CDH1 Promoter Research Articles

Combined analysis of E-cadherin gene (CDH1) promoter hypermethylation and E-cadherin protein expression in patients with gastric cancer: implications for treatment with demethylating drugs

Hypermethylation is studied as a new, relevant mechanism for silencing tumor suppressor genes. It is a potentially reversible epigenetic change and it is the target of novel anticancer compounds with demethylating activity. In this perspective, we investigated E-cadherin gene (CDH1) promoter hypermethylation in gastric carcinomas and its correlation with E-cadherin protein expression. Consecutive cases of gastric carcinoma with assessable paraffin-embedded tumor blocks and paired normal mucosa were considered eligible for study entry. CDH1 promoter hypermethylation and E-cadherin protein expression were determined by methylation-specific polymerase chain reaction and immunohistochemistry, respectively. CDH1 promoter hypermethylation was found in 20 out of 70 gastric carcinomas and the epigenetic change occurred in the early, as well as in the locally advanced disease. In five cases, hypermethylation was also detected in the normal mucosa. Eighteen out of 20 hypermethylated tumors were of the diffuse histotype (P=0.0001). Of 24 tumors with reduced or negative E-cadherin expression, 19 were hypermethylated and 5 were unmethylated (P=0.0001). CDH1 promoter hypermethylation frequently occurs in gastric carcinomas of the diffuse histotype and it is significantly associated with downregulated E-cadherin expression. The knowledge on the hypermethylation status of tumor suppressor genes may be relevant to the development of demethylating drugs and novel chemopreventive strategies in solid tumors.

Promoter hypermethylation of MGMT, CDH1, RAR-beta and SYK tumour suppressor genes in granulosa cell tumours (GCTs) of ovarian origin.

Ovarian carcinoma (OC) is a leading cause of death among women throughout the world. A number of cancer-associated genes have been shown to be inactivated by hypermethylation of CpG islands during tumorigenesis. We tested the hypothesis that methylation status of MGMT, CDH1, RAR-β and SYK could be important in the ovarian tumorigenic process and can lead to the gene(s) inactivation. Therefore, we assessed the promoter hypermethylation of MGMT, CDH1, RAR-β and SYK in 43 ovarian granulosa cell tumours (GCTs) (adult type) using methylation-specific PCR. These tumours are relatively rare, accounting for approximately 3% of all ovarian cancers. Hypermethylation of MGMT (in 14 tumours), CDH1 (in nine tumours), RAR-β (in eight tumours) and SYK (in seven tumours) have been found. Selective loss of RAR-β and RAR-β2 mRNA has been found in seven patients, while that of MGMT and SYK in three patients who also show aberrant methylation in promoter region of RAR-β in addition to MGMT, SYK and CDH1 genes. Promoter CpG hypermethylation may be an alternative to mutation(s) to inactivate tumour suppressor genes such as MGMT, CDH1, RAR-β and SYK, and this can also be an early event in the pathogenesis of OCs. Moreover, hypermethylation of the MGMT and CDH1, MGMT and RAR-β and CDH1 and RAR-β promoters occurred concordantly (P< 0.001, 0.0421 and 0.0005 respectively; Fischer's exact test). In addition to this, monosomy 22 and trisomy 14 have also been found in 10 tumours. It is clear from the results that hypermethylation of the promoter region of these tumour suppressor genes, monosomy 22 and trisomy 14, may be critical steps in the tumorigenesis, which consequently play a permissive role for tumour aggressiveness. All these events might play an important role in the early clinical diagnosis of the disease. Our results, therefore, suggest a potential role for epigenetic modification of these critical tumour suppressor genes in pathways relevant to the transformation and differentiation of rare type of ovarian cancer (GCTs).

Epigenetic and genetic alterations of APC and CDH1 genes in lobular breast cancer: relationships with abnormal E-cadherin and catenin expression and microsatellite instability.

