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

ABSTRACT: 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.

SUBMITTER: Messenger SW

PROVIDER: S-EPMC6080937 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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

Messenger Scott W SW Woo Sang Su SS Sun Zhongze Z Martin Thomas F J TFJ

The Journal of cell biology 20180621 8

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 rel ...[more]

PMID: 29930202

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Project description:BackgroundExosomes are nanovesicles secreted by tumour cells which have roles in paracrine signalling during tumour progression, including tumour-stromal interactions, activation of proliferative pathways and bestowing immunosuppression. Hypoxia is an important feature of solid tumours which promotes tumour progression, angiogenesis and metastasis, potentially through exosome-mediated signalling.MethodsBreast cancer cell lines were cultured under either moderate (1% O2) or severe (0.1% O2) hypoxia. Exosomes were isolated from conditioned media and quantitated by nanoparticle tracking analysis (NTA) and immunoblotting for the exosomal protein CD63 in order to assess the impact of hypoxia on exosome release. Hypoxic exosome fractions were assayed for miR-210 by real-time reverse transcription polymerase chain reaction and normalised to exogenous and endogenous control genes. Statistical significance was determined using the Student T test with a P value of < 0.05 considered significant.ResultsExposure of three different breast cancer cell lines to moderate (1% O2) and severe (0.1% O2) hypoxia resulted in significant increases in the number of exosomes present in the conditioned media as determined by NTA and CD63 immunoblotting. Activation of hypoxic signalling by dimethyloxalylglycine, a hypoxia-inducible factor (HIF) hydroxylase inhibitor, resulted in significant increase in exosome release. Transfection of cells with HIF-1α siRNA prior to hypoxic exposure prevented the enhancement of exosome release by hypoxia. The hypoxically regulated miR-210 was identified to be present at elevated levels in hypoxic exosome fractions.ConclusionsThese data provide evidence that hypoxia promotes the release of exosomes by breast cancer cells, and that this hypoxic response may be mediated by HIF-1α. Given an emerging role for tumour cell-derived exosomes in tumour progression, this has significant implications for understanding the hypoxic tumour phenotype, whereby hypoxic cancer cells may release more exosomes into their microenvironment to promote their own survival and invasion.

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

Publications

A Ca<sup>2+</sup>-stimulated exosome release pathway in cancer cells is regulated by Munc13-4.

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