SphingoDR

Sphingolipids (SLs) are ubiquitous ceramide-derived lipids, which play a dual function as modulators of membrane function and mediators of cancer cell growth and cell death. Death receptor (DR) ligands (e.g., CD95L and TRAIL) play crucial roles in anticancer immune surveillance. Many cancer cells resist DR-mediated apoptosis. Activation of these DRs triggers apoptosis that is accompanied by changes in SL metabolism, leading to the production of ceramide. Our project aims at understanding the molecular mechanisms which control the resistance of tumor cells towards DR ligands, and at evaluating the possibility to (re)sensitize cancer cells to CD95L and TRAIL by inhibiting the synthesis of complex SLs, i.e., sphingomyelin (SM) and glucosylceramide (GlcCer), from ceramide. Ongoing collaborative work between the 4 partners indicate that SM synthases 1 and 2 (SMS), GlcCer synthase (GCS) and their metabolic products may modulate DR-triggered cell signaling. Indeed, (i) SMS over-expression in HeLa cells inhibits apoptosis in response to CD95L (Lafont et al., in press) and TRAIL (unpublished); (ii) most of CD95L-resistant tumor cells exhibit higher SMS and GCS activities than CD95L-sensitive cells do (unpublished); (iii) whereas in resistant cells, CD95 is found enriched into lipid rafts, which contain high levels of SM and glycolipids, CD95 is partitioned outside lipid raft membranes in sensitive cells (unpublished). Working hypothesis: The relative composition of SM, GlcCer and ceramide in the plasma membrane may modulate (i) its biophysical and biochemical properties, (ii) the partition of DRs at the plasma membrane level, (iii) Death inducing signaling complex (DISC) formation, and (iv) the apoptotic response to DR ligands. Our study will focus on GCS and on recently identified proteins (CERT, SMS1 and SMS2) required for GlcCer and SM synthesis, respectively, to evaluate their roles in DR partitioning and DR-induced apoptosis. Our study will be carried out on cancer cell lines of various histological origins. Project: Task 1: SL modulation of Epithelial to Mesenchymal Transition (EMT) by SLs: EMT acts as an essential process in cancer progression and modulates CD95 signal. The role of SM/GlcCer/ceramide on the EMT will be evaluated. Task 2: development of novel approaches to manipulate SL metabolism: the biosynthesis of SM and GlcCer will be inhibited using RNA interference strategy as well as novel pharmacological tools to be developed. Conversely, CERT, SMS1, SMS2 or GCS will be over-expressed using retroviral vectors. Task 3: how does SL metabolism influence membrane fluidity, DR distribution and signaling?: we will address the impact of SM/GlcCer/ceramide levels on membrane fluidity, the membrane distribution of DRs, and the sensitivity of tumor cells to CD95L or TRAIL. Task 4: can modulation of SL metabolism and chemotherapy synergize to increase the sensitivity of tumor cells to DR ligands?: we will explore whether the alterations of SL metabolism are involved in (and/or synergize with) chemotherapy-induced priming and activation of the TRAIL apoptotic signaling pathway. Task 5: can modulation of SL metabolism affect the sensitivity of cancer cells to DR ligands in mouse models?: The role of SL metabolites in tumor formation/growth and response to DR ligands in vivo will be evaluated after transplantation to mice. This proposal seeks to bring scientists from different background and expertise in various fields together in a unique, original and timely program. Deciphering the role of SLs in the regulation of cancer cell death and tumor progression should be of particular interest for the identification of novel and original targets and future therapeutic intervention