Aberrant DNA methylation precedes loss of heterozygosity on chromosome 16 in chronic hepatitis and liver cirrhosis

The aim of this study was to examine the significance of aberrant DNA methylation, the participation of which in genetic instability is controversial, in hepatocarcinogenesis. The DNA methylation status of the region around the promoter of the E-cadherin tumor suppressor gene, which is located on 16q22.1, and the allelic status at the D16S421 locus, which is adjacent to the E-cadherin locus, were examined using microdissected liver specimens from 38 hepatocellular carcinoma (HCC) patients. Almost all of the non-cancerous liver tissues showed histological findings compatible with chronic hepatitis and cirrhosis, which are considered to be precancerous conditions. DNA hypermethylation was detected in 61% of the non-cancerous liver tissues. The incidence of DNA hypermethylation in the non-cancerous liver tissues of patients with HCCs also showing DNA hypermethylation (72%) was significantly higher than that of patients without DNA hypermethylation in their HCCs (53%, P<0.05). Loss of heterozygosity (LOH) at the D16S421 locus was detected in 35% of the non-cancerous liver tissues. The incidence of LOH in the non-cancerous liver tissues of patients with HCCs also showing LOH was 78%, whereas LOH was not detected in non-cancerous liver tissues of patients without LOH in their HCCs. Fifty-two percent of the non-cancerous liver tissues showed both or neither of DNA hypermethylation and LOH; the incidence of DNA hypermethylation alone in noncancerous liver tissue was 41%. The incidence of LOH alone in non-cancerous liver tissue (7%) was significantly lower compared to those of the former two cases (P<0.0001). These data suggest that aberrant DNA methylation participates in the precancerous stage of hepatocarcinogenesis by preceding, or causing, LOH.     

Aberrant DNA Methylation on Chromosome 16 Is an Early Event in Hepatocarcinogenesis

In order to clarify the significance of DNA methylation in both earlier and later stages of hepatocarcinogenesis, the DNA methylation state on chromosome 16, on which loss of heterozygosity (LOH) has frequently been detected in human hepatocellular carcinomas (HCCs), was examined. DNA from primary HCCs and tissues showing chronic hepatitis and liver cirrhosis, which are considered to be precancerous conditions, was analyzed by digestion with methylation-sensitive and non-sensitive restriction enzymes. DNA hypermethylation at the D16S32, tyrosine aminotransferase (TAT) and D16S7 loci and hypomethylation at the D16S4 locus were detected in 18%, 58%, 20% and 48% of examined HCCs, respectively. Aberrant DNA methylation occurred more frequently in advanced HCCs than in early HCCs. Moreover, DNA hypermethylation at the D16S32, TAT and D16S7 loci was frequently observed in chronic hepatitis and liver cirrhosis. The incidence of DNA hypermethylation was higher than that of LOH (42% at the TAT locus). These data suggest that DNA hypermethylation might predispose the locus to allelic loss. Aberrant DNA methylation is a significant change which may participate in the early developmental stages of HCCs.     

Increased protein expression of DNA methyltransferase (DNMT) 1 is significantly correlated with the malignant potential and poor prognosis of human hepatocellular carcinomas

Alteration of DNA methylation is one of the most consistent epigenetic changes in human cancers. DNA methyltransferase (DNMT) 1 is a major enzyme involved in establishing genomic methylation patterns. Most of the studies concerning DNMT1 expression in human cancers have been performed only at the mRNA level. To directly examine DNMT1 protein expression levels during human hepatocarcinogenesis, 16 histologically normal liver tissues, 51 noncancerous liver tissues exhibiting chronic hepatitis or cirrhosis, which are considered to be precancerous conditions, and 53 hepatocellular carcinomas (HCCs) were subjected to immunohistochemic examination. If more than 20% of the cells exhibited nuclear DNMT1 staining, the tissue sample was considered to be DNMT1-positive. DNMT1 immunoreactivity was observed in 23 (43%) of the HCCs, but in none (0%) of the histologically normal liver or noncancerous liver tissues exhibiting chronic hepatitis or cirrhosis. The incidence of increased DNMT1 protein expression in HCCs correlated significantly with poor tumor differentiation (p = 0.0006) and portal vein involvement (p = 0.0002). Moreover, the recurrence-free (p = 0.0001) and overall (p < 0.0001) survival rates of patients with HCCs exhibiting increased DNMT1 protein expression were significantly lower than those of patients with HCCs that did not exhibit increased expression. Increased DNMT1 protein expression may play a critical role in the malignant progression of HCCs and be a biologic predictor of both HCC recurrence and a poor prognosis in HCC patients.     

