Expression Of EphB2 And Mechanisms For Its Downregulation In Colorectal Cancer

EphB2 receptor coded by EPHB2 gene is a member of receptor tyrosine kinase. They are located on the cell surface, and transduce bi-directional signals when binding with their ligands on adjacent cell surface. Being cleaved by transmembrane proteases or removed from the cell surface by trans-endocytosis results in cell-cell repulsion. EphB2 was found at high levels in diverse human tumor types, including colorectal cancer (CRC). Elevated expression has been proposed to promote tumorigenesis by modulating cell motility and angiogenesis. But recent studies have found most human CRCs lose expression of EphB2 at the adenoma-carcinoma transition. It represents a critical step in CRC progression. EphB2-mediated compartmentalization by binding with its ligand restricts the spreading of tumor cells.The mechanisms of EphB2 expression silencing in CRC remain uncharacterized. Genetic changes, such as point mutations and loss of heterozygosity, were found in a fraction of CRCs. There are conflicting data regarding epigenetic regulation of EPHB2 in CRCs. Anyway, a further research on the expression and its regulation is helpful.In this study, we examined the expression of EphB2 in four colon cancer cells, 13 normal rectal mucosas, 30 CRCs and matched normal colorectal mucosa samples by using western blot, immunohistochemistry or real-time PCR. We found that expression of EphB2 was silenced in most of the CRCs. A significant association (P<0.05) was found between EphB2 expression and histological differentiation. Tumors with negative EphB2 expression were more often poorly differentiated. Methylation status of promoter CpG island of EPHB2 in all tissues and 4 colon cancer cells were determined by methylation specific PCR, and showed a complete absence of methylation. These results were confirmed in 17 CRCs with negative expression of EphB2 and the four colon cancer cells by bisulfite genomic sequencing.For the importance of transcriptional regulation in gene expression but the lack of detailed experimental data in the human EPHB2 promoter, it promoted us to not only investigate the responsible regions in the 5'flanking region of the human EPHB2, but also the transcription factors associated with the human EPHB2 transcript expression. Transfection assay using a series of deletion constructs of the 5'-flanking region fused to the luciferase reporter gene revealed that a 508-bp (-425/+83) fragment are necessary for high transcriptional activity, and identified a 205-bp (-1174/-970) fragment with silencer activity. The computer analysis predicted the presence of a cis-element (GC-box) in promoter region for Sp1 and a cis-element in silencer region for c-Rel. Mutation of GC-box resulted in a decline of about 15% in EPHB2 promoter activity compared to wild type promoter activity (P>0.05). Mutation of c-Rel binding site resulted in a highly significant increase of about 85% in EPHB2 promoter activity compared to wild type promoter activity (P<0.01).Our data indicated that expression of EphB2 was silenced at different degree in most CRCs. The silencing occured at both transcriptional and protein levels, and was not associated with methylati4on of EPHB2 promoter CpG island, but with transcriptional regulation. We postulated that transcription of EPHB2 gene is regulated negatively by binding c-Rel transcription factor on the silencer, which results in the silencing of EphB2 in CRCs. These results provide a new clew to investigate the mechanisms of EphB2 silencing in CRC, as well as a new target for gene therapy and drug treatment of CRCs.