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Hepatitis B virus (HBV) infection is a major driver of hepatocarcinogenesis. Ferroptosis is a type of iron-mediated cell death that can suppress liver transformation. Previous studies have linked HBV to ferroptosis in liver fibrosis and acute liver failure. However, whether ferroptosis is involved in HBV-mediated liver cancer is poorly understood. Here, we identified heat shock protein family A member 8 (HSPA8) as a crucial host factor that modulates HBV replication and ferroptosis in liver cancer. Hepatitis B X protein (HBx) upregulated HSPA8 by coactivating the transcription factor heat shock factor 1 (HSF1) in cells. HSPA8 enhanced HBV replication by recruiting hepatitis B core protein (HBc) to the HBV covalently closed circular DNA (cccDNA) minichromosome, forming a positive feedback loop. Moreover, HSPA8 suppressed ferroptosis in liver cancer cells by upregulating the expression of SLC7A11/GPX4 and decreasing erastin-mediated reactive oxygen species and Fe2+ accumulation in cells in vitro and in vivo. Inhibition of HSPA8 reduced the growth of HBV-positive liver tumors and increased sensitivity to erastin. In conclusion, HBx-elevated HSPA8 regulates both HBV replication and ferroptosis in liver cancer. Targeting HSPA8 could be a promising strategy for controlling HBV and hepatocarcinogenesis. Significance: HBV-induced upregulation of HSPA8 promotes hepatocarcinogenesis by suppressing ferroptosis and stimulating HBV replication, identifying HSPA8 as a potential therapeutic target in liver cancer.

Hepatitis B virus (HBV) infection is a major driver of hepatocarcinogenesis. Ferroptosis is a type of iron-mediated cell death that can suppress liver transformation. Previous studies have linked HBV to ferroptosis in liver fibrosis and acute liver failure. However, whether ferroptosis is involved in HBV-mediated liver cancer is poorly understood. Here, we identified heat shock protein family A member 8 (HSPA8) as a crucial host factor that modulates HBV replication and ferroptosis in liver cancer. Hepatitis B X protein (HBx) upregulated HSPA8 by coactivating the transcription factor heat shock factor 1 (HSF1) in cells. HSPA8 enhanced HBV replication by recruiting hepatitis B core protein (HBc) to the HBV covalently closed circular DNA (cccDNA) minichromosome, forming a positive feedback loop. Moreover, HSPA8 suppressed ferroptosis in liver cancer cells by upregulating the expression of SLC7A11/GPX4 and decreasing erastin-mediated reactive oxygen species and Fe2+ accumulation in cells in vitro and in vivo. Inhibition of HSPA8 reduced the growth of HBV-positive liver tumors and increased sensitivity to erastin. In conclusion, HBx-elevated HSPA8 regulates both HBV replication and ferroptosis in liver cancer. Targeting HSPA8 could be a promising strategy for controlling HBV and hepatocarcinogenesis.Significance:HBV-induced upregulation of HSPA8 promotes hepatocarcinogenesis by suppressing ferroptosis and stimulating HBV replication, identifying HSPA8 as a potential therapeutic target in liver cancer.

Supplementary materials and methods, Supplementary figures, Supplementary figure legends, Supplementary tables

Rationale: Hepatitis B x protein (HBx) is required to initiate and maintain the replication of hepatitis B virus (HBV). Protein arginine methyltransferases 5 (PRMT5) negatively regulates HBV transcription. WD repeat domain 77 protein (WDR77) greatly enhances the methyltransferase activity of PRMT5. However, the role of WDR77 in the modulation of cccDNA transcription and HBV replication is poorly understood. In this study, we investigated the mechanism by which HBx modulated HBV replication involving WDR77 in the liver. Methods: A human liver-chimeric mouse model was established. Immunohistochemistry (IHC) staining, Western blot analysis, Southern blot analysis, Northern blot analysis, immunofluorescence assays, ELISA, RT-qPCR, CoIP assays, and ChIP assays were performed in human liver-chimeric mouse model, primary human hepatocytes (PHHs), HepG2-NTCP, dHepaRG and HepG2 cell lines. Results: HBV infection and HBx expression remarkably reduced the protein levels of WDR77 in human liver-chimeric mice and HepG2-NTCP cells. WDR77 restricted cccDNA transcription and HBV replication in PHHs and HepG2-NTCP cells. Mechanically, WDR77 enhanced PRMT5-triggered symmetric dimethylation of arginine 3 on H4 (H4R3me2s) on the cccDNA minichromosome to control cccDNA transcription. HBx drove the cellular DDB1-containing E3 ubiquitin ligase to degrade WDR77 through recruiting WDR77, leading to the disability of methyltransferase activity of PRMT5. Thus, HBx promoted HBV replication by driving a positive feedback loop of HBx-DDB1/WDR77/PRMT5/H4R3me2s/cccDNA/HBV/HBx in the liver. Conclusions: HBx attenuates the WDR77-mediated HBV repression by driving DDB1-induced WDR77 degradation in the liver. Our finding provides new insights into the mechanism by which HBx enhances HBV replication in the liver.

