Identification of an invasion and tumor-suppressing gene, Endoglin (ENG), silenced by both epigenetic inactivation and allelic loss in esophageal squamous cell carcinoma

Abstract

Endoglin (ENG) has been identified as a candidate tumor-suppressor gene in esophageal squamous cell carcinoma (ESCC). Earlier microcell-mediated chromosome transfer (MMCT) studies of chromosome 9 in ESCC narrowed down a tumor-suppressive critical region to 9q33-34. ENG maps to 9q34-qter and encodes a transformation growth factor beta (TGFb) superfamily auxiliary receptor. This study aims to identify the potential role for ENG in ESCC development. Significant downregulation of ENG was detected at frequencies of 87.5% in 16 ESCC cell lines, 39.1% directly in 23 ESCC tumor specimens from Hong Kong, and 33.4% in 18 ESCC tumor specimens from the high-risk ESCC region of Henan, China. By methylation-specific PCR, methylated sequences were detected in an ESCC cell line panel and in clinical specimens. Following demethylation treatment in 9 ESCC cell lines, ENG expression was obviously restored. Loss of heterozygosity (LOH) in a 4.7 Mb region on 9q32-q34, where ENG maps, was observed directly in ESCC tumor tissues. Both epigenetic methylation and allelic loss appear to contribute to ENG downregulation in tumor cells. In vitro and in vivo functional studies such as colony formation, Matrigel culture, invasion and tumorigenicity assays were performed. Colony formation efficiency was significantly reduced by overexpression of ENG. In addition, significantly smaller colonies of ENG stable transfectants were formed in Matrigel culture. Significant suppression of invasion efficiency and tumorigenicity were also observed, when comparing the ENG stable transfectants with the vector-alone transfectants. This study provides evidence supporting ENG, as a cell invasion and tumor-suppressing gene in ESCC. © 2008 Wiley-Liss, Inc.