Abstract 273: Exosome targeted receptor activation (ExTRAcrine) signaling: A new mode of EGF receptor ligand signaling

Abstract

Abstract Epidermal growth factor receptor (EGFR) ligands have been shown to act in an autocrine, paracrine and juxtacrine manner. We report a new mode of signaling - via exosomes. Exosomes are 30-100 nm nano-vesicles that are produced by intralumenal budding into multivesicular bodies (MVB); they are released from many cell types after fusion of the MVB with the plasma membrane. Our studies showed that human breast and colorectal cancer cell lines differ in the composition of EGFR ligands in exosomes isolated from conditioned medium by established methods. By fluorescent-activated vesicle sorting (FAVS; Mol Cell Proteomics 7: 1651-1667, 2008), we show that a single HCA-7-derived exosome contains TGF-α, amphiregulin (AR) and HB-EGF. Ligands localized in exosomes are signaling competent with the ectodomains presented on the outside and cytoplasmic tails on the inside, and exist as stable transmembrane proteins. To explore the biological function of individual ligands, we isolated exosomes from MDCK cells individually stably expressing TGF-α, AR or HB-EGF. We assessed the invasive capacity of these exosomes by incubating them with a breast cancer-derived cell line (LM2-4175) in a Boyden chamber assay and measured the number of invading cells 72 hours later. Exosomes purifed from MDCK cells singly expressing AR induced invasion of LM2-4175 recipient cells 4-fold more than TGF-α or HB-EGF exosomes and 15-30-fold more than recombinant ligands TGF-α, HB-EGF or AR). The number of invading cells exposed to AR exosomes was significantly inhibited by an AR neutralizing antibody, suggesting that the invasive capacity of recipient cells is dependent upon exosomal AR engagement with recipient cell EGFR. Fixed and live cell imaging by confocal microscopy showed that AR exosomes are internalized by recipient cells. Further analysis showed that exosomes isolated from mutant KRAS DLD-1 colon cancer cells increased invasion of LM2-4175 cells 5-fold more than exosomes isolated from cells in which mutant KRAS was eliminated by homologous recombination (DKs-8 cells) and this correlated with increased levels of exosomal AR present in DLD-1 cells compared to DKs-8 cells. We provide the first evidence that EGFR ligands are present in exosomes and demonstrate that AR exosomes confer a greater invasive effect on recipient cells than TGF-α and HB-EGF exosomes or recombinant EGFR ligands. We propose ExTRAcrine signaling as a novel mode of EGFR ligand signaling with important diagnostic and therapeutic implications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 273.