E-cadherin Promoter Hypermethylation Research Articles

Hepatitis B virus X protein and hepatitis C virus core protein cooperate to repress E-cadherin expression via DNA methylation

Dual infection of hepatitis B virus (HBV) and hepatitis C virus (HCV) is closely associated with an increased risk of hepatocellular carcinoma; however, the underlying mechanism is poorly understood. In the present study, we found that HBV X protein (HBx) and HCV core protein work together to inhibit E-cadherin expression in human hepatoma cells. For this effect, they additively increased both the level and activity of enzymes, DNA methyltransferase 1, 3a, and 3b to induce promoter hypermethylation of E-cadherin in a p53-dependent fashion. Their additive effect on E-cadherin levels was reproduced in an in vitro HBV/HCV dual infection system using Huh7D-NTCP cells. As a result, HBV and HCV additively upregulated mesenchymal marker such as N-cadherin, Snail, Twist and Vimentin but cooperatively downregulated epithelial markers such as E-cadherin, Slug and Plakoglobin. In addition, the coinfected cells exhibited faster cell migration and higher invasion ability, as compared with monoinfection, which are hallmarks of epithelial-mesenchymal transition required for the initiation of metastasis in cancer progression.

MiR-770-5p regulates EMT and invasion in TNBC cells by targeting DNMT3A

MicroRNAs (miRNAs) are shown to regulate various processes in cancer like motility and invasion that are key features of the metastatic triple negative breast cancer (TNBCs). Epithelial-mesenchymal transition (EMT) is one of the well-defined cellular transitioning processes characterized with reduced E-cadherin expression and increased mesenchymal molecules such as Vimentin or Snail thereby gives the cells mobility and invasive character. Aberrant DNA methylation by DNA methyltransferases (DNMTs) plays an important role in carcinogenesis. It is well known that DNMTs are required for transcriptional silencing of tumor-associated genes. DNMT3A-induced promoter hypermethylation of E-cadherin has also been known to improve cancer metastasis. Our results indicated that miR-770-5p could downregulate Vimentin and Snail expression levels, while increasing or restoring the expression of E-Cadherin hence, leading to inhibition of EMT phenotypes along with motility and invasion. Specifically, we showed that overexpression of miR-770-5p restored the expression of E-Cadherin in MDA-MB-231 cells via directly targeting DNMT3A. We also observed the change in the spindled shapes showing the loss of mesenchymal characteristics and gain of epithelial phenotype in miR-770-5p overexpressing cells. When considered together, our results show that miR-770-5p could effectively inhibit invasion potential driven by EMT.

Does hypermethylation of CpG island in the promoter region of the E-cadherin gene increase the risk of lung cancer? A meta-analysis.

BackgroundHypermethylation of the CpG island in the promoter regions of tumor suppressor genes is common in the cancer tissue of non‐small cell lung cancer (NSCLC) patients. Epithelial cadherin (E‐cadherin) is a classic tumor suppressor gene of the cadherin superfamily and its promoter region is usually hypermethylated in malignant carcinomas. However, whether hypermethylation of the CpG island in the promoter region of E‐cadherin increases the risk of lung cancer is unknown. We conducted a meta‐analysis of E‐cadherin gene promoter methylation status in cancer tissue (CT) and autologous controls (AC).MethodsElectronic databases were searched for E‐cadherin gene promoter methylation and NSCLC. The hypermethylation status between CT and AC of NSCLC patients were compared and pooled by random or fixed effect models according to statistical heterogeneity across the included studies.ResultsEleven publications relevant to E‐cadherin gene promoter hypermethylation and lung cancer risk were identified and included. E‐cadherin gene promoter hypermethylation frequency in CT and AC was 0.32 (95% confidence interval [CI] 0.22–0.41) and 0.12 (95% CI 0.04–0.20), respectively, with statistical difference (P < 0.05). Significant statistical heterogeneity was found across the 11 studies (I2 = 54.5, P < 0.05). The data was pooled through a random effect model with an odds ratio of 4.21 (95% CI 2.33–7.58) in CT compared to AC.ConclusionThe frequency of E‐cadherin promoter hypermethylation in CT is significantly higher than in AC, indicating that promoter hypermethylation of E‐cadherin plays an important role in NSCLC carcinogenesis.

