Treatment of HCC with claudin-1-specific antibodies suppresses carcinogenic signaling and reprograms the tumor microenvironment.

BACKGROUND & AIMS: Despite recent approvals, the response to treatment and prognosis of patients with advanced hepatocellular carcinoma (HCC) remain poor. Claudin-1 (CLDN1) is a membrane protein that is expressed at tight junctions, but it can also be exposed non-junctionally, such as on the basolateral membrane of the human hepatocyte. While CLDN1 within tight junctions is well characterized, the role of non-junctional CLDN1 and its role as a therapeutic target in HCC remains unexplored. METHODS: Using humanized monoclonal antibodies (mAbs) specifically targeting the extracellular loop of human non-junctional CLDN1 and a large series of patient-derived cell-based and animal model systems we aimed to investigate the role of CLDN1 as a therapeutic target for HCC. RESULTS: Targeting non-junctional CLDN1 markedly suppressed tumor growth and invasion in cell line-based models of HCC and patient-derived 3D ex vivo models. Moreover, the robust effect on tumor growth was confirmed in vivo in a large series of cell line-derived xenograft and patient-derived xenograft mouse models. Mechanistic studies, including single-cell RNA sequencing of multicellular patient HCC tumorspheres, suggested that CLDN1 regulates tumor stemness, metabolism, oncogenic signaling and perturbs the tumor immune microenvironment. CONCLUSIONS: Our results provide the rationale for targeting CLDN1 in HCC and pave the way for the clinical development of CLDN1-specific mAbs for the treatment of advanced HCC. IMPACT AND IMPLICATIONS: Hepatocellular carcinoma (HCC) is associated with high mortality and unsatisfactory treatment options. Herein, we identified the cell surface protein Claudin-1 as a treatment target for advanced HCC. Monoclonal antibodies targeting Claudin-1 inhibit tumor growth in patient-derived ex vivo and in vivo models by modulating signaling, cell stemness and the tumor immune microenvironment. Given the differentiated mechanism of action, the identification of Claudin-1 as a novel therapeutic target for HCC provides an opportunity to break the plateau of limited treatment response. The results of this preclinical study pave the way for the clinical development of Claudin-1-specific antibodies for the treatment of advanced HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology.

A monoclonal antibody targeting nonjunctional claudin-1 inhibits fibrosis in patient-derived models by modulating cell plasticity.

Tissue fibrosis is a key driver of end-stage organ failure and cancer, overall accounting for up to 45% of deaths in developed countries. There is a large unmet medical need for antifibrotic therapies. Claudin-1 (CLDN1) is a member of the tight junction protein family. Although the role of CLDN1 incorporated in tight junctions is well established, the function of nonjunctional CLDN1 (njCLDN1) is largely unknown. Using highly specific monoclonal antibodies targeting a conformation-dependent epitope of exposed njCLDN1, we show in patient-derived liver three-dimensional fibrosis and human liver chimeric mouse models that CLDN1 is a mediator and target for liver fibrosis. Targeting CLDN1 reverted inflammation-induced hepatocyte profibrogenic signaling and cell fate and suppressed the myofibroblast differentiation of hepatic stellate cells. Safety studies of a fully humanized antibody in nonhuman primates did not reveal any serious adverse events even at high steady-state concentrations. Our results provide preclinical proof of concept for CLDN1-specific monoclonal antibodies for the treatment of advanced liver fibrosis and cancer prevention. Antifibrotic effects in lung and kidney fibrosis models further indicate a role of CLDN1 as a therapeutic target for tissue fibrosis across organs. In conclusion, our data pave the way for further therapeutic exploration of CLDN1-targeting therapies for fibrotic diseases in patients.

A human liver cell-based system modeling a clinical prognostic liver signature for therapeutic discovery.

Chronic liver disease and hepatocellular carcinoma (HCC) are life-threatening diseases with limited treatment options. The lack of clinically relevant/tractable experimental models hampers therapeutic discovery. Here, we develop a simple and robust human liver cell-based system modeling a clinical prognostic liver signature (PLS) predicting long-term liver disease progression toward HCC. Using the PLS as a readout, followed by validation in nonalcoholic steatohepatitis/fibrosis/HCC animal models and patient-derived liver spheroids, we identify nizatidine, a histamine receptor H2 (HRH2) blocker, for treatment of advanced liver disease and HCC chemoprevention. Moreover, perturbation studies combined with single cell RNA-Seq analyses of patient liver tissues uncover hepatocytes and HRH2+, CLEC5Ahigh, MARCOlow liver macrophages as potential nizatidine targets. The PLS model combined with single cell RNA-Seq of patient tissues enables discovery of urgently needed targets and therapeutics for treatment of advanced liver disease and cancer prevention.

