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.

Targeting clinical epigenetic reprogramming for chemoprevention of metabolic and viral hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is the fastest-growing cause of cancer-related mortality with chronic viral hepatitis and non-alcoholic steatohepatitis (NASH) as major aetiologies. Treatment options for HCC are unsatisfactory and chemopreventive approaches are absent. Chronic hepatitis C (CHC) results in epigenetic alterations driving HCC risk and persisting following cure. Here, we aimed to investigate epigenetic modifications as targets for liver cancer chemoprevention. DESIGN: Liver tissues from patients with NASH and CHC were analysed by ChIP-Seq (H3K27ac) and RNA-Seq. The liver disease-specific epigenetic and transcriptional reprogramming in patients was modelled in a liver cell culture system. Perturbation studies combined with a targeted small molecule screen followed by in vivo and ex vivo validation were used to identify chromatin modifiers and readers for HCC chemoprevention. RESULTS: In patients, CHC and NASH share similar epigenetic and transcriptomic modifications driving cancer risk. Using a cell-based system modelling epigenetic modifications in patients, we identified chromatin readers as targets to revert liver gene transcription driving clinical HCC risk. Proof-of-concept studies in a NASH-HCC mouse model showed that the pharmacological inhibition of chromatin reader bromodomain 4 inhibited liver disease progression and hepatocarcinogenesis by restoring transcriptional reprogramming of the genes that were epigenetically altered in patients. CONCLUSION: Our results unravel the functional relevance of metabolic and virus-induced epigenetic alterations for pathogenesis of HCC development and identify chromatin readers as targets for chemoprevention in patients with chronic liver diseases.

Combined Analysis of Metabolomes, Proteomes, and Transcriptomes of Hepatitis C Virus-Infected Cells and Liver to Identify Pathways Associated With Disease Development.

BACKGROUND & AIMS: The mechanisms of hepatitis C virus (HCV) infection, liver disease progression, and hepatocarcinogenesis are only partially understood. We performed genomic, proteomic, and metabolomic analyses of HCV-infected cells and chimeric mice to learn more about these processes. METHODS: Huh7.5.1dif (hepatocyte-like cells) were infected with culture-derived HCV and used in RNA sequencing, proteomic, metabolomic, and integrative genomic analyses. uPA/SCID (urokinase-type plasminogen activator/severe combined immunodeficiency) mice were injected with serum from HCV-infected patients; 8 weeks later, liver tissues were collected and analyzed by RNA sequencing and proteomics. Using differential expression, gene set enrichment analyses, and protein interaction mapping, we identified pathways that changed in response to HCV infection. We validated our findings in studies of liver tissues from 216 patients with HCV infection and early-stage cirrhosis and paired biopsy specimens from 99 patients with hepatocellular carcinoma, including 17 patients with histologic features of steatohepatitis. Cirrhotic liver tissues from patients with HCV infection were classified into 2 groups based on relative peroxisome function; outcomes assessed included Child-Pugh class, development of hepatocellular carcinoma, survival, and steatohepatitis. Hepatocellular carcinomas were classified according to steatohepatitis; the outcome was relative peroxisomal function. RESULTS: We quantified 21,950 messenger RNAs (mRNAs) and 8297 proteins in HCV-infected cells. Upon HCV infection of hepatocyte-like cells and chimeric mice, we observed significant changes in levels of mRNAs and proteins involved in metabolism and hepatocarcinogenesis. HCV infection of hepatocyte-like cells significantly increased levels of the mRNAs, but not proteins, that regulate the innate immune response; we believe this was due to the inhibition of translation in these cells. HCV infection of hepatocyte-like cells increased glucose consumption and metabolism and the STAT3 signaling pathway and reduced peroxisome function. Peroxisomes mediate ß-oxidation of very long-chain fatty acids; we found intracellular accumulation of very long-chain fatty acids in HCV-infected cells, which is also observed in patients with fatty liver disease. Cells in livers from HCV-infected mice had significant reductions in levels of the mRNAs and proteins associated with peroxisome function, indicating perturbation of peroxisomes. We found that defects in peroxisome function were associated with outcomes and features of HCV-associated cirrhosis, fatty liver disease, and hepatocellular carcinoma in patients. CONCLUSIONS: We performed combined transcriptome, proteome, and metabolome analyses of liver tissues from HCV-infected hepatocyte-like cells and HCV-infected mice. We found that HCV infection increases glucose metabolism and the STAT3 signaling pathway and thereby reduces peroxisome function; alterations in the expression levels of peroxisome genes were associated with outcomes of patients with liver diseases. These findings provide insights into liver disease pathogenesis and might be used to identify new therapeutic targets.