Associations of RASSF1A, RARβ, and CDH1 promoter hypermethylation with oral cancer risk: A PRISMA-compliant meta-analysis.

Background:Oral tumor is a heterogeneous group of tumors, in which it has several different histopathological and molecular features. Recently, genetic and epigenetic alterations are often detected in the development of oral cancer. Gene promoter hypermethylation leads to the silencing of cancer related genes without changes of genes sequence. To clarify the effect of RAS association domain family protein 1a (RASSF1A), retinoic acid receptor beta (RARβ), and E-cadherin (CDH1) promoter hypermethylation on the risk of oral cancer, we performed this meta-analysis.Methods:PubMed, Web of Science, Embase, and Chinese National Knowledge Infrastructure (CNKI) databases were retrieved to identify eligible articles. Stata 12.0 software was used to analyze extracted data of the included articles. Odds ratios (ORs) with the corresponding 95% confidence interval (95% CI) were calculated to evaluate the associations of RASSF1A, RARβ, and CDH1 promoter hypermethylation with oral cancer risk.Results:Around 23 literatures with 29 studies were included in the final meta-analysis, in which 12 studies were about RASSF1A promoter methylation, 4 studies were about RARβ promoter methylation, and 13 studies were about CDH1 promoter methylation. Overall, the results of this meta-analysis showed that there were significant associations between RASSF1A, RARβ, and CDH1 promoter hypermethylation and oral cancer risk (RASSF1A, OR = 11.8, 95% CI = 6.14–22.66; RARβ, OR = 20.35, 95% CI = 5.64–73.39; CDH1, OR = 13.46, 95% CI = 5.31–34.17). In addition, we found that RASSF1A promoter hypermethylation exerted higher frequency in the tongue tumor than other site tumor in mouth (RASSF1A, tongue tumor vs other site tumor in mouth, unmethylation vs methylation, OR = 0.65, 95%CI = 0.44–0.98).Conclusion:RASSF1A, RARβ, and CDH1 promoter hypermethylation might significantly increase the risk of oral cancer.

Transcriptional Suppression of E-Cadherin by HPV-16 E6 and E7 Oncogenes is Independent of Hypermethylation of E-Cadherin Promoter

Background: Cervical cancer is one of the most common cancers observed in women worldwide, and its development is related to E6 and E7 two viral oncoproteins of high-risk human papillomavirus (HPV) types. Aberrant expression of E-cadherin, which is associated with epithelial-to-mesenchymal transition (EMT), is frequently observed in cervical cancer. Objectives: The mechanisms underlying E-cadherin suppression in cervical cancer are not clear; therefore, this experimental study from Iran was designed to elucidate the relationship of DNA methyltransferase expression and E-cadherin promoter methylation with E-cadherin expression in HPV-16 E6- and E7-expressing cells. Materials and Methods: Real-time PCR and western blot were used to determine the effects of HPV-16 E6 and E7 on E-cadherin, DNMT1, DNMT3a, and DNMT3b expression in HCT-116 cell line. We also analyzed E-cadherin promoter methylation in cells expressing HPV-16 E6 and E7 oncoproteins by bisulfite sequencing. Results: HPV-16 E6 and E7 proteins reduced E-cadherin expression 3.7 and 2.2 times when compared with control cells (P = 0.0221 and P = 0.0461, respectively). This reduction was greater in HPV-16 E6-expressing cells than in HPV-16 E7-expressing cells. Although HPV-16 E6 and E7 increased DNA methyltransferase 1 expression 2.6 and 3.4 times, respectively (P = 0.0133 and P = 0.0113) when compared with control cells, they was no E-cadherin promoter methylation. Conclusions: Unlike other cancer-associated viruses (HBV, HCV, and EBV), reduction in E-cadherin expression in HPV-16 E6- and E7-expressing cells is not due to hypermethylation of the E-cadherin promoter.

