Current antiviral agents can control but not eliminate hepatitis B virus (HBV), because HBV establishes a stable nuclear covalently closed circular DNA (cccDNA). Interferon-α treatment can clear HBV but is limited by systemic side effects. We describe how interferon-α can induce specific degradation of the nuclear viral DNA without hepatotoxicity and propose lymphotoxin-β receptor activation as a therapeutic alternative. Interferon-α and lymphotoxin-β receptor activation up-regulated APOBEC3A and APOBEC3B cytidine deaminases, respectively, in HBV-infected cells, primary hepatocytes, and human liver needle biopsies. HBV core protein mediated the interaction with nuclear cccDNA, resulting in cytidine deamination, apurinic/apyrimidinic site formation, and finally cccDNA degradation that prevented HBV reactivation. Genomic DNA was not affected. Thus, inducing nuclear deaminases-for example, by lymphotoxin-β receptor activation-allows the development of new therapeutics that, in combination with existing antivirals, may cure hepatitis B.
Hepatocellular carcinoma (HCC) is among the leading causes of cancer-related death. Despite the advances in diagnosis and management of HCC, the biology of this tumor remains poorly understood. Recent evidence highlighted long non-coding RNAs (lncRNAs) as crucial determinants of HCC development. In this study, we report the lncRNA HOTTIP as significantly up-regulated in HCC specimens. HOTTIP gene is located in physical contiguity with HOXA13 and directly controls the HOXA locus gene expression via interaction with the WDR5 / MLL complex. HOX genes encode transcription factors regulating embryonic development and cell fate. We previously described HOX genes deregulation to be involved in hepatocarcinogenesis. Indeed, we observed the marked up-regulation of HOXA13 in HCC. Here, by correlating clinico-pathological and expression data, we demonstrate that the levels of HOTTIP and HOXA13 are associated with HCC patients’ clinical progression and predict disease outcome. In contrast to the majority of similar studies, our data are obtained from snap-frozen needle HCC biopsies (n=52) matched with their non-neoplastic counterparts collected from patients that had not yet received any HCC-tailored therapeutic treatments at the time of biopsy. In addition, taking advantage of gain and loss of function experiments in liver cancer-derived cell lines (HuH-6 and HuH-7), we uncover a novel bidirectional regulatory loop between HOTTIP / HOXA13. Conclusion: our study highlights the key role of HOTTIP and HOXA13 in HCC development by associating their expression to metastasis and survival in HCC patients, provides novel insights on the function of lncRNA-driven hepatocarcinogenesis and paves the way for further investigation about the possible role of HOTTIP as predictive biomarker of HCC.
Background and Rationale Approximately 50% of patients with chronic hepatitis C (CHC) have ongoing expression of interferon stimulated genes (ISGs) in the liver. It is unclear why this endogenous antiviral response is inefficient in eradicating the infection. Several viral escape strategies have been identified in vitro, including inhibition of interferon (IFN) induction and ISG mRNA translation. The in vivo relevance of these mechanisms is unknown, because reliable methods to identify hepatitis C virus (HCV)-infected cells in human liver are lacking. We developed a highly sensitive in situ hybridization (ISH) system capable of HCV RNA and ISG mRNA detection in human liver biopsies and applied it to study the interaction of HCV with endogenous IFN system. Main Results We simultaneously monitored HCV RNA and ISG mRNA using HCV isolate- and ISG mRNA-specific probes in liver biopsy sections from 18 CHC patients. The signals were quantified at the single cell resolution in a series of random high-power fields. The proportion of infected hepatocytes ranged from 1 to 54% and correlated with viral load, but not with HCV genotype or ISG expression. Infected cells occurred in clusters, pointing to cell-to-cell spread as the predominant mode of HCV transmission. ISG mRNAs were readily detected in HCV-infected cells, challenging previously proposed mechanisms of viral interference with the immune system. Conversely, infected cells and neighboring cells showed increased ISG mRNA levels, demonstrating that the stimulus driving ISG expression originates from HCV infected hepatocytes. Conclusion HCV infection in human hepatocytes during CHC does not efficiently interfere with IFN induction, IFN signaling or transcription of ISG mRNA.
The Yes-associated protein (YAP)/Hippo pathway has been implicated in tissue development, regeneration, and tumorigenesis. However, its role in cholangiocarcinoma (CC) is not established. We show that YAP activation is a common feature in CC patient biopsies and human CC cell lines. Using microarray expression profiling of CC cells with overexpressed or down-regulated YAP, we show that YAP regulates genes involved in proliferation, apoptosis, and angiogenesis. YAP activity promotes CC growth in vitro and in vivo by functionally interacting with TEAD transcription factors (TEADs). YAP activity together with TEADs prevents apoptosis induced by cytotoxic drugs, whereas YAP knockdown sensitizes CC cells to drug-induced apoptosis. We further show that the proangiogenic microfibrillar-associated protein 5 (MFAP5) is a direct transcriptional target of YAP/TEAD in CC cells and that secreted MFAP5 promotes tube formation of human microvascular endothelial cells. High YAP activity in human CC xenografts and clinical samples correlates with increased MFAP5 expression and CD31 1 vasculature. Conclusions: These findings establish YAP as a key regulator of proliferation and antiapoptotic mechanisms in CC and provide first evidence that YAP promotes angiogenesis by regulating the expression of secreted proangiogenic proteins. (HEPATOLOGY 2015;62:1497-1510 C holangiocarcinoma (CC) represents the second most frequent primary liver cancer, with increasing incidence and mortality rates worldwide. The majority of CC is detected at advanced stages, preventing curative surgery and leaving patients with a median survival of only 24 months. Systemic chemotherapy (cisplatin plus gemcitabine) is currently the first-line treatment for CC with limited success.
Therapy of chronic hepatitis C with pegylated interferon a (pegIFN-a) 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-a, because prolonged stimulation of cells with IFN-a 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-a, IFN-b, IFN-c, and IFNk. Pretreatment of mice with IFN-a, IFN-b, and IFN-k induced a strong expression of the negative regulator ubiquitin-specific peptidase 18 in the liver and gut. As a result, IFN-a signaling was significantly reduced when mice where reinjected 16 hours after the first injection. Surprisingly, both IFN-b and IFN-k 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-k treatment of human liver biopsies ex vivo resulted in strong and maintained phosphorylation of STAT1, whereas IFN-ainduced STAT1 activation was transient. Conclusion: Contrary to the action of IFN-a, IFNb, and IFN-k signaling in the liver does not become refractory during repeated stimulation of the IFN signal transduction pathway. The sustained efficacy of IFN-b and IFN-k could be an important advantage for the treatment patients who are nonresponders to pegIFN-a, through a preactivated endogenous IFN system.
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