Abstract
Tumour cells secrete exosomes that are involved in the remodelling of the tumour–stromal environment and promoting malignancy. The mechanisms governing tumour exosome release, however, remain incompletely understood. Here we show that tumour cell exosomes secretion is controlled by pyruvate kinase type M2 (PKM2), which is upregulated and phosphorylated in tumours. During exosome secretion, phosphorylated PKM2 serves as a protein kinase to phosphorylate synaptosome-associated protein 23 (SNAP-23), which in turn enables the formation of the SNARE complex to allow exosomes release. Direct phosphorylation assay and mass spectrometry confirm that PKM2 phosphorylates SNAP-23 at Ser95. Ectopic expression of non-phosphorylated SNAP-23 mutant (Ser95→Ala95) significantly reduces PKM2-mediated exosomes release whereas expression of selective phosphomimetic SNAP-23 mutants (Ser95→Glu95 but not Ser20→Glu20) rescues the impaired exosomes release induced by PKM2 knockdown. Our findings reveal a non-metabolic function of PKM2, an enzyme associated with tumour cell reliance on aerobic glycolysis, in promoting tumour cell exosome release.
Highlights
Tumour cells secrete exosomes that are involved in the remodelling of the tumour–stromal environment and promoting malignancy
In agreement with previous reports, we found that tumour cells (SW480, Hela, A549 and HepG2 cells) generally displayed more active exosome secretion than non-tumour mammalian cells, mouse primary myoblast and mammary epithelial cell (MEC) (Fig. 1d)
In line with the positive correlation between aerobic glycolysis and exosome secretion observed in Fig. 1f, we found that glycolysis level was positively correlated with the amount of exosome release in tumour cells (Supplementary Fig. 1)
Summary
Tumour cells secrete exosomes that are involved in the remodelling of the tumour–stromal environment and promoting malignancy. During exosome secretion, phosphorylated PKM2 serves as a protein kinase to phosphorylate synaptosome-associated protein 23 (SNAP-23), which in turn enables the formation of the SNARE complex to allow exosomes release. Cancer cellderived microparticles bearing P-selectin glycoprotein ligand 1 accelerate thrombus formation in vivo, and by targeting P-selectin glycoprotein ligand 1 researchers were able to prevent thrombosis[5] While these studies are exciting and suggest potential strategies for blocking metastasis, the mechanism underlying the active exocytosis of exosomes by tumour cells, remains unclear. 14) and VAMP-8 (refs 12,15) represent candidates for v-SNAREs. Phosphorylation of SNAP-23 directly increases cell exocytosis[16,17] and promotes association with other SNARE proteins, thereby allowing the formation of the stable SNARE complex to enhance cell exocytosis[18]. We conclude that PKM2, following phosphorylation and dimerization, plays an essential role in switching tumour cell metabolism from oxidative phosphorylation to aerobic glycolysis, and promoting tumour cell exosome secretion via directly phosphorylating SNAP-23
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