Abnormal lipid metabolism plays crucial roles in the development of cancer. Spindlin 1 (SPIN1) involving the process of spindle organization and chromosomal stability serves as an important player in the carcinogenesis. In this study, we try to identify the new function of SPIN1 in lipid metabolism of liver cancer. Tissue microarray showed that 75% (60/80) of hepatocellular carcinoma (HCC) tissues were positive for SPIN1, which was highly expressed in clinical HCC samples and positively associated with malignancy of HCC. Strikingly, SPIN1 could modulate abnormal lipid metabolism by increasing intracellular triglycerides, cholesterols, and lipid droplets in hepatoma cells, which could remarkably enhance the proliferation of hepatoma cells. Mechanistically, SPIN1 up-regulated FASN in hepatoma cells. SPIN1 co-activated transcriptional factor SREBP1c in the promoter of FASN through interaction with SREBP1c. Moreover, SPIN1 promoted the growth of liver cancer in vitro and in vivo and the levels of intracellular triglycerides, cholesterols and lipid droplets were increased in the tumor tissues from mice. In conclusion, SPIN1 modulates abnormal lipid metabolism and enhances growth of liver cancer through SREBP1c-triggered FASN signaling. Therapeutically, SPIN1 may serve as a novel target for HCC.

The epigenetic modification of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a crucial role in cccDNA transcription and viral persistence. Interferon-α (IFN-α) is a pivotal agent against HBV cccDNA. However, the mechanism by which IFN-α modulates the epigenetic regulation of cccDNA remains poorly understood. In this study, we report that IFN-α2b enhances the histone deacetylase 3 (HDAC3)-mediated de-2-hydroxyisobutyrylation of histone H4 lysine 8 (H4K8) on HBV cccDNA minichromosome to restrict the cccDNA transcription in liver. By screening acetyltransferases and deacetylases, we identified that HDAC3 was an effective restrictor of HBV transcription and replication. Moreover, we found that HDAC3 was able to mediate the de-2-hydroxyisobutyrylation of H4K8 in HBV-expressing hepatoma cells. Then, the 2-hydroxyisobutyrylation of histone H4K8 (H4K8hib) was identified on the HBV cccDNA minichromosome, promoting the HBV transcription and replication. The H4K8hib was regulated by HDAC3 depending on its deacetylase domain in the system. The low level of HDAC3 and high level of H4K8hib were observed in the liver tissues from HBV-infected human liver-chimeric mice. The levels of H4K8hib on HBV cccDNA minichromosome were significantly elevated in the liver biopsy specimens from clinical hepatitis B patients, which was consistent with the high transcriptional activity of cccDNA. Strikingly, IFN-α2b effectively facilitated the histone H4K8 de-2-hydroxyisobutyrylation mediated by HDAC3 on the HBV cccDNA minichromosome in primary human hepatocytes and hepatoma cells, leading to the inhibition of HBV transcription and replication. Our finding provides new insights into the mechanism by which IFN-α modulates the epigenetic regulation of HBV cccDNA minichromosome.

