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Clinical Cancer Research Vol. 7, 2765-2769, September 2001
© 2001 American Association for Cancer Research

Regular Articles

Frequent Hypermethylation of the 5' CpG Island of E-Cadherin in Esophageal Adenocarcinoma

Paul G. Corn1,, 2, Elisabeth I. Heath1, Richard Heitmiller, Franz Fogt, Arlene A. Forastiere, James G. Herman and Tsung-Teh Wu

University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104 [P. G. C., F. F.]; Johns Hopkins Oncology Center, Baltimore, Maryland 21231 [E. I. H., R. H., A. A. F., J. G. H.]; and Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205 [T-T. W.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: E-cadherin, a Mr 120,000 transmembrane glycoprotein, mediates calcium-dependent intercellular adhesion that is essential for normal tissue homeostasis. Loss of E-cadherin occurs in a variety of epithelial tumors and is correlated with invasion and metastasis. In esophageal adenocarcinoma, reduction of E-cadherin expression has been demonstrated previously, but mutations of the gene (CDH1) are rare.

Experimental Design: In this study, we used a nested PCR approach to examine the methylation status of the 5' CpG island of E-cadherin in esophageal specimens obtained from individuals with and without a history of esophageal cancer.

Results: In four individuals without esophageal cancer, E-cadherin was completely unmethylated in normal squamous cell-lined esophageal mucosa. In contrast, in patients with esophageal adenocarcinoma, E-cadherin was methylated in 26 of 31 (84%) tumor specimens. In the majority of cases, matched normal tissue (esophagus or stomach) from each patient was completely unmethylated. By immunostaining, methylated tumor samples demonstrated heterogeneously decreased membranous E-cadherin staining.

Conclusions: These data suggest that epigenetic silencing via aberrant methylation of the E-cadherin promoter is a common cause of inactivation of this gene in esophageal adenocarcinoma.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
E-cadherin is a Mr 120,000 transmembrane glycoprotein expressed on the surface of epithelial cells. In epithelial tissues, E-cadherin mediates homophilic, Ca2+-dependent intercellular adhesion that is essential for the maintenance of normal tissue architecture (1) . The cytoplasmic domain of E-cadherin is linked to actin in the cytoskeleton via ß- and {alpha}-catenins, and this interaction is critical for its function (2) . Loss of E-cadherin expression occurs in a variety of human tumors and is hypothesized to be an important step in the progression from tumor formation to invasion and metastasis (3) . In cancers originating from the breast, lung, nasopharynx, bladder, stomach, and esophagus, reduced E-cadherin expression has been associated with an unfavorable prognosis (4, 5, 6, 7, 8, 9) .

There are several mechanisms for abnormal E-cadherin function in tumors. It has recently been shown that germ-line mutations of the gene (CDH1), although rare, predispose individuals to familial gastric and colorectal cancer (10 , 11) . Similarly, whereas allelic loss of the E-cadherin locus at 16q22.1 has been reported in different epithelial tumors (breast, ovarian, endometrial, and prostate carcinomas), somatic mutations occur infrequently (12) . One important mechanism for loss of E-cadherin expression is methylation of the 5' CpG island within the promoter, which results in transcriptional repression of the gene (13) . In neoplasia, aberrant methylation serves as an alternative to point mutation and chromosomal deletion for inactivation of tumor suppressor genes and genes that preserve normal cell function, such as VHL and the DNA mismatch repair gene MLH1 (14 , 15) . Indeed, methylation-associated silencing of E-cadherin represents the most common cause for its inactivation and has been observed in cancers of the liver, prostate, breast, oral cavity, and stomach (16, 17, 18, 19) .

The incidence of adenocarcinoma of the esophagus has been rising dramatically in the United States and Western Europe over the last two decades (20 , 21) . The majority of these tumors arise from Barrett’s esophagus, a premalignant condition characterized by columnar epithelial metaplasia caused by chronic gastroesophageal reflux (22 , 23) . The progression from Barrett’s esophagus to adenocarcinoma is a multistep process involving nonrandom loss of heterozygosity, aneuploidy, and alterations of p53 and p16 (24, 25, 26) . Interestingly, E-cadherin expression is reduced in both Barrett’s esophagus and esophageal adenocarcinoma and in the latter has been correlated with a greater frequency of lymph node metastasis and shorter patient survival (27 , 28) . Mutations of the E-cadherin gene are rare in adenocarcinoma of the esophagus. Thus, the mechanism for its down-regulation in this tumor type has not been explained (29) . In this study, we demonstrate that aberrant methylation of the 5' CpG island of E-cadherin occurs in a majority of esophageal adenocarcinomas, leading to reduced E-cadherin expression.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Source of Tissue Samples.
Normal control cases consisted of paraffin-embedded sections of benign esophageal tissue from autopsy cases of individuals (mean age = 60 years) without a history of gastrointestinal malignancy. Histological sections of these autopsy specimens showed normal squamous cell-lined esophageal mucosa without acute or chronic inflammation. Tumor and paired normal tissue specimens were obtained from 31 patients with esophageal adenocarcinoma who underwent surgical resection at Johns Hopkins Oncology Center between March 1991 and August 1998. None of the patients received irradiation or chemotherapy preoperatively. For each patient, frozen sections were available from both the tumor and either normal esophageal or gastric mucosa. An H&E stain was performed to confirm the presence of carcinoma in each case. Normal specimens were obtained at least 1 cm from the distal negative margin of the tumor.

