Highlights from the 2023 International Meeting on the Molecular Biology of Hepatitis B virus.

Since its discovery in 1965, our understanding of the hepatitis B virus (HBV) replication cycle and host immune responses has increased markedly. In contrast, our knowledge of the molecular biology of hepatitis delta virus (HDV), which is associated with more severe liver disease, is less well understood. Despite the progress made, critical gaps remain in our knowledge of HBV and HDV replication and the mechanisms underlying viral persistence and evasion of host immunity. The International HBV Meeting is the leading annual scientific meeting for presenting the latest advances in HBV and HDV molecular virology, immunology, and epidemiology. In 2023, the annual scientific meeting was held in Kobe, Japan and this review summarises some of the advances presented at the Meeting and lists gaps in our knowledge that may facilitate the development of new therapies.

Major HBV splice variant encoding a novel protein important for infection.

BACKGROUND & AIMS: HBV expresses more than 10 spliced RNAs from the viral pregenomic RNA, but their functions remain elusive and controversial. To address the function of HBV spliced RNAs, we generated splicing-deficient HBV mutants and conducted experiments to assess the impact of these mutants on HBV infection. METHODS: HepG2-NTCP cells, human hepatocyte chimeric FRG mice (hu-FRG mice), and serum from patients with chronic hepatitis B were used for experiments on HBV infection. Additionally, SHifter assays and cryo-electron microscopy were performed. RESULTS: We found the infectivity of splicing-deficient HBV was decreased 100-1,000-fold compared with that of wild-type HBV in hu-FRG mice. Another mutant, A487C, which loses the most abundant spliced RNA (SP1), also exhibits severely impaired infectivity. SP1 hypothetically encodes a novel protein HBcSP1 (HBc-Cys) that lacks the C-terminal cysteine from full-length HBc. In the SHifter assay, HBcSP1 was detected in wild-type viral particles at a ratio of about 20-100% vs. conventional HBc, as well as in the serum of patients with chronic hepatitis B, but not in A487C particles. When infection was conducted with a shorter incubation time of 4-8 h at lower PEG concentrations in HepG2-NTCP cells, the entry of the A487C mutant was significantly slower. SP1 cDNA complementation of the A487C mutant succeeded in rescuing its infectivity in hu-FRG mice and HepG2-NTCP cells. Moreover, cryo-electron microscopy revealed a disulfide bond between HBc cysteine 183 and 48 in the HBc intradimer of the A487C capsid, leading to a locked conformation that disfavored viral entry in contrast to the wild-type capsid. CONCLUSIONS: Prior studies unveiled the potential integration of the HBc-Cys protein into the HBV capsid. We confirmed the proposal and validated its identity and function during infection. IMPACT AND IMPLICATIONS: HBV SP1 RNA encodes a novel HBc protein (HBcSP1) that lacks the C-terminal cysteine from conventional HBc (HBc-Cys). HBcSP1 was detected in cell culture-derived HBV and confirmed in patients with chronic infection by both immunological and chemical modification assays at 10-50% of capsid. The splicing-deficient mutant HBV (A487C) impaired infectivity in human hepatocyte chimeric mice and viral entry in the HepG2-NTCP cell line. Furthermore, these deficiencies of the splicing-deficient mutant could be rescued by complementation with the SP1-encoded protein HBcSP1. We confirmed and validated the identity and function of HBcSP1 during infection, building on the current model of HBV particles.

Serum MYCN as a predictive biomarker of prognosis and therapeutic response in the prevention of hepatocellular carcinoma recurrence.

