Syntenin-1 regulates the expression of adhesion proteins on small extracellular vesicles

Irmer, B; Egea-Jiménez, A; Audebert, S; Camoin, L; Bleckmann, A; Zimmermann, P; Menck, K

Zusammenfassung

Introduction: Tumour-derived small extracellular vesicles (sEVs) are known as potent promotors of tumour progression and metastasis. However, the attribution of such effects to specific sEV subpopulations remains challenging. Syntenin-1 (ST1) is a key regulator for the biogenesis of an endosomal-derived sEV subpopulation and overexpressed in many tumour entities. Moreover,ST1 contributes to the assembly of protein complexes at the membrane. Indeed, the PDZ domains of ST1 directly bind various transmembrane interactors like syndecans and tetraspanins supporting the formation of higher order signalling nexi. Therefore, this study aimed to characterize the ST1-dependent sEV protein signature and its functional relevance in breast cancer. Methods: sEVs from murine wildtype and ST1 knockout breast cancer cells (4T1) were isolated by serial ultracentrifugation and classified by proteomics and nanoparticle tracking analysis. Several differentially expressed proteins (DEPs) were selected forimmunoblot validation, database screening and further functional analysis. Direct protein interaction was quantified via surfaceplasmon resonance (SPR) spectroscopy. Results: Proteomic analysis revealed a subset of 52 ST1-dependent sEV marker proteins including direct ST1-binding partners like ALIX, CD63 and SDC4 as well as tumour-associated proteins with clinical impact on patient survival. Intriguingly, many of the downregulated DEPs on ST1-deficient sEVs were attributed to cell adhesion, extracellular matrix assembly and cell-cell contacts via pathway enrichment analysis. A ST1-dependent expression on sEVs was validated in 4T1 and human MCF 7 breast cancer cells for selected DEPs with such adhesive potential including the epithelial cell adhesion molecule (EpCAM). By SPR technology we show that EpCAM directly binds to ST1 via its PDZ-binding motif. Functionally, EpCAM seemed to act merely as a cargo protein without evident contribution to sEV biogenesis. Summary/Conclusion: ST1 regulates the formation of a sEV subpopulation associated with cell adhesion. Thus, ST1-dependent sEVs might be potential drivers for enhanced tumour-derived sEV distribution and pre-metastatic niche formation in breast cancer by promoting the disappearance of adhesion complexes from cancer cells and their secretion in the matrix on sEVs.