Transcriptional silencing of EphB6 receptor tyrosine kinase in invasive breast carcinoma cells and detection of methylated promoter by methylation specific PCR.

The receptor tyrosine kinase EphB6 is expressed at reasonable levels in normal breast cells. It shows decreased abundance in non-invasive breast carcinoma cells and is transcriptionally silenced in invasive breast carcinoma cells. We have characterized EphB6 promoter and correlated the expression of EphB6 transcript to differential methylation of the promoter region. The demethylation of promoter sequence in vivo by growth in media containing 5-aza-2'-deoxycytidine restores the expression of EphB6 to normal levels in breast carcinoma cells, and the ability of the promoter to initiate transcription of a reporter gene is lost after methylation of the promoter sequence. The promoter region has binding sites for various factors such as SP1 and p300. The specific methylation of CpG dinucleotides has allowed us to design primers that can selectively amplify the methylated promoter and thus facilitate identification of normal, non-invasive, and invasive breast cells. The potential significance of EphB6 to serve as a diagnostic and prognostic indicator is discussed.