Ryosuke Muroyama

Genome-wide association study identifies a susceptibility locus for HCV-induced hepatocellular carcinoma

To identify the genetic susceptibility factor(s) for hepatitis C virus–induced hepatocellular carcinoma (HCV-induced HCC), we conducted a genome-wide association study using 432,703 autosomal SNPs in 721 individuals with HCV-induced HCC (cases) and 2,890 HCV-negative controls of Japanese origin. Eight SNPs that showed possible association (P < 1 × 10−5) in the genome-wide association study were further genotyped in 673 cases and 2,596 controls. We found a previously unidentified locus in the 5′ flanking region of MICA on 6p21.33 (rs2596542, Pcombined = 4.21 × 10−13, odds ratio = 1.39) to be strongly associated with HCV-induced HCC. Subsequent analyses using individuals with chronic hepatitis C (CHC) indicated that this SNP is not associated with CHC susceptibility (P = 0.61) but is significantly associated with progression from CHC to HCC (P = 3.13 × 10−8). We also found that the risk allele of rs2596542 was associated with lower soluble MICA protein levels in individuals with HCV-induced HCC (P = 1.38 × 10−13).

MDM2 Promoter SNP309 Is Associated with the Risk of Hepatocellular Carcinoma in Patients with Chronic Hepatitis C

Purpose: A single nucleotide polymorphism (SNP) in the promoter region of MDM2 gene, SNP309, has recently been shown to be associated with accelerated tumor formation in both hereditary and sporadic cancers in humans. However, the association of SNP309 with hepatocellular carcinoma is unknown. We evaluated the association of SNP309 with the risk of hepatocellular carcinoma development among Japanese patients with chronic hepatitis C virus infection. Experimental Design: We genotyped the SNP309 at the MDM2 promoter in 435 Japanese patients with chronic hepatitis C virus infection, including 187 patients with hepatocellular carcinoma and 48 healthy subjects, using a fluorogenic PCR. Presence of SNP was also confirmed by direct sequencing of the MDM2 promoter region. Results: The proportion of G/G genotype of the SNP309 in patients with hepatocellular carcinoma (33%) was significantly higher than that in patients without hepatocellular carcinoma (23%), with an odds ratio (95% confidence interval) of 2.28 (1.30-3.98). A multivariate analysis revealed that MDM2 SNP309 (G/G versus T/T), age >60 years, male gender, presence of cirrhosis, serum α-fetoprotein >20 μg/L, and serum albumin <3.2 g/dL were independently associated with the hepatocellular carcinoma development at odds ratio of 2.27, 2.46, 3.08, 4.15, 4.87, and 6.33, respectively. Conclusions: The MDM2 promoter SNP309 is associated with the presence of hepatocellular carcinoma in Japanese patients with chronic hepatitis C.

Down-regulation of hedgehog-interacting protein through genetic and epigenetic alterations in human hepatocellular carcinoma.

Purpose: Hedgehog (Hh) signaling is activated in several cancers. However, the mechanisms of Hh signaling activation in hepatocellular carcinoma (HCC) have not been fully elucidated. We analyzed the involvement of Hh-interacting protein (HHIP) gene, a negative regulator of Hh signaling, in HCC. Experimental Design: Glioma-associated oncogene homologue (Gli) reporter assay, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, and quantitative real-time reverse transcription–PCR for the target genes of the Hh signals were performed in HHIP stably expressing hepatoma cells. Quantitative real-time PCR for HHIP was performed in hepatoma cells and 36 HCC tissues. The methylation status of hepatoma cells and HCC tissues was also analyzed by sodium bisulfite sequencing, demethylation assay, and quantitative real-time methylation-specific PCR. Loss of heterozygosity (LOH) analysis was also performed in HCC tissues. Results:HHIP overexpression induced significant reductions of Gli reporter activity, cell viability, and transcription of the target genes of the Hh signals. HHIP was hypermethylated and transcriptionally down-regulated in a subset of hepatoma cells. Treatment with a demethylating agent led to the HHIP DNA demethylation and restoration of HHIP transcription. HHIP transcription was also down-regulated in the majority of HCC tissues, and more than half of HCC tissues exhibited HHIP hypermethylation. The HHIP transcription level in HHIP-methylated HCC tissues was significantly lower than in HHIP-unmethylated HCC tissues. More than 30% of HCC tissues showed LOH at the HHIP locus. Conclusions: The down-regulation of HHIP transcription is due to DNA hypermethylation and/or LOH, and Hh signal activation through the inactivation of HHIP may be implicated in the pathogenesis of human HCC.

