Aberrant hypermethylation in the promoter regions

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1 Aberrant Methylation of Multiple Tumor Suppressor Genes in Aging Liver, Chronic Hepatitis, and Hepatocellular Carcinoma Naoshi Nishida, 1, Takeshi Nagasaka, 1 Takafumi Nishimura, Iwao Ikai, 3 C. Richard Boland, 1 and Ajay Goel 1 Aberrant DNA methylation is an important epigenetic alteration in hepatocellular carcinoma (). However, the molecular processes underlying the methylator phenotype and the contribution of hepatitis viruses are poorly understood. The current study is a comprehensive methylation analysis of human liver tissue specimens. A total of 176 liver tissues, including 77 pairs of s and matching noncancerous liver and normal livers, were analyzed for methylation. Methylation of 19 epigenetic markers was quantified, and the results were correlated with different disease states and the presence or absence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Based on methylation profiles, the 19 loci were categorized into 3 groups. Normal liver tissues showed methylation primarily in group 1 loci (HIC-1, CASP8, GSTP1, SOCS1, RASSF1A, p16, APC), which was significantly higher than group (CDH1, RUNX3, RIZ1, SFRP, MINT31) and group 3 markers (COX, MINT1, CACNA1G, RASSF, MINT, Reprimo, DCC)(P <.1). Noncancerous livers demonstrated increased methylation in both group 1 and group loci. Methylation was significantly more abundant in HCV-positive livers compared with normal liver tissues. Conversely, showed frequent methylation at each locus investigated in all 3 groups. However, the group 3 loci showed more dense and frequent methylation in HCV-positive cancers compared with both HBV-positive cancers and virus-negative cancers (P <.1). Conclusion: Methylation in is frequent but occurs in a gene-specific and disease-specific manner. Methylation profiling allowed us to determine that aberrant methylation is commonly present in normal aging livers, and sequentially progresses with advancing sts of chronic viral infection. Finally, our data provide evidence that HCV infection may accelerate the methylation process and suggests a continuum of increasing methylation with persistent viral infection and carcinogenesis in the liver. (HEPATOLOGY 8;47: ) Aberrant hypermethylation in the promoter regions of tumor suppressor genes is a crucial epigenetic alteration that involves the deregulation of many cellular processes that lead to the initiation and progres- Abbreviations: ANOVA, analysis of variance; CI, confidence interval;, hepatocellular carcinoma; COBRA, combined bisulfite restriction analysis; HBV, hepatitis B virus; HCV, hepatitis C virus; PCR, polymerase chain reaction. From the 1 Division of Gastroenterology, Department of Internal Medicine and Charles A. Sammons Cancer Center and Baylor Research Institute, Baylor University Medical Centre, Dallas, TX; and the Department of Gastroenterology and Hepatology and the 3 Department of Gastroenterological Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan. Received August 6, 7; accepted October 4, 7. Supported by grants R1 CA7851 and R1 CA9857 from the National Cancer Institute, National Institutes of Health, and funds from the Baylor Research Institute. Address reprint requests to: C. Richard Boland, M.D., or Ajay Goel, Ph.D., Baylor University Medical Centre, 35 Gaston Avenue, Gastrointestinal Cancer Research Laboratory, Suite H-5, Dallas, TX rickbo@baylorhealth.edu or ajayg@baylorhealth.edu; fax: Copyright 8 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI 1.1/hep.11 Potential conflict of interest: Nothing to report. Supplementary material for this article can be found on the HEPATOLOGY Web site ( sion of human cancers. 1-3 Several studies have suggested that methylation of multiple tumor suppressor genes in hepatocellular carcinomas (s) may contribute to the pathogenesis of this disease. 4-6 Such epigenetic defects also have been observed in noncancerous liver tissues of patients, supporting the concept that methylationinduced silencing may play a role in the early sts of. 7 Because the non-neoplastic liver tissue in patients is usually accompanied by chronic inflammation, it is conceivable that aberrant methylation seen in the surrounding liver tissue corresponds to the field defect that has been reported for colon and lung cancers. 8,9 Unlike genetic alterations such as mutations and deletions, epigenetic changes are potentially reversible. Exploiting this epigenetic characteristic, several clinical trials are underway evaluating the potential for cancer prevention and therapy through the reversal of methylationinduced alterations. 1-1 Our current understanding suggests that some of the aberrant methylation observed in human cancer may be a consequence of the normal aging process, persistent viral infections, and chronic in- 98

