Abstract 4856: Survivin packaging into exosomes is lipid raft-dependent

Abstract

Abstract We have shown that Survivin is exported out of the cancer cell via exosomes despite its lack of a secretory signaling peptide. However, the mechanism of Survivin exosomal loading is not yet known. Exosomal protein sorting can be endosomal sorting complex required for transport (ESCRT)-dependent or ESCRT-independent. ESCRT dependent protein sorting requires monoubiquitination of the target protein, which serves as an exosome sorting signal. The ESCRT independent mechanism is through preferential aggregation of the proteins which is based on intrinsic physical properties to lipid-rafts present in exosomes. Our lab has recently shown that exosomal Survivin is associated with both Hsp70 and Hsp90, as well as PRDX1 which have also been shown associated with detergent-resistant lipid rafts. Here, we determine whether Survivin is lipid-raft dependent and whether Hsp70 and Hsp90 and/or PRDX1 are required for exosomal Survivin loading. Exosomes were collected from conditioned media taken from HeLaS cells and isolated by centrifugation. Colocalizations of Survivin with Hsp70, Hsp90 and PRDX1 in the cell and exosomes were detected by immnocytochemistry visualized by confocal microscopy and immunoprecipitation. HeLaS cells were treated with increasing concentrations of methyl-β-cyclodextrin to disrupt lipid rafts as a way to determine protein lipid raft association. Hsp70, Hsp90 and PRDX1 knock-down using siRNAs will be accomplished to verify the requirement of these proteins for Survivin exosome loading and lipid-raft dependents. Here we show that Survivin colocalizes with Hsp70, Hsp90 and PRDX1 in HeLaS cells as well as their exosomes. Increasing concentrations of methyl-β-cyclodextrin disrupts lipid rafts in the HeLaS cells and releases exosomes which also subsequently contain decreasing PRDX1 and Survivin protein amounts. The release of Survivin into the tumor microenvironment is an important finding as this IAP may contribute to the aggressiveness of the cancer as well as the development of chemoresistance. Despite lacking a secretory signaling peptide, Survivin is being released into the extracellular space via exosomes using chaperones. Knowing how Survivin is exosomally packaged may prove useful in selecting patient specific treatment regimens. Citation Format: Malyn May Asuncion Valenzuela, Heather R. Ferguson Bennit, Carlos Joel Diaz Osterman, Salma Khan, Carlos Casiano, Nathan R. Wall. Survivin packaging into exosomes is lipid raft-dependent. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4856. doi:10.1158/1538-7445.AM2014-4856