Establishment of a patient-derived mucoepidermoid carcinoma cell line with the CRTC1-MAML2 fusion gene.

Mucoepidermoid carcinoma (MEC) is the most common malignant tumor of the major and minor salivary glands. Surgical resection is the only curative treatment and there is no effective post-operative therapy for MEC. The present study reports an Institutional Review Board-approved case of a 45-year-old Japanese female diagnosed with low-grade MEC in the hard palate. Radical resection, supraomohyoid neck dissection and antero-lateral thigh flap reconstruction was performed. A MEC cell line was then established from the resected tumor tissue. Short tandem repeat profiling confirmed the origin and authenticity of the cell line, that harbors a CRTC1-MAML2 translocation, which is frequently observed in MEC. Amphiregulin (AREG), identified as one of the targets of the CRTC1-MAML2 fusion gene, was expressed in the cell line. The AREG receptor, epidermal growth factor receptor (EGFR) was also highly phosphorylated. The results predicted that AREG-EGFR signaling, which is required for tumor growth and survival, might be activated in the cell line in a cell-autonomous manner. As AREG expression is associated with EGFR-targeted drug resistance, this cell line might assist with the identification of novel strategies for MEC treatment.

Serum interleukin-6 levels correlate with resistance to treatment of chronic hepatitis C infection with pegylated-interferon-α2b plus ribavirin.

BACKGROUND: Interleukin (IL)-6, a pleiotropic cytokine, is increased in various types of chronic liver disease, including chronic hepatitis C (CHC). It was reported recently that IL-6 is associated with insulin resistance, iron metabolism and interferon resistance, which may affect the outcome of antiviral treatment. In this study, we investigated the association of serum IL-6 levels with outcomes of pegylated interferon (PEG-IFN) plus ribavirin (RBV) combination therapy. METHODS: We included 149 CHC patients and measured serum IL-6 levels at baseline and at 4, 8 and 12 weeks, and the end of treatment in 49 patients. We performed univariate and multivariate regression analyses for the association of IL-6 levels and clinical and laboratory parameters and treatment responses. RESULTS: Serum IL-6 levels were significantly higher in CHC patients than healthy subjects. Pretreatment IL-6 levels of male patients were inversely correlated with sustained virological response (SVR) in univariate analysis (P=0.012). In male patients with SVR, serum IL-6 levels decreased significantly at 4 weeks of treatment (P=0.029) and remained significantly lower than those of non-SVR patients after 4, 8 and 12 weeks of PEG-IFN plus RBV therapy. CONCLUSIONS: Our results suggest that baseline levels of IL-6, as well as their decrease during treatment, are correlated to outcomes of PEG-IFN plus RBV therapy in male patients. Further analyses of IL-6 may provide new strategies for difficult-to-treat CHC patients and prevention of hepatocarcinogenesis.

Analysis of interferon signaling by infectious hepatitis C virus clones with substitutions of core amino acids 70 and 91.

Substitution of amino acids 70 and 91 in the hepatitis C virus (HCV) core region is a significant predictor of poor responses to peginterferon-plus-ribavirin therapy, while their molecular mechanisms remain unclear. Here we investigated these differences in the response to alpha interferon (IFN) by using HCV cell culture with R70Q, R70H, and L91M substitutions. IFN treatment of cells transfected or infected with the wild type or the mutant HCV clones showed that the R70Q, R70H, and L91M core mutants were significantly more resistant than the wild type. Among HCV-transfected cells, intracellular HCV RNA levels were significantly higher for the core mutants than for the wild type, while HCV RNA in culture supernatant was significantly lower for these mutants than for the wild type. IFN-induced phosphorylation of STAT1 and STAT2 and expression of the interferon-inducible genes were significantly lower for the core mutants than for the wild type, suggesting cellular unresponsiveness to IFN. The expression level of an interferon signal attenuator, SOCS3, was significantly higher for the R70Q, R70H, and L91M mutants than for the wild type. Interleukin 6 (IL-6), which upregulates SOCS3, was significantly higher for the R70Q, R70H, and L91M mutants than for the wild type, suggesting interferon resistance, possibly through IL-6-induced, SOCS3-mediated suppression of interferon signaling. Expression levels of endoplasmic reticulum (ER) stress proteins were significantly higher in cells transfected with a core mutant than in those transfected with the wild type. In conclusion, HCV R70 and L91 core mutants were resistant to interferon in vitro, and the resistance may be induced by IL-6-induced upregulation of SOCS3. Those mechanisms may explain clinical interferon resistance of HCV core mutants.