2 HEPATOLOGY, Vol. 47, No. 3, 8 NISHIDA ET AL. 99 Table 1. Clinicopathological Features of Patients with and Normal Liver Feature and Their Corresponding Noncancerous Liver HCV (n 46) HBV (n 15) NBNC (n 18) Normal Liver Cases (n ) Age (years) Distribution Mean 64. ( ) 53.8 ( ) 61.5 ( ) 56.5 (5. 63.) Median Sex Male Female Tumor size.9 cm NA 3. cm Missing Tumor differentiation Well 6 5 NA Moderately poorly Missing 1 HCV denotes HCVAb-positive; HBV denotes HBsAg-positive, and NBNC denotes virus-negative cases. Two cases with both HCVAb and HBsAg positive were counted in both the HCV-positive group and the HBV-positive group. Minimum and maximum of the group. NA, not applicable. Comparisons of distribution between every pair of 4 groups revealed no statistical differences between any pairs using Student t test as well as the Wilcoxon rank-sum test. Similarly, comparisons of sex, tumor size, and tumor differentiation between every pair of groups showed no statistical differences using the chi-squared test or Fisher s exact test. flammation.,13,14 Therefore, it is important to have a detailed understanding of the status of the promoters of genes known to be methylated in association with aging and chronic viral infection. In this study, we quantified methylation densities at 19 CpG loci using combined bisulfite restriction analysis (COBRA) in the liver tissues of patients at various s and compared this with the degrees of methylation seen in and the corresponding nonneoplastic liver tissues. Herein, we report that aberrant methylation of a limited number of loci is commonly seen in the normal aging liver and that these epigenetic alterations gradually progress and expand to a larger panel of methylation markers in. Additionally, we observed that persistent viral infection, particularly hepatitis C, accelerates -related methylation in the liver, suggesting that this may play an important role in the pathogenesis of. Patients and Methods Patients. We analyzed a total of 176 liver tissues, including 77 pairs of and the matched corresponding noncancerous liver tissues, and normal liver tissues. The and noncancerous liver tissues were frozen at 8 C immediately after resection. The hepatitis virus status of patients with were as follows. Thirteen patients were positive for hepatitis B virus surface antigen, 44 were hepatitis C virus (HCV) antibody positive, were positive for both hepatitis B surface antigen and HCV antibody, and 18 were negative for both. The details of the clinical data from the patients, classified by virus status, are listed in Table 1. Among the normal liver tissues, 19 specimens came from patients who had colon cancer with hepatic metastasis. The remaining normal liver tissues were from focal nodular hyperplasia, a hepatic hemangioma, and a hepatic adenoma. All normal liver tissues were confirmed to be free of serum hepatitis B surface antigen and HCV antibody, to have normal serum alanine aminotransferase levels, and to have normal blood platelet counts. Histological data were available for normal livers and demonstrated no evidence of fibrosis or inflammation. Nine of the normal livers were freshfrozen tissues that were stored at 8 C after surgery. The remaining 13 specimens were formalin-fixed paraffin-embedded samples. Written informed consent was obtained from each patient, and the study was approved by the institutional review boards of all the involved institutions. Although the s of the patients with hepatitis B virus (HBV)-related tended to be younger compared with the other groups, there were no statistical differences in the distributions of any clinical data among the HCV-related, HBV-related, virus-negative groups and the normal liver group (Table 1). Methylation Quantification Using COBRA. Genomic DNA was extracted using QIAamp DNA Mini Kit (Qin Inc., Valencia, CA) and TaKaRa DEXPAT kits (Takara Bio Inc., Otsu, Japan) for frozen tissues and

3 91 NISHIDA ET AL. HEPATOLOGY, March 8 paraffin-embedded samples, respectively. Approximately g DNA was subjected to bisulfite modification treatment to convert all the unmethylated cytosines to thymines. We have previously reported that the 19 methylation loci selected for this study exhibited significantly higher methylation levels in compared with corresponding noncancerous liver tissues. 15 The methylation loci included 16 gene promoters: HIC-1, CASP8, GSTP1, SOCS1, RASSF1A, p16, APC, RUNX3, RIZ1, SFRP, CDH1, COX, CACNA1G, RASSF, Reprimo, DCC, and 3 MINT loci: MINT1, MINT, MINT31. Primer sequences, polymerase chain reaction (PCR) conditions, and restriction enzymes used for methylation analysis of p16, CACNA1G, DCC, MINT1, MINT and MINT31, were reported previously. 