p16 Protein and Gigaxonin Are Associated with the Ubiquitination of NFκB in Cisplatin-induced Senescence of Cancer Cells

Background: Molecular mechanism of p16-mediated cellular senescence in cisplatin-treated cells is not known. Results: Cisplatin treatment leads to p16 nuclear transport and association with gigaxonin for the ubiquitination of NFB. Conclusion: A protein associated with neural diseases is involved in cisplatin-mediated cellular senescence. Significance: Nuclear expression of p16 and gigaxonin is a useful marker of cancer cell chemosensitivity. The molecular mechanism of p16-mediated senescence in cisplatin-treated cancer cells is not fully understood. Here we show that cisplatin treatment of head and neck cancer cells results in nuclear transport of p16 leading to a molecular modification of NFB. Chromatin immunoprecipitation assays show that this modification is associated with the inhibition of NFB interacting with its DNA binding sequences, leading to decreased expression of NFB-transcribed proteins. LCMS proteomic analysis of LAP-TAP-purified proteins from HeLa cells containing a tetracycline-inducible GFP-S peptide-NFB expression system identified gigaxonin, an ubiquitin E3 ligase adaptor, as an NFB-interacting protein. Immunoblotting and siRNA studies confirmed the NFB-gigaxonin interaction and the dependence of this binding on p16-NFB binding. Using gel shift assays, we have confirmed p16-NFB and gigaxonin-NFB interactions. Furthermore, we have observed increased NFB ubiquitination with cisplatin treatment that is abolished in the absence of p16 and gigaxonin expression. Analysis of 103 primary tumors has shown that increased nuclear p16 expression correlates with enhanced survival of head and neck cancer patients (p < 0.0000542), indicating the importance of nuclear p16 expression in prognosis. Finally, p16 expression is associated with reduced cytokine expression and the presence of human papilloma virus in chemoradiation-sensitive basaloid tumors. However, the absence of p16 expression is associated with enhanced cytokine expression and the absence of human papilloma virus in aggressive tumors. These results clearly demonstrate that nuclear p16 and gigaxonin play an important role in chemosensitivity of head and neck cancers through ubiquitination of NF kappa B.