Frequent Aberrant Methylation of the  CDH4  Gene Promoter in Human Colorectal and Gastric Cancer

Elena Miotto,Giovanni Lanza,Angelo Veronese,Roberta Gafà,Sergio Gullini,Laura Gramantieri,Luigi Bolondi,George A Calin,Massimo Negrini,Silvia Sabbioni,Eros Ferrazzi,Alberto Liboni

doi:10.1158/0008-5472.can-04-3000

Abstract

Gene promoter methylation causes loss of tumor suppressor genes function in human cancer. Here, we show that the CDH4 gene, a member of the cadherin family encoding for R-cadherin, contains a CpG island located at the 5' of the first exon, which functions as a promoter element and is frequently affected by methylation in human cancer. By using methylation-specific PCR and reverse transcription-PCR in human cancer cell lines, promoter methylation could be directly linked to loss of gene expression. After treatment with the demethylating agent 5-aza-2-deoxycytidine, expression could be restored. Analysis of human primary tumors revealed that the CDH4 gene is methylated in 78% (38 of 49) of colorectal and 95% (20 of 21) of gastric carcinomas. CDH4 methylation was not detected in nonneoplastic colonic (0 of 10) and stomach (0 of 10) tissues or in peripheral blood (0 of 17). CDH4 methylation was detected in histologically normal tissues located in proximity of the neoplasms, indicating that CDH4 methylation is an early event in gastrointestinal tumor progression. We also proved that CDH4 methylation can be revealed in the peripheral blood of cancer patients. Our results indicate that CDH4 may act as a tumor suppressor gene in human gastrointestinal tumors and can potentially be used as an early diagnostic marker for gastrointestinal tumorigenesis.

Highlights

Classical cadherins are membrane proteins characterized by five tandemly repeated extracellular cadherin domains and a wellconserved cytoplasmic domain that interact with the catenins ␤-catenin/Armadillo and p120ctn/␦-catenin. ␤-Catenin is connected to ␣catenin, which in turn links the cadherin-catenin complex to the actinHere, we report that a third member of this family, the CDH4 gene, encoding for the retinal cadherin (R-cadherin), is frequently silenced by promoter methylation in human gastrointestinal tumors, suggesting that it may play a previously unsuspected function of tumor suppressor gene
To determine whether aberrant methylation occurs at this region, we analyzed 21 human tumor cell lines by methylation-specific PCR with the primer set MF/MR (Table 1)
The CDH4 gene was silent in the methylated cell lines, whereas it was expressed in the unmethylated cell lines, implicating a direct relationship between promoter methylation and loss of CDH4 gene transcription (Fig. 1C)

Summary

Introduction

Classical cadherins are membrane proteins characterized by five tandemly repeated extracellular cadherin domains and a wellconserved cytoplasmic domain that interact with the catenins ␤-catenin/Armadillo and p120ctn/␦-catenin. ␤-Catenin is connected to ␣catenin, which in turn links the cadherin-catenin complex to the actinHere, we report that a third member of this family, the CDH4 gene, encoding for the retinal cadherin (R-cadherin), is frequently silenced by promoter methylation in human gastrointestinal tumors, suggesting that it may play a previously unsuspected function of tumor suppressor gene.

Methods

Results

Conclusion