HCV-Induced Epigenetic Changes Associated With Liver Cancer Risk Persist After Sustained Virologic Response.

BACKGROUND & AIMS: Chronic hepatitis C virus (HCV) infection is an important risk factor for hepatocellular carcinoma (HCC). Despite effective antiviral therapies, the risk for HCC is decreased but not eliminated after a sustained virologic response (SVR) to direct-acting antiviral (DAA) agents, and the risk is higher in patients with advanced fibrosis. We investigated HCV-induced epigenetic alterations that might affect risk for HCC after DAA treatment in patients and mice with humanized livers. METHODS: We performed genome-wide ChIPmentation-based ChIP-Seq and RNA-seq analyses of liver tissues from 6 patients without HCV infection (controls), 18 patients with chronic HCV infection, 8 patients with chronic HCV infection cured by DAA treatment, 13 patients with chronic HCV infection cured by interferon therapy, 4 patients with chronic hepatitis B virus infection, and 7 patients with nonalcoholic steatohepatitis in Europe and Japan. HCV-induced epigenetic modifications were mapped by comparative analyses with modifications associated with other liver disease etiologies. uPA/SCID mice were engrafted with human hepatocytes to create mice with humanized livers and given injections of HCV-infected serum samples from patients; mice were given DAAs to eradicate the virus. Pathways associated with HCC risk were identified by integrative pathway analyses and validated in analyses of paired HCC tissues from 8 patients with an SVR to DAA treatment of HCV infection. RESULTS: We found chronic HCV infection to induce specific genome-wide changes in H3K27ac, which correlated with changes in expression of mRNAs and proteins. These changes persisted after an SVR to DAAs or interferon-based therapies. Integrative pathway analyses of liver tissues from patients and mice with humanized livers demonstrated that HCV-induced epigenetic alterations were associated with liver cancer risk. Computational analyses associated increased expression of SPHK1 with HCC risk. We validated these findings in an independent cohort of patients with HCV-related cirrhosis (n = 216), a subset of which (n = 21) achieved viral clearance. CONCLUSIONS: In an analysis of liver tissues from patients with and without an SVR to DAA therapy, we identified epigenetic and gene expression alterations associated with risk for HCC. These alterations might be targeted to prevent liver cancer in patients treated for HCV infection.

miR-135a-5p-mediated downregulation of protein tyrosine phosphatase receptor delta is a candidate driver of HCV-associated hepatocarcinogenesis.

BACKGROUND AND AIMS: HCV infection is a leading risk factor of hepatocellular carcinoma (HCC). However, even after viral clearance, HCC risk remains elevated. HCV perturbs host cell signalling to maintain infection, and derailed signalling circuitry is a key driver of carcinogenesis. Since protein phosphatases are regulators of signalling events, we aimed to identify phosphatases that respond to HCV infection with relevance for hepatocarcinogenesis. METHODS: We assessed mRNA and microRNA (miRNA) expression profiles in primary human hepatocytes, liver biopsies and resections of patients with HCC, and analysed microarray and RNA-seq data from paired liver biopsies of patients with HCC. We revealed changes in transcriptional networks through gene set enrichment analysis and correlated phosphatase expression levels to patient survival and tumour recurrence. RESULTS: We demonstrate that tumour suppressor protein tyrosine phosphatase receptor delta (PTPRD) is impaired by HCV infection in vivo and in HCC lesions of paired liver biopsies independent from tissue inflammation or fibrosis. In liver tissue adjacent to tumour, high PTPRD levels are associated with a dampened transcriptional activity of STAT3, an increase of patient survival from HCC and reduced tumour recurrence after surgical resection. We identified miR-135a-5p as a mechanistic regulator of hepatic PTPRD expression in patients with HCV. CONCLUSIONS: We previously demonstrated that STAT3 is required for HCV infection. We conclude that HCV promotes a STAT3 transcriptional programme in the liver of patients by suppressing its regulator PTPRD via upregulation of miR-135a-5p. Our results show the existence of a perturbed PTPRD-STAT3 axis potentially driving malignant progression of HCV-associated liver disease.

