Abstract 5262: Resistance to fenretinide in neuroblastoma is associated with increased expression of sphingosine kinase and sphingosine-1-phosphate receptors and targeting sphingosine kinase reverses resistance

Abstract

Abstract The synthetic retinoid fenretinide, N-(4-hydroxyphenyl) retinamide (4-HPR), is selectively cytotoxic to cancer cells via increases in dihydroceramide levels and generation of reactive oxygen species and has clinical activity against recurrent neuroblastoma (J Clin Oncol 27: 15s abstr 10009, 2009). Metabolism of ceramides into various sphingolipid species, including sphingosine, and phosphorylation of dihydrosphingosine and sphingosine by sphingosine kinase 1 (SPHK1) are anti-apoptotic and thought to counter cytotoxicity mediated by ceramides. Multistep selection for resistance to 4-HPR was performed with a 4-HPR sensitive neuroblastoma cell line, SMS-KCNR (IC99=4.6 uM), resulting in the 4-HPR resistant line, KCNR-FR (IC99=19.9 uM). We generated a TaqMan low-density array (TLDA) that measures expression of 42 genes involved in ceramide synthesis and metabolism by quantitative RT-PCR. Genes overexpressed in KCNR-FR relative to SMS-KCNR included ceramide desaturase 2 (14.1-fold), sphingosine-1-phosphate receptors S1PR2, 3, and 4 (2.4, 1.6, and 1.3-fold, respectively), and SPHK1 (5.2-fold). These data point toward 4-HPR resistance being mediated by sphingosine-1-phosphate or dihydrosphingosine-1-phosphate, both of which are generated via phosphorylation of sphingosine by SPHK1. SPHK1-specific RNAi knockdown decreased SPHK1 mRNA expression (36% ± 0.007 of scrambled RNAi, p<0.05) and significantly increased apoptosis (35.8 ± 1.7%; p<0.05) in KCNR-FR treated with 4-HPR relative to scrambled RNAi controls (8.8 ± 6.5%; p<0.05). Safingol (L-threo-dihydrosphingosine), a putative sphingosine kinase inhibitor in phase I trials, partially reversed KCNR-FR resistance to 4-HPR (Combination Index = 0.56). Thus, selection for resistance to 4-HPR in neuroblastoma was associated with increased expression of multiple components of the anti-apoptotic sphingosine kinase signaling pathway, and such resistance can be partially overcome by targeting sphingosine kinase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5262.