The proto-oncogene MYCN expression marked a cancer stem-like cell population in hepatocellular carcinoma (HCC) and served as a therapeutic target of acyclic retinoid (ACR), an orally administered vitamin A derivative that has demonstrated promising efficacy and safety in reducing HCC recurrence. This study investigated the role of MYCN as a predictive biomarker for therapeutic response to ACR and prognosis of HCC. MYCN gene expression in HCC was analyzed in the Cancer Genome Atlas and a Taiwanese cohort (N = 118). Serum MYCN protein levels were assessed in healthy controls (N = 15), patients with HCC (N = 116), pre- and post-surgical patients with HCC (N = 20), and a subset of patients from a phase 3 clinical trial of ACR (N = 68, NCT01640808). The results showed increased MYCN gene expression in HCC tumors, which positively correlated with HCC recurrence in non-cirrhotic or single-tumor patients. Serum MYCN protein levels were higher in patients with HCC, decreased after surgical resection of HCC, and were associated with liver functional reserve and fibrosis markers, as well as long-term HCC prognosis (>4 years). Subgroup analysis of a phase 3 clinical trial of ACR identified serum MYCN as the risk factor most strongly associated with HCC recurrence. Patients with HCC with higher serum MYCN levels after a 4-week treatment of ACR exhibited a significantly higher risk of recurrence (hazard ratio 3.27; p = .022). In conclusion, serum MYCN holds promise for biomarker-based precision medicine for the prevention of HCC, long-term prognosis of early-stage HCC, and identification of high-response subgroups for ACR-based treatment.

HBV DNA Integration into Telomerase or MLL4 Genes and TERT Promoter Point Mutation as Three Independent Signatures in Subgrouping HBV-Related HCC with Distinct Features.

Introduction: A set of genetic mutations to classify hepatocellular carcinoma (HCC) useful to clinical studies is an unmet need. Hepatitis B virus-related HCC (HBV-HCC) harbors a unique genetic mutation, namely, the HBV integration, among other somatic endogenous gene mutations. We explored a combination of HBV DNA integrations and common somatic mutations to classify HBV-HCC by using a capture-sequencing platform. Methods: A total of 153 HBV-HCCs after surgical resection were subjected to capture sequencing to identify HBV integrations and three common somatic mutations in genomes. Three mutually exclusive mutations, HBV DNA integration into the TERT promoter, HBV DNA integration into MLL4, or TERT promoter point mutation, were identified in HBV-HCC. Results: They were used to classify HBV-HCCs into four groups: G1 with HBV-TERT integration (25.5%); G2 with HBV-MLL4 integration (10.5%); G3 with TERT promoter mutation (30.1%); and G4 without these three mutations (34.0%). Clinically, G3 has the highest male-to-female ratio, cirrhosis rate, and associated with higher early recurrence and mortality after resection, but G4 has the best outcome. Transcriptomic analysis revealed a grouping different from the published ones and G2 with an active immune profile related to immune checkpoint inhibitor response. Analysis of integrated HBV DNA provided clues for HBV genotype and variants in carcinogenesis of different HCC subgroup. This new classification was also validated in another independent cohort. Conclusion: A simple and robust genetic classification was developed to aid in understanding HBV-HCC and in harmonizing clinical studies.

ICTV Virus Taxonomy Profile: Coronaviridae 2023.

The family Coronaviridae includes viruses with positive-sense RNA genomes of 22-36 kb that are expressed through a nested set of 3' co-terminal subgenomic mRNAs. Members of the subfamily Orthocoronavirinae are characterized by 80-160 nm diameter, enveloped virions with spike projections. The orthocoronaviruses, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome-related coronavirus are extremely pathogenic for humans and in the last two decades have been responsible for the SARS and MERS epidemics. Another orthocoronavirus, severe acute respiratory syndrome coronavirus 2, was responsible for the recent global COVID-19 pandemic. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Coronaviridae which is available at www.ictv.global/report/coronaviridae.

Hepatitis B Virus DNA Integration Drives Carcinogenesis and Provides a New Biomarker for HBV-related HCC.