SOCS1 is an inducible negative regulator of interferon λ (IFN-λ)-induced gene expression in vivo.

Type I (α and ß) and type III (λ) IFNs are induced upon viral infection through host sensory pathways that activate IFN regulatory factors (IRFs) and nuclear factor κB. Secreted IFNs induce autocrine and paracrine signaling through the JAK-STAT pathway, leading to the transcriptional induction of hundreds of IFN-stimulated genes, among them sensory pathway components such as cGAS, STING, RIG-I, MDA5, and the transcription factor IRF7, which enhance the induction of IFN-αs and IFN-λs. This positive feedback loop enables a very rapid and strong host response that, at some point, has to be controlled by negative regulators to maintain tissue homeostasis. Type I IFN signaling is controlled by the inducible negative regulators suppressor of cytokine signaling 1 (SOCS1), SOCS3, and ubiquitin-specific peptidase 18 (USP18). The physiological role of these proteins in IFN-γ signaling has not been clarified. Here we used knockout cell lines and mice to show that IFN-λ signaling is regulated by SOCS1 but not by SOCS3 or USP18. These differences were the basis for the distinct kinetic properties of type I and III IFNs. We found that IFN-α signaling is transient and becomes refractory after hours, whereas IFN-λ provides a long-lasting IFN-stimulated gene induction.

Hepatic Notch1 deletion predisposes to diabetes and steatosis via glucose-6-phosphatase and perilipin-5 upregulation.

Notch signaling pathways have recently been implicated in the pathogenesis of metabolic diseases. However, the role of hepatic Notch signaling in glucose and lipid metabolism remains unclear and needs further investigation as it might be a candidate therapeutic target in metabolic diseases such as nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD). We used hepatocyte-specific Notch1 knockout (KO) mice and liver biopsies from NASH and NAFLD patients to analyze the role of Notch1 in glucose and lipid metabolism. Hepatocyte-specific Notch1 KO mice were fed with a high fat diet (HFD) or a regular diet (RD). We assessed the metabolic phenotype, glucose and insulin tolerance tests, and liver histology. Hepatic mRNA expression was profiled by Affymetrix Mouse Gene arrays and validated by quantitative reverse transcription PCR (qPCR). Akt phosphorylation was visualized by immunoblotting. Gene expression was analyzed in liver biopsies from NASH, NAFLD, and control patients by qPCR. We found that Notch1 KO mice had elevated fasting glucose. Gene expression analysis showed an upregulation of glucose-6-phosphatase, involved in the final step of gluconeogenesis and glucose release from glycogenolysis, and perilipin-5, a regulator of hepatic lipid accumulation. When fed with an HFD KO mice developed overt diabetes and hepatic steatosis. Akt was highly phosphorylated in KO animals and the Foxo1 target gene expression was altered. Accordingly, a reduction in Notch1 and increase in glucose-6-phosphatase and perilipin-5 expression was observed in liver biopsies from NAFLD/NASH compared with controls. Notch1 is a regulator of hepatic glucose and lipid homeostasis. Hepatic impairment of Notch1 expression may be involved in the pathogenesis of human NAFLD/NASH.

Clearance of persistent hepatitis C virus infection in humanized mice using a claudin-1-targeting monoclonal antibody.

Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and cancer. Cell entry of HCV and other pathogens is mediated by tight junction (TJ) proteins, but successful therapeutic targeting of TJ proteins has not been reported yet. Using a human liver-chimeric mouse model, we show that a monoclonal antibody specific for the TJ protein claudin-1 (ref. 7) eliminates chronic HCV infection without detectable toxicity. This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events. Antibody treatment reduces the number of HCV-infected hepatocytes in vivo, highlighting the need for de novo infection by means of host entry factors to maintain chronic infection. In summary, we demonstrate that an antibody targeting a virus receptor can cure chronic viral infection and uncover TJ proteins as targets for antiviral therapy.

Reduced IFNλ4 activity is associated with improved HCV clearance and reduced expression of interferon-stimulated genes.

