Aberrant methylation of p16INK4a and deletion of p15INK4b are frequent events in human esophageal cancer in Linxian, China

Abstract

p16^INK4a and p15^INK4b genes, which encode two functionally related CDK inhibitors, recently emerged as candidate tumor suppressor genes since they were both localized to 9p21, which frequently undergoes hemizygous and homozygous deletion in a variety of tumor types. To determine the mode of inactivation of these two genes in human esophageal squamous cell carcinoma (ESCC), we performed multiple molecular analyses in 60 ESCC specimens from Linxian, China using DNA methylation assay, LOH analysis, deletion screening and SSCP-sequencing. We observed that p16^INK4a inactivation was predominantly associated with aberrant methylation in the CpG island of its promoter region, whereas p15^INK4b frequently had homozygous deletions. Compared with aberrant methylation, which occurred in 17 of 34 cases, homozygous deletion of p16^INK4a and LOH at its nearby D9S942 microsatellite marker were observed at a much lower frequency (17%). Intragenic mutation in p16^INK4a gene was rare. In contrast, homozygous deletion in p15^INK4b and LOH at the nearby D9S171 marker were observed at frequencies of 35 and 47%, respectively, and the two events were significantly associated with each other. On the other hand, aberrant methylation of p15^INK4b was relatively infrequent (6/34) and occurred concomitantly with p16^INK4a methylation. Among the 60 cases, only four contained a continuous homozygous deletion spanning both p15^INK4b and p16^INK4a. Six cases were exclusively deleted at p16^INK4a and 17 exclusively deleted at p15^INK4b. LOH at D9S942 and D9S171 was also found to be mutually exclusive. Our results suggest that the alteration mode at 9p21 was not uniform, and the two genes were inactivated by distinct mechanisms. Altogether, 68% of the samples harbor at least one type of alteration in p16^INK4a gene and 50% of the samples were altered in p15^INK4b gene, indicating that they are the frequent inactivating targets during ESCC development.