The causes and functional consequences of E-cadherin (E-CD) loss in breast cancer are poorly understood. E-CD loss might act in concert with alterations in the APC/beta-catenin pathway to permit oncogenic beta-catenin signaling. To test this hypothesis, we have analyzed the presence of genetic and epigenetic alterations affecting E-CD (CDH1), APC and beta-catenin (CTNNB1) genes and the immunohistochemical expression of E-CD, beta- and gamma-catenin in a series of 46 infiltrating lobular breast carcinomas (ILCs). Since 80% of ILCs featured complete loss of E-CD expression, we analyzed the molecular alterations responsible for E-CD inactivation in these tumors. We found that 10 of 46 (22%) cases harbored mutations in CDH1, including 1 case with 2 different mutations (1 of which was germline). CDH1 was also inactivated by loss of heterozygosity (LOH; 30/41, 73%) and promoter hypermethylation (19/46, 41%). Interestingly, LOH and mutations were also detected in the corresponding in situ lesions of the ILCs, implying that these alterations are early events in lobular cancer tumorogenesis. Additionally, the presence of a polymorphism in the CDH1 promoter was found to be inversely correlated with CDH1 mutations, but not with E-CD levels. We next examined whether alterations in the APC/beta-catenin pathway also occurred in the same series of ILCs. Although no CTNNB1 or APC mutations were detected, promoter methylation (25/46, 52%) and LOH (7/30, 23%) of APC were found. Moreover, methylation of APC and CDH1 occurred concordantly. However, beta- and gamma-catenin were severely reduced or absent in 90% of these tumors, implying that alterations in CDH1 and APC genes do not promote beta-catenin accumulation in ILC. These molecular alterations were not associated with microsatellite instability. In summary, several different mechanisms (mutations, LOH, methylation) are involved in the frequent CDH1 inactivation in invasive and in situ lobular breast cancer. The same tumors also show genetic and epigenetic alterations of APC gene. However, altered CDH1 and APC genes do not promote beta-catenin accumulation in this tumor type.

Expression of DNA (5-cytosin)-methyltransferases (DNMTs) in hepatocellular carcinomas

Down-regulation of tumor suppressor genes by hypermethylation of 5′-CpGs is one of the important mechanisms involved in tumor development. DNA (5-cytosine)-methyltransferases (DNMTs) are enzymes that methylate the cytosine residue of CpGs, and four types have been identified (DNMT1, 2, 3a and 3b). To examine the involvement of DNMTs in hepatocellular carcinogenesis, we measured DNMT mRNAs in hepatocellular carcinomas (HCCs). mRNAs of DNMT1, 2, 3a and 3b were detected by reverse transcription-PCR analysis and quantified by a real-time PCR method in surgically resected HCCs and adjacent non-tumorous liver tissue. DNMT1 was expressed in all tissues and at a significantly higher level in HCCs than in non-tumorous liver tissue ( P=0.01). DNMT2 was expressed at a low level in all tissues. DNMT3a and DNMT3b mRNA were undetectable in normal liver. DNMT3a was expressed in all HCCs and was expressed at similar levels in 60% of the non-tumorous liver tissues. DNMT3b mRNA was detected at a significantly higher level ( P=0.002) in HCCs than in non-tumorous liver tissues. The amount of DNMT1, 3a and 3b mRNA was not different between HCCs with or without hypermethylation of the CDH1 promoter. These data suggest that overexpression of DNMT1 and DNMT3b contributes to hepatocellular carcinogenesis.

Abnormalities of E- and P-cadherin and catenin (beta-, gamma-catenin, and p120ctn) expression in endometrial cancer and endometrial atypical hyperplasia.