Hepatitis C virus (HCV) infections are the major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma worldwide. Both spontaneous and treatment-induced clearance of HCV depend on genetic variation within the interferon-lambda locus, but until now no clear causal relationship has been established. Here we demonstrate that an amino-acid substitution in the IFNλ4 protein changing a proline at position 70 to a serine (P70S) substantially alters its antiviral activity. Patients harbouring the impaired IFNλ4-S70 variant display lower interferon-stimulated gene (ISG) expression levels, better treatment response rates and better spontaneous clearance rates, compared with patients coding for the fully active IFNλ4-P70 variant. Altogether, these data provide evidence supporting a role for the active IFNλ4 protein as the driver of high hepatic ISG expression as well as the cause of poor HCV clearance.

IFN-λ receptor 1 expression is induced in chronic hepatitis C and correlates with the IFN-λ3 genotype and with nonresponsiveness to IFN-α therapies.

The molecular mechanisms that link IFN-λ3 genotypes to differential induction of interferon (IFN)-stimulated genes (ISGs) in the liver of patients with chronic hepatitis C (CHC) are not known. We measured the expression of IFN-λ and of the specific IFN-λ receptor chain (IFN-λR1) in 122 liver biopsies of patients with CHC and 53 control samples. The IFN-λ3 genotype was not associated with differential expression of IFN-λ, but rather IFN-λR1. In a series of 30 primary human hepatocyte (PHH) samples, IFN-λR1 expression was low but could be induced with IFN-α. IFN-α-induced IFN-λR1 expression was significantly stronger in PHHs carrying the minor IFN-λ3 allele. The analysis of liver biopsies of patients with CHC revealed a strong association of high IFN-λR1 expression with elevated ISG expression, with IFN-λ3 minor alleles, and with nonresponse to pegylated IFN-α and ribavirin. The findings provide a missing link between the IFN-λ3 genotype and the associated phenotype of treatment nonresponse.

Protein phosphatase 2A promotes hepatocellular carcinogenesis in the diethylnitrosamine mouse model through inhibition of p53.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Most HCCs develop in cirrhotic livers. Alcoholic liver disease, chronic hepatitis B and chronic hepatitis C are the most common underlying liver diseases. Hepatitis C virus (HCV)-specific mechanisms that contribute to HCC are presently unknown. Transgenic expression of HCV proteins in the mouse liver induces an overexpression of the protein phosphatase 2A catalytic subunit (PP2Ac). We have previously reported that HCV-induced PP2Ac overexpression modulates histone methylation and acetylation and inhibits DNA damage repair. In this study, we analyze tumor formation and gene expression using HCV transgenic mice that overexpress PP2Ac and liver tissues from patients with HCC. We demonstrate that PP2Ac overexpression interferes with p53-induced apoptosis. Injection of the carcinogen, diethylnitrosamine, induced significantly more and larger liver tumors in HCV transgenic mice that overexpress PP2Ac compared with control mice. In human liver biopsies from patients with HCC, PP2Ac expression was significantly higher in HCC tissue compared with non-tumorous liver tissue from the same patients. Our findings demonstrate an important role of PP2Ac overexpression in liver carcinogenesis and provide insights into the molecular pathogenesis of HCV-induced HCC.

IL28B expression depends on a novel TT/-G polymorphism which improves HCV clearance prediction.

Approximately 3% of the world population is chronically infected with the hepatitis C virus (HCV), with potential development of cirrhosis and hepatocellular carcinoma. Despite the availability of new antiviral agents, treatment remains suboptimal. Genome-wide association studies (GWAS) identified rs12979860, a polymorphism nearby IL28B, as an important predictor of HCV clearance. We report the identification of a novel TT/-G polymorphism in the CpG region upstream of IL28B, which is a better predictor of HCV clearance than rs12979860. By using peripheral blood mononuclear cells (PBMCs) from individuals carrying different allelic combinations of the TT/-G and rs12979860 polymorphisms, we show that induction of IL28B and IFN-γ-inducible protein 10 (IP-10) mRNA relies on TT/-G, but not rs12979860, making TT/-G the only functional variant identified so far. This novel step in understanding the genetic regulation of IL28B may have important implications for clinical practice, as the use of TT/G genotyping instead of rs12979860 would improve patient management.

