Suppressors of Cytokine Signaling and Hepatocellular Carcinoma.

Cytokines are secreted soluble glycoproteins that regulate cellular growth, proliferation, and differentiation. Suppressors of cytokine signaling (SOCS) proteins negatively regulate cytokine signaling and form a classical negative feedback loop in the signaling pathways. There are eight members of the SOCS family. The SOCS proteins are all comprised of a loosely conserved N-terminal domain, a central Src homology 2 (SH2) domain, and a highly conserved SOCS box at the C-terminus. The role of SOCS proteins has been implicated in the regulation of cytokines and growth factors in liver diseases. The SOCS1 and SOCS3 proteins are involved in immune response and inhibit protective interferon signaling in viral hepatitis. A decreased expression of SOCS3 is associated with advanced stage and poor prognosis of patients with hepatocellular carcinoma (HCC). DNA methylations of SOCS1 and SOCS3 are found in HCC. Precise regulation of liver regeneration is influenced by stimulatory and inhibitory factors after partial hepatectomy (PH), in particular, SOCS2 and SOCS3 are induced at an early time point after PH. Evidence supporting the important role of SOCS signaling during liver regeneration also supports a role of SOCS signaling in HCC. Immuno-oncology drugs are now the first-line therapy for advanced HCC. The SOCS can be potential targets for HCC in terms of cell proliferation, cell differentiation, and immune response. In this literature review, we summarize recent findings of the SOCS family proteins related to HCC and liver diseases.

Efficacy of Glecaprevir/Pibrentasvir for Real-World HCV Infected Patients in the Northern Part of Tokyo, Japan.

Hepatis virus C (HCV) infection causes liver cirrhosis and hepatocellular carcinoma (HCC) worldwide. The objective of our study was to examine the effects of the HCV nonstructural protein (NS) 3/4A inhibitor glecaprevir/NS5A inhibitor pibrentasvir on real-world HCV patients in the northern part of Tokyo, Japan. Although 106 patients were consecutively included, a total of 102 HCV-infected patients with chronic hepatitis or compensated cirrhosis, who received 8- or 12-week combination treatment with glecaprevir/pibrentasvir and were followed up to week 12 after the end of treatment were analyzed retrospectively. Only three patients discontinued treatment due to adverse events; however, they achieved a sustained virologic response at 12 weeks (SVR12). Finally, SVR rates were 99.0% (101/102). Only one patient without liver cirrhosis was a treatment relapser who received hepatic resection for HCC approximately two years after commencement of the 8-week combination treatment with glecaprevir/pibrentasvir. After the exclusion of patients with HCV genotype 1b and P32 deletion in the HCV NS5A region, a 12-week combination of glecaprevir/pibrentasvir led to SVR12 in all nine direct-acting antiviral-experienced patients. Glecaprevir/pibrentasvir had a high efficacy and an acceptable safety profile for real-world HCV patients in a single hospital in Japan.

Occurrence of hepatitis in an elderly woman during the treatment of pembrolizumab for right advanced renal pelvis, ureteral cancer, and bladder cancer.

Recently, the use of immune checkpoint inhibitors (ICIs) with or without chemotherapeutic agents has been increasing in the treatment for advanced cancer. Here, we report the occurrence of liver failure after the use of pembrolizumab in an 82-year-old woman with metastatic liver disease derived from right advanced renal pelvis, ureteral cancer, and bladder cancer. She was successfully treated with 0.6 mg/kg daily prednisolone. In patients treated with ICIs, ICI-induced hepatitis is occasionally observed. Even if patients are older, it appears important to diagnose and treat ICI-induced hepatitis earlier by multidisciplinary therapies including steroid treatment. This is a first report of pembrolizumab-induced liver failure in elder patient with age over 80 years. Even if patients are older, it appears important to diagnose and treat ICI-induced hepatitis earlier by multidisciplinary therapies including steroid treatment.

Male-Dominant Hepatitis A Outbreak Observed among Non-HIV-Infected Persons in the Northern Part of Tokyo, Japan.