Hepatitis B virus (HBV) DNA integration is an incidental event in the virus replication cycle and occurs in less than 1% of infected hepatocytes during viral infection. However, HBV DNA is present in the genome of approximately 90% of HBV-related HCCs and is the most common somatic mutation. Whole genome sequencing of liver tissues from chronic hepatitis B patients showed integration occurring at random positions in human chromosomes; however, in the genomes of HBV-related HCC patients, there are integration hotspots. Both the enrichment of the HBV-integration proportion in HCC and the emergence of integration hotspots suggested a strong positive selection of HBV-integrated hepatocytes to progress to HCC. The activation of HBV integration hotspot genes, such as telomerase (TERT) or histone methyltransferase (MLL4/KMT2B), resembles insertional mutagenesis by oncogenic animal retroviruses. These candidate oncogenic genes might shed new light on HBV-related HCC biology and become targets for new cancer therapies. Finally, the HBV integrations in individual HCC contain unique sequences at the junctions, such as virus-host chimera DNA (vh-DNA) presumably being a signature molecule for individual HCC. HBV integration may thus provide a new cell-free tumor DNA biomarker to monitor residual HCC after curative therapies or to track the development of de novo HCC.

Synergistic efficacy of telomerase-specific oncolytic adenoviral therapy and histone deacetylase inhibition in human hepatocellular carcinoma.

The telomerase-specific oncolytic adenovirus Telomelysin and the histone deacetylase inhibitor AR42 have demonstrated anticancer effects in preclinical models of human hepatocellular carcinoma (HCC). However, the clinical development of Telomelysin may be hindered by human antiviral immunity and tumor resistance. Combining oncolytic and epigenetic therapies is a viable approach for treating various cancers. This study investigated the potential synergism of Telomelysin and AR42 and the relevant underlying mechanisms. Telomelysin and AR42 exhibited synergistic antiproliferative effects in human HCC models in vitro and in vivo. Apoptosis induced by Telomelysin was significantly enhanced by AR42 in both PLC5 and Hep3B HCC cells. AR42 treatment unexpectedly attenuated the expression of the coxsackievirus and adenovirus receptor and the mRNA levels of human telomerase reverse transcriptase, which may be positively associated with the cytotoxicity of Telomelysin. Meanwhile, the cellular antiviral interferon response was not altered by AR42 treatment. Further, we found that Telomelysin enhanced Akt phosphorylation in HCC cells. AR42 reduced Telomelysin-induced phospho-Akt activation and enhanced Telomelysin-induced apoptosis. The correlation of Akt phosphorylation with drug-induced apoptosis was validated in HCC cells with upregulated or downregulated Akt signaling. Combination therapy with Telomelysin and AR42 demonstrated synergistic anti-HCC efficacy. Clinical trials investigating this new combination regimen are warranted.

Contrasting Patterns in the Early Stage of SARS-CoV-2 Evolution between Humans and Minks.

One of the unique features of SARS-CoV-2 is its apparent neutral evolution during the early pandemic (before February 2020). This contrasts with the preceding SARS-CoV epidemics, where viruses evolved adaptively. SARS-CoV-2 may exhibit a unique or adaptive feature which deviates from other coronaviruses. Alternatively, the virus may have been cryptically circulating in humans for a sufficient time to have acquired adaptive changes before the onset of the current pandemic. To test the scenarios above, we analyzed the SARS-CoV-2 sequences from minks (Neovision vision) and parental humans. In the early phase of the mink epidemic (April to May 2020), nonsynonymous to synonymous mutation ratio per site in the spike protein is 2.93, indicating a selection process favoring adaptive amino acid changes. Mutations in the spike protein were concentrated within its receptor-binding domain and receptor-binding motif. An excess of high-frequency derived variants produced by genetic hitchhiking was found during the middle (June to July 2020) and late phase I (August to September 2020) of the mink epidemic. In contrast, the site frequency spectra of early SARS-CoV-2 in humans only show an excess of low-frequency mutations, consistent with the recent outbreak of the virus. Strong positive selection in the mink SARS-CoV-2 implies that the virus may not be preadapted to a wide range of hosts and illustrates how a virus evolves to establish a continuous infection in a new host. Therefore, the lack of positive selection signal during the early pandemic in humans deserves further investigation.

Androgen receptor functions in pericentral hepatocytes to decrease gluconeogenesis and avoid hyperglycemia and obesity in male mice.