DNA Extraction.
Genomic DNA from esophageal adenocarcinoma was isolated from frozen tissue slides as described previously (30) . For the patients with adenocarcinoma, control DNA was isolated from normal esophageal (n = 21) or gastric (n = 10) mucosa.

Methylation Analysis.
Methylation patterns within the E-cadherin CpG island in Exon 1 (sequence -126 bp to +144 bp relative to transcription start, GenBank accession number D49685) were determined using a nested PCR approach that has been published previously (31) . In the first round of PCR, 100 ng of bisulfite-treated DNA were amplified using sequencing primers. The sequencing primers were 5'-GTTTAGTTTTGGGGAGGGGTT-3' (sense) and 5'-ACTACTACTCCAAAAACCCATAACTAA-3' (antisense), and the cycling conditions consisted of an initial denaturation step at 95°C for 5 min, followed by the addition of 1 unit of Taq polymerase and then 30 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 30 s. The size of the product after this initial PCR reaction was 270 bp. For the second round of PCR, this product was diluted 1:50 in water, and 2 µl of the dilution were used for MSP3 (32) . Nested primer sequences for E-cadherin for the methylated reaction were 5'-TGTAGTTACGTATTTATTTTTAGTGGCGTC-3' (sense) and 5'-CGAATACGATCGAATCGAACCG-3' (antisense), and primer sequences for the unmethylated reaction were 5'-TGGTTGTAGTTATGTATTTATTTTTAGTGGTGTT-3' (sense) and 5'-ACACCAAATACAATCAAATCAAACCAAA-3' (antisense). PCR parameters were as listed above, except that the annealing temperatures for the methylated and unmethylated reactions were 64°C and 62°C, respectively. The product sizes of the methylated and unmethylated reactions were 112 and 120 bp, respectively. As a positive control for methylation, we used the breast cancer cell line MB-MDA-231, which demonstrates methylation and silencing of CDH1. Samples were scored as methylated if there was visible PCR product after amplification with the methylated primers.

Immunohistochemistry for E-Cadherin.
Immunoperoxidase staining using diaminobenzidine as a chromogen was performed on parallel histopathological sections from ethanol-fixed frozen section tumor tissue, using the TechMate 1000 automatic staining system (BioTek Solutions, Tucson, AZ). E-cadherin gene product (mouse monoclonal antibody Clone 36; Transduction Laboratories, Lexington, KY; dilution = 1:400) was stained after antigen retrieval by a heat-induced epitope retrieval method.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Clinical and Pathological Features.
The clinical and pathological features of the patients with esophageal adenocarcinoma are shown in Table 1Citation . There were a total of 31 patients (25 men and 6 women) with a mean age of 67 years (range, 41–81 years). Patients with tumor-node-metastasis (TNM) stage I-IV cancers were represented in the group. There were 4 patients (13%) with stage I disease, 15 patients (48%) with stage II disease, 8 patients (26%) with stage III disease, and 4 patients (13%) with stage IV disease.


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  		Table 1 Clinical, pathological, and E-cadherin methylation characteristics of patients with esophageal adenocarcinoma

 
Methylation Analysis.
In this study, genomic DNA for analysis was extracted from a single slide of either frozen or paraffin-embedded tissue obtained from each surgical specimen. To detect E-cadherin methylation, we used a nested PCR approach that was developed previously in our laboratory (31) . Nested MSP is a two-step PCR that has the sensitivity to detect methylated alleles when they comprise as little as 5% of the total DNA sample, and it is well suited for the analysis of DNA obtained from paraffin-embedded material when the quantity and/or quality of the DNA substrate can be limiting. In addition, nested MSP preserves the specificity and reproducibility of standard (one-step) MSP (31) . For consistent analysis between DNA isolated from paraffin-embedded tissues and frozen tissues, the nested MSP approach was used on all of the samples.

