Die Rolle von Syntenin bei der Etablierung eines fördernden Tumormikromilieus im humanen Mammakarzinom
Hämatologie, Onkologie
Zusammenfassung der Projektergebnisse
Breast cancer is the second most common cancer worldwide. It is known that next to the tumor cells, the surrounding cells in the microenvironment play an important role in tumor progression. Using a combination of differential ultracentrifugation and size exclusion chromatography, this study shows that cancer cells secrete small extracellular vesicles (diameter <150nm), termed exosomes, into the extracellular space which interact with surrounding stroma cells. In particular, they enhance migration of endothelial cells and upregulate expression of the pro-tumorigenic factors Arg1, Il6 and Mmp9 in murine macrophages. Exosomes are derived from the endosomal system where they bud as small vesicles into the lumen of multivesicular bodies in which they are stored and finally released upon fusion of these bodies with the cellular plasma membrane. One protein which is essential for exosome biogenesis and cargo selection is the adaptor scaffolding protein syntenin/SDCBP which is often found to be overexpressed in cancer cells. Characterization of exosomes from syntenin-high and –low murine breast cancer cells by Proteomics demonstrated that syntenin is involved in the transport of many tumor proteins onto exosomes. Knockdown and overexpression experiments in human MCF7 breast cancer cells showed that the Epithelial Cell Adhesion Molecule (EpCAM) is one of them. Interestingly, EpCAM was not only found in its full length form on exosomes, but also as short C-terminal fragments (CTFs) whose expression depended on EpCAM’s C-terminal pdz binding motif. Whether exosomal EpCAM, full length or CTF, is important for the pro-tumorigenic phenotype of syntenin+ Exo is addressed at the moment. Looking closer at the molecular mechanism of exosome biogenesis by the syntenin/Alix pathway, this study further identified the intracellular adaptor protein CD2AP/CMS as novel regulator of the pathway. Using inhibitors for different degradative pathways, it was shown that CD2AP prevented the lysosomal degradation of syntenin and thereby controlled intracellular syntenin protein levels. Moreover, CD2AP was shown to control endosomal budding downstream of the tyrosine kinase SRC and upstream of Arf6/PLD2, three known members in the pathway. Since endosomal budding is a key process in the biogenesis of exosomes, it was not surprising that CD2AP influenced the composition of exosomes secreted by the cells, while it had no effect on the total number of vesicles being released. In particular, knockdown of CD2AP led to a significant reduction of syntenin, Alix and CD63 on exosomes, while overexpression of CD2AP enhanced their expression. By cloning and overexpressing several mutant forms of CD2AP, it was shown that its proline-rich domain is important for its function in exosome biogenesis. Taken together, the obtained results confirmed the initial hypothesis that syntenin is involved in the export of many tumor proteins onto exosomes, EpCAM being one of them. In search for a mechanism underlying this process, CD2AP was identified as a novel regulator of exosome composition by controlling syntenin degradation as well as endosomal budding. Further studies are required to define CD2AP interactors and which of the tumor proteins identified on breast cancer exosomes are involved in the observed effects on endothelial cells and macrophages.
Projektbezogene Publikationen (Auswahl)
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(2017) Syntenin mediates SRC function in exosomal cell-to-cell communication. Proceedings of the National Academy of Sciences of the United States of America 114 (47) 12495–12500
Imjeti, Naga Sailaja; Menck, Kerstin; Egea-Jimenez, Antonio Luis; Lecointre, Celine; Lembo, Frederique; Bouguenina, Habib; Badache, Ali; Ghossoub, Rania; David, Guido; Roche, Serge; Zimmermann, Pascale
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(2017), Characterisation of tumourderived microvesicles in cancer patients' blood and correlation with clinical outcome, J Extracell Vesicles 6(1): 1340745
Menck K, Bleckmann A, Wachter A, Hennies B, Ries L, Schulz M, Balkenhol M, Pukrop T, Schatlo B, Rost U, Wenzel D, Klemm F, Binder C
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Neutral sphingomyelinases control extracellular vesicle budding from the plasma membrane (2017), J Extracell Vesicles 6(1): 1378056
Menck K, Sönmezer C, Worst TS, Schulz M, Dihazi G, Streit F, Erdmann G, Kling S, Boutros M, Binder C, Gross JC
