Ceramide Mediates LC3B Dependent Mitophagy and Tumor Suppression

Ceramide is a signaling sphingolipid recognized for limiting tumor growth. Specifically, the product of Ceramide Synthase 1 (CerS1), C18-ceramide, regulates an unknown mechanism which limits head and neck squamous cell carcinoma growth. During the investigation of this mechanism we reconstituted C 18-ceramide by a tetracycline inducible system and treatment with the analog C18-pyridinium ceramide (C18PC). Exogenous treatment of 22A cells with 10muM C18PC induced LC3B-II protein levels in the presence and absence of Z-VAD-fmk, increased mature autophagosomes, and significantly increased trypan blue positive cells at 18 hr. Knockdown of two autophagy-related genes, Atg3 and Atg7, significantly decreased the number of trypan blue positive cells. Similarly, Atg5 -/- mouse embryonic fibroblasts showed significantly less trypan blue positive cells as compared to the wild type. Conversely; Bax-/- Bak -/- and caspase 3-/-7-/- showed no protection from C18PC treatment. C18PC treatment at 2hr showed a significant increase in mitochondria (MTR) merged with autophagosomes (LTR) that correlated with a decrease in respiration.;Next, we hypothesized that mitochondria) fission may be necessary for the decrease in respiration following wt-CerS 1 induction. To test this hypothesis we inhibited both mitochondria) fission and fusion proteins separately using knockout mouse embryonic fibroblasts and molecular techniques in order to dissect the role of this dynamic morphological change. Interestingly, only the inhibition of the fission protein Drpl by siRNA rescued the decrease of mitochondrial respiration induced by the induction of wt-CerS 1.;Reconstitution of wt-CerS1 increased protein levels of LC3B-II, LC3B-GFP punctate, and decreased respiration after 24 hr induction when compared with reconstitution of a catalytically inactive mutant CerS 1 (mut-CerS 1). Furthermore, a significant decrease in ATP production by wt-CerS 1 induced cells was rescued when the wt-CerS 1 cells constitutively expressed LC3B shRNA. Next, the necessity of CerS 1 was examined. Cells were treated with a common mitophagy inducer, Sodium Selenite (SS). Transient knockdown of CerS1 with siRNA prior to treatment with with 1011M of SS rescued the cells from increased autophagosome formation and engulfment of mitochondria resulting in decreased respiration. Confocal imaging following wt-CerSl induction showed that ceram.ide-specific antibody, cfp-LC3B, and the mitochondria (MTR) colocalized. Constitutive expression of LC3B shRNA and non-targeting shRNA in wt-CerS 1 cell lines showed a decrease in respiration rate that was rescued in the LC3B shRNA cell line. Xenografts with LC3B shRNA and non-targeting shRNA in combination with wt-CerS 1 were established on the flanks of SCID mice. In vivo results showed that knocking down LC3B with shRNA enhanced tumor growth in the presence of wt-CerS 1 while induction of wt-CerS 1 with non-targeting shRNA reduced tumor growth. Overall, these findings suggest that C18ceramide mediates an LC3B-dependent mitochondrial dysfunction pathway culminating in tumor suppression.