Analysis of Phosphotyrosine Signaling in Glioblastoma Identifies STAT5 as a Novel Downstream Target of ΔEGFR

An in-frame deletion mutation in Epidermal Growth Receptor (EGFR), ΔEGFR is a common and potent oncogene in glioblastoma (GBM), promoting growth and survival of cancer cells. This mutated receptor is ligand independent and constitutively active. Its activity is low in intensity and thought to be qualitatively different from acutely ligand stimulated wild-type receptor implying that the preferred downstream targets of ΔEGFR play a significant role in malignancy. To understand the ΔEGFR signal, we compared it to that of a kinase-inactivated mutant of ΔEGFR and wild-type EGFR with shotgun phosphoproteomics using an electron-transfer dissociation (ETD) enabled ion trap mass spectrometer. We identified and quantified 354 phosphopeptides corresponding to 249 proteins. Among the ΔEGFR-associated phosphorylations were the previously described Gab1, c-Met and Mig-6, and also novel phosphorylations including that of STAT5 on Y694/9. We have confirmed the most prominent phosphorylation events in cultured cells and in murine xenograft models of glioblastoma. Pathway analysis of these proteins suggests a preference for an alternative signal transduction pathway by ΔEGFR compared to wild-type EGFR. This understanding will potentially benefit the search for new therapeutic targets for ΔEGFR expressing tumors.

表皮生长因子受体（EGFR）的框内缺失突变体ΔEGFR是胶质母细胞瘤（GBM）中常见且强效的致癌基因，可促进癌细胞的增殖与存活。该突变受体不依赖配体且具备组成型激活活性，其激活强度较低，且在性质上与急性配体刺激的野生型EGFR存在显著差异，这提示ΔEGFR偏好的下游靶点在肿瘤恶性进程中发挥重要作用。为解析ΔEGFR的信号转导特征，我们采用搭载电子转移解离（ETD）功能的离子阱质谱仪，通过鸟枪法磷酸化蛋白质组学技术，将ΔEGFR的信号通路与其激酶失活突变体以及野生型EGFR的信号通路进行对比分析。本研究共鉴定并定量到对应249种蛋白质的354条磷酸肽。ΔEGFR相关的磷酸化修饰除已有报道的Gab1、c-Met及Mig-6外，还包含STAT5在Y694/9位点的新型磷酸化修饰。我们已在体外培养细胞及胶质母细胞瘤小鼠异种移植模型中验证了其中最显著的磷酸化事件。对上述蛋白质的通路富集分析显示，相较于野生型EGFR，ΔEGFR更偏好激活一条替代性的信号转导通路。该研究成果有望为表达ΔEGFR的肿瘤患者筛选新型治疗靶点提供理论支撑。