleocapsid protein (known as core protein or HBcAg) specifically. Compared with other HBV inhibitors, such as benzenesulfonamide (BS) and sulfanilamide (SA), CPC achieved significantly better reduction of HBV particle number in HepG2.2.15 cell line, a derivative of human HCC cells that stably expresses HBV. CPC also inhibited HBV replication in mouse hydrodynamic model system. Taken together, our results show that CPC inhibits capsid assembly and leads to reduced HBV biogenesis. Thus, CPC is an effective pharmacological agent that can reduce HBV particles.

PI3Kδ Is a Therapeutic Target in Hepatocellular Carcinoma.

Class I phosphoinositide 3-kinase (PI3K) signaling is a major pathway in human cancer development and progression. Among the four PI3K isoforms, PI3Kα and PI3Kß are ubiquitously expressed, whereas PI3Kγ and PI3Kδ are found primarily in leukocytes. Until now, PI3K targeting in solid tumors has focused on inhibiting PI3Kα-mediated and PI3Kß-mediated cancer cell-intrinsic PI3K activity. The role of PI3Kδ in solid tumors is unknown. Here, we evaluated the effects of PI3Kδ using established hepatocellular carcinoma (HCC) cells, malignant hepatocytes derived from patients with advanced HCC, murine models, and HCC tissues using RNA sequencing, quantitative PCR, immunoblotting, immunofluorescence, microarray, liquid chromatography-tandem mass spectrometry, and kinase assay. We established a chemical carcinogenesis model of liver malignancy that reflects the malignant phenotype and the in vivo environment of advanced HCC. In this in vivo advanced HCC-mimic system using HCC cells treated with hydrogen peroxide (H2 O2 ), we showed that H2 O2 selectively increases PI3Kδ activity while decreasing that of other class I PI3Ks. Blocking PI3Kδ activity with a PI3Kδ inhibitor or small interfering RNA-mediated PI3Kδ gene silencing inhibited HCC-cell proliferation and dampened key features of malignant HCC, including the up-regulation of telomerase reverse transcriptase (TERT). Mechanistically, H2 O2 induced oxidative modification of the serpin peptidase inhibitor, serpin peptidase inhibitor (SERPINA3), blocking its ubiquitin-dependent degradation and enhancing its activity as a transcriptional activator of PI3Kδ and TERT. High PI3Kδ levels in HCC were found to correlate with poor survival rates, with human advanced HCC showing positive correlations between the protein levels of oxidized SERPINA3, PI3Kδ, and TERT. Thus, PI3Kδ plays significant roles in malignant liver tumors. Conclusion: Our data identify PI3Kδ inhibition, recently approved for the treatment of human B-cell malignancies, as a potential treatment for HCC.

Heat shock protein 70 and heat shock protein 90 synergistically increase hepatitis B viral capsid assembly.

Hepatitis B virus (HBV) infection can cause chronic liver diseases, cirrhosis, and hepatocellular carcinoma (HCC). Heat shock proteins (Hsps) are important factors in the formation of the HBV capsid and in genome replication during the viral life cycle. Hsp90 is known to promote capsid assembly. However, the functional roles of Hsp70 in HBV capsid assembly with Hsp90 have not been studied so far. Using microscale thermophoresis analyses and in vitro nucleocapsid formation assays, we found that Hsp70 bound to a HBV core protein dimer and facilitated HBV capsid assembly. Inhibition of Hsp70 by methylene blue (MB) led to a decrease in capsid assembly. Moreover, Hsp70 inhibition reduced intracellular capsid formation and HBV virus particle number in HepG2.2.15 cells. Furthermore, we examined synergism between Hsp70 and Hsp90 on HBV capsid formation in vitro. Our results clarify the role of Hsp70 in HBV capsid formation via an interaction with core dimers and in synergistically promoting capsid assembly with Hsp90.

Oxidatively Modified Protein-Disulfide Isomerase-Associated 3 Promotes Dyskerin Pseudouridine Synthase 1-Mediated Malignancy and Survival of Hepatocellular Carcinoma Cells.

