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The escape of a subset of prostate tumor cells from ionizing radiation (IR)-induced killing may lead to disease progression and relapse.Bioactive sphingolipids such as ceramide and sphingosine 1-phosphate (S 1 P) influence signal transduction pathways regulating stress response.In particular, enhanced ceramide metabolism into sphingosine and S 1 P constitutes an important cell survival adaptation of irradiated cells, and places ceramidase activity at the crux of this process.Enzyme activity assays, and mRNA and protein analyses demonstrated selective up regulation of the lysosomal ceramide-metabolizing enzyme acid ceramidase (AC) in cancer cells exposed to IR.In vitro modeling of fractionated radiotherapy (RT) applied to prostate cancer cells demonstrated logarithmic kill, but a majority of surviving clonogens constitutively over-expressed AC.Moreover, levels of AC expression correlated with radioresistance and proliferation rate, suggesting AC plays a role in response to and relapse after RT.Immunohistochemical analyses of prostate cancer tissues from failed RT patients revealed higher levels of AC than adenocarcinoma tissues at diagnosis,PIN, or normal tissues.Promoter-reporter and ChIP-qPCR analyses demonstrated IR transactivated AC through AP1 activity that is sensitive to inhibition by myriocin or furmonisin, elaborating a critical feedback trigger of IR-induced ceramide generation via de novo biosynthesis.Radiosensitization of prostate cancer cells in vivo and in vitro by a dominant negative mutant of JUN was rescued, in part, by forced over expression of AC.Work with tumor xenografts exposed to IR plus or minus use of an acid ceramidase inhibitor revealed enhanced radiation killing when AC was inhibited.This work was sponsored by NCI P01 CA097132 and NCRR UL1 RR029882.