A279: Exosome switching: an mTORC1-dependent cellular stress response
1Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK,2The Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
Secreted exosomes are postulated to promote several cancer-induced activities, including angiogenesis and pre-metastatic niche formation. Classical exosomes are produced within late endosomes, and provide a multimolecular signal that reprogrammes target cells. Late endosomes and lysosomes are also intracellular signalling hubs that respond to the microenvironment and growth factors to modulate cell growth and metabolism via the amino acid-sensitive kinase complex, mechanistic Target of Rapamycin Complex 1 (mTORC1). Cells can release different types of exosomes in response to microenvironmental stress, but how this is regulated and whether mTORC1 is involved is unknown.
We combine molecular genetic analysis in a unique Drosophila model of exosome secretion, which we have recently developed1, with both genetic and pharmacological manipulation of mTORC1 activity and exosome secretion in human cancer cell lines, followed by proteomics, Nanosight Tracker Analysis, Tunable Resistive Pulse Sensing and confocal/super-resolution (3D-SIM) microscopy.
We demonstrate that in HCT116 colorectal cancer cells, a rapamycin-resistant form of mTORC1 regulates exosome cargo without affecting exosome number or size. In this so-called exosome switch, levels of the tetraspanin CD63 are reduced and the lipid raft-associated protein Caveolin-1 increased, an observation also made in MDA-MB-231 breast cancer cells. In both HCT116 cells and the fly model1, the switched exosomes are formed in recycling endosomal compartments, unlike classical exosomes. We observe exosome formation and differential loading of exosomes for the first time using live imaging. Furthermore, we demonstrate that stress-induced exosome changes lead to different cancer-relevant activities, with switched exosomes potentially driving compensatory changes in the tumour microenvironment.
We conclude that rapamycin-resistant mTORC1 controls the type of exosomes secreted by altering trafficking through endolysosomes. The switched exosome secretion pathway is evolutionarily conserved and drives cell-cell signalling changes in response to microenvironmental stress and drug treatment.