Dyskerin pseudouridine synthase 1 (DKC1) is a conserved gene encoding the RNA-binding protein dyskerin, which is an essential component of the telomerase holoenzyme. DKC1 up-regulation is frequently observed in many different human cancers including hepatocellular carcinoma (HCC); however, its regulatory mechanisms remain unclear. Thus, we investigated the regulatory mechanism of DKC1 in HCC progression. We found that protein-disulfide isomerase-associated 3 (PDIA3) interacted with the DKC1 regulatory DNA in HCC cells but not in HCC cells with elevated reactive oxygen species (ROS) levels, using liquid chromatographic-tandem mass spectrometric analysis after isolating the DKC1 regulatory region binding proteins. PDIA3 repressed DKC1 expression in HCC cells by recognizing the G-quadruplex DNA at the DKC1 location. However, oxidative modification of PDIA3 induced by ROS redistributed this protein into the cytosolic regions, which stimulated DKC1 expression. We also identified Met338 in PDIA3 as the oxidatively modified residue and validated the effect of oxidative modification using an ectopic expression system, a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 knock-in system, and a xenograft mouse model. We observed that oxidatively modified PDIA3 promoted DKC1-mediated malignancy and survival of HCC cells in vitro and in vivo. HCC tissues showed a positive association with ROS, cytoplasmic PDIA3, and nuclear DKC1 levels. HCC patients with high PDIA3 protein and DKC1 mRNA levels also displayed reduced recurrence-free survival rates. Cumulatively, the results showed that cytoplasmic PDIA3 activity could be essential in raising DKC1 expression in HCC progression and predicting poor prognoses in HCC patients. Conclusion: Our study indicates that the elevated ROS levels in HCC modulate cytoplasmic PDIA3 levels, resulting in HCC cell survival through DKC1 up-regulation.

WITHDRAWN: Cetylpyridinium chloride as a novel inhibitor of hepatitis B viral capsid assembly.

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Telomere length and reactive oxygen species levels are positively associated with a high risk of mortality and recurrence in hepatocellular carcinoma.

Telomeres protect chromosomal ends from deterioration and have been shown to be susceptible to shortening by reactive oxygen species (ROS)-induced damage. ROS levels increase during the progression from early to advanced hepatocellular carcinoma (HCC). An independent study found that the telomeres in most HCC tissues lengthened during carcinogenic advancement. Activated telomerase has been hypothesized to elongate telomeres during the progression of malignant HCC, but it remains unclear which signaling pathway is necessary for telomerase activation in HCC. Here, we showed using cell lines derived from human HCC that H2 O2 , which is a major component of ROS in living organisms, elongates telomeres by increasing telomerase activity through protein kinase B (AKT) activation. The AKT inhibitor, perifosine, decreased telomere length, cellular viability, and H2 O2 -mediated migration and invasion capacity in HCC cells while also inhibiting AKT activation, telomere maintenance, and tumor growth in nude mice. Advanced HCC tissues showed a positive correlation among ROS levels, phosphorylated AKT (pAKT) levels, and telomere length. Furthermore, patients with HCC tumors that have high ROS levels and long telomeres displayed poorer survival rates. These data demonstrate the significant utilities of ROS levels, pAKT levels, and telomere length for predicting a poor prognosis in patients with HCC. Taken together, AKT activation could be essential for telomere maintenance in advanced HCC tumors as well as being an important contributor to malignant HCC progression. CONCLUSION: We showed that H2 O2 contributes to telomere elongation through AKT activation in advanced HCC, suggesting that an AKT inhibitor such as perifosine may be useful for treating patients with malignant HCC. (Hepatology 2018;67:1378-1391).

Epigenetic regulation of Kcna3-encoding Kv1.3 potassium channel by cereblon contributes to regulation of CD4+ T-cell activation.

The role of cereblon (CRBN) in T cells is not well understood. We generated mice with a deletion in Crbn and found cereblon to be an important antagonist of T-cell activation. In mice lacking CRBN, CD4(+) T cells show increased activation and IL-2 production on T-cell receptor stimulation, ultimately resulting in increased potassium flux and calcium-mediated signaling. CRBN restricts T-cell activation via epigenetic modification of Kcna3, which encodes the Kv1.3 potassium channel required for robust calcium influx in T cells. CRBN binds directly to conserved DNA elements adjacent to Kcna3 via a previously uncharacterized DNA-binding motif. Consequently, in the absence of CRBN, the expression of Kv1.3 is derepressed, resulting in increased Kv1.3 expression, potassium flux, and CD4(+) T-cell hyperactivation. In addition, experimental autoimmune encephalomyelitis in T-cell-specific Crbn-deficient mice was exacerbated by increased T-cell activation via Kv1.3. Thus, CRBN limits CD4(+) T-cell activation via epigenetic regulation of Kv1.3 expression.

The TERT promoter SNP rs2853669 decreases E2F1 transcription factor binding and increases mortality and recurrence risks in liver cancer.