Recently, we experienced an outbreak of acute hepatitis A virus (HAV) infection between 2018 and 2020. Herein, we describe this male-dominant HAV infection outbreak observed among non-human immunodeficiency virus (HIV)-infected persons in the northern part of Tokyo, Japan. Clinical information was collected from patient interviews and from medical record descriptions. In the present study, 21 patients were retrospectively analyzed. A total of 90.4 and 33.3% of patients were males, and men who have sex with men (MSM), respectively. The total bilirubin levels and platelet counts tended to be lower in the MSM group than in the non-MSM group. C-reactive protein (CRP) levels tended to be higher in acute liver failure (ALF) patients than in non-ALF patients. Prolonged cholestasis was observed in one patient (4.8%). We also found that 18 HAV isolates belonged to HAV subgenotype IA/subgroup 13 (S13), which clustered with the HAV isolate (KX151459) that was derived from an outbreak of HAV infection among MSM in Taiwan in 2015. Our results suggest that the application of antivirals against HAV, as well as HAV vaccines, would be useful for the treatment and prevention of severe HAV infection.

Japanese Man with HCV Genotype 4 Infection and Cirrhosis Who Was Successfully Treated by the Combination of Glecaprevir and Pibrentasvir.

A 74-year-old man with a history of transfusion at 35 years old in Egypt was referred to our hospital. He was infected with hepatitis C virus (HCV) genotype 4 (GT4), which is a rare HCV GT in Japan, and was also diagnosed with hepatic compensated cirrhosis. We safely treated the patient for 12 weeks with the combination of glecaprevir and pibrentasvir, and a sustained virologic response (SVR) was achieved. This is the first report of HCV GT4 infection in a treatment-naïve Japanese patient with cirrhosis in whom SVR was achieved with the combination treatment of glecaprevir and pibrentasvir.

Additive Effects of Zinc Chloride on the Suppression of Hepatitis A Virus Replication by Interferon in Human Hepatoma Huh7 Cells.

BACKGROUND/AIM: Hepatitis A virus (HAV) infection is still one of the serious health problems worldwide, despite the existence of effective vaccines for HAV. Zinc compounds have antiviral activities against various DNA and RNA viruses. Therefore, we investigated the effects of zinc compounds on the antiviral activity of interferon against HAV. MATERIALS AND METHODS: The effects of zinc compounds with or without interferon on HAV genotype IIIA HA11-1299 replication were examined in human hepatoma Huh7 cells. Cell viability was examined by the MTS assay. Inflammasome associated gene expression was examined by real-time reverse transcription-polymerase chain reaction. RESULTS: Both zinc sulfate and zinc chloride had an inhibitory effect on HAV replication. Zinc sulfate tended to enhance while zinc chloride significantly enhanced the anti-HAV effect induced by interferon-alpha-2a. Zinc chloride significantly up-regulated mitogen-activated protein kinase 12 (MAPK12) and down-regulated 6 related genes [baculoviral IAP repeat containing 3 (BIRC3), interleukin 1 beta (IL1B), proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1), prostaglandin-endoperoxide synthase 2 (PTGS2), PYD and CARD domain containing (PYCARD), and tumor necrosis factor (TNF)]. CONCLUSION: Zinc chloride inhibits HAV replication and has additive effects on the anti-HAV activities of interferon.

Co-Occurrence of Hepatitis A Infection and Chronic Liver Disease.

Hepatitis A virus (HAV) infection occasionally leads to a critical condition in patients with or without chronic liver diseases. Acute-on-chronic liver disease includes acute-on-chronic liver failure (ACLF) and non-ACLF. In this review, we searched the literature concerning the association between HAV infection and chronic liver diseases in PubMed. Chronic liver diseases, such as metabolic associated fatty liver disease and alcoholic liver disease, coinfection with other viruses, and host genetic factors may be associated with severe hepatitis A. It is important to understand these conditions and mechanisms. There may be no etiological correlation between liver failure and HAV infection, but there is an association between the level of chronic liver damage and the severity of acute-on-chronic liver disease. While the application of an HAV vaccination is important for preventing HAV infection, the development of antivirals against HAV may be important for preventing the development of ACLF with HAV infection as an acute insult. The latter is all the more urgent given that the lives of patients with HAV infection and a chronic liver disease of another etiology may be at immediate risk.

Involvement of the Interferon Signaling Pathways in Pancreatic Cancer Cells.

