Proteomics

Dataset Information

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Chloroquine-mediated lysosomal inhibition alters composition and function of cancer-derived extracellular vesicles


ABSTRACT: Extracellular vesicles (EV) are signaling entities that are released by most, if not all, eukaryotic cells. EV are of special interest in cancer due to their reported roles in modulating the cancer microenvironment and facilitating invasion. Macroautophagy is a catabolic process initially known for the recycling of cytosolic cargos through lysosomal degradation, while its non-degradative functions have only begun to emerge. To better understand the relationships between autophagy machinery and small EV (sEV) biogenesis, we utilized chloroquine (CQ) to inhibit lysosomal degradation and autophagy turnover in triple-negative breast cancer (TNBC) cell lines and characterized its effects on sEV content and function. We performed quantitative mass spectrometry analysis of the sEV proteome, and discovered a CQ-induced enrichment of mammalian ATG8 paralogs and their adaptor proteins that coincided with increased levels of K48-specific polyubiquitination in sEV, as well as with the co-localization of ATG8s and endolysosomal markers in the cytoplasm. Using ATG4B knockout cells, we established the lipidation-dependent luminal localization of sEV-associated ATG8s. We demonstrated CQ-mediated enrichment of MAP1LC3B and GABARAP in CD63-high but not CD9-high sEV subpopulations. sEV isolated from CQ treated versus untreated cells had differential effects on recipient cell growth and HMEC-1 tube formation, suggesting sEV are an avenue of CQ mediated non-cell-autonomous biological effects. Our study demonstrates the flexibility of sEV composition in response to perturbation of intracellular trafficking pathways, and has implications for CQ efficacy in therapeutic settings.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Sharon Gorski  

LAB HEAD: Sharon Gorski

PROVIDER: PXD015614 | Pride | 2023-03-10

REPOSITORIES: Pride

Dataset's files

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Action DRS
SC_20Nov2016_MX_Exo1_TMT9_1.raw Raw
SC_20Nov2016_MX_Exo1_TMT9_2.raw Raw
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SC_20Nov2016_MX_Exo1_TMT9_5.raw Raw
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Publications

Chloroquine treatment induces secretion of autophagy-related proteins and inclusion of Atg8-family proteins in distinct extracellular vesicle populations.

Xu Jing J   Yang Kevin C KC   Go Nancy Erro NE   Colborne Shane S   Ho Cally J CJ   Hosseini-Beheshti Elham E   Lystad Alf H AH   Simonsen Anne A   Guns Emma Tomlinson ET   Morin Gregg B GB   Gorski Sharon M SM  

Autophagy 20220228 11


Chloroquine (CQ), a lysosomotropic agent, is commonly used to inhibit lysosomal degradation and macroautophagy/autophagy. Here we investigated the cell-extrinsic effects of CQ on secretion. We showed that lysosomal and autophagy inhibition by CQ altered the secretome, and induced the release of Atg8 orthologs and autophagy receptors. Atg8-family proteins, in particular, were secreted inside small extracellular vesicles (sEVs) in a lipidation-dependent manner. CQ treatment enhanced the release of  ...[more]

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