BACKGROUND & AIMS:Hepatocellular carcinoma (HCC) is a model of a diverse spectrum of cancers because it is induced by well-known etiologies, mainly hepatitis C virus (HCV) and hepatitis B virus. Here, we aimed to identify HCV-specific mutational signatures and explored the link between the HCV-related regional variation in mutations rates and HCV-induced alterations in genome-wide chromatin organization.METHODS:To identify an HCV-specific mutational signature in HCC, we performed high-resolution targeted sequencing to detect passenger mutations on 64 HCC samples from 3 etiology groups: hepatitis B virus, HCV, or other. To explore the link between the genomic signature and genome-wide chromatin organization we performed chromatin immunoprecipitation sequencing for the transcriptionally permissive H3K4Me3, H3K9Ac, and suppressive H3K9Me3 modifications after HCV infection.RESULTS:Regional variation in mutation rate analysis showed significant etiology-dependent regional mutation rates in 12 genes: LRP2, KRT84, TMEM132B, DOCK2, DMD, INADL, JAK2, DNAH6, MTMR9, ATM, SLX4, and ARSD. We found an enrichment of C->T transversion mutations in the HCV-associated HCC cases. Furthermore, these cases showed regional variation in mutation rates associated with genomic intervals in which HCV infection dictated epigenetic alterations. This signature may be related to the HCV-induced decreased expression of genes encoding key enzymes in the base excision repair pathway.CONCLUSIONS:We identified novel distinct HCV etiology-dependent mutation signatures in HCC associated with HCV-induced alterations in histone modification. This study presents a link between cancer-causing mutagenesis and the increased predisposition to liver cancer in chronic HCV-infected individuals, and unveils novel etiology-specific mechanisms leading to HCC and cancer in general.

Hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis, which often results in liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCV possesses an RNA genome and its replication is confined to the cytoplasm. Yet, infection with HCV leads to global changes in gene expression, and chromosomal instability (CIN) in the host cell. The mechanisms by which the cytoplasmic virus affects these nuclear processes are elusive. Here, we show that HCV modulates the function of the Structural Maintenance of Chromosome (SMC) protein complex, cohesin, which tethers remote regions of chromatin. We demonstrate that infection of hepatoma cells with HCV leads to up regulation of the expression of the RAD21 cohesin subunit and changes cohesin residency on the chromatin. These changes regulate the expression of genes associated with virus-induced pathways. Furthermore, siRNA downregulation of viral-induced RAD21 reduces HCV infection. During mitosis, HCV infection induces hypercondensation of chromosomes and the appearance of multi-centrosomes. We provide evidence that the underlying mechanism involves the viral NS3/4 protease and the cohesin regulator, WAPL. Altogether, our results provide the first evidence that HCV induces changes in gene expression and chromosome structure of infected cells by modulating cohesin.

The increasing worldwide prevalence of Hepatocellular carcinoma (HCC), characterized by resistance to conventional chemotherapy, poor prognosis and eventually mortality, place it as a prime target for new modes of prevention and treatment. Hepatitis C Virus (HCV) is the predominant risk factor for HCC in the US and Europe. Multiple epidemiological studies showed that sustained virological responses (SVR) following treatment with the powerful direct acting antivirals (DAAs), which have replaced interferon-based regimes, do not eliminate tumor development. We aimed to identify an HCV-specific pathogenic mechanism that persists post SVR following DAAs treatment. We demonstrate that HCV infection induces genome-wide epigenetic changes by performing chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) for histone post-translational modifications that are epigenetic markers for active and repressed chromatin. The changes in histone modifications correlate with reprogramed host gene expression and alter signaling pathways known to be associated with HCV life cycle and HCC. These epigenetic alterations require the presence of HCV RNA or/and expression of the viral proteins in the cells. Importantly, the epigenetic changes induced following infection persist as an "epigenetic signature" after virus eradication by DAAs treatment, as detected using in vitro HCV infection models. These observations led to the identification of an 8 gene signature that is associated with HCC development and demonstrate persistent epigenetic alterations in HCV infected and post SVR liver biopsy samples. The epigenetic signature was reverted in vitro by drugs that inhibit epigenetic modifying enzyme and by the EGFR inhibitor, Erlotinib. This epigenetic "scarring" of the genome, persisting following HCV eradication, suggest a novel mechanism for the persistent pathogenesis of HCV after its eradication by DAAs. Our study offers new avenues for prevention of the persistent oncogenic effects of chronic hepatitis infections using specific drugs to revert the epigenetic changes to the genome.