Abnormal expression of cadherins and catenins plays a critical role in the initiation and progression of multiple human tumours. This study aimed to evaluate the immunoreactivity of E- and P-cadherin, beta- and gamma-catenin, and p120ctn in premalignant and malignant endometrial lesions and to correlate their membranous expression with clinicopathological features. In addition, we examined whether or not LOH and promoter hypermethylation of the CDH1 gene were associated with E-cadherin expression and clinicopathological variables. Finally, we studied the frequency of beta-catenin mutations in premalignant endometrial lesions. Immunohistochemical staining was performed in 21 atypical endometrial hyperplasias (AEHs), 95 endometrioid carcinomas (EECs), and 33 non-endometrioid carcinomas (NEECs). Reduced E-cadherin expression was observed in 57.8% of the cases, being more frequent in NEECs (87.1%, p = 0.001) and carcinomas of more advanced stage (85.7% of stage III-IV carcinomas, p = 0.01). LOH of CDH1 gene was found in 57.1% of NEECs but only in 22.5% of EECs (p = 0.011) and showed a trend towards association with reduced E-cadherin expression (p = 0.089). CDH1 promoter hypermethylation was found in 21.2% of endometrial carcinomas but was not associated with clinicopathological or immunohistochemical variables. Reduced expression of beta- and gamma-catenin and p120ctn was found in 76.1%, 94.3%, and 63.6% of the cases, respectively, being more frequent in lesions with reduced E-cadherin expression. In addition, beta-catenin, but not gamma-catenin or p120ctn expression, was associated with the histology of the lesion, since it was reduced in 35% of AEHs, 80.3% of EECs, and 96.9% of NEECs (p = 0.000). Mutations in exon 3 of the beta-catenin gene, associated with beta-catenin nuclear expression, were detected in 3 (14.0%) AEH, a frequency similar to that previously reported in this series of ECs. Finally, upregulation of P-cadherin was observed in 28.6% of cases. This alteration was associated with the histology of the lesion, since it was found in 9.5% of AEHs, 27.7% of EECs, and 46.2% of NEECs (p = 0.021).

Frequent epigenetic inactivation of the RASSF1A tumour suppressor gene in testicular tumours and distinct methylation profiles of seminoma and nonseminoma testicular germ cell tumours.

Testicular germ cell tumours (TGCTs) are histologically heterogeneous neoplasms with variable malignant potential. Previously, we demonstrated frequent 3p allele loss in TGCTs, and recently we and others have shown that the 3p21.3 RASSF1A tumour suppressor gene (TSG) is frequently inactivated by promoter hypermethylation in a wide range of cancers including lung, breast, kidney and neuroblastoma. In order to investigate the role of epigenetic events in the pathogenesis of TGCTs, we analysed the promoter methylation status of RASSF1A and nine other genes that may be epigenetically inactivated in cancer (p16(INK4A), APC, MGMT, GSTP1, DAPK, CDH1, CDH13, RARbeta and FHIT) in 24 primary TGCTs (28 histologically distinct components). RASSF1A methylation was detected in four of 10 (40%) seminomas and 15 of 18 (83%) nonseminoma TGCT (NSTGCT) components (P=0.0346). None of the other nine candidate genes were methylated in seminomas, but MGMT (44%), APC (29%) and FHIT (29%) were frequently methylated in NSTGCTs. Furthermore, in two mixed germ cell tumours, the NSTGCT component for one demonstrated RASSF1A, APC and CDH13 promoter methylation, but the seminoma component was unmethylated for all genes analysed. In the second mixed germ cell tumour, the NSTGCT component was methylated for RASSF1A and MGMT, while the seminoma component was methylated only for RASSF1A. In all, 61% NSTGCT components but no seminoma samples demonstrated promoter methylation at two or more genes (P=0.0016). These findings are consistent with a multistep model for TGCT pathogenesis in which RASSF1A methylation occurs early in tumorigenesis and additional epigenetic events characterize progression from seminoma to NSTGCTs.

Abnormal methylation of several tumor suppressor genes in sporadic breast cancer

Multiplex methylation-sensitive PCR was employed in studying the methylation of CpG islands in the RB1, p16/CDKN2A, p15/CDKN2B, p14/ARF, CDH1, MGMT, HIC1, and N33 promoter regions in breast carcinoma (105 tumors). Methylation was often observed for the two major suppressor genes involved in cell-cycle control through the Cdk-Rb-E2F signaling pathway, RB1 (18/105, 17%) and p16 (59/105, 56%); both genes were methylated in 13 tumors. Methylation involved p15 in two (2%) tumors; CDH1, in 83 (79%) tumors; MGMT, in eight (8%) tumors, and N33, in nine (9%) tumors. The p14 promoter was not methylated in the tumors examined.