We initially examined tissues obtained from individuals without a history of esophageal cancer to determine whether E-cadherin was methylated in normal esophagus. Using nested MSP, all four normal esophageal samples were completely unmethylated at E-cadherin (Fig. 1A)Citation .



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  		Fig. 1. Methylation analysis of E-cadherin in esophageal specimens obtained from individuals with and without esophageal adenocarcinoma. Visible product in Lanes U indicates the presence of unmethylated genes; visible product in Lanes M indicates the presence of methylated genes. A, samples of normal squamous cell-lined esophageal mucosa in four individuals without esophageal cancer. E-cadherin was completely unmethylated in all cases. +, nested MSP analysis of the breast cancer cell line MB-MDA-231 served as a positive control. B, paired samples of normal esophagus or stomach tissue (N) and tumor (T) from eight patients are shown. There is tumor-specific methylation in Cases 2–4, 6, and 7. Note that in Case 5, the tumor is fully methylated, whereas the corresponding normal tissue has a very faint band demonstrating methylation.

 
We next examined paired tumor and normal specimens from patients with esophageal carcinoma. Twenty-six of 31 tumors (84%) obtained from these individuals demonstrated evidence of E-cadherin methylation using nested MSP (see Fig. 1BCitation and Table 1Citation ). Among these 26 cases, the methylation change was tumor specific in 20 cases, because normal esophagus tissues from the same patients were completely unmethylated. In the remaining six cases in which the tumor was methylated, normal esophagus (n = 3) or normal stomach (n = 3) tissues from the same patients were also methylated. Of note, E-cadherin methylation of the normal esophagus or stomach without corresponding methylation of the tumor was never observed.

E-cadherin methylation was relatively more frequent in poorly differentiated tumors, a parameter that has been associated with worse outcome in patients with esophageal adenocarcinoma (28) . With regard to tumor differentiation, 100% of poorly differentiated tumors were methylated versus approximately 72% of tumors of both poor-moderate and moderate differentiation. Whereas this observation suggests a correlation between E-cadherin methylation and other predictors of prognosis in esophageal adenocarcinoma, the sample size in this study was too small for these results to achieve statistical significance.

Immunostaining.
Immunostaining of E-cadherin was performed on six tumors [four tumors with corresponding normal mucosa (two esophageal and two gastric tumors)] with E-cadherin methylation and four tumors (one tumor with normal esophageal mucosa) without E-cadherin methylation. The staining was heterogeneous in all six tumors with E-cadherin methylation. Tumor cells with decreased membranous E-cadherin staining (ranging from 10%–70% of the tumor area) were mixed with tumor cells showing strong membranous staining (Fig. 2)Citation . In contrast, three of four tumors without E-cadherin methylation demonstrated diffuse strong membranous E-cadherin staining, with the remaining case showing focal (<5%) decreased staining in the tumor cells (Fig. 2)Citation .



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  		Fig. 2. E-cadherin immunostain in esophageal adenocarcinomas. A and B, E-cadherin expression is heterogeneous in a tumor with E-cadherin methylation. A, tumor cells with decreased membranous E-cadherin staining as compared with normal esophageal squamous epithelium. B, in another area, the same tumor shows a similar intensity of E-cadherin expression as compared with esophageal squamous epithelium. C and D, diffuse strong E-cadherin expression in a tumor without E-cadherin methylation. C, the normal esophageal squamous epithelium serves as an internal control. D, a higher power view of the membranous staining in tumor cells from a different area.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Perturbations of the cadherin-catenin complex occur frequently in esophageal adenocarcinoma, as evidenced by reduced expression of one or more proteins in the complex (33) . The biological consequences of this event include disruption of intercellular adhesion and impairment of ß-catenin-mediated transactivation (34 , 35) . To date, however, the regulatory mechanisms responsible for altered levels of these proteins in esophageal adenocarcinoma have not been elucidated.

In this study, we show that hypermethylation of the 5' CpG island of the E-cadherin promoter occurs frequently in esophageal adenocarcinoma and that this methylation change is associated with reduced expression of E-cadherin protein. In two previous studies of esophageal adenocarcinoma, reduced E-cadherin expression was noted in approximately 75% of the cases (28 , 33) . Although the etiology of E-cadherin down-regulation was not directly examined in these studies, mutations of the gene in esophageal adenocarcinoma were rare (29) . Our data suggest that epigenetic silencing of the E-cadherin promoter via hypermethylation is the critical mechanism for inactivation of this gene in esophageal adenocarcinoma. Gene silencing associated with hypermethylation is mediated by methyl-binding proteins that bind to methylated cytosines and recruit a complex of proteins that repress transcription, including histone deacetylases (36) .