16,17 The remaining assays were designed in our laboratory, and this information is available on request. Each PCR reaction was performed in a total volume of 5 L, which contained 1.5 L Hot- StarTaq Master Mix (Qin), 4 ng bisulfite-treated DNA template, and. M of each primer pair. After PCR amplification, 5 L of the amplified products were subjected to 5 units of restriction enzyme digestion for the determination of the degree of methylation at each CpG locus. Digested PCR products were electrophoresed on.5% agarose gels and subsequently visualized by ethidium bromide staining. Each assay included a positive control DNA sample that was treated with CpG methylase (CpGenome Universal Methylated DNA; Chemicon International Inc., Temecula, CA), as well as a negative control comprising normal lymphocytic and fibroblast DNA. Band intensities of digested or undigested PCR products were determined using a Kodak Gel-Logic Imaging System (Eastman Kodak Co., Rochester, NY). The band intensities of restriction enzyme digested PCR products (implying a methylated product) were divided by the total of both band intensities, and quantity of methylation was represented as percent density as described. 18 To account for differences attributable to incomplete digestion by the restriction enzyme, the percent methylation of each tumor DNA sample was normalized to that of CpG methylase-treated DNA, which should theoretically result in 1% methylation. 15 The sensitivity of COBRA to detect methylated alleles in our study was as low as %, because we could not distinguish band intensities less than % from the background staining. Therefore, percent methylation level of greater than % was defined as positive for methylation to determine frequencies of methylation of each locus. To validate and ensure the sensitivity and specificity of our COBRA analyses, we designed independent primer pairs (around the same CpG site) for multiple genes/loci. We compared methylation levels obtained from these assays and discovered that in each instance, both primers yielded concordant results regardless of their primer sequences and PCR condition, suggesting the specificity and sensitivity of our COBRA analyses (Supplementary Fig. 1). Additionally, we compared our COBRA data with pyrosequencing analyses for some of the genes and found concordant methylation results by these independent methodologies. Statistical Analysis. For comparing differences between groups of HCV-related, HBV-related, virusnegative patients with and normal liver cases, both Student t test and the Wilcoxon rank-sum test were used. For categorical comparisons of clinical data, the chisquared test or Fisher s exact test were used. The Wilcoxon rank-sum test was also applied to compare the methylation level of each locus between any categorical variables. To examine relationships between and methylation levels of each locus in normal liver, Pearson s correlation and Spearman correlation tests were applied. Spearman s rank correlation test was also used to examine the relationship between methylation densities at different loci. Because hierarchical clustering analysis is most appropriate to statistically discriminate according to methylation levels of 19 loci, we used this approach to classify all into distinct groups (group A and group B). Afterwards, the chi-squared test was used to compare the virus status in the groups. To compare the differences in methylation levels among 3 categorical variables, each of which contained methylation levels of different loci, we calculated Z-scores for normalization. Subsequently, 1-way factorial analysis of variance (ANOVA) and post hoc comparisons (Fisher s PLSD) as well as Kruskal-Wallis tests were performed based on these Z-scores. All P-values were -sided, and P.5 was considered statistically significant. All statistical analyses were calculated using JMP version 4.5J software (SAS Institute Inc., Cary, NC). Results Gene-Specific Methylation Patterns in Liver Tissues from Normal, Noncancerous, and Patients Allows Classification of Methylation Markers into Distinct Groups. In this study, we analyzed methylation frequencies and densities of 19 methylation targets in liver tissue DNA obtained from a series of normal and patients. Methylation analysis of normal liver tissues that were negative for the presence of both HBV and HCV, and showed neither inflammation nor fibrosis, demonstrated low levels of methylation at some of the loci. To determine whether differences in sample processing for frozen and paraffin-embedded tissues may have affected