Hepatitis C virus represses E-cadherin expression via DNA methylation to induce epithelial to mesenchymal transition in human hepatocytes

Hepatitis C virus (HCV) core protein is known to induce promoter hypermethylation of tumor suppressor genes including E-cadherin to repress their expression when overexpressed in human hepatocytes; however, its actual role during HCV infection is still unknown. Here, we report that infection with HCV derived from pJFH-1 replicon system that mimics natural infection elevates protein levels of DNA methyltransferase 1 and 3b to enhance DNMT activity in human hepatocytes. As a consequence, HCV induced promoter hypermethylation of E-cadherin, resulting in repression of its expression. In addition down-regulation of E-cadherin by HCV led to epithelial–mesenchymal transition that is known to be a critical event during the late stage of tumorigenesis.

E-cadherin gene promoter hypermethylation may contribute to the risk of bladder cancer among Asian populations

There are increasing scientific evidences suggesting that E-cadherin gene promoter hypermethylation may contribute to the development and progression of bladder cancer, but existing studies have yielded inconclusive results. This meta-analysis aims to assess the role of E-cadherin promoter hypermethylation in bladder carcinogenesis. We conducted an extensive literature search for relevant studies on PubMed, Embase, Web of Science, Cochrane Library, and CBM databases from their inception through May 1st, 2013. This meta-analysis was performed using the STATA 12.0 software. Crude risk ratio (RR) with 95% confidence interval (CI) was calculated. Ten clinical studies were included in this meta-analysis with a total of 620 bladder cancer samples, 199 normal adjacent samples and 131 normal urothelium tissue. Our meta-analysis revealed that the methylation frequencies in bladder cancer tissues were obviously higher than those in normal control tissues (RR=2.02, 95%CI: 1.00–4.12, P=0.050). Subgroup analysis by ethnicity indicated that higher methylation frequencies were observed in bladder cancer tissues among Asian populations (RR=2.35, 95%CI: 1.11–4.95, P=0.025), but not among Caucasian populations (RR=1.62, 95%CI: 0.48–5.53, P=0.439). Univariate and multivariate meta-regression analyses showed that ethnicity may be the major source of heterogeneity (P<0.05). No publication bias was detected in this meta-analysis (P=0.358). The present meta-analysis indicates that E-cadherin gene promoter hypermethylation may contribute to increased risk of bladder cancer among Asian populations.

MiRNA expression profiling of 51 human breast cancer cell lines reveals subtype and driver mutation-specific miRNAs

IntroductionBreast cancer is a genetically and phenotypically complex disease. To understand the role of miRNAs in this molecular complexity, we performed miRNA expression analysis in a cohort of molecularly well-characterized human breast cancer cell lines to identify miRNAs associated with the most common molecular subtypes and the most frequent genetic aberrations.MethodsUsing a microarray carrying LNA™ modified oligonucleotide capture probes), expression levels of 725 human miRNAs were measured in 51 breast cancer cell lines. Differential miRNA expression was explored by unsupervised cluster analysis and was then associated with the molecular subtypes and genetic aberrations commonly present in breast cancer.ResultsUnsupervised cluster analysis using the most variably expressed miRNAs divided the 51 breast cancer cell lines into a major and a minor cluster predominantly mirroring the luminal and basal intrinsic subdivision of breast cancer cell lines. One hundred and thirteen miRNAs were differentially expressed between these two main clusters. Forty miRNAs were differentially expressed between basal-like and normal-like/claudin-low cell lines. Within the luminal-group, 39 miRNAs were associated with ERBB2 overexpression and 24 with E-cadherin gene mutations, which are frequent in this subtype of breast cancer cell lines. In contrast, 31 miRNAs were associated with E-cadherin promoter hypermethylation, which, contrary to E-cadherin mutation, is exclusively observed in breast cancer cell lines that are not of luminal origin. Thirty miRNAs were associated with p16INK4 status while only a few miRNAs were associated with BRCA1, PIK3CA/PTEN and TP53 mutation status. Twelve miRNAs were associated with DNA copy number variation of the respective locus.ConclusionLuminal-basal and epithelial-mesenchymal associated miRNAs determine the subdivision of miRNA transcriptome of breast cancer cell lines. Specific sets of miRNAs were associated with ERBB2 overexpression, p16INK4a or E-cadherin mutation or E-cadherin methylation status, which implies that these miRNAs may contribute to the driver role of these genetic aberrations. Additionally, miRNAs, which are located in a genomic region showing recurrent genetic aberrations, may themselves play a driver role in breast carcinogenesis or contribute to a driver gene in their vicinity. In short, our study provides detailed molecular miRNA portraits of breast cancer cell lines, which can be exploited for functional studies of clinically important miRNAs.