BACKGROUND: Although the impact of hepatic androgen receptor (AR) pathway on liver pathogenesis was documented, its physiological function in normal liver is remained unclear. This study aims to investigate if hepatic AR acts on metabolism, the major liver function, using a hepatic-specific AR-transgenic (H-ARTG) mouse model. METHODS: We established the albumin promoter driven H-ARTG mice and included wild type (WT) and H-ARKO mice for study. The body weight, specific metabolic parameters and results from various tolerance tests were compared in different groups of mice fed a chow diet, from 2 to 18 months of age. Glucose feeding and insulin treatment were used to study the expression and zonal distribution pattern of AR and related genes in liver at different prandial stages. RESULTS: The body weight of H-ARTG mice fed a chow diet was 15 % lower than that of wild-type mice, preceded by lower blood glucose and liver triglyceride levels caused by AR reduced hepatic gluconeogenesis. The opposite phenotypes identified in H-ARKO and castrated H-ARTG mice support the critical role of activated AR in decreasing gluconeogenesis and triglyceride levels in liver. Hepatic AR acting by enhancing the expression of cytosolic glycerol-3-phosphate dehydrogenase (cGPDH), a key of glycerophosphate shuttle, was identified as one mechanism to decrease gluconeogenesis from glycerol. We further found AR normally expressed in zone 3 of hepatic lobules. Its level fluctuates dependent on the demand of glucose, decreased by fasting but increased by glucose uptake or insulin stimulation. CONCLUSION: AR is a newly identified zone 3 hepatic gene with function in reducing blood glucose and body weight in mice. It suggests that stabilization of hepatic AR is a new direction to prevent hyperglycemia, obesity and nonalcoholic fatty liver disease (NAFLD) in males.

Circulating Virus-Host Chimera DNAs in the Clinical Monitoring of Virus-Related Cancers.

The idea of using tumor-specific cell-free DNA (ctDNA) as a tumor biomarker has been widely tested and validated in various types of human cancers and different clinical settings. ctDNA can reflect the presence or size of tumors in a real-time manner and can enable longitudinal monitoring with minimal invasiveness, allowing it to be applied in treatment response assessment and recurrence monitoring for cancer therapies. However, tumor detection by ctDNA remains a great challenge due to the difficulty in enriching ctDNA from a large amount of homologous non-tumor cell-free DNA (cfDNA). Only ctDNA with nonhuman sequences (or rearrangements) can be selected from the background of cfDNA from nontumor DNAs. This is possible for several virus-related cancers, such as hepatitis B virus (HBV)-related HCC or human papillomavirus (HPV)-related cervical or head and neck cancers, which frequently harbor randomly integrated viral DNA. The junction fragments of the integrations, namely virus-host chimera DNA (vh-DNA), can represent the signatures of individual tumors and are released into the blood. Such ctDNA can be enriched by capture with virus-specific probes and therefore exploited as a circulating biomarker to track virus-related cancers in clinical settings. Here, we review virus integrations in virus-related cancers to evaluate the feasibility of vh-DNA as a cell-free tumor marker and update studies on the development of detection and applications. vh-DNA may be a solution to the development of specific markers to manage virus-related cancers in the future.

Drug Resistance Profile and Clinical Features for Hepatitis C Patients Experiencing DAA Failure in Taiwan.

About 4% of the population in Taiwan are seropositive for anti-HCV Ab and 70% with HCV RNA. To address this high chronic hepatitis C disease load, Taiwan National Health Insurance started reimbursing genotype-specific DAAs in 2017 and pangenotype DAAs in mid-2018. With a 97% SVR12 rate, there were still 2-3% of patients that failed to clear HCV. To understand the causes of DAA failure in Taiwan, we conducted a multi-center, clinical, and virologic study. A total of 147 DAA-failure patients were recruited, and we searched HCV NS3/4A, NS5A and NS5B for known resistance-associated substitutions (RASs) by population sequencing, and conducted whole genome sequencing (WGS) for those without known RASs. A total of 107 patients received genotype-specific DAAs while 40 had pangenotype DAAs. Clinically, the important cause of failure is poor adherence. Virologically, common RASs in genotype-specific DAAs were NS5A-L31, NS5A-Y93, and NS5B-C316, while common RASs in pangenotype DAAs were NS5A-L31, NS5A-A/Q/R30, and NS5A-Y93. Additionally, new amino acid changes were found by WGS. Finally, we identified 12 cases with inconsistent baseline and post-treatment HCV genotypes, which is suggestive of re-infection rather than treatment failure. Our study described the drug resistance profile for DAA failure in Taiwan, showing differences from other countries.