The E-cadherin gene is silenced by CpG methylation in human oral squamous cell carcinomas

Reduction of E-cadherin strongly relates to invasiveness and metastasis in vitro. To clarify CpG methylation around the promoter region of the E-cadherin gene in oral squamous cell carcinoma (SCC), we examined the DNA samples of various human SCC cell lines and primary oral SCC tissues by methylation-specific polymerase chain reaction (MSP). CpG methylation of the E-cadherin gene markedly correlated to the reduction of E-cadherin expression in human oral SCC cell lines. In primary oral SCC tissues, only 1 of 5 preserved E-cadherin-expressing tissues was methylated, whereas methylation was found in 17 (94.4%) of 18 E-cadherin-reduced tissues. Our results suggest that reduction of E-cadherin expression is associated with CpG methylation of the E-cadherin gene promoter. We recently established two cell lines with high and low metastatic potential, UM1 and UM2, from SCC primary tongue tissue of a patient. E-cadherin expression of high-metastatic UM1 was clearly lower than that of low-metastatic UM2, and MSP results showed CpG methylation in the UM1 but not the UM2 cell line. To investigate whether demethylation of CpG methylation of the E-cadherin gene could restore expression and function of E-cadherin, we treated UM1 with the demethylating agent 5-azacytidine (5-aza) and found that E-cadherin expression was indeed restored by demethylation. Moreover, in the demethylated UM1, invasion of the collagen gel was clearly suppressed compared with the untreated UM1. These results suggested that inactivation of E-cadherin expression resulted from CpG methylation of the gene promoter; a correlation between CpG methylation of the E-cadherin gene promoter and invasive potential was also suggested.     

Differential DNA methylation associated with hepatitis B virus infection in hepatocellular carcinoma

Gene inactivation through DNA hypermethylation plays a pivotal role in carcinogenesis. This study aimed to profile aberrant DNA methylation in different stages of liver disease, namely noncirrhosis, cirrhosis and hepatocellular carcinoma (HCC), and also to clarify the influence of hepatitis B virus (HBV) infection on the aberrant DNA methylation in HCCs. Promoter methylation in p14(ARF), p16(INK4a), O(6)-methylguanine-DNA methyltransferase (MGMT), glutathione S-transferase pi (GSTP1) and E-cadherin (E-Cad) genes of 58 HCCs paired with adjacent nontumorous tissues was assayed by methylation-specific PCR. HBV infection was determined using a hepatitis B virus surface antigen (HBsAg) serological assay. The frequency of p16(INK4a) promoter methylation increased from noncirrhotic, cirrhotic, to HCC tissues (noncirrhotic vs. HCC, p < 0.001), while that of GSTP1 promoter methylation increased in cirrhotic tissues compared to noncirrhotic ones (p = 0.029). The frequency of GSTP1 promoter hypermethylation is significantly higher in HCC than in nontumorous tissues (p = 0.022) from HBsAg-positive patients, but not the HBsAg-negative controls (p = 0.289). While the frequency of E-Cad promoter hypermethylation remained high in both nontumorous tissues and HCCs from HBsAg-positive patients (p = 0.438), it was lower in HCCs than in nontumorous tissues from HBsAg-negative patients (p = 0.002). In contrast, the frequency of p16(INK4a), MGMT and p14(ARF) promoter hypermethylation in HCCs was unrelated to HBsAg status. In conclusion, aberrant DNA methylation may begin at different stages of liver disease in a gene-dependent manner. Moreover, HBV infection may enhance or maintain GSTP1 and E-Cad promoter methylation and thereby affect hepatocarcinogenesis.     

