Targeting nitrative stress for treatment of cisplatin ototoxicity

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Abstract A critical gap exists in understanding how nitrative stress, which has been effectively targeted to inhibit cell death in other models, alters cochlear protein signaling to induce apoptosis in cisplatin-induced ototoxicity. Continued existence of this gap represents an important problem for the 40%-80% of cisplatin-treated cancer patients who suffer with significant and in some cases permanent hearing loss as a result of cisplatin use. Until the underlying nitrative stress mechanism is delineated the promise of this new interventional target for mitigating a dose-limiting side-effect of cisplatin likely will remain unrealized. The long-term goal is to better understand the functional as well as mechanistic role of cochlear nitrative stress in acquired hearing loss. The objective is to delineate signaling pathways by which cisplatin-induced nitrative stress, particularly nitration of cochlear LMO4, facilitates ototoxicity, because cisplatin treatment nitrates and downregulates LMO4 protein. LMO4 is a transcriptional regulator that controls pathways regulating cell survival and cell death. The central hypothesis is that cisplatin-induced nitrative stress downregulates cochlear LMO4 and compromises STAT3- mediated anti-apoptotic signaling to facilitate ototoxicity. Understanding the mechanisms whereby nitrated cochlear LMO4 promotes cisplatin-induced ototoxicity is likely to contribute to the development of strategies to prevent this debilitating adverse effect. Guided by strong preliminary data this study will pursue three specific aims: (1) establish the causal link between cisplatin-induced LMO4 nitration and ototoxicity; (2) determine the effects of cisplatin-induced LMO4 nitration on JAK/STAT signaling; and (3) determine the otoprotective efficacy of pharmacological inhibition of nitration. In Aim 1, cisplatin-induced apoptosis will be analyzed after blocking nitration of LMO4 by site-directed mutagenesis and inhibiting proteasomal degradation of nitrated-LMO4 by lactacystin. The link between LMO4 protein levels and cisplatin-induced ototoxicity will be ascertained by testing cochlear apoptosis/hearing loss in LMO4 knockout and overexpressing mice. In Aim 2, cisplatin- induced changes in protein-protein interactions of cochlear LMO4 will be analyzed using a mass spectrometry- based proteomics approach while JAK/STAT related apoptotic and inflammatory signaling will be analyzed using targeted gene arrays. In Aim 3, the otoprotective efficacy of SRI110, a peroxynitrite decomposition catalyst, will be assessed using CBA/J mice; potential interference of SRI110 with anti-cancer activity of cisplatin will be analyzed using SCID mice. This innovative research departs from the status quo by shifting the focus from oxidative stress to the pivotal role of nitrative stress in cisplatin ototoxicity. Significantly, outcomes are expected to vertically advance understanding of how nitrative stress regulates cochlear apoptosis in cisplatin-induced ototoxicity. Findings will have important translational applications in mitigating cisplatin- induced hearing loss and preventing other otopathologies where nitrative stress plays a crucial role.