Establishment and Validation of Real-Time Polymerase Chain Reaction Method for CDH1 Promoter Methylation

Aberrant methylation of the promoter region has emerged as the major mechanism for silencing tumor suppressor genes. However, for some genes, such as E-cadherin (CDH1), methylation and protein expression demonstrate considerable heterogeneity, making correlations difficult. We compared methylation and protein expression status of CDH1 in 56 primary breast carcinomas using semiquantitative assays. Aberrant CDH1 methylation was studied by methylation-specific polymerase chain reaction (MSP) and semiquantitative real-time MSP assays. The Cdh1 expression was investigated by immunostaining on archival formalin-fixed sections from 34 primary carcinomas and their accompanying normal epithelium and preinvasive and metastatic lesions. Membrane-specific Cdh1 expression in the neoplastic cells was quantified by image analysis using an automated cellular imaging system and a continuous score. Aberrant promoter methylation of the CDH1 was present in 24 of 56 (43%) breast carcinomas by MSP assay. There was excellent concordance between the standard MSP assay and the real-time assay (91%, P < 0.0001). The concordance between loss of Cdh1 expression and CDH1 methylation by standard MSP was 71% (P = 0.02). Furthermore, there was a strong correlation between the semiquantitative assays for methylation and protein expression (r = 0.47, P = 0.005). We conclude that promoter methylation of CDH1 significantly correlated with the Cdh1 expression level, demonstrating that epigenetic silencing is a valid pathway for silencing of tumor suppressor genes in primary breast carcinomas.

Epigenetic control of the E‐cadherin gene (CDH1) by CpG methylation in colectomy samples of patients with ulcerative colitis

E-cadherin belongs to the cadherin family of calcium-dependent cell-adhesion molecules. The cadherins play an essential role in biological processes such as ordering of cell sorting, migration, and differentiation, and their malfunctioning is connected with neoplasia. Neoplastic progression in patients with chronic ulcerative colitis is characterized by the development of epithelial dysplasia. Transcriptional silencing of tumor-suppressor genes by promoter methylation has been observed in different types of human cancers and dysplasia. To explore the mode of E-cadherin regulation, 156 biopsy samples from 26 patients with long-standing ulcerative colitis were screened. To detect the methylation status of our samples, a methylation-specific PCR was applied. Methylation of the E-cadherin (CDH1) promoter was detected in 93% of the patients with dysplastic biopsy samples, in contrast to only 6% of the patients without dysplasia (P < 0.001). We also examined the level of synthesis of E-cadherin protein by immunohistochemical staining in different paraffin-embedded samples of dysplastic and non-dysplastic origin in a subset of our patients. Samples with dysplasia displayed reduced levels, whereas samples without dysplasia revealed normal E-cadherin protein synthesis. These results show that the E-cadherin promoter is subjected to epigenetic control in colorectal ulceration. Obviously, this event may play an important role in the progression from chronic inflammation to colorectal cancer. For this reason, methylation of the CDH1 promoter is an attractive new biomarker for detecting ulcerative colitis patients with a high risk for developing colorectal cancers.

E-cadherin gene (CDH1) promoter methylation as the second hit in sporadic diffuse gastric carcinoma.

In diffuse gastric carcinoma, despite common E-cadherin gene (CDH1) mutations, tumors show absence of CDH1 loss of heterozigosity (LOH) in most cases. This observation challenges the classical two-hit model of tumor suppressor gene inactivation. In order to investigate whether or not CDH1 promoter methylation may function as the second hit we analysed a series of 23 sporadic gastric carcinomas for the presence of CDH1 mutations, CDH1 promoter methylation, LOH and E-cadherin expression. CDH1 mutations were detected in nine of the 16 (56.3%) diffuse gastric carcinomas and in none of the seven intestinal gastric carcinomas. In diffuse gastric carcinomas harboring CDH1 mutations, LOH was observed in a single case. Loss of plasma membrane E-cadherin expression was consistently found in all nine cases with CDH1 mutation, suggesting that tumors inactivated the remaining CDH1 allele via a different mechanism. CDH1 promoter methylation was observed in nine of the 16 (56.3%) diffuse-type gastric carcinoma cases, including six of the nine cases (66.7%) harboring CDH1 mutations. CDH1 promoter methylation was also seen in two (28.6%) intestinal-type cases. Our results show that CDH1 promoter methylation is the second hit in more than half of the sporadic diffuse gastric carcinoma cases harboring CDH1 mutations.