In the majority of the cases we examined, E-cadherin methylation was tumor specific. There were, however, six cases in which the methylation change was present in both tumor and normal esophagus or stomach tissues from the same patient. Given the sensitivity of nested MSP, it is possible that normal-appearing specimens contained a rare cancer cell that was undetectable by histomorphology. To avoid this, we selected histologically normal specimens as distant from the tumor as possible. Alternatively, it is possible that E-cadherin methylation occurs in normal tissues, albeit at a low frequency. If this latter possibility is true, it remains likely that E-cadherin methylation indicates pathology at the molecular level. In sections of benign esophageal tissue obtained from individuals without a history of esophageal cancer, we did not observe E-cadherin methylation. Furthermore, in previous studies investigating E-cadherin methylation in different tumor types, normal tissues from bone marrow, lymphocytes, thyroid, breast, and oral mucosa were unmethylated (17 , 31 , 37) . These data strongly suggest that the E-cadherin promoter methylation is an aberrant event. The fact that we only detected methylation in normal esophagus or stomach tissues from patients in whom the corresponding tumor was also methylated is consistent with the hypothesis that the cancer in these individuals arose from a methylated clonal precursor. In support of this hypothesis, in a recent study of neoplastic progression in Barrett’s esophagus, hypermethylation of the tumor suppressor gene p16 was detected in pathologically normal-appearing specimens obtained from a patient who later developed dysplasia (26) . Thus, epigenetic inactivation of tumor suppressor genes may be an early feature of esophageal tumorigenesis.

Our results differ from those recently reported by Eads et al. (38) during the preparation of this article. In their study, there was no evidence of E-cadherin methylation in 22 esophageal adenocarcinomas. There are several possible reasons for the different results. First, Eads et al. (38) used a different technique for methylation determination (real-time PCR) and did not examine methylation and gene expression. Second, the positive control for methylation in that study was in vitro-methylated DNA, not the well-characterized breast cancer cell line we used (MB-MDA-231) that demonstrates methylation and silencing of the CDH1 gene. Third, the region of the E-cadherin 5' CpG island that was examined differed from that examined in our study (-180 to -111 bp versus -126 to +144 bp relative to transcription start, respectively). Nonetheless, we feel that our study provides a molecular explanation for the loss of E-cadherin in esophageal adenocarcinoma described in the literature.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 These authors contributed equally to this work. Back

2 To whom requests for reprints should be addressed, at University of Pennsylvania, Division of Hematology/Oncology, Room 437A CRB, 415 Curie Boulevard, Philadelphia, PA 19104-4218. E-mail: pcorn{at}mail.med.upenn.edu Back

3 The abbreviation used is: MSP, methylation-specific PCR. Back

Received 2/21/01; revised 6/ 5/01; accepted 6/ 6/01.


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 DISCUSSION
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H.-T. Zhang, X.-F. Chen, M.-H. Wang, J.-C. Wang, Q.-Y. Qi, R.-M. Zhang, W.-Q. Xu, Q.-Y. Fei, F. Wang, Q.-Q. Cheng, et al.
Defective Expression of Transforming Growth Factor {beta} Receptor Type II Is Associated with CpG Methylated Promoter in Primary Non-Small Cell Lung Cancer
Clin. Cancer Res., April 1, 2004; 10(7): 2359 - 2367.
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 Ann. Thorac. Surg.Home page
P. S. Dahlberg, L. F. Ferrin, S. M. Grindle, C. M. Nelson, C. D. Hoang, and B. Jacobson
Gene expression profiles in esophageal adenocarcinoma
Ann. Thorac. Surg., March 1, 2004; 77(3): 1008 - 1015.
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M. V. Brock, M. Gou, Y. Akiyama, A. Muller, T.-T. Wu, E. Montgomery, M. Deasel, P. Germonpre, L. Rubinson, R. F. Heitmiller, et al.
Prognostic Importance of Promoter Hypermethylation of Multiple Genes in Esophageal Adenocarcinoma
Clin. Cancer Res., August 1, 2003; 9(8): 2912 - 2919.
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 CarcinogenesisHome page
P. G. Corn, M. K. Summers, F. Fogt, A. K. Virmani, A. F. Gazdar, T. D. Halazonetis, and W. S. El-Deiry
Frequent hypermethylation of the 5' CpG island of the mitotic stress checkpoint gene Chfr in colorectal and non-small cell lung cancer
Carcinogenesis, January 1, 2003; 24(1): 47 - 51.
[Abstract] [Full Text] [PDF]

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