Abstract 2875: Ceramide suffocates cancer cells to death: Regulation of mitophagy by ceramide signaling

Sphingolipid ceramide is a bioeffector molecule involved in the regulation of cell death. Endogenous ceramides contain different fatty acid chain lengths varying between C12-C26-ceramides. Ceramide synthase 1 (CerS1) selectively regulates the generation of C18-ceramide, which is down-regulated in the majority of tumors obtained from patients with head and neck squamous cell carcinomas (HNSCC). Reconstitution of C18-ceramide generation using a tetracycline inducible vector system for stable expression of CerS1 inhibits the growth of HNSCC cells and xenografts in culture and SCID mice, respectively. In this study, our goal was to determine mechanisms by which CerS1/C18-ceramide inhibits HNSCC tumor growth. Data revealed that reconstitution of C18-ceramide generation by wt-CerS1 induction, but not its catalytically inactive mutant, induces autophagy as detected by various techniques in HNSCC cells. Interestingly, soft agar studies showed that CerS1/C18-ceramide induction inhibits HNSCC cell growth, which is prevented by addition of the autophagy inhibitor 3-methyladenine. In addition, knockdown of Atg3 and Atg7 in HNSCC cells protected against cell death induced by exogenous addition of the C18-ceramide therapeutic analog, C18-pyridinium-ceramide (C18-Pyr-Cer). Additionally, Atg5-/- MEFs showed resistance to cell death when compared to wild type MEFs upon C18-Pyr-Cer exposure. Taken together these results suggest that autophagy is required for cell death. Electron microscopy showed that addition of C18-Pyr-Cer to UM-SCC-22A cells induced autophagosome formation and fusion with mitochondria. Mechanistically, our studies showed that CerS1/C18-ceramide-induced cell death/inhibition of tumor growth are mediated by mitochondrial damage and inhibition of respiration via induction of mitophagy. Inducible expression of CerS1/C18-ceramide and C18-Pyr-Cer treatment resulted in significant decreases in mitochondrial oxygen consumption rates. Furthermore, live and fixed cell imaging with mitochondrial markers MitoTracker Red colocalized with lysosomal marker LysoTracker Red upon C18-Pyr-Cer treatment and CerS1/C18-ceramide induction indicating mitochondrial fusion with lysosomes. Conversely, ceramide-mediated mitophagy was inhibited using LC3B siRNA, suggesting that mitochondria might be targeted to autophagosomes by ceramide signaling. In conclusion, we provide novel evidence here which suggest that C18-ceramide targets autophagosomes to mitochondria (mitophagy), which results in the modulation of respiration/oxygen consumption, leading to HNSCC growth inhibition and tumor suppression. 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 2875. doi:10.1158/1538-7445.AM2011-2875