Loss of E-cadherin expression in gastric intestinal metaplasia and later stage p53 altered expression in gastric carcinogenesis

Gastric cancers are commonly subdivided into intestinal and diffuse subtypes on a morphologic basis, supported by corollary evidence of differences at the pathogenetic and molecular levels. Chronic atrophic gastritis with intestinal metaplasia is a common precursor lesion for the intestinal type of carcinoma. To identify early molecular changes, in this study we have examined 13 surgical specimens both for the expression of E-cadherin, p53 and beta-catenin by immunohistochemistry and for methylation of the CDH1 promoter (E-cadherin) by bisulfite genomic sequencing of laser capture microdissected samples. Each specimen examined contained areas of normal (nonmetaplastic) gastric mucosa, as well as areas of intestinal metaplasia and/or carcinoma. Reduced or absent E-cadherin and partial to complete methylation of one to multiple CpG sites examined in the CDH1 promoter were observed in all of the metaplasia samples. Thus, the methylation status of the CDH1 promoter and expression of E-cadherin together provide strong evidence that loss of E-cadherin is an early event in intestinal type gastric carcinogenesis. In contrast, expression of p53, assumed to be mutant p53, was generally not detected (except for isolated cells) until the carcinoma stage in tissues from these patients. These results suggest that mutation of p53 is a late event in intestinal type gastric cancer. The level of beta-catenin expression did not appear to change between normal, metaplastic and carcinoma cells of intestinal type, and no nuclear staining was visible in any of the tissues. These results suggest that the Wnt signaling pathway is not upregulated in this type of cancer.

Multiple ways of silencing E-cadherin gene expression in lobular carcinoma of the breast.

The cell-cell adhesion receptor gene E-cadherin (CDH1) is expressed by epithelial cells, in which it mediates adhesion and morphogenesis. Invasive lobular carcinoma (ILC) characteristically infiltrates diffusely as single cells; by immunohistochemistry, many of these tumours lack E-cadherin expression. In the present study we investigated various ways in which loss of function of the E-cadherin gene could occur in ILCs, namely, promoter methylation, mutation and allelic loss. We analysed 22 ILCs and found 12 (55%) E-cadherin-negative samples by immunohistochemical analysis. Methylation-specific polymerase chain reaction (PCR) showed that 17/22 (77%) of these tumours had methylation of the CDH1 promoter, including 11/12 (91%) of the E-cadherin-negative tumours. All 16 exons of E-cadherin (including intron-exon boundaries) were amplified from chromosomal DNA and screened for mutations by conformation-sensitive gel electrophoresis (CSGE). Bands with altered mobility were analysed by direct sequencing. We identified five frameshift mutations, which resulted in downstream stop codons and one splice site mutation in six different tumours (29%). Loss of heterozygosity (LOH) was assessed using microsatellite markers, and 9/18 (50%) informative tumours showed LOH. We conclude that most ILCs show genetic or epigenetic changes affecting the E-cadherin gene and that many of these tumours lack E-cadherin expression. In all cases in which there was loss of expression, this was consistent with biallelic inactivation of CDH1 by promoter methylation, mutation or allelic loss in any combination.

Methylation of the CDH1 promoter as the second genetic hit in hereditary diffuse gastric cancer.

Aberrant promoter methylation and the associated loss of gene expression is a common accompaniment of human cancers. Nonetheless, it has been challenging to demonstrate in any given tumour that methylation of a specific gene was causal and not consequent to malignant transformation. In this regard, our attention was drawn to the genesis of gastric cancers in individuals with hereditary diffuse gastric cancer (HDGC). These individuals harbour germline mutations in the gene encoding E-cadherin, CDH1, but their cancers have consistently demonstrated absence of loss of heterozygosity at the CDH1 locus. These findings suggested the hypothesis that CDH1 promoter methylation might function as the 'second genetic hit' in the genesis of these cancers.