The ceramide-dependent EV secretome differentially affects prostate cancer cell migration

Tumor-derived extracellular vesicles (EVs) play an important role in cancer pro-gression. Neutral Sphingomyelinases (nSMases) are lipid modifying enzymes that modu-late the secretion of EVs from cells. How nSMase activity and therefore ceramide genera-tion affects the composition and functionality of secreted EV is not fully understood. Here, we aimed to investigated the expression of nSMases 1 and 2 in prostate cancer (PCa) tissue and their role in EV composition and secretion for prostate cancer cell migration. Reduced nSMase 1 and 2 expression was found in prostate cancer and correlated with age of pa-tients. When nSMase 2 was inhibited by GW4869 in PCa cells, PC3 and DU145, the EV se-cretome was significantly altered, while the number of EVs and total protein content of the released EVs were not significantly changed. Using proteomics analysis, we found extra-cellular matrix proteins, such as SDC4 (Syndecan-4) and SRPX-2 differentially secreted on EVs from GW4869-treated PC3 cells. In scratch wound migration assays, GW4869 signifi-cantly increased migration compared to control PC3 cells but not DU145 cells, while SDC4 knockdown significantly reduced migration of PC3 cells. These and other nSMase 2-dependent secreted proteins are interesting candidates to understand the role of stress-induced EV in the progression of prostate cancer.