Abstract 960: Ceramide-dependent release of nanovesicles is a class effect oflipid-based Akt inhibitors

Abstract Akt regulates many cellular processes and Akt inhibitors are being developed as cancer therapeutics. The mechanism of action of lipid-based Akt inhibitors (LBAIs) such as perifosine and phosphatidylinositol ether lipid analogues (PIAs) is incompletely understood. Here, we show that LBAIs quickly deplete cells of proteins such as EGFR, IGFR and p-Akt that can be recovered in tetraspanin-enriched 100,000 xg pellets from conditioned media. Electron micrographs of these pellets exhibited heterogenous vesicular structures whose diameter was less than 1 µM. In vitro, LBAI treatment increased vesicle shedding as revealed by live cell microscopy. In vivo, LBAIs increased plasma levels of nanovesicles derived from tumor xenografts. Several lines of evidence show that release of nanovesicles by LBAI depends upon ceramide generation. LBAIs increased levels of cellular ceramide, and addition of exogenous ceramide stimulated nanovesicle release. GW4869, an inhibitor of sphingomyelinase, an enzyme that generates ceramide by cleavage of sphingomyelin, blocked release of nanovesicles induced by LBAI. When transferred in vitro, purified nanovesicles increased ceramide levels and inhibited proliferation of naïve recipient cells, suggesting these contain residual LBAIs. Pre-treatment with GW4869 blocked ceramide induction in recipient cells and yielded greater retention of the donor nanovesicles. These studies identify ceramide-dependent release of cellular nanovesicles as a class effect of LBAI and a novel means to shed EGFR, IGF-IR and p-Akt from tumors that could perpetuate the cytotoxicity of LBAIs. We believe this is the first report of secondary pharmacologic effects via cell-mediated drug transfer in nanovesicles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 960. doi:10.1158/1538-7445.AM2011-960