Nickel chloride induced epithelial-mesenchymal transition by promoter hypermethylation of E-cadherin via ROS generation

Nickel chloride induced epithelial-mesenchymal transition by promoter hypermethylation of E-cadherin via ROS generation

Epigenetic inactivation of the E-cadherin gene in eyelid sebaceous gland carcinoma

E-cadherin and β-catenin are crucial components of the cell-cell adhesion complex. Their loss has often been associated with tumour metastasis and poor clinical outcome. Both loss of E-cadherin at the cell membrane and a stabilizing mutation in CTNNB1 (β-catenin gene) have been associated with ovarian, colorectal, hepatocellular and nonmelanoma skin cancer, such as squamous and basal cell carcinomas. Absence of E-cadherin may be caused by promoter hypermethylation of the E-cadherin gene (CDH1). To determine the role of E-cadherin promoter hypermethylation and CTNNB1 gene mutation in the aggressive behaviour of sebaceous gland carcinoma of the eyelid. Thirty-six cases of sebaceous gland carcinoma were subjected to E-cadherin methylation-specific polymerase chain reaction and mutational analysis for the CTNNB1 gene. E-cadherin and β-catenin staining was evaluated by immunohistochemistry. Results were correlated with the clinicopathological features of sebaceous gland carcinoma. nMethylation of the E-cadherin promoter region was detected in 72% of eyelid sebaceous gland carcinoma cases and loss of E-cadherin immunostaining in 83%. E-cadherin promoter hypermethylation showed a significant association with the loss of membranous E-cadherin (P = 0·038) and it was of borderline significance with reduced disease-free survival (P = 0·05). It was also found to be associated with advanced age (73%), tumour size ≥ 2 cm (77%), orbital invasion (83%), lymph node metastasis (60%), tumour recurrence (60%) and poor histological differentiation (90%). DNA sequencing revealed no stabilizing β-catenin gene mutation in sebaceous gland carcinoma. Loss of membranous β-catenin was observed in 61% cases, which associated significantly with both E-cadherin promoter methylation (P = 0·0262) and loss of E-cadherin membranous localization (P=0·0015). Epigenetic inactivation of the E-cadherin gene causes loss of membrane-bound E-cadherin and could contribute to the reduced disease-free survival in eyelid sebaceous gland carcinoma. Mutations in the β-catenin gene do not seem to be involved in the pathogenesis of eyelid sebaceous gland carcinoma.