Carcinogenetic risk estimation based on quantification of DNA methylation levels in liver tissue at the precancerous stage

For appropriate surveillance of patients at the precancerous stage for hepatocellular carcinomas (HCCs), carcinogenetic risk estimation is advantageous. The aim of our study was to establish criteria for such estimation based on DNA methylation profiling. The DNA methylation status of 203 CpG sites on 25 bacterial artificial chromosome (BAC) clones, whose DNA methylation status had been proven to discriminate samples of noncancerous liver tissue obtained from patients with HCC (N) from normal liver tissue (C) samples by BAC array-based methylated CpG island amplification, was evaluated quantitatively using pyrosequencing. The 45 CpG sites whose DNA methylation levels differed significantly between C and N in the learning cohort (n=22) were identified. The criteria combining DNA methylation status for the 30 regions including the 45 CpG sites were able to diagnose N as being at high risk of carcinogenesis with 100% sensitivity and specificity in the learning cohort and 95.6% sensitivity and 100% specificity in the validation (n=90) cohort. DNA methylation status for the 30 regions in N samples was significantly correlated with the outcome of patients with HCCs, indicating that clinicopathologically valid DNA methylation alterations have already accumulated at the precancerous stage. The DNA methylation status of the 30 regions did not depend on the presence or absence of hepatitis virus infection, or the status of noncancerous liver tissue (chronic hepatitis or cirrhosis). These criteria may be applicable for carcinogenetic risk estimation using liver biopsy specimens obtained from patients who are followed up because of chronic liver diseases.     

CpG methylation of MGMT and hMLH1 promoter in hepatocellular carcinoma associated with hepatitis viral infection

Inactivations of DNA repair genes, O(6)-methylguanine-DNA methyltransferase (MGMT) and hMLH1, by promoter hypermethylation have been reported in several types of primary human neoplasia. This epigenetic inactivation mechanism remains elusive in hepatocellular carcinoma (HCC). To investigate the relation between the expression of MGMT and hMLH1 and the CpG methylation within their promoters in HCCs with or without hepatitis viral infection, we performed immunohistochemistry and urea/bisulphite sequencing on 46 HCCs, corresponding noncancerous tissues, and 20 normal liver tissues. MGMT- and hMLH1-negative HCCs were 60.9% (28 out of 46) and 21.8% (10 out of 46), respectively. HCCs lacking both proteins were 10.9% (five out of 46). The frequency and extent of CpG methylation in the MGMT promoter increased along with hepatitis viral infection and pathological progression. MGMT-negative tumours showed very frequent and widespread methylation in the promoter compared with MGMT-positive tumours. Half of the hMLH1-negative HCCs showed promoter hypermethylation. These data suggested that MGMT gene silencing in a subset of HCCs was likely caused by epigenetic alteration, such as promoter hypermethylation, and that the promoter hypermethylation silenced the hMLH1 gene in half of the hMLH1-negative tumours. A correlation between the promoter methylation status and viral infection, although it was weak, intimated that hepatitis viral infections could play a role in the CpG methylation of the MGMT promoter.     

肺癌E-cadherin基因启动子CpG岛甲基化与蛋白表达的关系及其意义

背景与目的：目前认为CpG岛甲基化导致转录抑制是恶性肿瘤发生的重要机制之一.E-cadherin能抑制肿瘤细胞的浸润和转移,被公认为是浸润、转移抑制基因.本研究检测肺癌组织中E-cadherin基因启动子CpG岛甲基化的状况,并探讨基因异常甲基化与蛋白表达的关系及其意义.方法：采用甲基化特异性PCR技术,检测22例肺癌组织,相应的癌旁组织和9例正常肺组织中E-cadherin基因启动子CpG岛甲基化的状况.采用免疫组化S-P法相应检测了E-cadherin蛋白的表达.结果：肺癌中E-cadherin基因启动子CpG岛完全甲基化率为13.6%（3/22）,部分甲基化率为27.3%（6/22）,总甲基化率为40.9%（9/22）,显著高于相应癌旁组织中该基因的甲基化率9.1%（2/22） （P＜0.05）.9例正常肺组织中未检测到此基因的甲基化（0/9）.22例癌组织中E-cadherin蛋白表达减弱或缺失率59.1%（13/22）,显著高于相应癌旁组织蛋白表达减弱缺失率27.3%（6/22）.肺癌组织中发生甲基化者蛋白表达率及强度明显低于未甲基化者.结论：肺癌组织中存在E-cadherin基因启动子CpG岛的异常甲基化,E-cadherin基因启动子CpG岛的异常甲基化可能是E-cadherin蛋白表达下调的主要原因.     

