Abstract
The ether-phospholipid edelfosine, a prototype antitumor lipid (ATL), kills yeast cells and selectively kills several cancer cell types. To gain insight into its mechanism of action, we performed chemogenomic screens in the Saccharomyces cerevisiae gene-deletion strain collection, identifying edelfosine-resistant mutants. LEM3, AGP2, and DOC1 genes were required for drug uptake. Edelfosine displaced the essential proton pump Pma1p from rafts, inducing its internalization into the vacuole. Additional ATLs, including miltefosine and perifosine, also displaced Pma1p from rafts to the vacuole, suggesting that this process is a major hallmark of ATL cytotoxicity in yeast. Radioactive and synthetic fluorescent edelfosine analogues accumulated in yeast plasma membrane rafts and subsequently the endoplasmic reticulum. Although both edelfosine and Pma1p were initially located at membrane rafts, internalization of the drug toward endoplasmic reticulum and Pma1p to the vacuole followed different routes. Drug internalization was not dependent on endocytosis and was not critical for yeast cytotoxicity. However, mutants affecting endocytosis, vesicle sorting, or trafficking to the vacuole, including the retromer and ESCRT complexes, prevented Pma1p internalization and were edelfosine-resistant. Our data suggest that edelfosine-induced cytotoxicity involves raft reorganization and retromer- and ESCRT-mediated vesicular transport and degradation of essential raft proteins leading to cell death. Cytotoxicity of ATLs is mainly dependent on the changes they induce in plasma membrane raft-located proteins that lead to their internalization and subsequent degradation. Edelfosine toxicity can be circumvented by inactivating genes that then result in the recycling of internalized cell-surface proteins back to the plasma membrane.
Highlights
The antitumor lipid edelfosine kills yeast by inducing selective internalization of raft-associated proteins
In this study and the accompanying paper (46), we have found that following interaction of edelfosine with lipid rafts, resistance and sensitivity to this drug are mediated through two major processes, namely: (i) disturbance of pH homeostasis (46), and (ii) sorting of specific proteins at the endosome via sorting nexins and retromer at early and late endosomes, respectively, and by the ESCRT vesicular trafficking pathway
Despite the fact that several proteins would be affected, our results consistently point to a main role for the essential plasma membrane Hϩ-pump Pma1p in mediating the toxic effect of edelfosine, as well as of the clinically relevant antitumor lipid (ATL) miltefosine and perifosine
Summary
The antitumor lipid edelfosine kills yeast by inducing selective internalization of raft-associated proteins. In addition to the identification of three genes required for edelfosine uptake, our data indicate that edelfosine accumulates in lipid rafts and ER in S. cerevisiae and mediates its cytotoxic activity by affecting raft protein composition, which eventually leads to intracellular acidification (see accompanying paper (46)) This alteration in raft composition seems to be conserved within the ATL family, as it was observed for miltefosine and perifosine. The yeast genome-wide screen and subsequent experiments performed here revealed that vesicular trafficking is a critical process mediating edelfosine resistance, with mutants that result in increased recycling between endosomal compartments and the plasma membrane being resistant to edelfosine cytotoxicity This is a highly conserved process, and this mode of resistance might be extrapolated from yeast to tumor cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
