Sphingomyelin (SM) hydrolysis is a key pathway during several cellular stress responses, including apoptosis, cell cycle arrest. and senescence. Sphingomyelinases (SMases) are key enzymes in this pathway regulating conversion of membrane sphingomyelin to the lipid second messenger ceramide (Cer). Besides its well-established cytotoxic effects, other tumor suppressor properties of ceramide are still widely unknown. In this study, we investigated the role of the SM/Cer pathway in the cytoskeletal changes induced by the DNA-damaging agent cisplatin. Treatment of the breast cancer cell line, MCF-7, with cisplatin induced a transient activation of acidic sphingomyelinase (ASMase), with a concomitant elevation in total Cer levels. In addition, translocation of ASMase from endolysosomes to the plasma membrane (PM) was observed. This translocation process was blocked upon infection with a dominant negative PKCδ adenovirus or transfection of ASMaseS508A mutant. Interestingly, cisplatin treatment induced clear morphologic changes, including loss of cellular lamellipodia/filopodia and appearance of membrane ruffles. These changes were reproduced upon exogenous delivery of D-e-C16-Cer but not dihydro-C16-Cer. Phalloidin staining revealed that cisplatin and Cer treatment caused loss of filamentous actin, its dissociation from the PM, and the appearance of cortical stress fibers. Further investigations showed that Cer generated by cisplatin treatment induced dephosphorylation (inactivation) of the actin binding protein, ezrin. Indeed, immunofluorescence studies showed relocation of ezrin from membrane protrusions such as lamelli/filopodia to the cytosol after cisplatin treatment. Importantly, knockdown of ASMase using specific RNAi sequence protected MCF-7 cells from cisplatin-induced cytoskeletal changes, including ezrin dephosphorylation. Taken together, these results highlight a novel tumor suppressor property for Cer and an important role for ASMase in mediating cisplatin-induced cytoskeletal remodeling. On the translational level, our study indicates that modulation of the SM/Cer cycle offers a potential novel pathway for modulating tumor invasion and metastasis.
*Supported by NCI P01-CA97132 to Y.A.H.
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