Clinical outcomes of downregulation of E-cadherin gene expression in non-small cell lung cancer

Objective: To investigate the promoter methylation status of the E-cadherin gene in non-small cell lung cancer(NSCLC) and its association with clinical pathological parameters, and to explore the relationship betweendownregulation of E-cadherin gene expression and the methylation status of its promoter region. Methods:Nested methylation-specific PCR was performed to examine CpG methylation within the 5’ CpG island of theE-cadherin gene in lung cancer and para-cancerous tissue from 37 patients with primary non-small cell lungcancer. Quantitative real-time PCR was performed to measure the level of E-cadherin mRNA. Results: Ofthirty-seven cases, 12 (32.4%) samples showed aberrant CpG methylation in tumor tissues compared with thecorresponding normal tissues. In addition, a reduction in E-cadherin mRNA levels was observed in 11 of the12 (91.7%) tumor tissues carrying a methylated E-cadherin gene. However, only 10 (43.5%) cases displayedreduced mRNA levels in tumor tissues from the remaining 23 cases (excluding 2 samples from which mRNA wasunavailable) without methylation events. Downregulation of E-cadherin gene expression significantly correlatedwith the promoter methylation status of this gene. Conclusion: These results provide strong evidence that themethylation status of E-cadherin gene contributes to a reduction in the expression of E-cadherin mRNA, andmay play a role in the development and progression of NSCLC.     

Genome‐wide DNA methylation profiles in liver tissue at the precancerous stage and in hepatocellular carcinoma

To clarify genome‐wide DNA methylation profiles during hepatocarcinogenesis, bacterial artificial chromosome (BAC) array‐based methylated CpG island amplification was performed on 126 tissue samples. The average numbers of BAC clones showing DNA hypo‐ or hypermethylation increased from noncancerous liver tissue obtained from patients with hepatocellular carcinomas (HCCs) (N) to HCCs. N appeared to be at the precancerous stage, showing DNA methylation alterations that were correlated with the future development of HCC. Using Wilcoxon test, 25 BAC clones, whose DNA methylation status was inherited by HCCs from N and were able to discriminate 15 N samples from 10 samples of normal liver tissue obtained from patients without HCCs (C) with 100% sensitivity and specificity, were identified. The criteria using the 25 BAC clones were able to discriminate 24 additional N samples from 26 C samples in the validation set with 95.8% sensitivity and 96.2% specificity. Using Wilcoxon test, 41 BAC clones, whose DNA methylation status was able to discriminate patients who survived more than 4 years after hepatectomy from patients who suffered recurrence within 6 months and died within a year after hepatectomy, were identified. The DNA methylation status of the 41 BAC clones was correlated with the cancer‐free and overall survival rates of patients with HCC. Multivariate analysis revealed that satisfying the criteria using the 41 BAC clones was an independent predictor of overall outcome. Genome‐wide alterations of DNA methylation may participate in hepatocarcinogenesis from the precancerous stage, and DNA methylation profiling may provide optimal indicators for carcinogenetic risk estimation and prognostication. © 2009 UICC     

DNA methyltransferase 1 expression and promoter methylation of E-cadherin in mucoepidermoid carcinoma