A common single-nucleotide polymorphism in the telomerase reverse transcriptase (TERT) promoter, rs2853669 influences patient survival rates and the risk of developing cancer. Recently, several lines of evidence suggest that the rs2853669 suppresses TERT promoter mutation-mediated TERT expression levels and cancer mortality as well as recurrence rates. However, no reports are available on the impact of rs2853669 on TERT expression in hepatocellular carcinoma (HCC) and its association with patient survival. Here, we found that HCC-related overall and recurrence-free survival rates were not associated with TERT promoter mutation individually, but rs2853669 and the TERT promoter mutation in combination were associated with poor survival rates. TERT mRNA expression and telomere fluorescence levels were greater in patients with HCC who had both the combination. The combination caused TERT promoter methylation through regulating the binding of DNA methyltransferase 1 and histone deacetylase 1 to the TERT promoter in HCC cell lines. The TERT expression level was significantly higher in HCC tumor with a methylated promoter than in that with an unmethylated promoter. In conclusion, we demonstrate a substantial role for the rs2853669 in HCC with TERT promoter mutation, which suggests that the combination of the rs2853669 and the mutation indicate poor prognoses in liver cancer.

Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly.

Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study that Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly.

Reactive oxygen species increase HEPN1 expression via activation of the XBP1 transcription factor.

Hepatocellular carcinoma downregulated 1 (HEPN1), a cell growth arrest- and apoptosis-related gene, is suppressed in hepatocellular carcinoma (HCC). However, transcriptional control of HEPN1 has not been characterized. Here, we show that exposure to reactive oxygen species (ROS) leads to upregulation of the mRNA expression of HEPN1 in HCC cell lines. Mechanistically, ROS increase production of an alternately spliced form of X-box binding protein 1 (XBP1s) and XBP1s increases HEPN1 expression by binding to the HEPN1 promoter, thereby acting as a transcriptional activator. Finally, HEPN1 overexpression increases the expression of p53, p21, and Bax, all of which are ROS-upregulated proteins.

Positive association of long telomeres with the invasive capacity of hepatocellular carcinoma cells.

Invasion, the representative feature of malignant tumors, leads to an increase in mortality. The malignant liver tumor - hepatocellular carcinoma (HCC) - has an enhanced invasive capacity that results in increased patient mortality. Moreover, this enhanced invasive capacity is due to the up-regulation of invasion promoters such as zinc finger protein SNAI1 (Snail) and matrix metalloproteinases (MMPs), and the down-regulation of invasion suppressor molecules such as E-cadherin. Telomerase reverse transcriptase (TERT), which encodes the catalytic subunit of telomerase, is highly expressed in a variety of invasive cancers, including HCC. Telomerase activation induces telomere elongation, thereby leading to cell immortalization during malignant tumor progression. However, the relationship between telomere length and invasion is yet to be experimentally corroborated. In this paper, we revealed that invasive HCC cells passing through the Matrigel display significantly longer telomeres than non-invasive HCC cells. Moreover, we established a method that can distinguish and sort cells containing long telomeres and short telomeres. Using this system, we observed that the HCC cells containing long telomeres had a high-level expression of invasion-promoting genes and a low-level expression of invasion-suppressing E-cadherin. Furthermore, HCC cells containing long telomeres exhibited a higher invasive capacity than HCC cells containing short telomeres. Taken together, our findings suggest that long telomeres are positively associated with the invasive capacity of HCC cells and may be a potent target for malignant liver cancer treatment.

Interaction between nucleophosmin and HBV core protein increases HBV capsid assembly.

Host factors are involved in Hepatitis B virus (HBV) genome replication and capsid formation during the viral life cycle. A host factor, nucleophosmin (B23), was found to bind to HBV core protein dimers, but its functional role has not been studied. This interaction promoted HBV capsid assembly and decreased the degree of capsid dissociation when subjected to denaturant treatments in vitro. In addition, inhibition of B23 reduced intracellular capsid formation resulting in a decrease of HBV production in HepG2.2.15 cells. These results provide important evidence that B23 acts on core capsid assembly via its interaction with HBV core dimers.

Snail inhibits Notch1 intracellular domain mediated transcriptional activation via competing with MAML1.

Notch1 intracellular domain (NICD) is the transcription factor which controls cell fate and differentiation in embryonic and tumor cells. Snail has a critical role which increases invasion and metastasis of cancer cell as a transcription factor and epigenetic regulator. Recently, we discovered NICD induced Snail degradation by direct binding interaction with Snail. In this experiment, we found that Snail suppressed transcriptional activity of the protein complex formed with NICD and RBPJk in nucleus. Moreover, Snail decreased transcription of NICD target genes via competing with MAML1, co-activator, in NICD complex. In conclusion, Snail inhibited NICD-mediated transcriptional activation of target genes by physical interaction with NICD.

