BACR6
The mechanisms and consequences of p53 inactivation in laryngeal squamous cell carcinoma
Anna Behrendt1, Terrence Jones2, Mark Boyd1
1University of Liverpool, Liverpool, Merseyside, UK, 2University Hospital Aintree, Liverpool, Merseyside, UK
Background
Loss of p53 function is a critical event in tumour development. p53 mutation can result in oncogenic gain-of-function (GOF) leading to both poorer prognosis and therapeutic response. Such p53 mutants exert their gain-of-function properties by modifying cells through altered transcriptional activity or novel protein-protein interactions. Our goal is to define the mechanism of action of mutant p53 by identifying the protein interactions of two GOF mutants that represent two distinct mutational classes; structural (R175H) and contact (R273H). To avoid the potentially confounding impact of studying cells from biologically distinct anatomical sub-sites we have studied laryngeal squamous cell carcinoma (LSCC) cells.
Method
Stable clones of a p53 null LSCC cell line have been generated which stably express p53 mutants cloned into Tandem Affinity Purification (TAP)-tag vectors to permit purification of protein complexes. TAP-tag p53 (wt and GOF) has been tested for expression and dominant-negative activity by western-blot and luciferase-reporter assays respectively. p53-binding proteins have been purified by tandem affinity chromatography and individual peptides are being identified by mass spectrometry.
Results
p53 GOF mutants display dominant-negative activity when expressed and tested in a p53 transcriptional assay. A well established p53-interacting protein, MDM2 has been identified by immunoblotting in the purified complexes containing mutant p53, validating the TAP-tag purification system. The individual proteins are being identified by mass spectrometry.
Conclusion
GOF mutant p53 promotes carcinogenesis through as yet poorly defined mechanisms. We are using the TAP-tag system to identify mutant p53 target protein interactions in laryngeal cancer cells to enable elucidation of the mechanisms of action of GOF mutant p53. Since p53 GOF mutants are typically linked with poor prognosis these novel interacting proteins may represent important new therapeutic targets.