Potential contribution of tumor suppressor p53 in the host defense against hepatitis C virus.

Infection by hepatitis C virus (HCV) usually results into chronic hepatitis that can ultimately lead to cirrhosis and hepatocellular carcinoma. Type 1 interferons (IFN‐α/β) constitute the primary cellular defense against viral infection including HCV. IFN binding to their receptors activates associated Jak1 and Tyk2 kinases, which ultimately leads to phosphorylation and assembly of a signal transducer and activator of transcription protein (STAT)1‐STAT2‐interferon regulatory factor (IRF)9 trimetric complex called interferon‐stimulated gene factor 3 that translocates into the nucleus and binds to the interferon‐ stimulated response elements (ISRE), leading to transcriptional induction of several antiviral genes, including double‐stranded RNA‐activated protein kinase (PKR), 2′,5′‐ oligoadenylate synthetase (OAS), and myxovirus resistance protein A (MxA). Understanding the mechanisms of how the virus evades this cellular innate defense and establishes a chronic infection is the key for the development of better therapeutics against HCV infection. Here, we demonstrate that p53 could have a crucial role in the cellular innate defense against HCV. We observed significantly higher levels of HCV RNA replication and viral protein expression in the Huh7 cells when their p53 expressions were knocked down. Moreover, IFN treatment was less effective in inhibiting the HCV RNA replication in the p53–knocked‐down (p53kd) Huh7 cells. In fact, the activation of the ISRE and the induction of ISGs were significantly attenuated in the p53kd Huh7 cells and p53 was found to directly interact with IRF9. Conclusion: These observations underscore the potential contributions of the tumor suppressor p53 in cellular antiviral immunity against HCV with possible therapeutic implications. (HEPATOLOGY 2008.)

Soluble MICA and a MICA Variation as Possible Prognostic Biomarkers for HBV-Induced Hepatocellular Carcinoma

MHC class I polypeptide-related chain A (MICA) molecule is induced in response to viral infection and various types of stress. We recently reported that a single nucleotide polymorphism (SNP) rs2596542 located in the MICA promoter region was significantly associated with the risk for hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) and also with serum levels of soluble MICA (sMICA). In this study, we focused on the possible involvement of MICA in liver carcinogenesis related to hepatitis B virus (HBV) infection and examined correlation between the MICA polymorphism and the serum sMICA levels in HBV-induced HCC patients. The genetic association analysis revealed a nominal association with an SNP rs2596542; a G allele was considered to increase the risk of HBV-induced HCC (P = 0.029 with odds ratio of 1.19). We also found a significant elevation of sMICA in HBV-induced HCC cases. Moreover, a G allele of SNP rs2596542 was significantly associated with increased sMICA levels (P = 0.009). Interestingly, HCC patients with the high serum level of sMICA (>5 pg/ml) exhibited poorer prognosis than those with the low serum level of sMICA (≤5 pg/ml) (P = 0.008). Thus, our results highlight the importance of MICA genetic variations and the significance of sMICA as a predictive biomarker for HBV-induced HCC.

A genome-wide association study of HCV-induced liver cirrhosis in the Japanese population identifies novel susceptibility loci at the MHC region

BACKGROUND & AIMS We performed a genome-wide association study (GWAS) of hepatitis C virus (HCV)-induced liver cirrhosis (LC) to identify predictive biomarkers for the risk of LC in patients with chronic hepatitis C (CHC). METHODS A total of 682 HCV-induced LC cases and 1045 CHC patients of Japanese origin were genotyped by Illumina Human Hap 610-Quad bead Chip. RESULTS Eight SNPs which showed possible associations (p<1.0 × 10(-5)) at the GWAS stage were further genotyped using 936 LC cases and 3809 CHC patients. We found that two SNPs within the major histocompatibility complex (MHC) region on chromosome 6p21, rs910049 and rs3135363, were significantly associated with the progression from CHC to LC (pcombined=9.15 × 10(-11) and 1.45 × 10(-10), odds ratio (OR)=1.46 and 1.37, respectively). We also found that HLA-DQA1(*)0601 and HLA-DRB1(*)0405 were associated with the progression from CHC to LC (p=4.53 × 10(-4) and 1.54 × 10(-4) with OR=2.80 and 1.45, respectively). Multiple logistic regression analysis revealed that rs3135363, rs910049, and HLA-DQA1(*)0601 were independently associated with the risk of HCV-induced LC. In addition, individuals with four or more risk alleles for these three loci have a 2.83-fold higher risk for LC than those with no risk allele, indicating the cumulative effects of these variations. CONCLUSIONS Our findings elucidated the crucial roles of multiple genetic variations within the MHC region as prognostic/predictive biomarkers for CHC patients.