D614G Substitution of SARS-CoV-2 Spike Protein Increases Syncytium Formation and Virus Titer via Enhanced Furin-Mediated Spike Cleavage.

Since the D614G substitution in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, the variant strain has undergone a rapid expansion to become the most abundant strain worldwide. Therefore, this substitution may provide an advantage for viral spreading. To explore the mechanism, we analyzed 18 viral isolates containing S proteins with either G614 or D614 (S-G614 and S-D614, respectively). The plaque assay showed a significantly higher virus titer in S-G614 than in S-D614 isolates. We further found increased cleavage of the S protein at the furin substrate site, a key event that promotes syncytium formation, in S-G614 isolates. The enhancement of the D614G substitution in the cleavage of the S protein and in syncytium formation has been validated in cells expressing S protein. The effect on the syncytium was abolished by furin inhibitor treatment and mutation of the furin cleavage site, suggesting its dependence on cleavage by furin. Our study pointed to the impact of the D614G substitution on syncytium formation through enhanced furin-mediated S cleavage, which might increase the transmissibility and infectivity of SARS-CoV-2 strains containing S-G614. IMPORTANCE Analysis of viral genomes and monitoring of the evolutionary trajectory of SARS-CoV-2 over time has identified the D614G substitution in spike (S) as the most prevalent expanding variant worldwide, which might confer a selective advantage in transmission. Several studies showed that the D614G variant replicates and transmits more efficiently than the wild-type virus, but the mechanism is unclear. By comparing 18 virus isolates containing S with either D614 or G614, we found significantly higher virus titers in association with higher furin protease-mediated cleavage of S, an event that promotes syncytium formation and virus infectivity, in the S-G614 viruses. The effect of the D614G substitution on furin-mediated S cleavage and the resulting enhancement of the syncytium phenotype has been validated in S-expressing cells. This study suggests a possible effect of the D614G substitution on S of SARS-CoV-2; the antiviral effect through targeting furin protease is worthy of being investigated in proper animal models.

Unique Features of Hepatitis B Virus-Related Hepatocellular Carcinoma in Pathogenesis and Clinical Significance.

Hepatitis B virus (HBV) infection is one of the important risk factors for hepatocellular carcinoma (HCC) worldwide, accounting for around 50% of cases. Chronic hepatitis B infection generates an inflammatory microenvironment, in which hepatocytes undergoing repeated cycles of damage and regeneration accumulate genetic mutations predisposing them to cancer. A striking male dominance in HBV-related HCC highlights the influence of sex hormones which interact with viral factors to influence carcinogenesis. HBV is also considered an oncogenic virus since its X and surface mutant proteins showed tumorigenic activity in mouse models. The other unique mechanism is the insertional mutagenesis by integration of HBV genome into hepatocyte chromosomes to activate oncogenes. HCC survival largely depends on tumor stages at diagnosis and effective treatment. However, early diagnosis by the conventional protein biomarkers achieves limited success. A new biomarker, the circulating virus-host chimera DNA from HBV integration sites in HCC, provides a liquid biopsy approach for monitoring the tumor load in the majority of HBV-HCC patients. To maximize the efficacy of new immunotherapies or molecular target therapies, it requires better classification of HCC based on the tumor microenvironment and specific carcinogenic pathways. An in-depth study may benefit both the diagnosis and treatment of HBV-related HCC.

Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects.

Development of specific antiviral agents is an urgent unmet need for SARS-coronavirus 2 (SARS-CoV-2) infection. This study focuses on host proteases that proteolytically activate the SARS-CoV-2 spike protein, critical for its fusion after binding to angiotensin-converting enzyme 2 (ACE2), as antiviral targets. We first validate cleavage at a putative furin substrate motif at SARS-CoV-2 spikes by expressing it in VeroE6 cells and find prominent syncytium formation. Cleavage and the syncytium are abolished by treatment with the furin inhibitors decanoyl-RVKR-chloromethylketone (CMK) and naphthofluorescein, but not by the transmembrane protease serine 2 (TMPRSS2) inhibitor camostat. CMK and naphthofluorescein show antiviral effects on SARS-CoV-2-infected cells by decreasing virus production and cytopathic effects. Further analysis reveals that, similar to camostat, CMK blocks virus entry, but it further suppresses cleavage of spikes and the syncytium. Naphthofluorescein acts primarily by suppressing viral RNA transcription. Therefore, furin inhibitors may be promising antiviral agents for prevention and treatment of SARS-CoV-2 infection.

The origin and underlying driving forces of the SARS-CoV-2 outbreak.

BACKGROUND: SARS-CoV-2 began spreading in December 2019 and has since become a pandemic that has impacted many aspects of human society. Several issues concerning the origin, time of introduction to humans, evolutionary patterns, and underlying force driving the SARS-CoV-2 outbreak remain unclear. METHOD: Genetic variation in 137 SARS-CoV-2 genomes and related coronaviruses as of 2/23/2020 was analyzed. RESULT: After correcting for mutational bias, the excess of low frequency mutations on both synonymous and nonsynonymous sites was revealed which is consistent with the recent outbreak of the virus. In contrast to adaptive evolution previously reported for SARS-CoV during its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation. The sequence similarity in the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression that occurred approximately 40 years ago. The current outbreak of SARS-CoV-2 was estimated to have originated on 12/11/2019 (95% HPD 11/13/2019-12/23/2019). The effective population size of the virus showed an approximately 20-fold increase from the onset of the outbreak to the lockdown of Wuhan (1/23/2020) and ceased to increase afterwards, demonstrating the effectiveness of social distancing in preventing its spread. Two mutations, 84S in orf8 protein and 251 V in orf3 protein, occurred coincidentally with human intervention. The former first appeared on 1/5/2020 and plateaued around 1/23/2020. The latter rapidly increased in frequency after 1/23/2020. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China is two times higher than those derived from the rest of the world. A network analysis found that haplotypes collected from Wuhan were interior and had more mutational connections, both of which are consistent with the observation that the SARS-CoV-2 outbreak originated in China. CONCLUSION: SARS-CoV-2 might have cryptically circulated within humans for years before being discovered. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and determine the critical steps required for effective spreading.

Cell-Free Virus-Host Chimera DNA From Hepatitis B Virus Integration Sites as a Circulating Biomarker of Hepatocellular Cancer.

BACKGROUND AND AIMS: Early recurrence of hepatocellular carcinoma (HCC) after surgical resection compromises patient survival. Timely detection of HCC recurrence and its clonality is required to implement salvage therapies appropriately. This study examined the feasibility of virus-host chimera DNA (vh-DNA), generated from junctions of hepatitis B virus (HBV) integration in the HCC chromosome, as a circulating biomarker for this clinical setting. APPROACH AND RESULTS: HBV integration in 50 patients with HBV-related HCC was determined by the Hybridization capture-based next-generation sequencing (NGS) platform. For individual HCC, the vh-DNA was quantified by specific droplet digital PCR (ddPCR) assay in plasma samples collected before and 2 months after surgery. HBV integrations were identified in 44 out of 50 patients with HBV-related HCC. Tumor-specific ddPCR was developed to measure the corresponding vh-DNA copy number in baseline plasma from each patient immediately before surgery. vh-DNA was detected in 43 patients (97.7%), and the levels correlated with the tumor sizes (detection limit at 1.5 cm). Among the plasma collected at 2 months after surgery, 10 cases (23.3%) still contained the same signature vh-DNA detected at baseline, indicating the presence of residual tumor cells. Nine of them (90%) experienced HCC recurrence within 1 year, supporting vh-DNA as an independent risk factor in predicting early recurrence. Analysis of circulating vh-DNA at recurrence further helped identify the clonal origin. A total of 81.8% of recurrences came from original HCC clones sharing the same plasma vh-DNA, whereas 18.2% were from de novo HCC. CONCLUSIONS: vh-DNA was shown to be a circulating biomarker for detecting the tumor load in majority of patients with HBV-related HCC and aided in monitoring residual tumor and recurrence clonality after tumor resection.