Structure-based design and biochemical evaluation of sulfanilamide derivatives as hepatitis B virus capsid assembly inhibitors.

Virus capsid structure is essential in virion maturation and durability, so disrupting capsid assembly could be an effective way to reduce virion count and cure viral diseases. However, currently there is no known antiviral which affects capsid inhibition, and only a small number of assembly inhibitors were experimentally successful. In this present study, we aimed to find hepatitis B virus (HBV) capsid assembly inhibitor which binds to the HBV core protein and changes protein conformation. Several candidate molecules were found to bind to certain structure in core protein with high specificity. Furthermore, these molecules significantly changed the protein conformation and reduced assembly affinity of core protein, leading to decrease of the number of assembled capsid or virion, both in vitro and in vivo. In addition, prediction also suggests that improvements in inhibition efficiency could be possible by changing functional groups and ring structures.

Co-expression patterns of Notch1, Snail, and p53 in grade III hepatocellular carcinoma with postoperative recurrence: a preliminary study.

BACKGROUND/AIMS: We aimed to determine the association between the co-expression patterns of Notch1, Snail, and p53 proteins (NSP) and the postoperative prognosis of hepatocellular carcinoma (HCC). METHODS: The immunoblot data for molecular expression (147 HCC/corresponding non-HCC tissues and 15 dysplastic nodules) and the sequencing data for p53 mutations (110 HCCs) were obtained from our previous study. Data analyses were restricted to cases with HCC differentiation grade III (n=47), due to its high p53 mutation rate. RESULTS: Nineteen of the 47 patients (40.4%) -comprising 12 in the liver and 7 in distant organs-had relapsed at 1-2 years after surgery. There was no relationship between p53 mutation and postoperative recurrence in the grade III HCCs. Seven (87.5%) of the eight relapsed cases with Notch1, Snail, and p53 (wild) co-expression experienced recurrence only within the liver, and all tumors were smaller than 5 cm in diameter. Extrahepatic relapse occurred mostly in HCC patients with tumors larger than 5 cm in diameter, without any deviation in the NSP pattern. CONCLUSIONS: The results of this preliminary study suggest that the co-expression of Notch1, Snail, and p53 (wild) is not inferior to the patterns with p53 mutation as an indicator of postoperative recurrence of grade III HCC.

Notch1 binds and induces degradation of Snail in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common, highly invasive malignant tumor associated with a high mortality rate. We previously reported that the aberrant expression of Snail via activation of reactive oxygen species contributes to the invasive property of HCC, in part by downregulation of E-cadherin through both transcriptional repression and epigenetic modification of the E-cadherin promoter. Having demonstrated the ability of Snail to bind and recruit histone deacetylase 1 and DNA methyltransferase 1 in this context, we set out to look for other interactions that could affect its ability to promote oncogenic transformation and cancer cell invasion. RESULTS: Using cells that stably expressed Snail, we characterized Snail protein interactors by tandem affinity purification and mass spectrometry. Immunoprecipitation and subcellular colocalization studies were performed to confirm our identification of the Notch1 intracellular domain (NICD) as a novel Snail-binding partner. NICD interaction with Snail was found to induce ubiquitination and MDM2-dependent degradation of Snail. Interestingly, NICD inhibited Snail-dependent invasive properties in both HCC cells and mouse embryonic fibroblasts. CONCLUSIONS: Our study demonstrates that NICD can oppose Snail-dependent HCC cell invasion by binding and inducing proteolytic degradation of Snail. Although Notch signaling and Snail are both widely considered tumor-promoting factors, our findings indicate that the individual oncogenic contribution of Notch1 and Snail in malignant systems should be interpreted carefully, particularly when they are conjointly expressed.

Reactive oxygen species downregulate catalase expression via methylation of a CpG island in the Oct-1 promoter.

Reactive oxygen species (ROS) caused oxidative stress plays a key role in carcinogenesis. The POU domain transcription factor Oct-1 and catalase is closely associated with ROS. However, a correlation between these two key proteins has not been demonstrated before. In this report, we show that Oct-1 acts as an activator of catalase, by binding to the catalase promoter in hepatocellular carcinoma (HCC) cell lines. In addition, we suggest that Oct-1 is downregulated by ROS via CpG island methylation in its promoter. These findings contribute to a better understanding of the epigenetic changes induced by ROS in the process of carcinogenesis.