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Mechanisms of p16INK4A inactivation in non small-cell lung cancers
S Gazzeri1, V Gouyer, C Vour'ch
1Groupe de recherche sur le cancer du poumon, DYOGEN, Institut Albert Bonniot, La Tronche, France.
Abstract:
The cyclin-dependent kinase inhibitor p16 (p16INK4A/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 p16INK4A as a tumor suppressor in the genesis of non small cell lung cancers (NSCLC) and correlate loss of p16INK4A 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 p16INK4A protein expression with immunohistochemistry, deletions of the gene by FISH, and determined the methylation status of exon 1alpha 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 p16INK4A nuclear staining. Of these, three (14%) had frameshift or missense mutations, seven (33%) displayed methylation of exon 1alpha and 10 (48%) displayed homozygous deletions. In total, 95% of the tumors with p16INK4A 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 p16INK4A 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 p16INK4A gene inactivation events that result in loss of protein expression.
Insights
The cyclin-dependent kinase inhibitor p16INK4A is frequently inactivated in non-small cell lung cancers (NSCLC) through genetic or epigenetic changes. Immunohistochemistry effectively screens for p16INK4A loss, indicating tumor suppressor function in NSCLC development.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- p16INK4A acts as a tumor suppressor by inhibiting cyclin D-dependent phosphorylation of the retinoblastoma protein (Rb).
- Inactivation of p16INK4A leads to loss of G1 cell cycle arrest, contributing to cancer development.
- Understanding p16INK4A deregulation is crucial for analyzing non-small cell lung cancer (NSCLC) genesis.
Purpose of the Study:
- To investigate the role of p16INK4A as a tumor suppressor in NSCLC.
- To correlate the loss of p16INK4A protein expression with underlying genetic or epigenetic mechanisms.
- To assess the utility of immunohistochemistry for detecting p16INK4A inactivation.
Main Methods:
- Analysis of p16INK4A protein expression using immunohistochemistry in 43 NSCLC samples.
- Detection of gene deletions via fluorescence in situ hybridization (FISH).
- Assessment of exon 1alpha methylation status using PCR-based assays.
- Identification of mutations through single-strand conformation polymorphism (SSCP) and sequencing.
Main Results:
- Absence of p16INK4A nuclear staining was observed in 49% of NSCLC cases.
- Mechanisms of inactivation included rare point mutations (14%), exon 1alpha methylation (33%), and frequent homozygous deletions (48%).
- Approximately 95% of tumors with negative p16INK4A staining showed one of these alterations; 58% exhibited chromosome 9 polysomy.
Conclusions:
- Deregulation of the p16INK4A gene locus is a common event in NSCLC, occurring via point mutations, promoter methylation, and homozygous deletions.
- Loss of p16INK4A protein expression is a reliable indicator of gene locus inactivation.
- Immunohistochemistry serves as a rapid and accurate method for screening p16INK4A inactivation in NSCLC.