HRas signal transduction promotes hepatitis C virus cell entry by triggering assembly of the host tetraspanin receptor complex.

Hepatitis C virus (HCV) entry is dependent on coreceptor complex formation between the tetraspanin superfamily member CD81 and the tight junction protein claudin-1 (CLDN1) on the host cell membrane. The receptor tyrosine kinase EGFR acts as a cofactor for HCV entry by promoting CD81-CLDN1 complex formation via unknown mechanisms. We identify the GTPase HRas, activated downstream of EGFR signaling, as a key host signal transducer for EGFR-mediated HCV entry. Proteomic analysis revealed that HRas associates with tetraspanin CD81, CLDN1, and the previously unrecognized HCV entry cofactors integrin ß1 and Ras-related protein Rap2B in hepatocyte membranes. HRas signaling is required for lateral membrane diffusion of CD81, which enables tetraspanin receptor complex assembly. HRas was also found to be relevant for entry of other viruses, including influenza. Our data demonstrate that viruses exploit HRas signaling for cellular entry by compartmentalization of entry factors and receptor trafficking.

Epidermal growth factor receptor signaling impairs the antiviral activity of interferon-alpha.

UNLABELLED: Interferon-alpha (IFN-α) exhibits its antiviral activity through signal transducer and activator of transcription protein (STAT) signaling and the expression of IFN response genes (IRGs). Viral infection has been shown to result in activation of epidermal growth factor receptor (EGFR)-a host cell entry factor used by several viruses, including hepatitis C virus. However, the effect of EGFR activation for cellular antiviral responses is unknown. Here, we uncover cross-talk between EGFR and IFN-α signaling that has a therapeutic effect on IFN-α-based therapies and functional relevance for viral evasion and IFN resistance. We show that combining IFN-α with the EGFR inhibitor, erlotinib, potentiates the antiviral effect of each compound in a highly synergistic manner. The extent of the synergy correlated with reduced STAT3 phosphorylation in the presence of erlotinib, whereas STAT1 phosphorylation was not affected. Furthermore, reduced STAT3 phosphorylation correlated with enhanced expression of suppressors of cytokine signaling 3 (SOCS3) in the presence of erlotinib and enhanced expression of the IRGs, radical S-adenosyl methionine domain containing 2 and myxovirus resistance protein 1. Moreover, EGFR stimulation reduced STAT1 dimerization, but not phosphorylation, indicating that EGFR cross-talk with IFN signaling acts on the STATs at the level of binding DNA. CONCLUSIONS: Our results support a model where inhibition of EGFR signaling impairs STAT3 phosphorylation, leading to enhanced IRG expression and antiviral activity. These data uncover a novel role of EGFR signaling in the antiviral activity of IFN-α and open new avenues of improving the efficacy of IFN-α-based antiviral therapies.

Interferon-γ-stimulated genes, but not USP18, are expressed in livers of patients with acute hepatitis C.

BACKGROUND & AIMS: Approximately 50% of patients with chronic hepatitis C (CHC) have a sustained virologic response to treatment with pegylated interferon (pegIFN)-α and ribavirin. Nonresponse to treatment is associated with constitutively increased expression of IFN-stimulated genes (ISGs) in the liver. Treatment of patients with acute hepatitis C (AHC) is more effective, with sustained virologic response rates greater than 90%. We investigated mechanisms of the different responses of patients with CHC and AHC to pegIFN-α therapy. METHODS: We analyzed IFN signaling and ISG expression in liver samples from patients with AHC, patients with CHC, and individuals without hepatitis C (controls) using microarray, immunohistochemical, and protein analyses. Findings were compared with those from primary human hepatocytes stimulated with IFN-α or IFN-γ, as reference sets. RESULTS: Expression levels of hundreds of genes, primarily those regulated by IFN-γ, were altered in liver samples from patients with AHC compared with controls. Expression of IFN-γ-stimulated genes was induced in liver samples from patients with AHC, whereas expression of IFN-α-stimulated genes was induced in samples from patients with CHC. In an expression analysis of negative regulators of IFN-α signaling, we did not observe differences in expression of suppresor of cytokine signaling 1 or SOCS3 between liver samples from patients with AHC and those with CHC. However, USP18 (another negative regulator of IFN-α signaling), was up-regulated in liver samples of patients with CHC that did not respond to therapy, but not in AHC. CONCLUSIONS: Differences in expression of ISGs might account for the greater response of patients with AHC, compared with those with CHC, to treatment with pegIFN-α and ribavirin. Specifically, USP18 is up-regulated in liver samples of patients with CHC that did not respond to therapy, but not in patients with AHC.

