Epigenetic inactivation of the LRFN5 gene encoding type I transmembrane glycoprotein in esophageal squamous cell carcinoma

Alcohol drinking and cigarette smoking are significantly associated with an increased risk of esophageal cancer. Promoter hypermethylation of tumor suppressor genes plays an important role in esophageal squamous cell carcinoma (ESCC). To profile methylation changes and to identify methylated genes in clinical ESCC samples in a high-throughput fashion, we carried out genomewide methylation analysis using the methylated CpG island amplifi cation microarray (MCAM) technique in paired samples of ESCC and adjacent non-cancerous tissue and normal esophageal mucosa. As a surrogate of genome-wide hypomethylation, levels of LINE-1 methylation were analyzed using bisulfi te pyrosequencing. The degree of genome-wide methylation of the majority of non-cancerous esophageal tissues was intermediate between that of ESCC tissues and that of normal esophageal mucosa of healthy volunteers. A total of 210 candidate genes that are hypermethylated in ESCCs were identifi ed by using the MCAM technique. Among the candidate genes, we found that the LRFN5 gene coding for a transmembrane protein was transcriptionally silenced by promoter CpG island hypermethylation in ESCC. LRFN5 CpG island hypermethylation was a relatively common event even in patients without drinking habits. LRFN5 expression levels were reduced in most of the ESCC cell lines and ESCC tissues, being correlated with hypemethylation.Methylation levels of LINE-1 were significantly lower in tumor tissues than in adjacent non-tumor tissues of ESCC and inversely correlated with LRFN5 methylation. Importantly, LRFN5 immunostaining was undetectable in 5/7 (71%) intramucosal ESCC tissues,suggesting that LRFN5 silencing is an early event in ESCC pathogenesis. Hypermethylation of the LRFN5 gene may provide a useful marker for detecting preinvasive and early-stage ESCC, especially in individuals without drinking habits.