The major goal of the Hepatic Pathogenesis Section is to conduct translational research on the pathogenesis of acute and chronic liver disease, with a major focus on viral hepatitis and its long-term sequelae, cirrhosis and hepatocellular carcinoma (HCC), which contribute to a very large burden of disease worldwide. 1. Molecular Pathogenesis of Hepatitis B Virus (HBV)-Associated Acute Liver Failure (ALF) HBV-associated ALF is a dramatic clinical syndrome leading to death or liver transplantation in 80% of cases. However, the pathogenesis of ALF remains largely unknown. To investigate the molecular pathogenesis of this disease, we performed a comprehensive genetic and functional characterization of the virus and the host in liver tissue from HBV-associated ALF, and compared the results with those of classic acute hepatitis B in chimpanzees. In contrast with acute hepatitis B, HBV strains detected in ALF livers displayed highly mutated HBV core antigen (HBcAg), associated with increased HBcAg expression ex vivo, which was independent of viral replication levels. Combined gene and microRNA expression profiling revealed a dominant B-cell-driven disease signature, with massive intrahepatic production of IgM and IgG in germline configuration exclusively targeting HBcAg with subnanomolar affinities, and complement deposition. Thus, HBV-ALF appears to be an anomalous T-cell-independent, HBV core-driven B-cell disease, which results from the rare and unfortunate encounter between a host with an unusual nave B-cell repertoire and an infecting virus with highly mutated core antigen. 2. Host and viral factors in the pathogenesis of chronic viral hepatitis, cirrhosis and hepatocellular carcinoma (HCC), and search for biomarkers for the early detection of HCC HCC is the third leading cause of cancer-related death worldwide, and chronic infection with hepatitis viruses accounts for over 80% of cases. Cirrhosis is the single most important risk factor for HCC being present in 80-90% of the cases. Although the major etiologic agents and risk factors for HCC are well defined, the molecular mechanisms of hepatocarcinogenesis remain unclear. a) Determinants of fibrogenesis and disease progression in chronic hepatitis C In collaboration with Dr. Alter, we investigated the association between circulating microRNA (miRNA) levels and disease progression in chronic hepatitis C (CHC). We studied 130 patients with chronic hepatitis C prospectively followed over several decades. A comprehensive miRNA profile identified miRNAs as candidate predictors of disease progression. Longitudinal analyses showed that let-7s levels in plasma markedly declined over time in parallel with fibrosis progression, thereby providing the best correlation with hepatic fibrosis in CHC. Pathway analysis suggested that low levels of let-7 may influence hepatic fibrogenesis through activation of TGF- signaling in hepatic stellate cells. b) Diminished Replication and Viral Compartmentalization of Hepatitis C Virus (HCV) in HCC We recently demonstrated that HCV replication is severely impaired within HCC tissue in vivo, but the mechanisms responsible for this restriction are presently unknown. Tracking of individual variants demonstrated changes in the viral population between tumorous and non-tumorous areas whose extent correlated with the decline in HCV RNA, suggesting HCV compartmentalization within the tumor. c) Identification of TACSTD2 as a novel host cofactor for HCV entry To investigate the nature of factors that restrict HCV replication in primary HCC tissue, we analyzed expression of the four major HCV co-receptors, SR-B1, CD81, CLDN1 and OCLN, but none of them was differentially expressed between tumor and non-tumorous tissue. We identified tumor-associated calcium signal transducer 2 (TACSTD2), one of the most downregulated genes in primary HCC tissue, as a novel host co-factor for HCV entry that interacts with the HCV co-receptors CLDN1 and OCLN, and is involved in the regulation of their cellular localization. TACSTD2 gene silencing disrupts the typical linear distribution of CLDN1 and OCLN along the cellular membrane in both hepatoma cells and primary human hepatocytes, recapitulating the pattern observed in vivo in primary HCC tissue. Mechanistic studies suggest that TACSTD2 is involved in the phosphorylation of CLDN1 and OCLN, which is required for their proper cellular localization. Silencing of TACSTD2 dramatically inhibits HCV infection with a pan-genotype effect. HCV-entry assays using genotype 1a and 1b HCV pseudoparticles demonstrated that the inhibitory effect occurs at the level of viral entry. These results identify TACSTD2 as a novel host cofactor for HCV entry, which may have relevance for the pathogenesis, treatment and prevention of HCV infection. d) Molecular Signature of Hepatitis D Virus (HDV)-Associated Hepatocellular Carcinoma HDV causes the least common but most severe and rapidly progressive form of hepatitis, leading to cirrhosis in about 80% of cases. Although HCC develops in a high proportion of patients with HDV infection, there are no data on the molecular mechanisms of HDV-induced hepatocarcinogenesis. It is also unknown whether HCC is an effect of the underlying cirrhosis or a direct oncogenic effect of HDV, or a cumulative oncogenic effect of HBV and HDV, given the vital dependence of HDV on HBV. Gene expression profiling was performed on laser capture-microdissected hepatocytes (LCM, malignant and non-malignant hepatocytes) and whole liver tissue (WLT, tumor and non-tumor tissue) from patients with HDV-HCC, and on WLT from non-HCC HDV cirrhosis. Malignant hepatocytes showed an enrichment of upregulated genes involved in cell cycle/DNA replication, damage and repair. Most differentially expressed genes were co-regulated and over-expressed when compared to non-malignant hepatocytes, as well as to malignant hepatocytes from HCC associated with HBV alone. Activation and co-regulation of genes critically associated with DNA replication, damage and repair point to genetic instability as an important mechanism of HDV hepatocarcinogenesis. Thus, despite the dependence of HDV on HBV, HDV and HBV appear to promote carcinogenesis by distinct molecular mechanisms. d) Identification of Long Noncoding RNA Associated with HCC of Different Viral Etiology. Several long noncoding (lnc) RNAs have been reported to play a role in HCC. However, a major limitation of previous studies is that they either analyzed lncRNAs selectively in HBV-related HCC or considered HCC as a single group regardless of the hepatitis virus involved. We analyzed lncRNAs expression in HBV-, HCV- and HDV-related HCC samples with the aim of investigating the differential role of lncRNAs in relation to the different viral etiology of HCC. We studied 88 paired liver specimens from HCC patients (tumor and non-tumorous tissue), individual specimens from 20 patients with non-HCC cirrhosis, and 18 controls with normal liver. We identified 8 novel HCC-related lncRNAs that were significantly dysregulated in HCC tissue compared to their surrounding non-tumorous tissue. Some of these lncRNAs were significantly dysregulated only in one specific hepatitis virus-related HCC, including PCAT-29 in HBV-related HCC, aHIF and PAR5 in HCV-related HCC, and Y3 in HDV-related HCC. To our knowledge, this is the first study in which the expression of lncRNAs was analyzed in paired liver specimens obtained from patients with HBV-, HCV- and HDV-associated HCC. Our study suggests that HCC of different viral etiology is regulated, at least in part, by different lncRNAs.
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