Mechanisms of inactivation of p14(ARF), p15(INK4b), and p16(INK4a) genes in human esophageal squamous cell carcinoma

The 9p21 gene cluster, harboring growth suppressive genes p14(ARF), p15(INK4b), and p16(INK4a), is one of the major aberration hotspots in human cancers. It was shown that p14(ARF) and p16(INK4a) play active roles in the p53 and Rb tumor suppressive pathways, respectively, and p15(INK4b) is a mediator of the extracellular growth inhibition signals. To elucidate specific targets and aberrations affecting this subchromosomal region, we constructed a detailed alteration map of the 9p21 gene cluster by analyzing homozygous deletion, hypermethylation, and mutation of the p14(ARF), p15(INK4b), and p16(INK4a) genes individually in 40 esophageal squamous cell carcinomas (ESCCs) and compared the genetic alterations with mRNA expression in 18 of these samples. We detected aberrant promoter methylation of the p16(INK4a) gene in 16 (40%), of p14(ARF) in 6 (15%), and of p15(INK4b) in 5 (12.5%) tumor samples. Most p16(INK4a) methylations were exclusive, whereas all but one of the p14(ARF)/p15(INK4b) methylations were accompanied by concomitant p16(INK4a) methylation. We detected homozygous deletion of p16(INK4a) in 7 (17.5%), of p14(ARF)-E1β in 13 (33%), and of p15(INK4b) in 16 (40%) tumor samples. Most deletions occurred exclusively on the E1β- p15(INK4b) loci. Two samples contained p14(ARF) deletion but with p16(INK4a) and p15(INK4b) intact. No mutation was detected in the p14(ARF) and p16(INK4a) genes. Comparative RT-PCR showed good concordance between suppressed mRNA expression and genetic alteration for p15(INK4b) and p16(INK4a) genes in the 18 frozen samples, whereas 5 of the 13 cases with suppressed p14(ARF) mRNA expression contained no detectable E1β alteration but aberrations in the p16(INK4a) locus. Our results show that in human ESCCs, p14(ARF) is a primary target of homozygous deletion along with p15(INK4b), whereas p16(INK4a) is the hotspot of hypermethylation of the 9p21 gene cluster. The frequent inactivation of the p14(ARF) and p16(INK4a) genes may be an important mechanism for the dysfunction of both the Rb and p53 growth regulation pathways during ESCC development.