Complement C1q mediates the expansion of periportal hepatic progenitor cells in senescence-associated inflammatory liver.

Most hepatocellular carcinomas (HCCs) develop in patients with chronic hepatitis, which creates a microenvironment for the growth of hepatic progenitor cells (HPCs) at the periportal area and subsequent development of HCCs. We investigated the signal from the inflammatory liver for this pathogenic process in the hepatic conditional ß-catenin knockout mouse model. Senescent ß-catenin-depleted hepatocytes in aged mice create an inflammatory microenvironment that stimulates periportal HPC expansion but arrests differentiation, which predisposes mice to the development of liver tumors. The release of complement C1q from macrophages in the inflammatory niche was identified as the unorthodox signal that activated the ß-catenin pathway in periportal HPCs and was responsible for their expansion and de-differentiation. C1q inhibitors blocked the ß-catenin pathway in both the expanding HPCs and the liver tumors but spared its orthodox pathway in pericentral normal hepatocytes. This mechanism has been validated in human liver specimens from patients with chronic hepatitis. Taken together, these results demonstrate that C1q- mediated activation of ß-catenin pathway in periportal HPCs is a previously unrecognized mechanism for replenishing hepatocytes in the inflammatory liver and, if unchecked, for promoting hepatocarcinogenesis. C1q may become a new target for blocking carcinogenesis in patients with chronic hepatitis.

Specific diacylglycerols generated by hepatic lipogenesis stimulate the oncogenic androgen receptor activity in male hepatocytes.

BACKGROUND: Obesity-induced hepatocellular carcinoma (HCC) is more prevalent in males than in females, but the underlying mechanism remains unclear. The influence of hepatic androgen receptor (AR) pathway on the gender difference of HCC has been well documented. Here we investigated the role of hepatic lipogenesis, which is elevated in the livers of obese and nonalcoholic fatty liver disease (NAFLD) patients, in stimulating the AR pathway for the male preference of obesity induced HCC. METHODS: Male C57BL/6J mice were fed a fructose-rich high carbohydrate diet (HCD) to induce hepatic lipogenesis. The effect of hepatic lipogenesis on AR was examined by the expression of hydrodynamically injected AR reporter and the endogenous AR target gene; the mechanism was delineated in hepatoma cell lines and validated in male mice. RESULTS: The hepatic lipogenesis induced by a fructose-rich HCD enhanced the transcriptional activity of hepatic AR in male mice, which did not happen when fed a high fat diet. This AR activation was blocked by sh-RNAs or inhibitors targeting key enzymes in lipogenesis, either acetyl-CoA carboxylase subunit alpha (ACCα), or fatty acid synthase (FASN), in vivo and in vitro. Further mechanistic study identified that specific unsaturated fatty acid, the oleic acid (C18:1 n-9), incorporated DAGs produced by hepatic lipogenesis are the key molecules to enhance the AR activity, through activation of Akt kinase, and this novel mechanism is targeted by metformin. CONCLUSIONS: Our study elucidates a novel mechanism underlying the higher risk of HCC in obese/NAFLD males, through specific DAGs enriched by hepatic lipogenesis to increase the transcriptional activity of hepatic AR, a confirmed risk factor for male HCC.