BACKGROUND: Loss of E-cadherin expression is found frequently in many types of human malignancies, including mucoepidermoid carcinoma (MEC). CpG methylation in the promoter region has proven important in the regulation of gene expression implicated in malignant transformation. DNA methyltransferases (DNMTs) are the major enzymes involved in establishing genomic methylation patterns. The current study was designed to test the hypothesis that CpG methylation of the promoter region of the E-cadherin gene may inactivate its expression and to examine DNMT 1 (DNMT1) protein expression in MEC. METHODS: Genomic DNA was obtained from paraffin embedded sections by laser microdissection in 46 MEC specimens. Methylation status of the E-cadherin promoter was examined by utilizing the methylation-specific polymerase chain reaction assay. To examine E-cadherin and DNMT1 proteins expression levels, the MEC specimens and adjacent epithelial tissues were studied immunohistochemically. Chi-square analysis was used to evaluate the correlation of protein expression and E-cadherin methylation status with clinicopathologic parameters. Comparisons of the survival rate between patients with DNMT1-positive and DNMT1-negative patients were made using Kaplan-Meier analysis. RESULTS: The data showed that all normal tissues expressed E-cadherin, and no promoter methylation was detected. Of the MEC samples analyzed, methylation allele was found in 33 of 46 samples (72%), and reduced E-cadherin expression was found in 21 of 46 samples (45%). DNMT1-positive expression was observed in 29 of 46 MEC samples (63%). A significant correlation was found between E-cadherin expression and the methylation status of E-cadherin promoter (P = 0.021). In addition, increased DNMT1 expression was correlated with histologic grade, clinical stage, and a poor prognosis in patients with MEC. CONCLUSIONS: Hypermethylation of CpG sites at the 5' promoter of E-cadherin was a common event associated with E-cadherin expression levels in MEC, suggesting an epigenetically mediated loss of E-cadherin function in these tumors. Increased DNMT1 protein expression may play a critical role in the carcinogenesis and disease progression of MEC.     

DNA binding protein A expression and methylation status in hepatocellular carcinoma and the adjacent tissue

We investigated the expression and promoter methylation of dbpA in human hepatocellular carcinoma (HCC) and examined their correlation with clinicopathological features. In 96 paired samples of HCC and adjacent non-tumorous liver, and 10 normal liver specimens, dbpA mRNA was quantified by real-time RT-PCR, and promoter methylation was examined by methylation-specific polymerase chain reaction and bisulfite sequencing. The results showed that dbpA mRNA expression levels were higher in HCC compared to corresponding non-tumor tissues (P<0.01) and higher in non-virus-associated HCC compared to virus-associated cases (P<0.01). dbpA promoter was methylated in 37.7% of HCC samples and the promoter methylation was significantly correlated with the low expression of dbpA in non-virus-associated HCC (P<0.01), but not in virus-associated HCC. Surprisingly, poor prognosis was more significantly associated with high dbpA expression in non-tumorous liver (P=0.018) but not with that in HCC. Non-tumorous tissues consist of chronic hepatitis or liver cirrhosis, and these conditions are the background of hepatocarcinogenesis, defined as the hypercarcinogenic state. Our results suggest that the high expression of dbpA in the hypercarcinogenic state is an indicator of poor prognosis.     

Variable DNA methylation patterns associated with progression of disease in hepatocellular carcinomas

Hepatocellular carcinoma (HCC) most commonly arises from chronic inflammation due to viral infection, as a result of genetic and epigenetic abnormalities. A global picture of epigenetic changes in HCC is lacking. We used methylated CpG island amplification microarrays (MCAMs) to study 6458 CpG islands in HCC and adjacent preneoplastic tissues [chronic hepatitis (CH) or liver cirrhosis (LC)] in comparison with normal liver tissues where neither viral infection nor hepatitis has existed. MCAM identified 719 (11%) prominent genes of hypermethylation in HCCs. HCCs arising from LC had significantly more methylation than those arising from CH (1249 genes or 19% versus 444 genes or 7%, P < 0.05). There were four patterns of aberrant methylation: Type I (4%, e.g. matrix metalloproteinase 14) shows a substantially high methylation level in adjacent tissue and does not increase further in cancer. Type II (55%, e.g. RASSF1A) shows progressively increasing methylation from adjacent tissue to HCC. Type III (4%, e.g. GNA14) shows decreased methylation in adjacent tissue but either similar or increased methylation in HCC. Type IV (37%, e.g. CDKN2A) shows low levels of methylation in normal tissue and adjacent tissue but high levels in HCC. These DNA methylation changes were confirmed by quantitative pyrosequencing methylation analysis in representative 24 genes and were analyzed for correlation with clinicopathological parameters in 38 patients. Intriguingly, methylation in the Type IV genes is characteristic of moderately/poorly differentiated cancer. Our global epigenome analysis reveals distinct patterns of methylation that are probably to represent different pathophysiologic processes in HCCs.     