Nickel-induced Epithelial-Mesenchymal Transition by Reactive Oxygen Species Generation and E-cadherin Promoter Hypermethylation

Epithelial-mesenchymal transition (EMT) is considered a critical event in the pathogenesis of lung fibrosis and tumor metastasis. During EMT, the expression of differentiation markers switches from cell-cell junction proteins such as E-cadherin to mesenchymal markers such as fibronectin. Although nickel-containing compounds have been shown to be associated with lung carcinogenesis, the role of nickel in the EMT process in bronchial epithelial cells is not clear. The aim of this study was to examine whether nickel contributes to EMT in human bronchial epithelial cells. We also attempted to clarify the mechanisms involved in NiCl(2)-induced EMT. Our results showed that NiCl(2) induced EMT phenotype marker alterations such as up-regulation of fibronectin and down-regulation of E-cadherin. In addition, the potent antioxidant N-acetylcysteine blocked EMT and expression of HIF-1α induced by NiCl(2), whereas the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine restored the down-regulation of E-cadherin induced by NiCl(2). Promoter hypermethylation of E-cadherin, determined by quantitative real time methyl-specific PCR and bisulfate sequencing, was also induced by NiCl(2). These results shed new light on the contribution of NiCl(2) to carcinogenesis. Specifically, NiCl(2) induces down-regulation of E-cadherin by reactive oxygen species generation and promoter hypermethylation. This study demonstrates for the first time that nickel induces EMT in bronchial epithelial cells.

Association Between p16, hMLH1 and E-cadherin Promoter Hypermethylation and Intake of Local Hot Salted Tea and Sun-dried Foods in Kashmiris with Gastric Tumors

The aim of this study was to evaluate the methylation status of three important cancer related genes viz. p16, E-cadherin and hMLH1 promoters and to associate the findings with specific dietary habits in Kashmiris, a culturally distinct population in India, with gastric cancer. The study subjects were divided into three age groups viz. 0-30 yrs (1st), 31-60 yrs (2nd) and 61-90 yrs (3rd). A highly significant association between the intake of local hot salted tea in 2nd (p=0.001) and 3rd (p=0.009) age groups was observed with the promoter hypermethylation of E cadherin. Again a highly significant association between the aberrant methylation of hMLH1 (p=0.000) and p16 (p=0.000) promoters and the intake of local hot salted tea was observed in the 2nd age group of gastric cancer patients. The intake of sun-dried food was also significantly associated with the promoter hypermethylation of E cadherin (p=0.003) and p16 (p=0.015) genes in 3rd age group. The results of the present study suggest a close association between the aberrant methylation of p16, E-cadherin and hMLH1 promoters and the intake of local hot salted tea and sun-dried foods in Kashmiri population.

Link of Metabolic Phenotypes of Gastric Cancer Tissue and Blood Plasma

G A A b st ra ct s were 0.674, 0.727, 0.680, 0.739, respectively. Two peaks were found in the autofluorescence spectrum of gastric juice from all patients. The first peak (P1FI) was distinctively higher in gastric cancer than that in benign lesion (P<0.01). The AUC from ROC for P1FI was 0.872, the sensitivity, specificity, and accuracy of P1FI were 83.8%, 75.2%, and 78.8%, respectively, while the false positive rate was 24.8% (41/165). Furthermore, all of the four candidate genes and P1FI were evaluated in univariate and multivariate logistic regression (LR) models. The multivariate model included logarithmic transformations of E-cadherin, DAPK and P1FI, and the AUC from ROC was 0.933, which was statistically superior to either parameter alone (P<0.01), the sensitivity, specificity and accuracy were 89.7%, 86.7% and 87.9%, respectively, the false positive rate was 13.3% (22/165). Additionally, CART (Classification and Regression Trees) (Salford Systems) was carried out to set up the diagnostic model for gastric cancer and similar to the results of LR, P1FI, E-cadherin and DAPK were identified as tree nodes(Fig.1). The sensitivity, specificity, and accuracy were 81.2 %, 91.5%, and 87.2%, respectively. It is noticeable that the false positive rate was 8.5% (14/165). Conclusions The complementary use of both LR and CART models could be a promising approach to analyze multiple markers. The model, which combining the autofluorescene spectrum with the DNA promoter hypermethylation of E-cadherin and DAPK of gastric juice, could be a potential promising diagnostic model for better decision making for gastric cancer screening.

