Ataxia telangiectasia mutated nuclear localization in head and neck cancer cells is PPP2R2B-dependent

Abstract

Abstract Background: Protein phosphatase 2A (PP2A) has been implicated in radiation-induced activation of cellular responses, likely by its ability to regulate the autophosphorylation of the ataxia telangiectasia mutated (ATM) protein, a key molecule involved in the DNA damage response initiated by double-stranded DNA breaks. Interestingly, a hereditary defect in the PPP2R2B gene, which encodes the beta isoform of PP2A regulatory subunit B, causes autosomal dominant spinocerebellar ataxia 12, a clinical condition resembling that of ataxia telangiectasia patients. Moreover, PPP2R2B is significantly down-regulated in many human cancers, including head and neck squamous cell carcinomas (HNSCCs). Objective: Examine whether PPP2R2B regulates ATM function, thereby contributing to tumor progression due to the resulting defective DNA repair. Methods: The roles of PPP2R2B were evaluated in irradiated HNSCC cell lines, siRNAPPP2R2B cells and okadaic acid treated cells. Expression of PPP2R2B was measured by microarray, Western blot analysis and real time quantitative rtPCR. ATM quantity and localization, ATM phosphorylation and γ-H2AX were determined by Western blot analysis and/or immunofluorescence assay. Clonogenic cell survival assay was performed to determine ionizing radiation sensitivity. Results: PPP2R2B expression is reduced in multiple tumor types, including HNSCCs. Indeed, HNSCC cell lines that have lower PPP2R2B mRNA expression and siRNAPPP2R2B cells lower basal and radiation-induced levels of phosphorylated ATM and the consequent reduction in the levels of phosphorylation of the downstream ATM target, γ-H2AX. Depletion of PPP2R2B and inhibition of PP2A with okadaic acid resulted in limited ATM nuclear localization. Finally, siRNAPPP2R2B cells displayed enhanced sensitivity to death after radiation. Conclusion: In HNSCCs, ATM nuclear localization is PPP2R2B dependent, and decreased PPP2R2B expression may result in limited ATM activation by preventing its nuclear accumulation and ATM-chromatin interaction. Therefore, decreased PPP2R2B expression in HNSCCs may contribute to genomic instability, cancer development and radiation sensitivity by limiting ATM functions.