Targeting OCT2 to ameliorate cisplatin‐induced ototoxicity in zebrafish and mice

Abstract

BackgroundThe organic cation transporter OCT2 is expressed in renal tubular cells, dorsal root ganglia, and outer hair cells, where it regulates uptake of xenobiotic substrates such as the widely used anticancer drug, cisplatin. We previously reported that various tyrosine kinase inhibitors (TKIs) with YES1‐kinase inhibitory ability, including dasatinib, can potently reduce OCT2 function (Sprowl et al, Nat Commun 2016). Against this background, we here explored the utility of zebrafish (Danio rerio), compared with mice, as in vivo screening tools to identify effective modulators of OCT2‐dependent, debilitating side effect associated with cisplatin treatment, such as ototoxicity (hearing loss).MethodsIn vitro uptake studies were performed with HeLa cells expressing human OCT2, mouse Oct2 or the single zebrafish OCT (zfOct) with high genomic similarity to mammalian OCTs. HEK293T cells were used for transfection of mutated zfOct using different substrates (ASP+, cisplatin) with or without inhibitors. In vivo studies were done in ABxLF zebrafish (5 dpf) and wild‐type or OCT2‐deficient mice on an FVB background. Cisplatin‐induced hearing loss in zebrafish was evaluated by hair cell death in the lateral line after treatment with YO‐PRO1, and rheotaxis movement was captured by video camera. Auditory brainstem responses (8, 16, 32 kHz) were measured to assess hearing loss associated with cisplatin (10 mg/kg, i.p.) in mice with and without dasatinib (15 mg/kg, p.o.) pretreatment.ResultsA comparative in vitro screen of OCT2 and zfOct using the prototypical fluorescent substrate ASP showed that both transporters could be inhibited by the same TKIs, including dasatinib (63.6% vs 66.5%) and nilotinib (53.6% vs 68.8%), with comparable IC50 values. Hela cells expressing OCT2 or zfOct accumulated cisplatin by >2‐fold compared to control cells, and these processes were sensitive to inhibition by the same TKIs (inhibition from 40 to 100% of the uptake). Tyr to Phe mutagenesis in zfOct at previously confirmed YES1‐regulated tyrosine phosphorylation sites in OCT2 was associated with impaired transport function. In addition, silencing YES1 expression with siRNA impaired zfOct function, suggesting that a similar regulatory mechanism exists for both the mammalian and zebrafish transporters. In vivo, neuromasts, consisting of receptive hair cells, accumulated OCT2/zfOct specific substrates such as ASP+, and this process could be inhibited by pretreatment with cisplatin (100 μM, 6‐h pre‐incubation) or dasatinib (10 μM, 6 h). In addition, cisplatin treatment was associated with a disruption of neuromast‐related control of hearing and balance, as determined from a seeker response assay. In mice, pretreatment with dasatinib protected against cisplatin‐induced high frequency hearing loss in a manner similar to that observed in OCT2‐deficient mice.ConclusionThese studies provide new insights into the regulatory mechanism and role of OCT2 in cisplatin‐induced toxicities in zebrafish and mice, and support the further translational exploration of OCT2 inhibitors as a preventative strategy to ameliorate a debilitating toxicity associated with a commonly used chemotherapeutic agent.