4 HEPATOLOGY, Vol. 47, No. 3, 8 NISHIDA ET AL. 911 Fig. 1. Distribution of percent methylation levels (box and whiskers plots) and their frequencies (gray bar) at each of the 19 methylation loci (A, B, and C), and overall distribution of methylation level expressed as Z-scores in 3 groups (D, E, and F) as observed in normal liver (A, D), noncancerous liver from patients (B, E), and (C, F). Red boxes and whiskers plots denote 75% and 95% distribution, respectively, and the red lines in the boxes show the median values. Statistically significant by ANOVA and posthoc comparisons. (D) Differences were significant between group 1 versus group, and group 1 versus group 3 [ANOVA; F(, 415) 46.7; P.1, Kruskal-Wallis test; P.1]. (E) Differences were significant between every pair [ANOVA; F(, 146) 153.5, P.1, Kruskal-Wallis test; P.1]. (F) Differences were significant between every pair [ANOVA; F(, 1536) 7.4, P.1, Kruskal-Wallis test; P.1] our results, we obtained both types of tissues from a subset of the patients and performed COBRA analyses on some of the Group 1 loci/markers. We noted that the methylation levels at each marker for a given patient were comparable in DNA obtained from either type of tissue, suggesting that sample processing did not affect our methylation results (data not shown). The overall methylation frequencies and densities in normal liver tissues were significantly lower in comparison with as well as noncancerous liver tissues from patients with. Analyzing normal livers for methylation at 19 loci, we found that 7 of 19 markers (HIC-1, CASP8, GSTP1, SOCS1, RASSF1A, p16, and APC) showed methylation, and the frequencies of methylation ranged between 7.3% and 7.7%, and the mean percent methylation ranged between.7 [95% confidence interval (CI), for p16] and 1.5 (95% CI, for SOCS1) (Table ). Conversely, the remaining 1 methylation loci were either completely unmethylated or showed a negligible degree of methylation. Based on the clear distinction between frequently methylated and unmethylated loci in normal liver tissues, initially, we categorized the 7 loci with methylation as group 1 methylation markers. The distributions of percent methylation of group 1 markers A B C HIC-1 CASP8 Group-1 Group- Group-3 HIC-1 CASP8 GSTP1 SOCS1 RASSF1A p16 APC GSTP1 SOCS1 RASSF1A p16 APC CDH1 RUNX3 RIZ1 SFRP MINT31 Group-1 Group- Group-3 HIC-1 CASP8 GSTP1 SOCS1 RASSF1A p16 APC CDH1 COX MINT1 CACNA1G RASSF MINT Reprimo DCC Group-1 Group- Group-3 CDH1 RUNX3 RIZ1 RUNX3 RIZ1 SFRP MINT31 SFRP MINT31 COX MINT1 CACNA1G RASSF MINT Reprimo DCC COX MINT1 CACNA1G RASSF MINT Reprimo DCC were clearly different from those of the remaining 1 loci (Fig. 1A). Calculating the Z-scores for methylation (Fig. 1D), we found that the methylation of group 1 loci in normal livers was significantly higher compared with the other groups [analysis of variance (ANOVA); F (, 415) 46.7, P.1, Kruskal-Wallis test; P.1]. We next categorized the remaining 1 methylation loci into groups based on methylation frequencies of the noncancerous liver tissues from patients. Methylation analysis of 77 noncancerous liver tissues revealed that only 5 of 1 loci (CDH1, RUNX3, RIZ1, SFRP, and MINT31) were more frequently methylated in these tissues, with the frequencies ranging from 11.7% to 7.8%, and the mean methylation ranging from.4 (95% CI, for RIZ1) to 1.3 (95% CI,.4-. for RUNX3) (Table, Fig. 1B). Conversely, limited evidence of methylation was observed at the remaining 7 of 1 markers (COX, MINT1, CACNA1G, RASSF, MINT, Reprimo and DCC) in these tissues. Comparing Z-scores, it was clear that the methylation frequencies of these 5 markers segregated with noncancerous liver tissues, because the methylation levels of these markers were significantly higher than those of the remaining 7 markers (ANOVA; F D E F Group-1 Group-1 Group-1 Group- Group- Group- Group-3 Group-3 Group-3

5 91 NISHIDA ET AL. HEPATOLOGY, March 8 Table. Comparison of Methylation Status Between Normal Liver and Noncancerous Liver in Patients Normal Liver (n ) Noncancerous Liver From Patients (n 77) Locus Samples Samples P Group 1 HIC-1 16 (7.7) 7. ( ) 49 (63.6) 6.9 ( ) NS CASP8 13 (53.1) 5.8 (.9 8.8) 6 (33.8) 7. ( ) NS GSTP1 13 (53.1) 5. (.8 7.5) 7 (35.1) 7.8 ( ) NS SOCS1 1 (45.5) 1.5 ( ) 53 (68.9) 16.9 (13.5.3) NS RASSF1A 1 (45.5) 6.3 (.1 1.4) 43 (55.8) 11.9 ( ) NS P16 7 (31.8).7 (.6 4.7) 5 (3.5) 3. ( ) NS APC 6 (7.3) 3.3 (.7 5.9) 3 (3.) 7.1 ( ) NS Group CDH1 4 (18.) 1. (.4.8) 1 (7.8) 1.8 (1..5) NS RUNX3 14 (18.1) 1.3 (.4.).34 RIZ1 1 (4.5). (..6) 1 (15,6).4 (1. 