胃癌E-cadherin基因启动子CpG岛甲基化的研究

背景与目的:目前认为CpG岛甲基化导致转录抑制是恶性肿瘤发生的重要机制之一。E-cadherin能抑制肿瘤细胞的浸润和转移,被公认为是浸润、转移抑制基因。低分化胃癌常表现有E-cadherin基因失活。我们通过检测胃癌及癌旁组织中E-cadherin基因启动子CpG岛甲基化水平,初步探讨胃癌的发病机制。方法:采用特异性甲基化PCR(MSP)法检测51例胃癌组织及37例癌旁组织中的E-cadherin基因启动子CpG岛甲基化水平,采用免疫组织化学染色法检测E-cadherin表达。结果:健康对照组织中未检测到CpG岛甲基化,4例(10.8%)癌旁组织检测到CpG岛甲基化,32例(62.7%)胃癌组织中检测到CpG岛甲基化。低分化腺癌(72.2%,26/36)CpG岛甲基化显著高于高分化腺癌(33.3%,6/15)(P<0.01),T1/T2期(55.6%,10/18)与T3/T4期胃癌(66.7%,22/33)的CpG岛甲基化率无显著性差异(P>0.05)。32例CpG岛甲基化胃癌中27例(84.5%)E-cadherin表达下调,19例非甲基化胃癌中5例(26.3%)E-cadherin表达下调。未发现E-cadherin基因CpG岛甲基化与幽门螺杆菌感染有关。结论:胃癌、尤其是低分化腺癌广泛存在E-cadherin基因启动子CpG岛甲基化,启动子CpG岛甲基化可能参与胃癌的早期过程,E-cadherin基因CpG岛甲基化与幽门螺杆菌感染无相关关系。     

Frequent trefoil factor 3 (TFF3) overexpression and promoter hypomethylation in mouse and human hepatocellular carcinomas

Expression profiling analysis revealed ectopic high expression of mouse TFF3 in non-tumor liver tissues from the hepatocellular carcinoma (HCC) susceptible PWK/Rbrc strain. TFF3 is a member of the trefoil factor family peptides, which are small secreted proteins regulating mucosal regeneration and repair, and which are overexpressed during inflammatory processes and cancer progression. We, therefore, analyzed the TFF3 expression extensively in mouse and human HCCs. Expression of the mouse TFF3 gene was significantly increased in 6 out of 7 HCCs from a PWK spontaneous tumor model and in all 7 HCCs from an SV40T antigen-induced transgenic MT-D2C57BL/6 model. In humans, 8 of 20 HCCs (40%) had overexpression of TFF3 in both mRNA level and protein level. We then analyzed DNA methylation patterns of the TFF3 promoter region to evaluate expression regulation of promoter methylation. In mouse HCCs, we demonstrated that two CpGs, at positions -992 and +109, were hypomethylated in 13 of 14 mouse HCCs. In human HCCs, hypomethylation at CpG -260 was associated with TFF3 overexpression (p=0.04). These results indicate that TFF3 overexpression may be a critical process in mouse and human hepatocellular carcinogenesis, and the specific promoter CpG hypomethylation may be one of the regulation mechanisms of TFF3 overexpression in HCCs.     

肝癌及癌前病变中p27蛋白和mRNA的表达

[目的]研究肝细胞癌(HCC)及癌前病变中细胞周期负性调控因子p27的表达及其意义.[方法]应用免疫组织化学S-P染色法和原位分子杂交法检测正常肝组织、慢性肝炎、肝硬化、癌周肝硬化和肝癌组织中p27蛋白及其mRNA的表达,并对结果进行计算机图像定量分析.[结果]p27蛋白在正常肝组织和慢性肝炎中呈低表达,在肝硬化和癌周肝硬化中表达明显增强,而在肝癌中表达明显下降,与前4组比较差异有显著性(P＜0.05).p27mRNA在非肝癌组织中表达均较强,在肝癌中表达明显减弱,与前4组比较差异有显著性(P＜0.05).正常肝组织、慢性肝炎和肝硬化中p27蛋白阳性信号主要定位于胞核,而癌周肝硬化和HCC中主要定位于胞浆.随着HCC的分化程度降低,p27蛋白和p27mRNA的表达减弱,差异有显著性(P＜0.05).[结论]p27表达下降可能参与了HCC的发生,且与HCC的分化有关.p27的胞浆定位可能与HCC的早期发生有关.