BACKGROUND/AIM: To examine interferon (IFN) signaling pathways in human pancreatic cancer cells and their therapeutic application for pancreatic ductal adenocarcinoma (PDAC). MATERIALS AND METHODS: We examined the effects of IFNα on cytotoxicity, migration, as well as on the levels of toll-like receptor (TLR) signaling pathway-associated genes expression in pancreatic cancer cells. We also examined the additive effects of IFNα and poly(I-C) on tyrosine kinase inhibitor (TKI)-induced cytotoxicity. We performed transcriptome analysis (RNA-Seq) of clinical samples and compared the profile between pancreatic intraepithelial neoplasias (PanINs) and PDACs. RESULTS: IFNα suppressed cell viability and cell migration, and affected TLR signaling pathways, in pancreatic cancer cells. TLR3 is one of the potential genes involved in IFN-treated pancreatic cancer cells. Furthermore, similar to IFN, extracellular addition of poly(I-C) enhanced TKI-induced cytotoxicity in pancreatic cancer cells. RNA-Seq analysis demonstrated that IFN signaling is one of the potential pathways involved in the progression of PanIN to PDAC. CONCLUSION: IFN signaling may be involved in the development of PDAC. Treatments that target the IFN and TLR3 signaling pathways may be therapeutic options against PDAC.

Noninvasive Assessment of Liver Fibrosis: Current and Future Clinical and Molecular Perspectives.

Liver fibrosis is one of the risk factors for hepatocellular carcinoma (HCC) development. The staging of liver fibrosis can be evaluated only via a liver biopsy, which is an invasive procedure. Noninvasive methods for the diagnosis of liver fibrosis can be divided into morphological tests such as elastography and serum biochemical tests. Transient elastography is reported to have excellent performance in the diagnosis of liver fibrosis and has been accepted as a useful tool for the prediction of HCC development and other clinical outcomes. Two-dimensional shear wave elastography is a new technique and provides a real-time stiffness image. Serum fibrosis markers have been studied based on the mechanism of fibrogenesis and fibrolysis. In the healthy liver, homeostasis of the extracellular matrix is maintained directly by enzymes called matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs). MMPs and TIMPs could be useful serum biomarkers for liver fibrosis and promising candidates for the treatment of liver fibrosis. Further studies are required to establish liver fibrosis-specific markers based on further clinical and molecular research. In this review, we summarize noninvasive fibrosis tests and molecular mechanism of liver fibrosis in current daily clinical practice.

Cell Culture Systems and Drug Targets for Hepatitis A Virus Infection.

Hepatitis A virus (HAV) infection is one of the major causes of acute hepatitis, and this infection occasionally causes acute liver failure. HAV infection is associated with HAV-contaminated food and water as well as sexual transmission among men who have sex with men. Although an HAV vaccine has been developed, outbreaks of hepatitis A and life-threatening severe HAV infections are still observed worldwide. Therefore, an improved HAV vaccine and anti-HAV drugs for severe hepatitis A should be developed. Here, we reviewed cell culture systems for HAV infection, and other issues. This review may help with improving the HAV vaccine and developing anti-HAV drugs.

Different Mechanisms of Action of Regorafenib and Lenvatinib on Toll-Like Receptor-Signaling Pathways in Human Hepatoma Cell Lines.

Multiple kinase inhibitors are available for patients with advanced hepatocellular carcinoma (HCC). It is largely unknown whether regorafenib or lenvatinib modulates innate immunity including Toll-like receptor (TLR)-signaling pathways in HCC. We performed real-time RT-PCR to investigate 84 TLR-associated gene expression levels and compared these gene expression levels in each hepatoma cells treated with or without regorafenib or lenvatinib. In response to regorafenib, nine and 10 genes were upregulated in Huh7 and HepG2 cells, respectively, and only C-X-C motif chemokine ligand 10 was upregulated in both cell lines. A total of 14 and 12 genes were downregulated in Huh7 and HepG2 cells, respectively, and two genes (Fos proto-oncogene, AP-1 transcription factor subunit, and ubiquitin conjugating enzyme E2 N) were downregulated in both cell lines. In response to lenvatinib, four and 16 genes were upregulated in Huh7 and HepG2 cells, respectively, and two genes (interleukin 1 alpha and TLR4) were upregulated in both cells. Six and one genes were downregulated in Huh7 and HepG2, respectively, and no genes were downregulated in both cell lines. In summary, regorafenib and lenvatinib affect TLR signaling pathways in human hepatoma cell lines. Modulation of TLR signaling pathway may improve the treatment of HCC patients with refractory disease.