3.7) NS SFRP 3 (13.6) 1.5 (.3 3.3) 1 (13.). (.7 3.7) NS MINT31 (9.1) 1.7 ( ) 9 (11.7).3 (.6 3.9) NS Group3 COX (9.1).4 (..9) NS MINT1 (9.1).8 (.3 1.9) NS CACNA1G 1 (4.5). (..5) NS RASSF (.6).3 (.1.8) NS MINT NS Reprimo (.6).4 (..9) NS DCC 1 (1.3).1 (.1.4) NS P value was calculated by the Wilcoxon rank-sum test. Loci that showed methylation of more than 6 samples in normal liver were segregated as group 1. Similarly, loci that showed methylation of more than 9 noncancerous livers were segregated as Group (bold). (, 146) 153.5; P.1, Kruskal-Wallis test; P.1) (Fig. 1E). We classified these 5 loci as group markers and the remaining 7 markers as group 3. Analysis of showed frequent and dense methylation at all loci. Of note, only showed methylation of the 7 loci categorized as group 3. However, collective analysis of the methylation data from all 19 markers revealed that the methylation frequencies were highest in group 1 and least in group 3 loci (ANOVA; F (, 1536) 7.4, P.1, Kruskal-Wallis test; P.1; the difference was significant in every pair by post hoc comparisons) (Fig. 1C, F). Age-Related Methylation Is Present in Normal Liver. As discussed previously, only group 1 loci demonstrated methylation in pathologically and clinically normal liver tissues. These data supported the hypothesis that low levels of methylation accrue as a function of in the normal liver. We therefore analyzed the data using Pearson s as well as Spearman s correlation tests and calculated the and values respectively. Interestingly, we observed that the percent methylation positively correlated with at all 7 group 1 loci (r values ranged from.65 to.44; values ranging from.73 to.8) (Fig. ). In addition, we observed that methylation levels of all group 1 loci in patients s 65 or older were higher compared with those who were younger than 65 years of in normal liver samples (statistically significant for all markers but GSTP1; Supplementary Table 1). These data demonstrated that the methylation of tumor suppressor genes that are reportedly responsible for the development of also takes place in the normal liver as it s. Simultaneous HCV Infection Enhances Age-Related Methylation. We next determined whether hepatitis viruses have any influence on the epigenetic alterations in hepatocarcinogenesis. Figure 3 illustrates the status at all methylation loci within the 3 groups in each of the noncancerous livers, as well as tissues. These tissues have further been classified according to presence or absence of the hepatitis viruses, HCV and HBV. We observed that HCV-positive noncancerous liver tissues were more highly methylated at group 1 and group loci in comparison with HBV-positive or virus-negative cases. In addition, methylation of group 3 loci was rare in noncancerous liver tissues regardless of the viral status. In tissues, although methylation of group 1 loci was more frequent and dense regardless of viral status, HCV-related tended to have higher methylation levels at both group and group 3 loci compared with virus-negative. To determine whether the presence of viral hepatitis might enhance -related methylation detected in normal liver, we compared methylation of group 1 and group loci between normal liver and noncancerous liver tissues classified according to viral status using the Wilcoxon

6 HEPATOLOGY, Vol. 47, No. 3, 8 NISHIDA ET AL r =.57 ρ =.6 HIC r =.61 ρ = r =.48 1 ρ = CASP8 GSTP r =.49 ρ =.45 SOCS r =.44 ρ =.8 RASSF1A r =.5 ρ =.54 p r =.65 ρ =.73 APC r =.46 1 ρ =.55 SFRP Fig.. Correlation between methylation densities at 7 group 1 loci (HIC-1, CASP8, GSTP1, SOCS1, RASSF1A, p16, and APC) and 1 group locus (SFRP ) in normal liver. The r value of Pearson correlation test and value of Spearman correlation test were calculated. rank-sum test. Only 1 locus showed significant differences in methylation levels between normal liver and HBV-positive noncancerous livers (SOCS1; P.193, Supplementary Table ), whereas none of the loci showed a significantly higher methylation level in virus-negative noncancerous liver when compared with normal liver. Conversely, 3 of 7 group 1 loci (SOCS1, RASSF1A, and APC) and 3 of 5 group loci (CDH1, RUNX3, and RIZ1) showed significantly higher levels of methylation in HCV-positive noncancerous tissues than in normal liver (Table 3). Although it did not reach significance, the distribution of methylation levels (mean, median, and maximum percent methylation) at the remaining 4 group 1 and group loci was higher in HCV-positive noncancerous liver than in normal liver (data not shown). Next, to determine whether HCV infection dominates the effect in terms of progression of methylation, we made separate comparisons; first, we examined the relationship between and total number of methylation events in group 1 loci for HCV-positive noncancerous tissues; secondly, we analyzed the relationship between blood platelet counts and methylation events in group 1 loci. We did not find any significant relationship between the number of group 1 methylated loci and in HCV-positive cases (categorized as 65 years of versus 65 years of ; P.937 by Kruskal-Wallis test). However, interestingly enough, patients with lower platelet counts tended to carry increased numbers of methylated loci in their HCV-positive noncancerous livers (platelet count of more than /ml versus less than /ml; P.858 by Kruskal-Wallis test). Because platelet count is known to correlate inversely with increased fibrosis st in patients with chronic hepatitis C, it