Hepatocellular carcinoma (HCC) represents the fifth most common cancer worldwide and the third cause of cancer-related mortality. Hepatitis C virus (HCV) is the leading cause of chronic hepatitis, which often results in liver fibrosis, cirrhosis, and eventually HCC. HCV is the most common risk factor for HCC in western countries and leads to a more aggressive and invasive disease with poorer patient survival rates. However, the mechanism by which the virus induces the metastatic spread of HCC tumor cells through the regulation of invadopodia, the key features of invasive cancer, is still unknown. Here, the integration of transcriptome with functional kinome screen revealed that HCV infection induced invasion and invadopodia-related gene expression combined with activation of host cell tyrosine kinases, leading to invadopodia formation and maturation and consequent cell invasiveness in vitro and in vivo. The promotion of invadopodia following HCV infection was mediated by the sustained stimulation of epidermal growth factor receptor (EGFR) via the viral NS3/4A protease that inactivates the T-cell protein tyrosine phosphatase (TC-PTP), which inhibits EGFR signaling. Characterization of an invadopodia-associated gene signature in HCV-mediated HCC tumors correlated with the invasiveness of HCC and poor patient prognosis. These findings might lead to new prognostic and therapeutic strategies for virus-mediated invasive cancer.

Hepatitis C virus (HCV) is a major public health concern, with over 70 million people infected worldwide, who are at risk for developing life-threatening liver disease. No vaccine is available, and immunity against the virus is not well-understood. Following the acute stage, HCV usually causes chronic infections. However, ~30% of infected individuals spontaneously clear the virus. Therefore, using HCV as a model for comparing immune responses between spontaneous clearer (SC) and chronically infected (CI) individuals may empower the identification of mechanisms governing viral infection outcomes. Here, we provide the first in-depth analysis of adaptive immune receptor repertoires in individuals with current or past HCV infection. We demonstrate that SC individuals, in contrast to CI patients, develop clusters of antibodies with distinct properties. These antibodies' characteristics were used in a machine learning framework to accurately predict infection outcome. Using combinatorial antibody phage display library technology, we identified HCV-specific antibody sequences. By integrating these data with the repertoire analysis, we constructed two antibodies characterized by high neutralization breadth, which are associated with clearance. This study provides insight into the nature of effective immune response against HCV and demonstrates an innovative approach for constructing antibodies correlating with successful infection clearance. It may have clinical implications for prognosis of the future status of infection, and the design of effective immunotherapies and a vaccine for HCV.

Background: The PIM-2 kinase is a potent survival factor; its role in the DNA damage response has never been addressed. Results: PIM-2 promotes DNA lesions repair in an E2F-1 and ATM-dependent manner; Pim-2-silenced cells are more susceptible to UV damage. Conclusion: PIM-2 is an upstream activator of E2F-1 and ATM in the UV damage response. Significance: PIM-2 is an essential component of the UV damage response. The oncogenic nature ascribed to the PIM-2 kinase relies mostly on phosphorylation of substrates that act as pro-survival/anti-apoptotic factors. Nevertheless, pro-survival effects can also result from activating DNA repair mechanisms following damage. In this study, we addressed the possibility that PIM-2 plays a role in the cellular response to UV damage, an issue that has never been addressed before. We found that in U2OS cells, PIM-2 expression and activity increased upon exposure to UVC radiation (2–50 mJ/cm2), and Pim-2-silenced cells were significantly more sensitive to UV radiation. Overexpression of PIM-2 accelerated removal of UV-induced DNA lesions over time, reduced γH2AX accumulation in damaged cells, and rendered these cells significantly more viable following UV radiation. The protective effect of PIM-2 was mediated by increased E2F-1 and activated ATM levels. Silencing E2F-1 reduced the protective effect of PIM-2, whereas inhibiting ATM activity abrogated this protective effect, irrespective of E2F-1 levels. The results obtained in this study place PIM-2 upstream to E2F-1 and ATM in the UV-induced DNA damage response.

Potent survival effects have been ascribed to the serine/threonine kinase proto-oncogene PIM-2. Elevated levels of PIM-2 are associated with various malignancies. In human cells, a single Pim-2 transcript gives rise mainly to two protein isoforms (34, 41 kDa) that share an identical catalytic site but differ at their N-terminus, due to in-frame alternative translation initiation sites. In this study we observed that the 34 kDa PIM-2 isoform has differential nuclear and cytoplasmic forms in all tested cell lines, suggesting a possible role for the balance between these forms for PIM-2's function. To further study the cellular role of the 34 kDa isoform of PIM-2, an N-terminally HA-tagged form of this isoform was transiently expressed in HeLa cells. Surprisingly, this resulted in increased level of G1 arrested cells, as well as of apoptotic cells. These effects could not be obtained by a Flag-tagged form of the 41 kDa isoform. The G1 arrest and apoptotic effects were associated with an increase in T14/Y15 phosphorylation of CDK2 and proteasom-dependent down-regulation of CDC25A, as well as with up-regulation of p57, E2F-1, and p73. No such effects were obtained upon over-expression of a kinase-dead form of the HA-tagged 34 kDa PIM-2. By either using a dominant negative form of p73, or by over-expressing the 34 kDa PIM-2 in p73-silenced cells, we demonstrated that these effects were p73-dependent. These results demonstrate that while PIM-2 can function as a potent survival factor, it can, under certain circumstances, exhibit pro-apoptotic effects as well.