The Plasma Membrane Monoamine Transporter (PMAT) is Highly Expressed in Neuroblastoma and Functions as a Mitochondrial mIBG Transporter

Abstract

<b>Abstract ID 20301</b> <b>Poster Board 183</b> Neuroblastoma (NB) is a pediatric cancer with low survival rates in high-risk patients. High dose ¹³¹I-mIBG therapy has emerged as a promising therapy that kills tumor cells by β radiation. Consequently, tumor uptake and retention of ¹³¹I-mIBG are a major determinant for its therapeutic efficacy. ¹³¹I-mIBG enters NB cells through the norepinephrine transporter (NET) but NET expression alone cannot predict its clinical response. mIBG accumulates in mitochondria in NB cells but the underlying transport mechanism is unknown. Previously our laboratory showed that mIBG is a substrate of the polyspecific organic cation transporters (OCTs, MATEs), suggesting that transporters other than NET could play a role in tumor disposition and response to ¹³¹I-mIBG. We hypothesized that besides NET, additional mIBG transporter(s) are expressed in NB cells and affect intracellular disposition and efficacy of ¹³¹I-mIBG. In this study, we profiled the expression of potential mIBG transporters in NB tumor samples, explored potential association with patient overall survival (OS), and determined transporter subcellular localization and interaction with mIBG. Furthermore, using mitochondrial fraction isolated from two NB cell lines (SH-SY5Y and SK-N-BE(2)), the mechanism of mitochondrial mIBG transport was investigated. Analysis of mRNAseq data from 156 NB tissue samples from the NCI TARGET database, as well as qPCR results from NB samples from Seattle Children9s Hospital consistently showed that NB tumor cells mainly expresses NET, the plasma membrane monoamine transporter (PMAT), and the vesicular membrane monoamine transporter 1/2 (VMAT1/2), with PMAT expression being the highest among all transporters analyzed. In contrast, OCTs and MATEs are minimally expressed in NB tumors. Kaplan Meier survival analysis using gene expression array data matched to clinical outcome of 243 NB patients from the TARGET database revealed that PMAT expression strongly correlates with OS of high-risk NB patients without the amplification of the MYCN oncogene. Uptake studies in PMAT-transfected HEK293 cells showed that mIBG is efficiently transported by PMAT with a K_m of 151.7 ± 50.7 μM. Immunostaining studies further revealed that the PMAT protein is mainly localized intracellularly in NB cells and co-localizes largely with mitochondria. Importantly, uptake studies using mitochondrial fraction isolated from cultured NB cells demonstrated that mIBG is taken up by mitochondria, and decynium-22 (a PMAT inhibitor) reduced mitochondrial mIBG uptake by 51% and 42% in SH-SY5Y and SK-N-BE(2) cells respectively. Together, our data demonstrated that PMAT is localized intracellularly in NB cells and plays a role in mitochondrial uptake of mIBG. Our results suggest that PMAT is a previously unrecognized transporter highly expressed in NB tumors and may play an important role in tumor retention, exposure, and therapeutic response to ¹³¹I-mIBG. This work is supported by NIH Grant R01GM066233