Alteration of Ceramide Synthase 6/C16-Ceramide Induces Activating Transcription Factor 6-mediated Endoplasmic Reticulum (ER) Stress and Apoptosis via Perturbation of Cellular Ca2+ and ER/Golgi Membrane Network

Mechanisms that regulate endoplasmic reticulum (ER) stress-induced apoptosis in cancer cells remain enigmatic. Recent data suggest that ceramide synthase1-6 (CerS1-6)-generated ceramides, containing different fatty acid chain lengths, might exhibit distinct and opposing functions, such as apoptosis versus survival in a context-dependent manner. Here, we investigated the mechanisms involved in the activation of one of the major ER stress response proteins, ATF-6, and subsequent apoptosis by alterations of CerS6/C-16-ceramide. Induction of wild type (WT), but not the catalytically inactive mutant CerS6, increased tumor growth in SCID mice, whereas siRNA-mediated knockdown of CerS6 induced ATF-6 activation and apoptosis in multiple human cancer cells. Down-regulation of CerS6/C-16-ceramide, and not its further metabolism to glucosylceramide or sphingomyelin, activated ATF-6 upon treatment with ER stress inducers tunicamycin or SAHA (suberoylanilide hydroxamic acid). Induction of WT-CerS6 expression, but not its mutant, or ectopic expression of the dominant-negative mutant form of ATF-6 protected cells from apoptosis in response to CerS6 knockdown and tunicamycin or SAHA treatment. Mechanistically, ATF-6 activation was regulated by a concerted two-step process involving the release of Ca2+ from the ER stores ([Ca2+](ER)), which resulted in the fragmentation of Golgi membranes in response to CerS6/C16-ceramide alteration. This resulted in the accumulation of pro-ATF-6 in the disrupted ER/Golgi membrane network, where pro-ATF6 is activated. Accordingly, ectopic expression of a Ca2+ chelator calbindin prevented the Golgi fragmentation, ATF-6 activation, and apoptosis in response to CerS6/C16-ceramide down-regulation. Overall, these data suggest a novel mechanism of how CerS6/C-16-ceramide alteration activates ATF6 and induces ER-stress-mediated apoptosis in squamous cell carcinomas.