C18-ceramide mediates lethal autophagy by anchoring LC3B-II (lipidated LC3) to mitochondrial membranes during mitochondrial fission and thereby recruiting autophagosomes. Mechanisms by which autophagy promotes cell survival or death are unclear. We provide evidence that C18-pyridinium ceramide treatment or endogenous C18-ceramide generation by ceramide synthase 1 (CerS1) expression mediates autophagic cell death, independent of apoptosis in human cancer cells. C18-ceramide–induced lethal autophagy was regulated via microtubule-associated protein 1 light chain 3 β-lipidation, forming LC3B-II, and selective targeting of mitochondria by LC3B-II–containing autophagolysosomes (mitophagy) through direct interaction between ceramide and LC3B-II upon Drp1-dependent mitochondrial fission, leading to inhibition of mitochondrial function and oxygen consumption. Accordingly, expression of mutant LC3B with impaired ceramide binding, as predicted by molecular modeling, prevented CerS1-mediated mitochondrial targeting, recovering oxygen consumption. Moreover, knockdown of CerS1 abrogated sodium selenite–induced mitophagy, and stable LC3B knockdown protected against CerS1- and C18-ceramide–dependent mitophagy and blocked tumor suppression in vivo. Thus, these data suggest a new receptor function of ceramide for anchoring LC3B-II autophagolysosomes to mitochondrial membranes, defining a key mechanism for the induction of lethal mitophagy.

Sphingolipids have emerged as bioeffector molecules, controlling various aspects of cell growth and proliferation in cancer, which is becoming the deadliest disease in the world. These lipid molecules have also been implicated in the mechanism of action of cancer chemotherapeutics. Ceramide, the central molecule of sphingolipid metabolism, generally mediates antiproliferative responses, such as cell growth inhibition, apoptosis induction, senescence modulation, endoplasmic reticulum stress responses and/or autophagy. Interestingly, recent studies suggest de novo-generated ceramides may have distinct and opposing roles in the promotion/suppression of tumors, and that these activities are based on their fatty acid chain lengths, subcellular localization and/or direct downstream targets. For example, in head and neck cancer cells, ceramide synthase 6/C16-ceramide addiction was revealed, and this was associated with increased tumor growth, whereas downregulation of its synthesis resulted in ER stress-induced ...

Mechanisms that alter protein phosphatase 2A (PP2A)‐dependent lung tumour suppression via the I2PP2A/SET oncoprotein are unknown. We show here that the tumour suppressor ceramide binds I2PP2A/SET selectively in the nucleus and including its K209 and Y122 residues as determined by molecular modelling/simulations and site‐directed mutagenesis. Because I2PP2A/SET was found overexpressed, whereas ceramide was downregulated in lung tumours, a sphingolipid analogue drug, FTY720, was identified to mimick ceramide for binding and targeting I2PP2A/SET, leading to PP2A reactivation, lung cancer cell death, and tumour suppression in vivo . Accordingly, while molecular targeting of I2PP2A/SET by stable knockdown prevented further tumour suppression by FTY720, reconstitution of WT‐I2PP2A/SET expression restored this process. Mechanistically, targeting I2PP2A/SET by FTY720 mediated PP2A/RIPK1‐dependent programmed necrosis (necroptosis), but not by apoptosis. The RIPK1 inhibitor necrostatin and knockdown or genetic loss of RIPK1 prevented growth inhibition by FTY720. Expression of WT‐ or death‐domain‐deleted (DDD)‐RIPK1, but not the kinase‐domain‐deleted (KDD)‐RIPK1, restored FTY720‐mediated necroptosis in RIPK1 ^−/− MEFs. Thus, these data suggest that targeting I2PP2A/SET by FTY720 suppresses lung tumour growth, at least in part, via PP2A activation and necroptosis mediated by the kinase domain of RIPK1.

Emerging results suggest that ceramides with different fatty acid chain lengths might play distinct functions in the regulation of tumor growth and therapy. Here we report that de novo ‐generated C ₁₈ ‐ and C ₁₆ ‐ceramides by ceramide synthases 1 and 6 (CerS1 and CerS6) play opposing proapoptotic and prosurvival roles, respectively, in human head and neck squamous cell carcinomas (HNSCCs). Unexpectedly, knockdown of CerS6/C ₁₆ ‐ceramide using small interfering RNA induced endoplasmic reticulum (ER)‐stress‐mediated apoptosis. Reconstitution of C ₁₆ ‐ceramide generation by induced expression of wild‐type CerS6, but not its catalytically inactive mutant, protected cells from cell death induced by knockdown of CerS6. Moreover, using molecular tools coupled with analysis of sphingo‐lipid metabolism showed that generation of C ₁₆ ‐cer‐amide, and not dihydro‐C ₁₆ ‐ceramide, by induced expression of CerS6 rescued cells from ER stress and apoptosis. Mechanistically, regulation of ER‐stress‐in‐duced apoptosis by CerS6/C ₁₆ ‐ceramide was linked to the activation of a specific arm, ATF6/CHOP, of the unfolded protein response pathway. Notably, while expression of CerS1/C ₁₈ ‐ceramide inhibited HNSCC xenograft growth, CerS6/C ₁₆ ‐ceramide significantly protected ER stress, leading to enhanced tumor development and growth in vivo , consistent with their pro‐ and antiapoptotic roles, respectively. Thus, these data reveal an unexpected and novel prosur‐vival role of CerS6/C ₁₆ ‐ceramide involved in the protection against ER‐stress‐induced apoptosis and induction of HNSCC tumor growth.—Senkal, C. E., Ponnusamy, S., Bielawski, J., Hannun, Y. A., Ogret‐men, B. Antiapoptotic roles of ceramide‐synthase‐6‐generated C16‐ceramide via selective regulation of the ATF6/CHOP arm of ER‐stress‐response pathways. FASEB J . 24, 296–308 (2010). www.fasebj.org

Treatment of A549 cells with C(6)-ceramide resulted in a significant increase in the endogenous long chain ceramide levels, which was inhibited by fumonisin B1 (FB1), and not by myriocin (MYR). The biochemical mechanisms of generation of endogenous ceramide were investigated using A549 cells treated with selectively labeled C(6)-ceramides, [sphingosine-3-(3)H]d-erythro-, and N-[N-hexanoyl-1-(14)C]d-erythro-C(6)-ceramide. The results demonstrated that (3)H label was incorporated into newly synthesized long chain ceramides, which was inhibited by FB1 and not by MYR. Interestingly, the (14)C label was not incorporated into long chain ceramides. Taken together, these results show that generation of endogenous ceramide in response to C(6)-ceramide is due to recycling of the sphingosine backbone of C(6)-ceramide via deacylation/reacylation and not due to the elongation of its fatty acid moiety. Moreover, the generation of endogenous long chain ceramide in response to C(6)-ceramide was completely blocked by brefeldin A, which causes Golgi disassembly, suggesting a role for the Golgi in the metabolism of ceramide. In addition, the generation of endogenous ceramide in response to short chain exogenous ceramide was induced by d-erythro- but not l-erythro-C(6)-ceramide, demonstrating the stereospecificity of this process. Interestingly, several key downstream biological activities of ceramide, such as growth inhibition, cell cycle arrest, and modulation of telomerase activity were induced by d-erythro-C(6)-ceramide, and not l-erythro-C(6)-ceramide (and inhibited by FB1) in A549 cells, suggesting a role for endogenous long chain ceramide in the regulation of these responses.