Inhibitory effect of a triterpenoid compound, with or without alpha interferon, on hepatitis C virus infection.

A lack of patient response to alpha interferon (α-IFN) plus ribavirin (RBV) treatment is a major problem in eliminating hepatitis C virus (HCV). We screened chemical libraries for compounds that enhanced cellular responses to α-IFN and identified a triterpenoid, toosendanin (TSN). Here, we studied the effects and mechanisms of action of TSN on HCV replication and its effect on α-IFN signaling. We treated HCV genotype 1b replicon-expressing cells and HCV-J6/JFH-infected cells with TSN, with or without α-IFN, and the level of HCV replication was quantified. To study the effects of TSN on α-IFN signaling, we detected components of the interferon-stimulated gene factor 3 (ISGF3), phosphorylated signal transducer and activator of transcription 1 (STAT1), and STAT2 by Western blotting analysis; expression levels of mRNA of interferon regulatory factor 9 using real-time reverse transcription-PCR (RT-PCR); and interferon-stimulated response element reporter activity and measured the expression levels of interferon-inducible genes for 2',5'-oligoadenylate synthetase, MxA, protein kinase R, and p56 using real-time RT-PCR. TSN alone specifically inhibited expression of the HCV replicon (50% effective concentration = 20.6 nM, 50% cytotoxic concentration > 3 µM, selectivity index > 146). Pretreatment with TSN prior to α-IFN treatment was more effective in suppressing HCV replication than treatment with either drug alone. Although TSN alone did not activate the α-IFN pathway, it significantly enhanced the α-IFN-induced increase of phosphorylated STATs, interferon-stimulated response element activation, and interferon-stimulated gene expression. TSN significantly increased baseline expression of interferon regulatory factor 9, a component of interferon-stimulated gene factor 3. Antiviral effects of treatment with α-IFN can be enhanced by pretreatment with TSN. Its mechanisms of action could potentially be important to identify novel molecular targets to treat HCV infection.

Cholestatic liver fibrosis and toxin-induced fibrosis are exacerbated in matrix metalloproteinase-2 deficient mice.

Matrix metalloproteinase (MMP) plays an important role in homeostatic regulation of the extracellular environment and degradation of matrix. During liver fibrosis, several MMPs, including MMP-2, are up-regulated in activated hepatic stellate cells, which are responsible for exacerbation of liver cirrhosis. However, it remains unclear how loss of MMP-2 influences molecular dynamics associated with fibrogenesis in the liver. To explore the role of MMP-2 in hepatic fibrogenesis, we employed two fibrosis models in mice; toxin (carbon tetrachloride, CCl4)-induced and cholestasis-induced fibrosis. In the chronic CCl4 administration model, MMP-2 deficient mice exhibited extensive liver fibrosis as compared with wild-type mice. Several molecules related to activation of hepatic stellate cells were up-regulated in MMP-2 deficient liver, suggesting that myofibroblastic change of hepatic stellate cells was promoted in MMP-2 deficient liver. In the cholestasis model, fibrosis in MMP-2 deficient liver was also accelerated as compared with wild type liver. Production of tissue inhibitor of metalloproteinase 1 increased in MMP-2 deficient liver in both models, while transforming growth factor ß, platelet-derived growth factor receptor and MMP-14 were up-regulated only in the CCl4 model. Our study demonstrated, using 2 experimental murine models, that loss of MMP-2 exacerbates liver fibrosis, and suggested that MMP-2 suppresses tissue inhibitor of metalloproteinase 1 up-regulation during liver fibrosis.

IL-6-mediated intersubgenotypic variation of interferon sensitivity in hepatitis C virus genotype 2a/2b chimeric clones.