The flavonoid apigenin inhibits hepatitis C virus replication by decreasing mature microRNA122 levels

Despite recent progress in the development of direct-acting antivirals against hepatitis C virus (HCV), chronic HCV infection remains an important health burden worldwide. MicroRNA122 (miR122), a liver-specific microRNA (miRNA), positively regulates HCV replication, and systemic application of antisense oligonucleotides against miR122 led to the long-lasting suppression of HCV viremia in human clinical trials. Here, we report that apigenin, a flavonoid and an inhibitor of maturation of a subset of miRNAs, inhibits HCV replication in vitro. Apigenin decreased the expression levels of mature miR122 without significantly affecting cell growth. Because supplementation of synthesized miR122 oligonucleotides or overexpression of constitutively active TRBP blocked these effects, the inhibitory effects of apigenin on HCV replication seemed to be dependent on the reduction of mature miR122 expression levels through inhibition of TRBP phosphorylation. Thus, apigenin intake, either through regular diet or supplements, may decrease HCV replication in chronically infected patients.

Characteristic mutations in hepatitis C virus core gene related to the occurrence of hepatocellular carcinoma

Chronic hepatitis C virus (HCV) infection often results in hepatocellular carcinoma (HCC). Previous studies have shown that there might be some characteristic mutations in the core region of HCV related to HCC. Thus, we downloaded and analyzed HCV genotype 1b core gene sequences from HCV databases online to identify them. Based on the information of the sequences, 63 from patients with HCC and 188 from non‐HCC were enrolled into our analysis. Then, the nucleotides at each position were compared by χ2‐test between the two groups, and 24 polymorphisms were found to be associated with HCC. Further analysis of these 24 polymorphisms by logistic regression indicated that eight were significantly related to the increased HCC risk: A028C, G209A, C219U/A, U264C, A271C/U, C378U/A, G435A/C, and G481A. Moreover, U303C/A was associated with the decreased HCC risk. These mutations could bring about four amino acid substitutions: K10Q, R70Q, M91L, and G161S. In conclusion, eight characteristic mutations in the HCV‐1b core gene related to the occurrence of HCC were identified. The structural and functional alterations of core protein due to these mutations and the relationship with the occurrence of HCC need to be further studied. (Cancer Sci 2009; 100: 2465–2468)

Polymorphism of OAS-1 determines liver fibrosis progression in hepatitis C by reduced ability to inhibit viral replication.

Background: Progression of disease after hepatitis C virus (HCV) infection differs among individuals, indicating a possibility of participation of host genetic factors. 2′‐5′‐oligoadenylate synthetase 1 (OAS‐1), an important component of the innate immune system, has an antiviral function, and may therefore have a certain relationship with progression of disease.

Absence of tyrosine kinase mutations in Japanese colorectal cancer patients

Tyrosine kinases, which are important regulators of intracellular signal-transduction pathways, have mutated forms that are often associated with oncogenesis and are attractive targets for therapeutic intervention. Recently, systematic mutational analyses of tyrosine kinases revealed that a minimum of 30% of colorectal cancer contain at least one mutation in the tyrosine kinases. To further explore these mutations, we examined all reported mutations of NTRK3, FES, KDR, EPHA3, NTRK2, JAK1, PDGFRA, EPHA7, EPHA8, ERBB4, FGFR1, MLK4 and GUCY2F genes in the 24 colorectal cancer cell lines. Unexpectedly, among 24 colorectal cancer cell lines, only two cell lines (LoVo and CaR1) harbored mutation C1408T (R470C) in MLK4 gene. The mutation rate was extremely low compared to that previously reported. Therefore, we analyzed mutations in 46 colorectal cancer samples resected from the same number of Japanese patients. Surprisingly, none of the 46 samples contained any of the mutations reported. Based on our study, we advise that a more comprehensive tyrosine kinase gene mutation assay is necessary in the future.