Rationale: Hepatitis B virus (HBV) is a major risk factor for liver cancer, in which HBV covalently closed circular DNA (cccDNA) plays crucial roles. However, the effect of pseudogene-derived long noncoding RNAs (lncRNAs) acting as functional regulators of their ancestral gene expression on HBV replication and hepatocellular carcinoma (HCC) remains unclear. In this study, we speculated that the pseudogene-derived lncRNA PCNAP1 and its ancestor PCNA might modulate HBV replication and promote hepatocarcinogenesis. Methods: We investigated the roles of lncRNA PCNAP1 in contribution of HBV replication through modulating miR-154/PCNA/HBV cccDNA signaling in hepatocarcinogenesis by using CRISPR/Cas9, Southern blot analysis, confocal assays, et al. in primary human hepatocytes (PHH), HepaRG cells, HepG2-NTCP cells, hepatoma carcinoma cells, human liver-chimeric mice model, transgenetic mice model, in vitro tumorigenicity and clinical patients. Results: Interestingly, the expression levels of PCNAP1 and PCNA were significantly elevated in the liver of HBV-infectious human liver-chimeric mice. Clinically, the mRNA levels of PCNAP1 and PCNA were increased in the liver of HBV-positive/HBV cccDNA-positive HCC patients. Mechanistically, PCNA interacted with HBV cccDNA in a HBc-dependent manner. PCNAP1 enhanced PCNA through sponging miR-154 targeting PCNA mRNA 3'UTR. Functionally, PCNAP1 or PCNA remarkably enhanced HBV replication and accelerated the growth of HCC in vitro and in vivo. Conclusion: We conclude that lncRNA PCNAP1 enhances the HBV replication through modulating miR-154/PCNA/HBV cccDNA signaling and the PCNAP1/PCNA signaling drives the hepatocarcinogenesis. Our finding provides new insights into the mechanism by which lncRNA PCNAP1 enhances HBV replication and hepatocarcinogenesis.

Rationale: Hepatitis B virus (HBV) is a leading cause of liver diseases. HBV covalently closed circular DNA (cccDNA) is a critical obstacle of complete elimination by anti-HBV therapy. HBV cccDNA accumulates in nucleus as a chromatin-like cccDNA minichromosome assembled by histones and non-histones. However, the underlying mechanism of modulation of cccDNA minichromosome in hepatocytes is poorly understood. Methods: A human liver-chimeric mouse model was established. The cccDNA-ChIP, Southern blot analysis, confocal assays, RIP assays and RNA pull-down assays, et al. were performed to assess the mechanism of assembly and epigenetic regulation of cccDNA minichromosome in human liver-chimeric mouse model, human primary hepatocytes (PHH), dHepaRG, HepG2-NTCP cell lines and clinical liver tissues. Results: Importantly, the expression levels of HAT1, CAF-1 and lncRNA HULC were significantly elevated in the liver from HBV-infected human liver-chimeric mice. Strikingly, the depletion of HAT1 reduced HBV replication and cccDNA accumulation, and impaired the assembly of histone H3/H4 and the deposition of HBx and p300 onto cccDNA to form cccDNA minichromosome in the cells. Mechanically, chromatin assembly factor-1 (CAF-1) was involved in the events. Interestingly, HAT1 modified the acetylation of histone H3K27/H4K5/H4K12 on cccDNA minichromosome. Moreover, lncRNA HULC-scaffold HAT1/HULC/HBc complex was responsible for the modification on cccDNA minichromosome. Additionally, HBV activated HAT1 through HBx-co-activated transcriptional factor Sp1 in a positive feedback manner. Conclusion: HAT1 signaling contributes to assembly and epigenetic regulation of HBV cccDNA minichromosome.

Aspirin can efficiently inhibit the glycolysis and proliferation of cancer cells, however, the underlying mechanism is poorly understood. Here, we report that aspirin attenuates the glycolysis and proliferation of hepatoma cells through modulating the levels of lysine 2-hydroxyisobutyrylation (Khib) of enolase 1 (ENO1). We found that aspirin decreased the levels of glucose consumption and lactate production in hepatoma cells. Moreover, 4 mM aspirin reduced the activities of ENO1, a key enzyme of glycolysis, and decreased the levels of ENO1 Khib in the cells. Interestingly, we identified that 4 mM aspirin could decrease the levels of Khib on many proteins by using pan Khib antibody in the cells. Interestingly, the activities of ENO1 could be rescued by the transient overexpression of ENO1, but not by ENO1 mutant (K281R). Moreover, we identified that the C646, an inhibitor of p300 which is a writer of Khib, could reduce the levels of ENO1 Khib, resulting in the decrease of ENO1 activities. The treatment with PDTC, an inhibitor of NF-κB which is a target of aspirin, could work well as C646 in the cells. Both of aspirin and C646 (or PDTC) displayed a stronger effect than the single treatment in the system. Functionally, ENO1, but not ENO1 mutant (K281R), could rescue the aspirin-induced inhibition of proliferation of liver cancer cells in vitro, suggesting that ENO1K281 is involved in the aspirin-mediated inhibition of liver cancer. Our finding provides new insights into the mechanism by which aspirin attenuates the glycolysis and proliferation of hepatoma cells.