Interferon-ß and interferon-λ signaling is not affected by interferon-induced refractoriness to interferon-α in vivo.

UNLABELLED: Therapy of chronic hepatitis C with pegylated interferon α (pegIFN-α) and ribavirin achieves sustained virological responses in approximately half of the patients. Nonresponse to treatment is associated with constitutively increased expression of IFN-stimulated genes in the liver already before therapy. This activation of the endogenous IFN system could prevent cells from responding to therapeutically injected (peg)IFN-α, because prolonged stimulation of cells with IFN-α induces desensitization of the IFN signal transduction pathway. Whether all types of IFNs induce refractoriness in the liver is presently unknown. We therefore treated mice with multiple injections and different combinations of IFN-α, IFN-ß, IFN-γ, and IFN-λ. Pretreatment of mice with IFN-α, IFN-ß, and IFN-λ induced a strong expression of the negative regulator ubiquitin-specific peptidase 18 in the liver and gut. As a result, IFN-α signaling was significantly reduced when mice where reinjected 16 hours after the first injection. Surprisingly, both IFN-ß and IFN-λ could activate the Janus kinase-signal transducer and activator of transcription (STAT) pathway and the expression of IFN-stimulated genes despite high levels of ubiquitin-specific peptidase 18. IFN-λ treatment of human liver biopsies ex vivo resulted in strong and maintained phosphorylation of STAT1, whereas IFN-α-induced STAT1 activation was transient. CONCLUSION: Contrary to the action of IFN-α, IFN-ß, and IFN-λ signaling in the liver does not become refractory during repeated stimulation of the IFN signal transduction pathway. The sustained efficacy of IFN-ß and IFN-λ could be an important advantage for the treatment patients who are nonresponders to pegIFN-α, through a preactivated endogenous IFN system.

Interferon-induced gene expression is a stronger predictor of treatment response than IL28B genotype in patients with hepatitis C.

BACKGROUND & AIMS: The host immune response during the chronic phase of hepatitis C virus infection varies among individuals; some patients have a no interferon (IFN) response in the liver, whereas others have full activation of IFN-stimulated genes (ISGs). Preactivation of this endogenous IFN system is associated with nonresponse to pegylated IFN-α (pegIFN-α) and ribavirin. Genome-wide association studies have associated allelic variants near the IL28B (IFNλ3) gene with treatment response. We investigated whether IL28B genotype determines the constitutive expression of ISGs in the liver and compared the abilities of ISG levels and IL28B genotype to predict treatment outcome. METHODS: We genotyped 109 patients with chronic hepatitis C for IL28B allelic variants and quantified the hepatic expression of ISGs and of IL28B. Decision tree ensembles, in the form of a random forest classifier, were used to calculate the relative predictive power of these different variables in a multivariate analysis. RESULTS: The minor IL28B allele was significantly associated with increased expression of ISG. However, stratification of the patients according to treatment response revealed increased ISG expression in nonresponders, irrespective of IL28B genotype. Multivariate analysis of ISG expression, IL28B genotype, and several other factors associated with response to therapy identified ISG expression as the best predictor of treatment response. CONCLUSIONS: IL28B genotype and hepatic expression of ISGs are independent predictors of response to treatment with pegIFN-α and ribavirin in patients with chronic hepatitis C. The most accurate prediction of response was obtained with a 4-gene classifier comprising IFI27, ISG15, RSAD2, and HTATIP2.

S-adenosyl-methionine and betaine improve early virological response in chronic hepatitis C patients with previous nonresponse.

