Feasibility of phosphoproteomics to uncover oncogenic signalling in secreted extracellular vesicles using glioblastoma-EGFRVIII cells as a model

磷酸蛋白质组学 微泡 激酶 细胞生物学 PI3K/AKT/mTOR通路 磷酸化 癌细胞 生物 蛋白激酶B MAPK/ERK通路 癌症研究 癌症 信号转导 化学 小RNA 蛋白激酶A 蛋白质磷酸化 生物化学 基因 遗传学
作者
Irene V. Bijnsdorp,Tim Schelfhorst,Mark J. Luinenburg,Frank Rolfs,Sander R. Piersma,Richard R. de Haas,Thang V. Pham,Connie R. Jiménez
出处
期刊:Journal of Proteomics [Elsevier BV]
卷期号:232: 104076-104076 被引量:9
标识
DOI:10.1016/j.jprot.2020.104076
摘要

Cancer cells secrete extracellular vesicles (EVs) that contain molecular information, including proteins and RNA. Oncogenic signalling can be transferred via the cargo of EVs to recipient cells and may influence the behaviour of neighbouring cells or cells at a distance. This cargo may contain cancer drivers, such as EGFR, and also phosphorylated (activated) components of oncogenic signalling cascades. Till date, the cancer EV phosphoproteome has not been studied in great detail. In the present study, we used U87 and U87EGFRvIII cells as a model to explore EV oncogenic signalling components in comparison to the cellular profile. EVs were isolated using the VN96 ME-kit and subjected to LC-MS/MS based phosphoproteomics and dedicated bioinformatics. Expression of (phosphorylated)-EGFR was highly increased in EGFRvIII overexpressing cells and their secreted EVs. The increased phosphorylated proteins in both cells and EVs were associated with activated components of the EGFR-signalling cascade and included EGFR, AKT2, MAPK8, SMG1, MAP3K7, DYRK1A, RPS6KA3 and PAK4 kinases. In conclusion, EVs harbour oncogenic signalling networks including multiple activated kinases including EGFR, AKT and mTOR. SIGNIFICANCE: Extracellular vesicles (EVs) are biomarker treasure troves and are widely studied for their biomarker content in cancer. However, little research has been done on the phosphorylated protein profile within cancer EVs. In the current study, we demonstrate that EVs that are secreted by U87-EGFRvIII mutant glioblastoma cells contain high levels of oncogenic signalling networks. These networks contain multiple activated (phosphorylated) kinases, including EGFR, MAPK, AKT and mTOR.
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