Abstract LB-279: Proteasome inhibitor bortezomib induces E-cadherin reexpression through inhibiting NF- B mediated DNA methylation in esophageal cancer

Abstract Background: Esophageal cancer represents a significant challenge as most patients present with advanced stage disease. This is due to the development of early nodal or distant metastasis. Metastatic progression is initiated by a process termed epithelial-mesenchymal transition (EMT). Recently, bortezomib, as an anti-tumor reagent, has been used as first-line regimen for patients with metastatic esophageal cancer in a Phase II clinical trial. However, the molecular mechanisms through which bortezomib inhibits tumor progression have not been well described. E-cadherin is a hallmark of EMT in tumor metastatic progression. Its aberrant hypermethylation is significantly associated with a poor prognosis in esophageal cancer. Our prior work indicated that bortezomib increased E-cadherin at transcriptional level in esophageal cancer. We hypothesized that NF-κB inhibition with bortezomib would reduce DNA methylation of E-cadherin and result in increased E-cadherin expression in esophageal cancer. Methods: Esophageal squamous and adenocarcinoma cancer cells were treated with TNF to simulate the pro-inflammatory tumor milieu and TGF-ß, a cytokine critical for the induction of EMT, followed by treatment with bortezomib (50nM) for 24 hrs. Cellular morphological changes were evaluated. E-cadherin promoter methylation status and mRNA was assessed using Methylation Specific PCR (MSP) and real-time PCR, respectively. To evaluate the effect of NF-κB on the methylation of the E-cadherin promoter in esophageal cancer, cells were either stimulated with TNF or transfected with an expression vector encoding the NF-κB subunit RelA/p65 or a super-suppressor IκB (SR- IκB), then treated with or without bortezomib. Results: We examined the effects of bortezomib on cellular morphology and found that bortezomib completely reverses TNF/TGF -induced EMT of esophageal cancer cells, correlating with an increase of E-cadherin transcript (5∼6 fold). Simultaneously, bortezomib dramatically inhibits methylation of the E-cadherin promoter in esophageal cancer cells. Furthermore, treatment with TNF or overexpression of the RelA/p65 induced hypermethylation of E-cadherin promoter. While inactivating NF-κB using SR- IκB expression vector significantly inhibits methylation of E-cadherin promoter in esophageal cancer cell lines. Interestingly, bortezomib failed to induce either hypomethylation or transactivation of E-cadherin in the absence of RelA/p65 when using SR-IκB in esophageal cancer cell lines. Conclusion: These findings indicate that bortezomib inhibits EMT of esophageal cancer cells by demethylation of the E-cadherin promoter, and that this function is NF- B dependent. In addition to functioning as a transcription activator, RelA/p65 appears to function as a transcription repressor by participating in methylation and silencing of E-cadherin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-279.

Modulation of E-cadherin expression by K-Ras; involvement of DNA methyltransferase-3b

E-cadherin, as a tumor suppressor, plays an important role for intercellular adhesion involved in metastasis. Although K-Ras is highly expressed in a variety of cancers, the regulation of E-cadherin expression by K-Ras in association with DNA methylation and cell metastasis has not been completely clarified. In this study, E-cadherin expression was repressed in 267B1/K-Ras human epithelial prostate cancer cells stably overexpressing K-Ras, resulting from hypermethylation of E-cadherin promoter as evidenced by methylation-specific polymerase chain reaction (PCR), bisulfite sequencing, real-time reverse transcription-PCR and western blot analysis. The increased level of DNA methyltransferase (DNMT) 3b in 267B1/K-Ras cells was reduced by small interfering RNA-mediated knockdown of k-ras, whereas DNMT1 and DNMT3a did not change regardless of K-Ras or 5-aza-2'-deoxycytidine (5'-AzaC) treatment. Furthermore, binding of DNMT3b to E-cadherin promoter was increased in 267B1/K-Ras cells but was reduced by 5'-AzaC, as revealed by chromatin immunoprecipitation assay, which was in agreement with cell aggregation and invasive mobilization of the cells. Hence, our data suggest that increased binding of DNMT3b to E-cadherin promoter region by K-Ras cause promoter hypermethylation for reduced expression of E-cadherin, leading to the decreased cell aggregation and increased metastasis of human prostate cancer cells overexpressing K-Ras.

