639 Glutathione Peroxidase 7 is a Potential Tumor Suppressor Gene Silenced by Location-Specific Promoter Methylation in Barrett's Tumorigenesis

Abstract

Background: Esophageal adenocarcinoma (EAC) is considered to be developed from a precancerous condition Barrett's esophagus through dysplasia. Glutathione peroxidase 7 (GPX7) is a recent member of the GPX family with unknown biological functions, showing frequent downregulation in EACs. Promoter DNA hypermethylation is one of the mechanisms that silence tumor suppressor genes. Methods and Results: GPX7 gene expression is significantly down-regulated in Barrett's dysplasia and EACs as compared to normal esophagus. Using pyrosequencing technology, we examined the GPX7 promoter region (-162 to +138 from TSS) in esophageal cell lines and 78 primary EACs. We observed a significant increase in DNA methylation levels in Barrett's esophagus in most of the CpG sites examined as compared to normal samples, except for the CpG sites from +13 to +64, suggesting that DNA methylation of GPX7 may be an early event in Barrett's carcinogenesis. DNA hypermethylation of the CpG sites from +13 to +64 are only seen in EACs, not in normal samples and Barrett's esophagus, suggesting that this region may be more important in Barrett's carcinogenesis than other sites. DNA hypermethylation of this region was detected in 69% (54/78) of primary EACs and 5 out of 5 cancer cell lines, and are inversely correlated to the GPX7 gene expression (P<.01). On the other hand, mutations in the coding exons of GPX7 (examined by direct sequencing) and copy number losses (examined by real-time qPCR) were rarely seen (<5%) in the 30 EACs that we analyzed. Confirming that promoter DNA hypermethylation is the predominant molecular mechanism for inactivation of GPX7 in EACs. To understand the biological functions of GPX7 in EAC, we reconstituted GPX7 expression in two esophageal adenocarcinoma cell lines; OE33 and FLO-1. The reconstitution of GPX7 led to a significant reduction in growth rate on both cancer cell lines as demonstrated by colony formation and soft agar assays (P<0.05). Using mouse xenograft models, the growth of the GPX7-expressing OE33 cells was significantly hampered as compared to controls (p<0.01). Consistent with these findings, GPX7-expressing cells had a significantly lower EdU incorporation rate (indicating lower cell proliferation) and showed impairment in the G1/S progression as compared to control cells. Analyses of cell cycle regulators by Western blotting displayed high levels of p73, p27 and p21 proteins in GPX7-expressing cells. In addition, GPX7-expressing cells showed increased activity of pRB tumor suppressor gene as indicated by lower levels of phosphorylated pRB. Conclusions: GPX7 is a novel tumor suppressor gene in esophageal adenocarcinoma. Silencing of GPX7 by promoter DNA methylation unleashes the tumorigenic capacity through deregulating the levels of p73, p27, p21, and pRB.