Mechanisms of p16^INK4A inactivation in non small-cell lung cancers

Abstract

The cyclin-dependent kinase inhibitor p16 (p16^INK4A/CDKN2/MTS1) is a potent inhibitor of the cyclin D-dependent phosphorylation of the retinoblastoma gene (Rb) product, the inactivation of which induces loss of Rb-dependent G1 arrest through inappropriate phosphorylation of the Rb protein. To analyse the role of p16^INK4A as a tumor suppressor in the genesis of non small cell lung cancers (NSCLC) and correlate loss of p16^INK4A protein expression to genetic or epigenetic mechanisms, we have performed a comprehensive study of p16 status in a series of 43 NSCLC. To this end, we have investigated p16^INK4A protein expression with immunohistochemistry, deletions of the gene by FISH, and determined the methylation status of exon 1α using a PCR-based methylation assay. Finally, possible mutations were studied by SSCP and subsequent sequencing. Twenty one of the 43 (49%) NSCLC studied exhibited an absence of p16^INK4A nuclear staining. Of these, three (14%) had frameshift or missense mutations, seven (33%) displayed methylation of exon1α and 10 (48%) displayed homozygous deletions. In total, 95% of the tumors with p16^INK4A negative staining carried one of these three alternative genetic or epigenetic alterations. Furthermore, a high degree of chromosome 9 polysomy was found (58%) in those tumors with p16^INK4A inactivation. Taken together these results suggest that deregulation of the p16 gene locus is a frequently occurring event in NSCLC through distinct mechanisms including rare point mutations, promotor methylation and frequent homozygous deletions. Furthermore, our data show that immunohistochemistry is a rapid and an accurate technique for screening of p16^INK4A gene inactivation events that result in loss of protein expression.