Superinfection of hepatitis A virus in hepatocytes infected with hepatitis B virus.

Hepatitis A virus (HAV) infection is a major cause of acute hepatitis including acute liver failure. Hepatitis B infection (HBV) occurs worldwide, with the highest rates in Asian and African countries, and there are several reports that HAV infection may have a more severe clinical course in patients with chronic HBV infection. We previously demonstrated that Japanese miso extracts have inhibitory effects on HAV replication. In the present study, we examined the replication of HAV and HBV in a hepatocyte superinfection model and the inhibitory effects of Japanese miso extracts on both viruses. According to the results, HAV infection inhibited HBV replication in superinfected hepatocytes, and Japanese rice-koji miso extracts had inhibitory effects on HAV replication. Our findings provide useful information for clinicians in managing HAV infection in patients with chronic HBV infection.

Repression of MicroRNA-30e by Hepatitis C Virus Enhances Fatty Acid Synthesis.

Chronic hepatitis C virus (HCV) infection often leads to end-stage liver disease, including hepatocellular carcinoma (HCC). We have previously observed reduced expression of microRNA-30e (miR-30e) in the liver tissues and sera of patients with HCV-associated HCC, although biological functions remain unknown. In this study, we demonstrated that HCV infection of hepatocytes transcriptionally reduces miR-30e expression by modulating CCAAT/enhancer binding protein ß. In silico prediction suggests that autophagy-related gene 5 (ATG5) is a direct target of miR-30e. ATG5 is involved in autophagy biogenesis, and HCV infection in hepatocytes induces autophagy. We showed the presence of ATG5 in the miR-30e-Argonaute 2 RNA-induced silencing complex. Overexpression of miR-30e in HCV-infected hepatocytes inhibits autophagy activation. Subsequent studies suggested that ATG5 knockdown in Huh7.5 cells results in the remarkable inhibition of sterol regulatory element binding protein (SREBP)-1c and fatty acid synthase (FASN) level. We also showed that overexpression of miR-30e decreased lipid synthesis-related protein SREBP-1c and FASN in hepatocytes. Conclusion: We show new mechanistic insights into the interactions between autophagy and lipid synthesis through inhibition of miR-30e in HCV-infected hepatocytes.

Transforming Growth Factor ß Acts as a Regulatory Molecule for Lipogenic Pathways among Hepatitis C Virus Genotype-Specific Infections.

Hepatitis C virus (HCV) infection promotes metabolic disorders, and the severity of lipogenic disease depends upon the infecting virus genotype. Here, we have examined HCV genotype 1-, 2-, or 3-specific regulation of lipid metabolism, involving transforming growth factor ß (TGF-ß)-regulated phospho-Akt (p-Akt) and peroxisome proliferator-activated receptor alpha (PPARα) axes. Since HCV core protein is one of the key players in metabolic regulation, we also examined its contribution in lipid metabolic pathways. The expression of regulatory molecules, TGF-ß1/2, phospho-Akt (Ser473), PPARα, sterol regulatory element-binding protein 1 (SREBP-1), fatty acid synthase (FASN), hormone-sensitive lipase (HSL), and acyl dehydrogenases was analyzed in virus-infected hepatocytes. Interestingly, HCV genotype 3a exhibited much higher activation of TGF-ß and p-Akt, with a concurrent decrease in PPARα expression and fatty acid oxidation. A significant and similar decrease in HSL, unlike in HCV genotype 1a, was observed with both genotypes 2a and 3a. Similar observations were made from ectopic expression of the core genomic region from each genotype. The key role of TGF-ß was further verified using specific small interfering RNA (siRNA). Together, our results highlight a significant difference in TGF-ß-induced activity for the HCV genotype 2a- or 3a-induced lipogenic pathway, exhibiting higher triglyceride synthesis and a decreased lipolytic mechanism. These results may help in therapeutic modalities for early treatment of HCV genotype-associated lipid metabolic disorders.IMPORTANCE Hepatic steatosis is a frequent complication associated with chronic hepatitis C virus (HCV) infection and is a key prognostic indicator for progression to fibrosis and cirrhosis. Several mechanisms are proposed for the development of steatosis, especially with HCV genotype 3a. Our observations suggest that transforming growth factor ß (TGF-ß) and peroxisome proliferator-activated receptor alpha (PPARα)-associated mechanistic pathways in hepatocytes infected with HCV genotype 2a and 3a differ from those in cells infected with genotype 1a. The results suggest that a targeted therapeutic approach for enhanced PPARα and lipolysis may reduce HCV genotype-associated lipid metabolic disorder in liver disease.