Mechanisms of difference in interferon sensitivity between hepatitis C virus (HCV) strains have yet to be clarified. Here, we constructed an infectious genotype2b clone and analyzed differences in interferon-alpha sensitivity between HCV-2b and 2a-JFH1 clones using intergenotypic homologous recombination. The HCV-2b/JFH1 chimeric virus able to infect Huh7.5.1 cells and was significantly more sensitive to IFN than JFH1. IFN-induced expression of MxA and 25-OAS was significantly lower in JFH1 than in 2b/JFH1-infected cells. In JFH1-infected cells, expression of SOCS3 and its inducer, IL-6, was significantly higher than in 2b/JFH1-infected cells. The IFN-resistance of JFH1 cells was negated by siRNA-knock down of SOCS3 expression and by pretreatment with anti-IL6 antibody. In conclusion, intergenotypic differences of IFN sensitivity of HCV may be attributable to the sequences of HCV structural proteins and can be determined by SOCS3 and IL-6 expression levels.

Comparison of HCV-associated gene expression and cell signaling pathways in cells with or without HCV replicon and in replicon-cured cells.

BACKGROUND: Hepatitis C virus (HCV) replication is affected by several host factors. Here, we screened host genes and molecular pathways that are involved in HCV replication by comprehensive analyses using two genotypes of HCV replicon-expressing cells, their cured cells and naïve Huh7 cells. METHODS: Huh7 cell lines that stably expressed HCV genotype 1b or 2a replicon were used. The cured cells were established by treating HCV replicon cells with interferon-alpha. Expression of 54,675 cellular genes was analyzed by GeneChip DNA microarray. The data were analyzed by using the KEGG Pathway database. RESULTS: Hierarchical clustering analysis showed that the gene-expression profiles of each cell group constituted clear clusters of naïve, HCV replicon-expressed, and cured cell lines. The pathway process analysis between the replicon-expressing and the cured cell lines identified significantly altered pathways, including MAPK, steroid biosynthesis and TGF-beta signaling pathways, suggesting that these pathways were affected directly by HCV replication. Comparison of cured and naïve Huh7 cells identified pathways, including steroid biosynthesis and sphingolipid metabolism, suggesting that these pathways were required for efficient HCV replication. Cytoplasmic lipid droplets were obviously increased in replicon-expressing and cured cells as compared to naïve cells. HCV replication was significantly suppressed by peroxisome proliferator-activated receptor (PPAR)-alpha agonists but augmented by PPAR-gamma agonists. CONCLUSION: Comprehensive gene expression and pathway analyses show that lipid biosynthesis pathways are crucial to support proficient virus replication. These metabolic pathways could constitute novel antiviral targets against HCV.

Antiviral effects of the interferon-induced protein guanylate binding protein 1 and its interaction with the hepatitis C virus NS5B protein.

UNLABELLED: Interferons (IFNs) and the interferon-stimulated genes (ISGs) play a central role in antiviral responses against hepatitis C virus (HCV) infection. We have reported previously that ISGs, including guanylate binding protein 1 (GBP-1), interferon alpha inducible protein (IFI)-6-16, and IFI-27, inhibit HCV subgenomic replication. In this study we investigated the effects of these ISGs against HCV in cell culture and their direct molecular interaction with viral proteins. HCV replication and virus production were suppressed significantly by overexpression of GBP-1, IFI-6-16, or IFI-27. Knockdown of the individual ISGs enhanced HCV RNA replication markedly. A two-hybrid panel of molecular interaction of the ISGs with HCV proteins showed that GBP-1 bound HCV-NS5B directly. A protein truncation assay showed that the guanine binding domain of GBP-1 and the finger domain of NS5B were involved in the interaction. Binding of NS5B with GBP-1 inhibited its guanosine triphosphatase GTPase activity, which is essential for its antiviral effect. Taken together, interferon-induced GBP-1 showed antiviral activity against HCV replication. CONCLUSION: Binding of the HCV-NS5B protein to GBP-1 countered the antiviral effect by inhibition of its GTPase activity. These mechanisms may contribute to resistance to innate, IFN-mediated antiviral defense and to the clinical persistence of HCV infection.