Veena MS, Wilken R, Zheng JY, Gholkar A, Venkatesan N, Vira D, Ahmed S, Basak SK, Dalgard CL, Ravichandran S, Batra RK, Kasahara N, Elashoff D, Fishbein MC, Whitelegge JP, Torres JZ, Wang MB, Srivatsan ES

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 NFκB. Chip (chromatin immunoprecipitation) assay shows that this modification is ...

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associated with the inhibition of NFκB interacting with its DNA binding sequences, leading to decreased expression of NFκB transcribed proteins. LCMS proteomic analysis of LAP-TAP purified proteins from HeLa cells containing a tetracycline inducible GFP-S peptide-NFκB expression system identified gigaxonin, a ubiquitin E3 ligase adaptor, as an NFκB interacting protein. Immunoblotting and siRNA studies confirmed NFκB- gigaxonin interaction and the dependence of this binding on p16-NFκB binding. Using gel shift assays, we have confirmed p16-NFκB and gigaxonin-NFκB interactions. Further, we have observed increased NFκB 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 HPV in chemo-radiation sensitive basaloid tumors. However, absence of p16 expression is associated with enhanced cytokine expression and absence of HPV in aggressive tumors. These results clearly demonstrate that nuclear p16 and gigaxonin play an important role in chemo-sensitivity of head and neck cancers through ubiquitination of NFκB.

Date: Oct. 20, 2014

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