Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Urology, Cancer Center Amsterdam, De Boelelaan 1117, Amsterdam, Netherlands; Amsterdam UMC, Vrije Universiteit Amsterdam, OncoProteomics Laboratory, Cancer Center Amsterdam, De Boelelaan 1117, Amsterdam, Netherlands.
Amsterdam UMC, Vrije Universiteit Amsterdam, OncoProteomics Laboratory, Cancer Center Amsterdam, De Boelelaan 1117, Amsterdam, Netherlands; Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Medical Oncology, Cancer Center Amsterdam, De Boelelaan 1117, Amsterdam, Netherlands.
J Proteomics. 2021 Feb 10;232:104076. doi: 10.1016/j.jprot.2020.104076. Epub 2020 Dec 8.
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.
癌细胞分泌包含分子信息的细胞外囊泡 (EVs)，包括蛋白质和 RNA。致癌信号可以通过 EV 的货物传递到受体细胞，并可能影响邻近细胞或远处细胞的行为。这种货物可能包含癌症驱动因子，如 EGFR，以及致癌信号级联的磷酸化（激活）成分。迄今为止，尚未对癌症 EV 磷酸蛋白质组进行详细研究。在本研究中，我们使用 U87 和 U87EGFRvIII 细胞作为模型，与细胞图谱相比，探索 EV 致癌信号成分。使用 VN96 ME-kit 分离 EVs，并进行基于 LC-MS/MS 的磷酸蛋白质组学和专用生物信息学分析。在 EGFRvIII 过表达细胞及其分泌的 EV 中，（磷酸化）-EGFR 的表达高度增加。在细胞和 EV 中增加的磷酸化蛋白与 EGFR 信号级联的激活成分相关，包括 EGFR、AKT2、MAPK8、SMG1、MAP3K7、DYRK1A、RPS6KA3 和 PAK4 激酶。总之，EV 包含致癌信号网络，包括多个激活的激酶，包括 EGFR、AKT 和 mTOR。意义：细胞外囊泡 (EVs) 是生物标志物的宝库，广泛研究其在癌症中的生物标志物含量。然而，关于癌症 EV 中的磷酸化蛋白质谱研究甚少。在当前的研究中，我们证明由 U87-EGFRvIII 突变胶质母细胞瘤细胞分泌的 EV 含有高水平的致癌信号网络。这些网络包含多个激活的（磷酸化）激酶，包括 EGFR、MAPK、AKT 和 mTOR。