BACKGROUND/AIMS: Treatment of chronic hepatitis C (CHC) with pegylated interferon α (pegIFNα) and ribavirin results in a sustained response in approximately half of patients. Viral interference with IFNα signal transduction through the Jak-STAT pathway might be an important factor underlying treatment failure. S-adenosyl-L-methionine (SAMe) and betaine potentiate IFNα signaling in cultured cells that express hepatitis C virus (HCV) proteins, and enhance the inhibitory effect of IFNα on HCV replicons. We have performed a clinical study with the aim to evaluate efficacy and safety of the addition of SAMe and betaine to treatment of CHC with pegIFNα/ribavirin. METHODS: In this open-label pilot study, 29 patients with CHC who failed previous therapy with (peg)IFNα/ribavirin were treated with SAMe, betaine, pegIFNα2b and ribavirin. Treatment duration was 6 or 12 months, depending on genotype, and the protocol comprised a stopping rule at week 12 if early virological response (EVR) was not achieved. Virological and biochemical response and safety were assessed throughout the treatment. RESULTS: 29 patients were enrolled and treated according to the study protocol. 79% of the patients were infected with genotype 1, 72% had advanced fibrosis, 76% had previously received pegIFNα/ribavirin, and only 14% achieved EVR to the previous treatment. When treated with the study medications, 17 patients (59%) showed an EVR, only 3 (10%) however achieved a sustained virological response (SVR). SAMe and betaine were found to be safe when used with pegIFNα/ribavirin. CONCLUSION: The addition of SAMe and betaine to pegIFNα/ribavirin improves early virological response in CHC. TRIAL REGISTRATION: ClinicalTrials.gov NCT00310336.

Hepatitis C virus-induced up-regulation of protein phosphatase 2A inhibits histone modification and DNA damage repair.

UNLABELLED: The molecular mechanisms underlying hepatocarcinogenesis in chronic viral hepatitis are poorly understood. A potential tumorigenic pathway could involve protein phosphatase 2A (PP2A) and protein arginine methyltransferase 1 (PRMT1), because both enzymes are dysregulated in chronic hepatitis C, and both enzymes have been involved in chromatin remodeling and DNA damage repair. We used cell lines that allow the inducible expression of hepatitis C virus proteins (UHCV57.3) and of the catalytic subunit of PP2A (UPP2A-C8) as well as Huh7.5 cells infected with recombinant cell culture-derived hepatitis C virus (HCVcc) to study epigenetic histone modifications and DNA damage repair. The induction of viral proteins, the overexpression of PP2Ac, or the infection of Huh7.5 cells with HCVcc resulted in an inhibition of histone H4 methylation/acetylation and histone H2AX phosphorylation, in a significantly changed expression of genes important for hepatocarcinogenesis, and inhibited DNA damage repair. Overexpression of PP2Ac in NIH-3T3 cells increased anchorage-independent growth. These changes were partially reversed by the treatment of cells with the methyl-group donor S-adenosyl-L-methionine (SAMe). CONCLUSION: Hepatitis C virus-induced overexpression of PP2Ac contributes to hepatocarcinogenesis through dysregulation of epigenetic histone modifications. The correction of defective histone modifications by S-adenosyl-L-methionine makes this drug a candidate for chemopreventive therapies in patients with chronic hepatitis C who are at risk for developing hepatocellular carcinoma.

Alpha interferon induces long-lasting refractoriness of JAK-STAT signaling in the mouse liver through induction of USP18/UBP43.

Recombinant alpha interferon (IFN-alpha) is used for the treatment of viral hepatitis and some forms of cancer. During these therapies IFN-alpha is injected once daily or every second day for several months. Recently, the long-acting pegylated IFN-alpha (pegIFN-alpha) has replaced standard IFN-alpha in therapies of chronic hepatitis C because it is more effective, supposedly by inducing a long-lasting activation of IFN signaling pathways. IFN signaling in cultured cells, however, becomes refractory within hours, and little is known about the pharmacodynamic effects of continuously high IFN-alpha serum concentrations. To investigate the behavior of the IFN system in vivo, we repeatedly injected mice with IFN-alpha and analyzed its effects in the liver. Within hours after the first injection, IFN-alpha signaling became refractory to further stimulation. The negative regulator SOCS1 was rapidly upregulated and likely responsible for early termination of IFN-alpha signaling. For long-lasting refractoriness, neither SOCS1 nor SOCS3 were instrumental. Instead, we identified the inhibitor USP18/UBP43 as the key mediator. Our results indicate that the current therapeutic practice using long-lasting pegIFN-alpha is not well adapted to the intrinsic properties of the IFN system. Targeting USP18 expression may allow to exploit the full therapeutic potential of recombinant IFN-alpha.