Exosomes and Hepatocellular Carcinoma: From Bench to Bedside.

As hepatocellular carcinoma (HCC) usually occurs in the background of cirrhosis, which is an end-stage form of liver diseases, treatment options for advanced HCC are limited, due to poor liver function. The exosome is a nanometer-sized membrane vesicle structure that originates from the endosome. Exosome-mediated transfer of proteins, DNAs and various forms of RNA, such as microRNA (miRNA), long noncoding RNA (lncRNA) and messenger RNA (mRNA), contributes to the development of HCC. Exosomes mediate communication between both HCC and non-HCC cells involved in tumor-associated cells, and several molecules are implicated in exosome biogenesis. Exosomes may be potential diagnostic biomarkers for early-stage HCC. Exosomal proteins, miRNAs and lncRNAs could provide new biomarker information for HCC. Exosomes are also potential targets for the treatment of HCC. Notably, further efforts are required in this field. We reviewed recent literature and demonstrated how useful exosomes are for diagnosing patients with HCC, treating patients with HCC and predicting the prognosis of HCC patients.

Rapid hepatitis C virus clearance by antivirals correlates with immune status of infected patients.

Altered immune parameters associated with hepatitis C virus (HCV) genotype 1b infection and their correlation with virus eradication in direct-acting antivirals (DAA)-treated patients were examined. Thirty-one HCV-infected patients were treated with DAAs for 12 weeks. Pre-DAA-treatment and post-DAA-treatment sera were analyzed for cytokines/chemokines using MILLIPLEX MAP. Serum complement level and antibody neutralization activity were measured separately. Sera from 11 spontaneously cleared HCV subjects were included for comparison. Rapid virological responders (RVR) or end-of-treatment responders (EOTR) were defined as patients with HCV RNA negative at week 4 or positive at week 4 and negative at week 12, respectively. HCV RNA eradication and a decrease in liver fibrosis-related cytokines after treatment were observed when compared with pretreatment sera from RVR and EOTR. In pretreatment sera, interferons and T-helper 1 or 2 cell-associated cytokines/chemokines were significantly higher among RVR as compared with EOTR. Furthermore, serum complement and virus neutralizing antibody levels were higher in pretreatment RVR sera. Eradication of HCV RNA by DAA decreased liver fibrosis-related cytokines. Pretreatment sera from RVR displayed an enhanced cytokine/chemokine, complement and virus neutralizing antibody response as compared with EOTR sera. Our results suggested that enhanced host immune status may play an additive role on HCV RNA clearance by DAA.

Association between MicroRNA-373 and Long Noncoding RNA NORAD in Hepatitis C Virus-Infected Hepatocytes Impairs Wee1 Expression for Growth Promotion.

