Abstract A26: Direct interaction between ceramide and SET/I2PP2A restores nuclear PP2A activity leading to the degradation of cMyc and suppression of lung tumor growth

In our recent studies, (Mukhopdahyay A, Saddoughi, S. et al., FASEB J, 2009) ceramide, a bioactive sphingolipid, has been shown to exert anti-proliferative functions against lung cancers via degradation of the cMyc proto-oncogene through directly binding to the inhibitor 2 of PP2A, (I2PP2A) also known as the SET oncoprotein. Data demonstrated that the interaction between ceramide and SET/I2PP2A restores PP2A activity in vitro. In addition, studies in A549 human lung cancer cells revealed that ceramide mediates c-Myc degradation via its PP2A-dependent dephosphorylation of S62, and treatment with okadiac acid and expression of cMyc mutant with S62A conversion resulted in resistance to ceramide-mediated degradation. The role of I2PP2A was elucidated via the down-regulation of I2PP2A, which enhanced PP2A mediated cMyc degradation in response to ceramide, and the overexpression of I2PP2A prevented the growth inhibitory effects of ceramide both in anchorage independent growth assays and xenograft-driven tumors. Our unpublished data suggests that SET/I2PP2A preferentially binds endogenous ceramides with C18 and longer fatty acid chains in A549 cells. However, the subcellular localization of endogenous ceramides/SET(I2PP2A) interaction in the regulation of nuclear versus cytoplasmic anti-proliferative targets of PP2A in lung cancer cells remains unknown. SET/I2PP2A is mainly a nuclear protein, while endogenous ceramides are mainly generated in the ER by the de novo pathway. Thus, a SET/I2PP2A mutant (I2PP2A-ER) was generated to accumulate specifically in the ER. Although, I2PP2A-ER mutant was still able to bind ceramide in vitro, its association with ceramide was almost completely inhibited in A549 cells in-situ, suggesting the requirement of the nuclear localization of SET/I2PP2A for ceramide binding. Therefore, our ongoing research efforts are focused on understanding how ceramides reach the nucleus for SET/I2PP2A binding in A549 cells. Two enzymes, Ceramide Synthase (CerS) and Neutral Sphingomyelinase (nSMase) are known to be the main generators of ceramide in cells. The activity of these enzymes in the nucleus of A549 compared to non-cancerous WI38 lung epithelial fibroblasts are being explored to determine the mechanism of ceramide generation in the nucleus. To this end, the overexpression of CerS1, which generates C18-ceramide, leads to the degradation of cMyc, which is prevented by T58A mutation of cMyc. Interestingly, the downregulation of nSMase1 leads to an increase in the endogenous levels of cMyc, and this increase is prevented with SET/I2PP2A RNAi. Thus, these data support the role for endogenous ceramide in the regulation of SET/I2PP2A and PP2A-dependent regulation of cMyc in A549 cells. The clinical relevance of the ceramide-SET/I2PP2A interaction in lung cancer progression was explored using 10 patient lung adenocarcinoma tissues, with their paired pathologically non-cancerous adjacent lung tissues. In these samples, it was demonstrated that I2PP2A/SET is overexpressed in about 60% of these tumors and the mRNA expression of the ceramide generating enzymes, CerS1 and nSMase1 are decreased within the majority of tumors. In summary, this research highlights the structural and biological roles of ceramide-SET/I2PP2A binding in the regulation of PP2A/cMyc axis leading to the suppression of human lung cancers. These studies will lead to the development of mechanism-based therapeutic strategies, by targeting SET/I2PP2A through ceramide signaling for the treatment of human lung cancers. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A26