STOREDB:STUDY1095 Radiation alters the cargo of exosomes released from squamous head and neck cancer cells to promote migration of recipient cells [DOI:10.20348/STOREDB/1095]
|Radiation alters the cargo of exosomes released from squamous head and neck cancer cells to promote migration of recipient cells|
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|MELODI RESEARCH PRIORITY|
|Identification, development and validation of biomarkers for radiation-induced health (cancer and non-cancer) effects through sound epidemiological studies|
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|Radiation is a highly efficient therapy in squamous head and neck carcinoma (HNSCC) treatment. However, local recurrence and metastasis are common complications. Recent evidence shows that cancer-cell-derived exosomes modify tumour cell movement and metastasis. In this study, we link radiation-induced changes of exosomes to their ability to promote migration of recipient HNSCC cells.
We demonstrate that exosomes isolated from irradiated donor cells boost the motility of the HNSCC cells BHY and FaDu. Molecular data identified enhanced AKT-signalling, manifested through increased phospho-mTOR and MMP2/9 protease activity, as underlying mechanism. Furthermore, AKT-inhibition blocked the pro-migratory action, suggesting AKT-signalling as key player in exosome-mediated migration. Proteomic analysis of exosomes isolated from irradiated and non-irradiated BHY donor cells identified 39 up- and 36 downregulated proteins. In line with the observed pro-migratory effect of exosomes isolated from irradiated cells STRING process enrichment analysis assigned the deregulated exosomal proteins to cell motility and AKT-signalling.
Together, our findings demonstrate that exosomes derived from irradiated HNSCC cells confer a migratory phenotype to recipient cancer cells. This is possibly due to radiation-regulated exosomal proteins that increase AKT-signalling. We conclude that exosomes may act as a driver of HNSCC progression during tumour radiotherapy and are therefore attractive targets to improve radiation therapy strategies.
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STOREDB:DATASET1135 exosome proteome from squamous head and neck cancer cells [DOI:10.20348/STOREDB/1095/1135]
Created on:2017-06-07 10:13:05 Modified On:2017-06-07 10:13:05
|exosome proteome from squamous head and neck cancer cells|
|Exosomal proteins were isolated by adding 20 µl of lysis buffer II (25 mM Tris pH 7.5, 120 mM NaCl, 1 % Triton X-100, 1 % PSMF, 1 mM NOV, 1 mM Leupeptin) to 40 µl of exosome suspension isolated from 1.5 x 107 cells. The samples were incubated for 4 hours on ice with repeated vortexing and the protein concentration was determined by the BCA assay (PierceTM BCA Protein Assay Kit, Thermo Fisher Scientific) following the manufacturer’s instructions.
Sample preparation, LC-MS/MS measurement, label-free quantitative analysis and database searches were performed as previously described13. Briefly, 5 µg of protein were digested using a modified filter-aided sample preparation (FASP), followed by the LC-MS/MS analysis performed on a LTQ OrbitrapXL (Thermo Fisher Scientific) coupled to an Ultimate3000 nano high-performance liquid chromatography system (Dionex). Alignment of peptides was set to at least 89.5 % and single charged features as well as features with charges higher than +7 were eliminated. The Mascot search engine (Matrix Science, version 2.5.0) with the Ensembl Human database (version 83, 31236148 residues, 83462 sequences) was used for identification.
To identify significantly changed proteins a FDR-adjusted p-value (q-value) of three independent biological replicates was calculated. Here peptides with ≥ 2 unique peptides, a fold-change between ≤ 0.7 and ≥ 1.3 plus a q-value of < 0.05 were considered as statistically significant deregulated.
In silico analysis was performed with several bioinformatics tools. The top exosomal protein candidates of ExoCarta, the web-based database of exosomal proteins, RNA and lipids, was used to compare the detected exosomal proteins from BHY cells with proteins recorded within exosomes ((http://exocarta.org/exosome_markers_new) accessed 09.03.2017)48. Protein subcellular localizations and functions were determined using STRING: functional protein association networks (http://STRING-db.org/)49. A pathway enrichment analysis (FDR ˂ 0.05) of the deregulated exosomal proteins was performed using the Reactome 5.1.0 application50 in the Cytoscape 3.2.1 software51.