A Ca²⁺-stimulated exosome release pathway in cancer cells is regulated by Munc13-4

Cancer cells secrete copious amounts of exosomes, and elevated intracellular Ca²⁺ is critical for tumor progression and metastasis, but the underlying cellular mechanisms are unknown. Munc13-4 is a Ca²⁺-dependent SNAP receptor- and Rab-binding protein required for Ca²⁺-dependent membrane fusion. Here we show that acute elevation of Ca²⁺ in cancer cells stimulated a fivefold increase in CD63⁺, CD9⁺, and ALIX⁺ exosome release that was eliminated by Munc13-4 knockdown and not restored by Ca²⁺ binding-deficient Munc13-4 mutants. Direct imaging of CD63-pHluorin exosome release confirmed its Munc13-4 dependence. Depletion of Munc13-4 in highly aggressive breast carcinoma MDA-MB-231 cells reduced the size of CD63⁺ multivesicular bodies (MVBs), indicating a role for Munc13-4 in MVB maturation. Munc13-4 used a Rab11-dependent trafficking pathway to generate MVBs competent for exosome release. Membrane type 1 matrix metalloproteinase trafficking to MVBs by a Rab11-dependent pathway was also Munc13-4 dependent, and Munc13-4 depletion reduced extracellular matrix degradation. These studies identify a novel Ca²⁺- and Munc13-4-dependent pathway that underlies increased exosome release by cancer cells.