Abstract 2497: Induction of AP-1 activity by ceramide results in ASAH1 upregulation: A mechanism of prostate cancer cell radioresistance

Background: Although radiation therapy is a mainstay of treatment for localized prostatic disease, the escape of a subset of tumor cells from ionizing radiation (IR)-induced killing may lead to disease relapse and progression. The metabolic balance between the pro-apoptotic sphingolipid, ceramide, and its anti-apoptotic metabolites has been characterized as a critical mediator of cell death under conditions of cell stress, including IR exposure. While the lysosomal ceramide-metabolizing enzyme, acid ceramidase (ASAH1), is constitutively expressed in all human tissues, ASAH1 has been found to be over-expressed in cancer cells exposed to IR. The goal of this study was to examine the biological impact of and possible mechanism for altered ASAH1 expression observed in irradiated prostate cancer cells. Methods: Sphingolipid expression was measured by LC-MS. Ceramidase activities were assessed by thin layer chromatography. ASAH1 mRNA and protein expression in cell lines were measured by qPCR and western blot, respectively. Transcription activity was assessed using a luciferase reporter driven by the 1.5kb proximal promoter region of ASAH1. AP-1 promoter occupancy was determined by chromatin immunoprecipitation (ChIP)-qPCR. Post-irradiative cell growth was determined by clonogenic survival assay. Results: IR induced nearly two-fold expression of ceramide, and between three- and four-fold expression of its metabolites, sphingosine and sphingosine-1-phosphate. Enzyme activity assays demonstrated specific increase of acid, but not neutral or alkaline, ceramidase in the irradiated prostate cancer cells, suggesting a prominent role for ASAH1 in IR-induced sphingolipid metabolism. Consistent with increased mRNA and protein levels, luciferase-promoter-reporter assay confirmed that ASAH1 regulation occurs at least in part at the transcriptional level. Interestingly, inhibition of de novo and salvage pathways of ceramide biogenesis abrogated IR-induced ASAH1 upregulation, implicating the involvement of ceramide in IR-induced ASAH1 overexpression. Through studies using protein-DNA binding arrays and ChIP-qPCR, we identified AP-1 as a direct, ceramide-sensitive transcription factor of ASAH1 in irradiated cells. Expression of a dominant negative mutant of c-Jun, a central component of the AP-1 complex, was able to abrogate IR-induced ASAH1 overexpression. Since ASAH1 silencing by shRNA sensitized prostate cancer cells to IR-induced cell killing, here we also observed radiosensitization by expression of the c-Jun dominant negative construct, which was rescued in part by forced overexpression of an ASAH1 transgene. Conclusion: The reported studies demonstrate that IR programs an unexpected phenotype of radiation resistance through ASAH1 upregulation mediated by ceramide-sensitive AP-1 activation. This study was supported in part by NIH/NCI P01 CA097132. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2497. doi:10.1158/1538-7445.AM2011-2497