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.

Extracellular lipid-free apolipoprotein E inhibits HCV replication and induces ABCG1-dependent cholesterol efflux.

OBJECTIVE: The HCV life cycle and the lipid metabolism are inextricably intertwined. In the blood, HCV virions are associated with lipoproteins, forming lipoviroparticles (LVPs), which are the most infectious form of the virus. Apolipoprotein E (apoE), a key LVP component, plays an essential role in HCV entry, assembly and egress. ApoE is also a cell host factor involved in lipoprotein homeostasis. Although the majority of apoE is associated with lipoproteins, a lipid-free (LF) form exists in blood. However, the role of LF-apoE in both lipid metabolism and HCV life cycle is poorly understood. DESIGN: In this study, using the cell culture-derived HCV model system in human hepatoma Huh7.5.1 cells and primary human hepatocytes (PHH), we investigated the effect of LF-apoE on the early steps of HCV life cycle and on the lipid metabolism of hepatic cells. RESULTS: A dose-dependent decrease in HCV replication was observed when Huh7.5.1 cells and PHH were treated with increasing amounts of LF-apoE. We showed that LF-apoE acts on HCV replication independently of previously described apoE receptors. We observed that LF-apoE induced a marked hepatic cholesterol efflux via the ATP-binding cassette subfamily G member 1 (ABCG1) protein that in turn inhibits HCV replication. LF-apoE also increases both apolipoprotein AI and high-density lipoprotein production. CONCLUSIONS: Our findings highlight a new mechanism in lipid metabolism regulation and interaction of the lipid metabolism with the HCV life cycle, which may be important for viral pathogenesis and might also be explored for antiviral therapy.

Hepatitis C Virus-Induced Upregulation of MicroRNA miR-146a-5p in Hepatocytes Promotes Viral Infection and Deregulates Metabolic Pathways Associated with Liver Disease Pathogenesis.

UNLABELLED: Hepatitis C virus (HCV)-induced chronic liver disease is a leading cause of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying HCC development following chronic HCV infection remain poorly understood. MicroRNAs (miRNAs) play an important role in homeostasis within the liver, and deregulation of miRNAs has been associated with liver disease, including HCC. While host miRNAs are essential for HCV replication, viral infection in turn appears to induce alterations of intrahepatic miRNA networks. Although the cross talk between HCV and liver cell miRNAs most likely contributes to liver disease pathogenesis, the functional involvement of miRNAs in HCV-driven hepatocyte injury and HCC remains elusive. Here we combined a hepatocyte-like cell-based model system, high-throughput small RNA sequencing, computational analysis, and functional studies to investigate HCV-miRNA interactions that may contribute to liver disease and HCC. Profiling analyses indicated that HCV infection differentially regulated the expression of 72 miRNAs by at least 2-fold, including miRNAs that were previously described to target genes associated with inflammation, fibrosis, and cancer development. Further investigation demonstrated that the miR-146a-5p level was consistently increased in HCV-infected hepatocyte-like cells and primary human hepatocytes, as well as in liver tissue from HCV-infected patients. Genome-wide microarray and computational analyses indicated that miR-146a-5p overexpression modulates pathways that are related to liver disease and HCC development. Furthermore, we showed that miR-146a-5p has a positive impact on late steps of the viral replication cycle, thereby increasing HCV infection. Collectively, our data indicate that the HCV-induced increase in miR-146a-5p expression both promotes viral infection and is relevant for pathogenesis of liver disease. IMPORTANCE: HCV is a leading cause of chronic liver disease and cancer. However, how HCV induces liver cancer remains poorly understood. There is accumulating evidence that a viral cure does not eliminate the risk for HCC development. Thus, there is an unmet medical need to develop novel approaches to predict and prevent virus-induced HCC. miRNA expression is known to be deregulated in liver disease and cancer. Furthermore, miRNAs are essential for HCV replication, and HCV infection alters miRNA expression. However, how miRNAs contribute to HCV-driven pathogenesis remains elusive. Here we show that HCV induces miRNAs that may contribute to liver injury and carcinogenesis. The miR-146a-5p level was consistently increased in different cell-based models of HCV infection and in HCV patient-derived liver tissue. Furthermore, miR-146a-5p increased HCV infection. Collectively, our data are relevant to understanding viral pathogenesis and may open perspectives for novel biomarkers and prevention of virus-induced liver disease and HCC.

