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 Ca(2+) from the ER stores ([Ca(2+)](ER)), which resulted in the fragmentation of Golgi membranes in response to CerS6/C(16)-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 Ca(2+) chelator calbindin prevented the Golgi fragmentation, ATF-6 activation, and apoptosis in response to CerS6/C(16)-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.
Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425.
Can E
CE
Senkal
Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425.
Suriyan
S
Ponnusamy
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina 29425.
Yefim
Y
Manevich
Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425.
Marisa
M
Meyers-Needham
Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425.
Sahar A
SA
Saddoughi
Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425.
Archana
A
Mukhopadyay
Department of Neurosurgery, Virginia Commonwealth University, Richmond, Virginia 23298.
Paul
P
Dent
Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425.
Jacek
J
Bielawski
Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425. Electronic address: ogretmen@musc.edu.
Besim
B
Ogretmen
D051702:Activating Transcription Factor 6 / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000378:metabolism; D045744:Cell Line, Tumor; D002518:Ceramides / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D010088:Oxidoreductases / Q000378:metabolism*; D013107:Sphingolipids / Q000378:metabolism; D051049:Sphingosine N-Acyltransferase / Q000378:metabolism*
C497111:ATF6 protein, human; D051702:Activating Transcription Factor 6; C497112:Atf6 protein, mouse; D002518:Ceramides; D008565:Membrane Proteins; D013107:Sphingolipids; D010088:Oxidoreductases; C108138:dihydroceramide desaturase; C506123:CERS6 protein, human; D051049:Sphingosine N-Acyltransferase; D002118:Calcium