would seem that methylation events may be influenced as a function of HCV infection rather than aging in HCV-positive cases. Increased Methylation Associates With HCV Infection in. It was critical to understand the impact of hepatitis virus infection on the progression of methylation from a noncancerous st to cancer. To answer this question, we calculated the difference of methylation between the noncancerous liver and in the same patient at every locus. Using hierarchical clustering analysis, we were able to classify all cases according to differences in methylation. As shown in Fig. 4, 39 of 75 s were classified as group A, and 36 were segregated into group B. We excluded the patients from analysis that were positive for the simultaneous presence of both HCV and HBV. We then analyzed differences in methylation between the and noncancerous tissue at each locus in group A and group B (Supplementary Table 3). All but 5 loci showed significantly more methylation in the group B cancers in comparison with group A neoplasms (P value ranged from.1 to.169), indicating that group B cancers represented a subset of with enhanced progression of methylation from surrounding noncancerous liver. Among 36 group B s, 9 (81%) were HCV positive, 5 (14%) were HBV positive, and (5%) were virusnegative. Among the 39 group A s, 15 (38%) were HCV positive, 8 were HBV positive (1%), and 16 (41%) were virus negative (P. by chi-squared test; Table 4). In addition, the proportion of HCV-related was significantly higher in group B compared with group A cancers (HCV-related versus HBV-related or virus-negative; P. by chi-squared test). Group

7 914 NISHIDA ET AL. HEPATOLOGY, March 8 Fig. 3. Distribution of methylation at various subgroups at methylation loci in noncancerous liver tissues of patients and their corresponding s classified according to virus status. Samples were sorted in ascending order based on the number of methylated loci. A hatched square denotes a locus with methylation ( % methylation density), and a solid square denotes a locus with 5% methylation. An open square indicates no methylation. B was predominantly virus positive compared with group A neoplasms. In group B, 34 of 36 (94%) were either HCV-positive or HBV-positive, and only of 36 (6%) were virus-negative, whereas in group A, 3 of 39 s (59%) were virus positive, and 16 of 39 (41%) were virus negative (P.1 by chi-squared test). Table 5 summarizes comparisons of methylation status between HCV-related and virus-negative s in all 19 loci. Among the 19 loci, 1 of 7 group 1 loci (GSTP1),3of 5 group loci (RIZ1, RUNX3, MINT31), and6of7 group 3 loci (CACNA1G, COX, RASSF, MINT1, MINT, and Reprimo) showed significantly higher levels Table 3. Comparison of Methylation Status Between Normal Liver and HCV-Positive Noncancerous Liver of Patients Normal Liver (n ) HCV-Positive Noncancerous Liver (n 46) Locus Samples Samples P Group 1 HIC-1 16 (7.7) 7. ( ) 36 (78.3) 9.1 ( ).1741 CASP8 13 (53.1) 5.8 (.9 8.8) (43.5) 1. ( ).767 GSTP1 13 (53.1) 5. (.8 7.5) 19 (41.3) 1.8 ( ).6545 SOCS1 1 (45.5) 1.5 ( ) 34 (73.9). ( ).4 RASSF1A 1 (45.5) 6.3 (.1 1.4) 8 (6.9) 16.1 ( ).353 P16 7 (31.8).7 (.6 4.7) 1 (45.7) 4.9 (.7 7.1).8 APC 6 (7.3) 3.3 (.7 5.9) 1 (45.7) 11.5 ( ).33 Group CDH1 4 (18.) 1. (.4.8) (43.5).9 ( ).354 RUNX3 11 (3.9) 1.9 (.4 3.4).134 RIZ1 1 (4.5). (..6) 11 (3.9) 3.8 (1.6 6.).411 SFRP 3 (13.6) 1.5 (.3 3.3) 9 (19.6) 3.4 (1. 5.9).4838 MINT31 (9.1) 1.7 ( ) 7 (15.).7 (.7 4.6).543 P value was calculated by Wilcoxon rank-sum test. Bold denotes P.5.

8 HEPATOLOGY, Vol. 47, No. 3, 8 NISHIDA ET AL. 915 Group-A Group-B HIC-1 CASP8 GSTP1 SOCS1 RASSF1A P16 APC CDH1 RUNX3 RIZ1 SFRP MINT31 COX MINT1 CACNA1G RASSF MINT Reprimo DCC HBV positive HCV positive Virus negative Fig. 4. Classification of cases according to differences in methylation between s and their matching noncancerous tissues using hierarchical clustering analysis. Green trees represent group 1 clusters, and red trees represent group clusters. A solid gray circle represents HBV-positive, solid black represent HCV-positive, and an open circle indicates virus-negative cases. of methylation in HCV-related s than virus-negative. Conversely, 1 group 1 locus (GSTP1) and 1 group locus (RIZ1), but no group 3 loci represented higher methylation levels in HBV-related s compared with virus-negative tumors (Supplementary Table 4). Next, we compared the overall distribution of methylation level at the 3 groups of methylation loci expressed as Z-scores in HCV, HBV, and virus-negative. Using ANOVA and post-hoc comparisons (Fisher s PLSD), methylation of group 1 loci was significantly higher in HCV-related than in virus-negative (Fig. 5A) (ANOVA; F(, 5) 4.34, P.134, Kruskal-Wallis test; P.168). Similarly, for group loci, methylation was more prominent in HCV-related or HBV-related compared with non-b, non-c related (Fig. 5B) (ANOVA; F(, 37) 1.17, P.1, Kruskal- Wallis test; P.1). Methylation of group 3 was more prominent in HCV-related compared with HBV-related or virus-negative (Fig. 5C) (ANOVA; F(, 5) 18.31, P.1, Kruskal-Wallis test, P.1). Discussion Epigenetic instability characterized by methylation of multiple cancer-related genes is gaining recognition as a key mechanism of tumor suppressor gene silencing in many human cancers, including. 