Hepatocellular carcinoma chemoprevention by targeting the angiotensin-converting enzyme and EGFR transactivation.

Hepatocellular carcinoma (HCC) is a leading cause of death among cirrhotic patients, for which chemopreventive strategies are lacking. Recently, we developed a simple human cell-based system modeling a clinical prognostic liver signature (PLS) predicting liver disease progression and HCC risk. In a previous study, we applied our cell-based system for drug discovery and identified captopril, an approved angiotensin converting enzyme (ACE) inhibitor, as a candidate compound for HCC chemoprevention. Here, we explored ACE as a therapeutic target for HCC chemoprevention. Captopril reduced liver fibrosis and effectively prevented liver disease progression toward HCC development in a diethylnitrosamine (DEN) rat cirrhosis model and a diet-based rat model for nonalcoholic steatohepatitis-induced (NASH-induced) hepatocarcinogenesis. RNA-Seq analysis of cirrhotic rat liver tissues uncovered that captopril suppressed the expression of pathways mediating fibrogenesis, inflammation, and carcinogenesis, including epidermal growth factor receptor (EGFR) signaling. Mechanistic data in liver disease models uncovered a cross-activation of the EGFR pathway by angiotensin. Corroborating the clinical translatability of the approach, captopril significantly reversed the HCC high-risk status of the PLS in liver tissues of patients with advanced fibrosis. Captopril effectively prevents fibrotic liver disease progression toward HCC development in preclinical models and is a generic and safe candidate drug for HCC chemoprevention.

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.

Interleukin-32 Contributes to Human Nonalcoholic Fatty Liver Disease and Insulin Resistance.

Nonalcoholic fatty liver disease (NAFLD) is a metabolic disorder due to increased accumulation of fat in the liver and in many cases to enhanced inflammation. Although the contribution of inflammation in the pathogenesis of NAFLD is well established, the cytokines that are involved and how they influence liver transformation are still poorly characterized. In addition, with other modifiers, inflammation influences NAFLD progression to liver cirrhosis and hepatocellular carcinoma, demonstrating the need to find new molecular targets with potential future therapeutic applications. We investigated gene signatures in 38 liver biopsies from patients with NAFLD and obesity who had received bariatric surgery and compared these to 10 control patients who had received a cholecystectomy, using DNA microarray technology. A subset of differentially expressed genes was then validated on a larger cohort of 103 patients who had received bariatric surgery for obesity; data were thoroughly analyzed in terms of correlations with NAFLD pathophysiological parameters. Finally, the impact of a specific cytokine, interleukin-32 (IL32), was addressed on primary human hepatocytes (PHHs). Transcript analysis revealed an up-regulation of proinflammatory cytokines IL32, chemokine (C-X-C motif) ligand 9 (CXCL9), and CXCL10 and of ubiquitin D (UBD), whereas down-regulation of insulin-like growth factor-binding protein 2 (IGFBP2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) was reported in patients with NAFLD. Moreover, IL32, which is the major deregulated gene, correlated with body mass index (BMI), waist circumference, NAFLD activity score (NAS), aminotransferases (alanine aminotransferase [ALAT] and aspartate aminotransferase [ASAT]), and homeostasis model assessment of insulin resistance (HOMA-IR) index in patients. Consistent with an instrumental role in the pathophysiology of NAFLD, treatment of control human hepatocytes with recombinant IL32 leads to insulin resistance, a hallmark metabolic deregulation in NAFLD hepatocytes. Conclusion: IL32 has a critical role in the pathogenesis of NAFLD and could be considered as a therapeutic target in patients.