Exosome secretion is enhanced by invadopodia and drives invasive behavior
Exosome secretion is enhanced by invadopodia and drives invasive behavior
Daisuke Hoshino1, Kellye C. Kirkbride1, Kaitlin Costello1, Emily S. Clark2, Seema Sinha1, Nathan Grega-Larson3, Matthew J. Tyska3 and Alissa M. Weaver1, 4
1 Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
2 Department of Microbiology and Immunology, University of Miami Medical School, Miami, FL 33101, USA
3 Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
4 Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
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Graphical Abstract
Highlights
• Invadopodia are key docking sites for exosome-containing multivesicular endosomes• Invadopodia regulators control the quantity of exosomes secreted from cancer cells• Exosome secretion controls invadopodia biogenesis and matrix-degrading activity• A synergistic relationship exists between exosomes and invadopodia
Summary
Unconventional secretion of exosome vesicles from multivesicular endosomes (MVEs) occurs across a broad set of systems and is reported to be upregulated in cancer, where it promotes aggressive behavior. However, regulatory control of exosome secretion is poorly understood. Using cancer cells, we identified specialized invasive actin structures called invadopodia as specific and critical docking and secretion sites for CD63- and Rab27a-positive MVEs. Thus, inhibition of invadopodia formation greatly reduced exosome secretion into conditioned media. Functionally, addition of purified exosomes or inhibition of exosome biogenesis or secretion greatly affected multiple invadopodia life cycle steps, including invadopodia formation, stabilization, and exocytosis of proteinases, indicating a key role for exosome cargoes in promoting invasive activity and providing in situ signaling feedback. Exosome secretion also controlled cellular invasion through three-dimensional matrix. These data identify a synergistic interaction between invadopodia biogenesis and exosome secretion and reveal a fundamental role for exosomes in promoting cancer cell invasiveness.
INTRODUCTION
Exosomes are small extracellular vesicles that carry functional protein and RNA cargoes and influence cell behavior (Théry, 2011). In cancer, exosomes are thought to promote tumor progression and metastasis (Bobrie et al., 2012,Peinado et al., 2012,Yang and Robbins, 2011). Although numerous proteomics studies have identified exosome cargoes, little is known about how exosomes are secreted from cells. Recent studies have identified critical docking factors for multivesicular endosomes (MVEs), including Rab27a, Rab27b (Ostrowski et al., 2010), Rab35, and TBC1D10A-C (Hsu et al., 2010). Nonetheless, how exosome-docking and secretion sites are specified at the plasma membrane is unknown.
Invadopodia are actin-rich subcellular structures formed by invasive cancer cells that protrude into and degrade extracellular matrix (ECM). Similar structures are used by normal cells to cross tissue barriers and resorb bone (Murphy and Courtneidge, 2011). Recent studies have shown that ECM-degrading proteinases are secreted preferentially at invadopodia (Artym et al., 2006,Clark and Weaver, 2008,Hoshino et al., 2012b,Steffen et al., 2008). Although originally it was assumed that invadopodia proteinases were transported directly from biosynthetic pathways, the late endosomal/lysosomal (LE/Lys) v-SNARE VAMP7 was found to be necessary for transport of the critical metalloproteinase MT1-MMP to invadopodia (Steffen et al., 2008). These findings raised the possibility that cargo destined for invadopodia may be routed to the plasma membrane via a specialized endolysosomal compartment, such as exosome-containing MVEs.
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