19, In this study, we performed a detailed quantitative methylation analysis of a large number of methylation loci in normal aging liver, noncancerous liver tissues from patients, and neoplastic tissues. The results presented herein clearly demonstrate that some degree of methylation occurs in the context of the normal aging process in the liver, and it is likely that some of these methylation events may sequentially progress and participate in the development of hepatic neoplasia. Another important observation is that the presence of hepatitis viruses, especially HCV, Table 4. Relationships Between Groups of Cancers Classified According to the Differences in Methylation Levels Between and the Matched Noncancerous Liver, and the Presence of Hepatitis Viruses Virus-Positive Group HCV-Related HBV-Related Virus-Negative P Group A (n 39) 15 (38%; 15/39) 8 (1%; 8/39) 16 (41%; 16/39) Group B (n 36) 9 (81%; 9/36) 5 (14%; 5/36) (5%; /36). P value by chi-squared test (HCV related versus HBV related versus virus-negative ). Difference was also significant between virus-positive versus virus-negative (P.1), and HCV-related versus HBV-related or virus-negative (P.).

9 916 NISHIDA ET AL. HEPATOLOGY, March 8 Table 5. Comparison of Methylation Status Between HCV-Related and Virus-Negative HCV-Related (n 44) Virus-Negative (n 18) Locus Samples Samples P Group 1 HIC-1 38 (86.4) 34. (7. 4.9) 14 (77.8) 5.7 ( ).19 CASP8 19 (43.) 17.8 (1. 5.3) 9 (5.) 15.5 ( ).956 GSTP1 38 (86.4) 59. ( ) 13 (7.) 34.8 ( ).155 SOCS1 9 (65.9) 4.1 ( ) 1 (66.7) 37.6 ( ).7758 RASSF1A 38 (86.4) 33.4 ( ) 16 (88.9) 8.3 ( ).5814 P16 35 (79.5) 3. ( ) 1 (66.7).1 ( ).3 APC 4 (9.9) 5.5 ( ) 14 (77.8) 41.6 ( ).1947 Group CDH1 (45.5) 4.5 (.5 6.5) 7 (38.9).8 (.8 4.9).564 RUNX3 36 (81.8) 3.7 ( ) 7 (38.9) 8.1 ( ).9 RIZ1 36 (4.5) 33.4 ( ) 6 (33.3) 8.8 ( ). SFRP 13 (9.5) 9.8 ( ) (11.1).4 ( ).1167 MINT31 33 (75.) 8.3 ( ) 7 (38.9) 9.9 ( ).63 Group 3 COX (45.5) 16. (8.9 3.) 3 (16.7) 4.1 (.8 9.).355 MINT1 (45.5) 1.9 ( ) 1 (5.6).7 (.7.1).4 CACNA1G 3 (88.6) 18.8 ( ) 6 (33.3).7 (.7 6.6).61 RASSF 1 (47.7) 13.4 ( ) 1 (5.6) 1.8 (. 5.5).9 MINT 17 (38.6) 9.4 ( ).5 Reprimo 13 (9.5) 6.4 (.6 1.) 1 (5.6).4 (.5 1.4).315 DCC 4 (9.1) 1.6 (.1 3.) 1 (5.6) 1.3 ( ).6694 Two cases that were both HBV positive and HCV-positive were excluded from this analysis. P value calculated by Wilcoxon rank-sum test. Bold denotes P value.5. could play a role in accelerating the methylation process that is involved in development. In this study, we first categorized all CpG methylation loci into 3 groups according to the frequencies of methylation in various subsets of liver tissues to better understand gene-specific characteristics of individual CpG loci. This categorization allowed us to appreciate that group 1 loci demonstrate methylation in the normal liver in association with increasing, suggesting that these loci may get methylated as a function of, because these may be weakly protected in the aging liver against methylation alterations. 1 Conversely, we noted hypermethylation of both group 1 and group loci in noncancerous liver. tissues carried much more frequent and dense methylation at all methylation markers, but the group 3 markers were solely methylated in. The differences in frequency and pattern of methylation among various sts of liver disease suggest that methylation progresses sequentially from group 1 to group 3 loci with the progression of disease. We observed significantly higher levels of methylation in as well as the corresponding non-cancerous liver tissues compared to normal tissue. We also found that the background liver tissues in patients, where chronic viral infection and inflammation are common, also carried concordant methylation of multiple genes. Because HCV is a common cause of chronic liver disease and worldwide, we speculated that chronic infection by HCV might accelerate the methylation process during hepatocarcinogenesis. Comparison of methylation levels between normal liver and noncancerous livers of HCVpositive patients demonstrated that several markers showed significantly higher methylation in HCV-positive cases, which contrasts with HBV-positive and virus-negative cases. More frequent and denser methylation of all 19 loci was a characteristic feature of ; however, clear differences in methylation were also evident between HCV-positive and virus-negative in all 3 subgroups of methylation loci. Previous reports have suggested that methylation of GSTP1 and p16 are frequent in HBV-related. 