Inactivation and growth suppression of CDKN2 and CDKN2B in esophageal cancer

Abstract

The genes CDKN2 (MTS1, CDK4I, p16{dollar}\rm\sp{lcub}INK4{rcub}){dollar} and CDKN2B (MTS2, CDK4Ib, p15{dollar}\rm\sp{lcub}INK4{rcub}){dollar} encoding the proteins p16 and p15 respectively, are both located on chromosomal band 9p21, a locus at which frequent homozygous and heterozygous deletions occur in many primary human tumors, including esophageal carcinoma. CDKN2 and CDKN2B belong to a family of cyclin-dependent kinase 4 inhibitors (INK4) and control cell proliferation by preventing entry into the S phase of the cell cycle. Their inactivation may contribute to uncontrolled growth in human cancers. To investigate whether CDKN2 and CDKN2B are involved in esophageal tumorigenesis, intragenic mutations of CDKN2 and CDKN2B in primary esophageal cancers were examined. Sixty primary esophageal cancers were analyzed for mutations in both exons 1 and 2 of CDKN2 and CDKN2B by direct sequencing of PCR-amplified genomic DNAs. Six sequence alterations of CDKN2 were observed in five squamous cell carcinomas and in one adenocarcinoma. All six nucleotide changes resulted in marked alterations in amino acid sequence. Four were nonsense mutations leading to premature termination codons; nucleotide substitutions identical to two of these stop codons were previously reported in other tumor types. One CDKN2B nonsense mutation which truncates the protein product by 75% and one silent CDKN2B mutation occurred somatically. In order to determine the tumor suppressive role of these genes in vitro, homozygous deletion, intragenic mutation, and messenger RNA (mRNA) expression of CDKN2 and CDKN2B in nine esophageal squamous cancer cell lines were studied. To establish whether CDKN2 and CDKN2B exert growth suppressive effect on esophageal cancer cell lines, CDKN2, CDKN2B, and a combination of these two genes were transfected into esophageal cancer cells that were homozygously deleted for CDKN2, CDKN2B or both genes. Polymerase chain reaction (PCR) amplification revealed that five of nine cell lines (55%) manifested homozygous deletions of CDKN2, CDKN2B, and/or flanking loci on chromosomal band 9p21. Reverse transcriptase-PCR (RT-PCR) was used to examine CDKN2 and CDKN2B mRNA in the nine cell lines. Lack of CDKN2 and CDKN2B mRNA correlated perfectly with homozygous deletion involving these genes. No subtle intragenic mutations of CDKN2 and CDKN2B were detected by DNA sequencing of their entire coding sequences in any cell lines lacking homozygous deletion. Two of the cell lines manifested homozygous deletions excluding CDKN2; one of these two deletions also excluded CDKN2B. Restoration of CDKN2 and/or CDKN2B into the cancer cell lines demonstrated marked growth suppression by CDKN2 and/or CDKN2B. Taken together, the data obtained about CDKN2 and CDKN2B in primary tumors and cell lines suggest that both genes are involved in the malignant phenotype in esophageal cells. Inhibition of cancer cell growth or proliferation by expression of transfected CDKN2 and CDKN2B is functional evidence that CDKN2 and CDKN2B are tumor suppressors in esophageal epithelium. The relative varity of point mutations and frequency of deletions suggest that homozygous deletion is the predominant mechanism of inactivation of CDKN2 and CDKN2B.