E-cadherin promoter hypermethylation induced by interleukin-1β treatment or H. pylori infection in human gastric cancer cell lines

Interleukin-1β is up-regulated in the presence of Helicobacter pylori infection. H. pylori infection was associated with E-cadherin methylation. In this study, we examined if IL-1β could induce promoter methylation of E-cadherin in human gastric cancer cell lines TMK-1, MKN-74 and MKN-7. Cells were treated with IL-1β (0.025, 0.1, 0.25, 1.0, 2.5 ng/mL) for 6, 12 and 24 h. Methylation status was determined by MSP and sequencing. The effects of IL-1β or H. pylori on the cells, and after blockade with interleukin-1 receptor antagonist (IL-1ra) were tested. Promoter methylation of E-cadherin was induced in all three cells treated with IL-1β or co-cultured with H. pylori. Treatment of IL-1ra could reverse the phenomena. Our study indicated that IL-1β is an important step in mediating E-cadherin methylation.

Absence of E-Cadherin Expression Distinguishes Noncohesive from Cohesive Pancreatic Cancer

The role of E-cadherin in carcinogenesis is of great interest, but few studies have examined its relevance to pancreatic carcinoma. We evaluated E-cadherin protein expression by immunohistochemistry in pancreatobiliary cancers having a noncohesive histologic phenotype (21 undifferentiated adenocarcinomas and 7 signet ring carcinomas), comparing the results with pancreatic cancers having a cohesive phenotype (25 moderately differentiated and 14 poorly differentiated adenocarcinomas). Twenty of 21 undifferentiated cancers had complete absence of E-cadherin expression, as did two signet ring carcinomas. In contrast, cohesive cancers (n = 39) had E-cadherin labeling at the plasma membrane (P < 0.001). Subsets of cancers were also evaluated for beta-catenin expression. All of the cohesive lesions (n = 28) showed a membranous beta-catenin expression pattern, whereas noncohesive foci (n = 7) were characterized by either cytoplasmic labeling or complete absence of beta-catenin protein expression, suggestive of a deficient zonula adherens in noncohesive cancers. E-cadherin promoter hypermethylation was observed in an undifferentiated pancreatic cancer cell line, MiaPaCa-2, whereas two pancreatic cancer cell lines derived from differentiated lesions lacked any evidence of E-cadherin promoter methylation. No pattern of E-cadherin promoter methylation could be determined in three primary cancers having mixed histologic patterns (contained both cohesive and noncohesive foci). No somatic mutations in E-cadherin were identified in noncohesive pancreatic cancers having inactivated E-cadherin. Noncohesive pancreatic cancers were characterized by the loss of E-cadherin protein expression. Promoter hypermethylation is a possible mechanism of E-cadherin gene silencing in a subset of these cancers.

Hypermethylation of epithelial-cadherin gene promoter is associated with Epstein-Barr virus in nasopharyngeal carcinoma