Chronic hepatitis C virus (HCV) infection may lead to end-stage liver disease, including hepatocellular carcinoma (HCC). We have shown previously that microRNA-373 (miR-373) is upregulated in HCV-infected human liver biopsy specimens. To gain insight into the role of miR-373 in HCV-mediated pathogenesis, we investigated its interacting partner for hepatocyte growth regulation. Transcriptome sequencing (RNA-seq) data revealed that Wee1 is associated with miR-373 and is a direct target. Interestingly, higher expression of Wee1 was noted in HCV-infected hepatocytes than in uninfected hepatocytes, suggesting that other factors may block miR-373-mediated Wee1 inhibition. We subsequently found an association between the long noncoding RNA NORAD (LINC00657) and miR-373, and we demonstrated that NORAD binds to miR-373 and Wee1 independently. However, the high level of Wee1 expression in HCV-infected hepatocytes suggested that miR-373 forms a complex with NORAD. Depletion of miR-373 or the inhibitor Wee1 reduces the growth of Huh7.5 cells harboring the HCV genome as well as reducing Wee1 expression. Taken together, our data demonstrate a novel mechanism of hepatocyte growth promotion during HCV infection involving a miR-373-NORAD-Wee1 axis, which may be a target for future therapy against HCV-associated HCC.IMPORTANCE The mechanism of HCV-mediated liver pathogenesis is poorly understood. In this study, we observed that HCV infection upregulates miR-373 and Wee1, a pivotal player in the G2 checkpoint in the cell cycle, although Wee1 is a direct target for miR-373. Subsequent investigation demonstrated that miR-373 forms a complex with the long noncoding RNA NORAD, resulting in the release of their common target, Wee1, in HCV-infected cells, which, in turn, favors uncontrolled cell growth. Our study suggested a previously unknown mechanism for hepatocyte growth promotion following HCV infection, and this pathway can be targeted for future therapy against HCV-mediated liver pathogenesis.

Successful retreatment with grazoprevir and elbasvir for patients infected with hepatitis C virus genotype 1b, who discontinued prior treatment with NS5A inhibitor-including regimens due to adverse events.

BACKGROUND: Sustained virologic response (SVR) by interferon and interferon-free treatment can results in the reduction of advanced liver fibrosis and the occurrence of hepatocellular carcinoma in patients infected with hepatitis C virus (HCV). Recent interferon-free treatment for HCV shortens the duration of treatment and leads to higher SVR rates, without any serious adverse events. However, it is important to retreat patients who have had treatment-failure with HCV non-structural protein 5A (NS5A) inhibitor-including regimens. Combination of sofosbuvir and ledipasvir only leads to approximately 100% SVR rates in HCV genotype (GT1b), NS5A inhibitor-naïve patients in Japan. This combination is not an indication for severe renal disease or heart disease, and these patients should be treated or retreated with a different regimen. CASE SUMMARY: Retreatment with HCV non-structural protein 3/4A inhibitor, grazoprevir, and HCV NS5A inhibitor, elbasvir, successfully eradicated HCV RNA in three patients with HCV genotype 1b infection who discontinued prior interferon-free treatments including HCV NS5A inhibitors due to adverse events within 2 weeks. CONCLUSION: Retreatment with the 12-week combination regimen of grazoprevir and elbasvir is effective for HCV GT1b patients who discontinue the HCV NS5A inhibitor-including regimens within 2 weeks. The treatment response may be related to the short duration of initial treatment, which did not produce treatment-emergent RASs.

Interferon-free treatment for patients with chronic hepatitis C and autoimmune liver disease: higher SVR rates with special precautions for deterioration of autoimmune hepatitis.

BACKGROUND: Interferon-free treatment can achieve higher sustained virological response (SVR) rates, even in patients in whom hepatitis C virus (HCV) could not be eradicated in the interferon treatment era. Immune restoration in the liver is occasionally associated with HCV infection. We examined the safety and effects of interferon-free regimens on HCV patients with autoimmune liver diseases. RESULTS: All 7 HCV patients with autoimmune hepatitis (AIH) completed treatment and achieved SVR. Three patients took prednisolone (PSL) at baseline, and 3 did not take PSL during interferon-free treatment. In one HCV patient with AIH and cirrhosis, PSL were not administered at baseline, but she needed to take 40 mg/day PSL at week 8 for liver dysfunction. She also complained back pain and was diagnosed with vasospastic angina by coronary angiography at week 11. However, she completed interferon-free treatment. All 5 HCV patients with primary biliary cholangitis (PBC) completed treatment and achieved SVR. Three of these HCV patients with PBC were treated with UDCA during interferon-free treatment. CONCLUSIONS: Interferon-free regimens could result in higher SVR rates in HCV patients with autoimmune liver diseases. As interferon-free treatment for HCV may have an effect on hepatic immunity and activity of the autoimmune liver diseases, careful attention should be paid to unexpected adverse events in their treatments. METHODS: Total 12 patients with HCV and autoimmune liver diseases [7 AIH and PBC], who were treated with interferon-free regimens, were retrospectively analyzed.