1, In our study, significant differences in methylation density were detected at GSTP1 and RIZ1 between HBVrelated and virus-negative. In addition, overall methylation levels of group loci were significantly higher in HBV-related than in virus-negative. Because HBV-related patients tend to be younger than virus-negative patients, and because methylation events could be affected by the aging process, HBV infection also may play a role in the progression of methylation in these patients. Conversely, the highest levels of methylation, which correlated with group 3 loci, appeared to be cancer-specific and were unique for HCVrelated only. Previous reports suggest that methyl-

10 HEPATOLOGY, Vol. 47, No. 3, 8 NISHIDA ET AL. 917 A 1-1 B 3 1 C 6 HCV-related HCV-related Group-1 locus HBV-related Group- locus HBV-related Group-3 locus virus-negative virus-negative ation of SOCS-1 and APC, p15 was more frequently observed in HCV-related s than in virus-negative s, 6 whereas another report failed to show a clear relationship between methylation of specific loci and HCV in tissues. 5 However, most of the previous studies were reported based on nonquantitative methylation assays. Because the biological meaning of methylation may be attributed according to its density at a given locus, in the current study we performed quantitative methylation analyses for individual genes. Based on our data, we could clearly demonstrate methylation differences in HCV-positive, HBV-positive, and virus-negative cases. In addition, we classified all s using hierarchical clustering analysis to account for the differences in methylation between s and the noncancerous liver tissues. Even using this approach, it was clear that infection with hepatitis virus, especially HCV, strongly associated with the progression of methylation. Our study showed that group 3 loci carried more methylation in HCV-related s than HBV-related and virus-negative s. This was not merely attributed to, because even though HCV-positive cases were older than HBV-positive cases, there were no differences between HCV-positive and virus-negative cases. In addition, we could not find any association between and methylation level of group 3 loci for tissues in the HCV-positive group (data not shown). Similarly, increased methylation levels at group 1 and group loci in HCV-positive noncancerous liver could not be attributed to because there were no differences between HCV-positive and virus-negative patients. As it is believed that disruption of the balance between methylation pressure and the protective mechanisms may be responsible for the induction of aberrant DNA methylation, 1 we can speculate that protection against the spread of methylation is weaker at group 1 loci and strongest at group 3 loci. However, chronic HCV infection may act as a powerful epi-mutn and may induce methylation 4 HCV-related HBV-related virus-negative 4 Fig. 5. Distribution of methylation density (box and whiskers plots) of group 1 (A), group (B), and group 3 loci (C) in classified as HCV-related, HBV-related, and virus-negative. Each density was expressed as a Z-score. Red boxes and whiskers denote 75% and 95% distributions, respectively, and the red lines in the boxes showed median values. Differences were significant by ANOVA and post hoc comparisons. Methylation of group 1 loci were significantly higher in HCV-related than in virus-negative (A) [ANOVA; F(, 5) 4.34, P.134, Kruskal-Wallis test; P.168]. Similarly, methylation was prominent in HCV-related and HBV-related compared with non-b non-c related at group loci. (B) [ANOVA; F(, 37) 1.17, P.1, Kruskal-Wallis test; P.1], and methylation of group 3 was prominent in HCV-related compared with HBV-related and virusnegative. (C) [ANOVA; F(, 5) 18.31, P.1, Kruskal- Wallis test, P.1].

11 918 NISHIDA ET AL. HEPATOLOGY, March 8 even at group 3 loci, making the group 3 loci unique HCV-associated events in hepatic carcinogenesis. The data presented here also suggest that methylation causes disruption of a variety of genes and pathways during hepatocarcinogenesis, such as RB-related (p16, RIZ1), p53-related (HIC-1, Reprimo), WNT/APC (APC, SFRP, CDH1), receptor-tyrosine kinase-associated (RASSF1A, RASSF, SOCS-1), transforming growth factor beta signaling (RUNX3), and apoptosis-related pathways (CASP8). These disruptions are supposed to act in concert and may play an active role in. It is well known that tumor cells need to accumulate several ratelimiting mutations for cancer development. In this regard, we can speculate that continuous exposure to an epi-mutn, such as HCV, will induce the disruption of multiple genes and pathways for cancer development. 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