Background: Epstein-Barr virus (EBV) is documented as the important etiologic agent of nasopharyngeal carcinoma (NPC) but the mechanism of development and pathogenesis induced by EBV is presently unclear. Hypermethylation of epithelial-cadherin (E-cadherin) promoter has been shown to be induced in NPC cell line by EBV LMP1 via DNA methyltransferase activation. EBV genomes and hypermethylation of E-cadherin promoter were investigated in NPC tissues to evaluate the role of EBV in the hypermethylation and pathogenesis of NPC. Methods: Methylation-specific polymerase chain reaction (MSP) was performed to detect E-cadherin promoter hypermethylation in paraffin embedded tissues from patients with NPC and normal nasopharyngeal tissues. EBV genomes were detected by PCR in the tissue samples. Results: Hypermethylation of E-cadherin promoter and EBV were predominantly detected in undifferentiated and non-keratinizing NPC compared to those in squamous cell NPC. Hypermethylation of E-cadherin was found in 28 of 38 (73.7%) patient samples. EBV was detected in 22 of the 28 (78.6%) NPC samples demonstrating E-cadherin hypermethylation. EBV genomes and hypermethylation were not detected in normal nasopharyngeal tissues. Significant association was found between E-cadherin hypermethylation and EBV genomes ( p < 0.001; Fisher's exact test). Hypermethylation of E-cadherin was more frequently detected in advanced stages compared to those in early stages of NPC ( p = 0.036; Fisher's exact test). Conclusions: The high incidence of EBV with the consistency of E-cadherin hypermethylation, particularly in undifferentiated and non-keratinizing NPC suggests the role of EBV in the hypermethylation. EBV exists at early stage of NPC that induces the hypermethylation and contributes to progression of the disease to the advanced stage of NPC.

A Correlation Between the Reduced Expression of E-Cadherin and Promoter Hypermethylation in Oral Squamous Cell Carcinoma

Statement: E-cadherin is a transmembrane glycoprotein that mediates epithelial cell-to-cell adhesion and plays an important role in maintaining epithelial stability. E-cadherin expression is greatly reduced in many types of human cancers including oral cancer. An epigenetic alteration in association with promoter hypermethylation is one important mechanism of E-cadherin gene silencing. Materials and Methods: In our previous immunohistochemical study, we evaluated the expression of E-cadherin by immunohistochemical staining of 50 oral squamous cell carcinomas and found that the expression of E-cadherin was reduced in 40/50 (80%) primary tumors and in 21/22 (95.5%) lymph nodes. To correlate E-cadherin expression and its mechanism of regulation, approximately 1000 cells from representative cancers were microdissected and used for methylation-specific PCR to analyze the status of CpG promoter hypermethylation of the E-cadherin gene. Method of Data Analysis: n/a Results: As a preliminary result, we found that the promoter region of E-cadherin was hypermethylated in one specimen of 20 primary oral cancers. Conclusion: Our findings showed that aberrant expression of E-cadherin was not strongly associated with promoter hypermethylation of E-cadherin. Further study is needed on tumor recurrences and nodal metastases.

E‐cadherin Gene Promoter Hypermethylation in H. pylori‐Induced Enlarged Fold Gastritis

Promoter hypermethylation of E-cadherin plays an important role on gastric carcinogenesis. We have previously reported that the odds ratio for gastric carcinoma and the prevalence of diffuse-type early gastric carcinoma in Helicobacter pylori-induced enlarged fold gastritis increased with increasing fold width. Thus, we examined E-cadherin methylation in gastric mucosa from H. pylori-induced enlarged fold gastritis before and after H. pylori eradication. Moreover, we analyzed the mechanism of H. pylori infection-induced E-cadherin hypermethylation. Twenty-three H. pylori-positive patients with enlarged folds, 18 H. pylori-positive and seven H. pylori-negative patients without enlarged folds, were involved in the study. E-cadherin promoter methylation was studied using quantitative methylation-specific polymerase chain reaction. We investigated methylation percentage and DNA methyltransferase activity in gastric cancer cell lines treated with EGF, TNFalpha, and MG132. E-cadherin methylation percentage of the gastric antral and body mucosa in H. pylori-positive patients with enlarged folds was much greater than that in both H. pylori-positive and -negative patients without enlarged folds. After H. pylori eradication, the methylation percentage in six patients with enlarged fold gastritis decreased significantly from 15.6 +/- 3.9 to 8.8 +/- 2.2 (p < .05). Moreover, the methylation was induced by TNFalpha, MG132, and EGF treatment, and DNA methyltransferase activity was induced by EGF treatment in MKN-1 cells. Our findings suggest that the hypermethylation of E-cadherin promoter might be involved in the process of gastric carcinoma through the specialized factors in H. pylori-induced enlarged fold gastritis.