Chronic infection of HBV is a serious global health issue. However, the HBV covalently closed circular DNA (cccDNA) cannot be completely cleaned in liver. Here, we engineer a novel polypeptide, termed D-TTK001, targeting HBV X protein (HBx) against HBV cccDNA. D-TTK001 is a D-type polypeptide, which has a high binding affinity to HBx. Interestingly, it is able to against HBV efficiently in primary human hepatocytes (PHHs) from three donors. D-TTK001 displays high activities against HBV, remarkably reducing the levels of cccDNA in the HBV-infected human liver-chimeric mice. Mechanically, D-TTK001 depresses cccDNA transcription by regulating the epigenetic modification of cccDNA minichromosome and disturbing the interaction of HBx with DDB1 in the cells. D-TTK001 decreases the levels of cccDNA by down-regulating MSL2, leading to the increase of APOBEC3B in liver. When combined with entecavir or interferon-α, D-TTK001 shows additive antiviral effects. Clinically, D-TTK001 is a strong candidate to clear cccDNA against HBV.

Background Hepatitis B virus covalently closed circular DNA (HBV cccDNA) is assembled by histones and non-histones into a chromatin-like cccDNA minichromosome in the nucleus. The cellular histone acetyltransferase GCN5, displaying succinyltransferase activity, is recruited onto cccDNA to modulate HBV transcription in cells. Clinically, IFN-α is able to repress cccDNA. However, the underlying mechanism of IFN-α in the depression of cccDNA mediated by GCN5 is poorly understood. Here, we explored the effect of IFN-α on GCN5-mediated succinylation in the epigenetic regulation of HBV cccDNA minichromosome. Results Succinylation modification of the cccDNA minichromosome has been observed in HBV-infected human liver-chimeric mice and HBV-expressing cell lines. Moreover, histone H3K79 succinylation by GCN5 was identified in the system. Interestingly, the mutant of histone H3K79 efficiently blocked the replication of HBV, and interference with GCN5 resulted in decreased levels of HBV DNA, HBsAg, and HBeAg in the supernatant from de novo HBV-infected HepaRG cells. Consistently, the levels of histone H3K79 succinylation were significantly elevated in the livers of HBV-infected human liver-chimeric mice. The knockdown or overexpression of GCN5 or the mutant of GCN5 could affect the binding of GCN5 to cccDNA or H3K79 succinylation, leading to a change in cccDNA transcription activity. In addition, Southern blot analysis validated that siGCN5 decreased the levels of cccDNA in the cells, suggesting that GCN5-mediated succinylation of histone H3K79 contributes to the epigenetic regulation of cccDNA minichromosome. Strikingly, IFN-α effectively depressed histone H3K79 succinylation in HBV cccDNA minichromosome in de novo HepG2-NTCP and HBV-infected HepaRG cells. Conclusions IFN-α epigenetically regulates the HBV cccDNA minichromosome by modulating GCN5-mediated succinylation of histone H3K79 to clear HBV cccDNA. Our findings provide new insights into the mechanism by which IFN-α modulate the epigenetic regulation of HBV cccDNA minichromosome.

Lysine succinylation (Ksucc) is an evolutionarily conserved and widespread post-translational modification. Histone acetyltransferase 1 (HAT1) is a type B histone acetyltransferase, regulating the acetylation of both histone and non-histone proteins. However, the role of HAT1 in succinylation modulation remains unclear. Here, we employ a quantitative proteomics approach to study succinylation in HepG2 cancer cells and find that HAT1 modulates lysine succinylation on various proteins including histones and non-histones. HAT1 succinylates histone H3 on K122, contributing to epigenetic regulation and gene expression in cancer cells. Moreover, HAT1 catalyzes the succinylation of PGAM1 on K99, resulting in its increased enzymatic activity and the stimulation of glycolytic flux in cancer cells. Clinically, HAT1 is significantly elevated in liver cancer, pancreatic cancer, and cholangiocarcinoma tissues. Functionally, HAT1 succinyltransferase activity and the succinylation of PGAM1 by HAT1 play critical roles in promoting tumor progression in vitro and in vivo. Thus, we conclude that HAT1 is a succinyltransferase for histones and non-histones in tumorigenesis.