Expression data from U87MG cells expressing EGFRvIII. Homo sapiens

EGFRvIII is the most common deletion mutant of EGFR in human cancer and its levels are highly correlated with poor prognosis in GBM. The deletion of exons 2-7 removes most of the extracellular ligand binding domain, so it is unable to bind EGF or other EGFR-binding ligands. Nevertheless, the mutant receptor is constitutively phosphorylated, and is capable of activating downstream signaling pathways at a low level. To comprehensively identify the downstream signaling consequences of the EGFRvIII, we incorporated phosphoproteomic, transcription profiling and DNase-Seq data from U87MG glioblastoma cells expressing titrated levels of this mutant receptor. Overall design: Total RNA were extracted from U87MG cells engineered to expressed different levels of EGFRvIII: medium (U87M; 1.5 million copies of EGFRvIII receptor per cell), high (U87H; 2 million copies per cell), super-high (U87SH; 2.5 million copies per cell), and kinase-dead EGFRvIII (U87DK; 2 million copies of kinase dead EGFRvIII per cell). RNA was hybridized to Affymetrix microarrays.

EGFRvIII是人类癌症中表皮生长因子受体（EGFR）最常见的缺失突变体，其表达水平与胶质母细胞瘤（GBM）的不良预后高度相关。其外显子2-7的缺失移除了大部分细胞外配体结合域，因此无法结合表皮生长因子（EGF）或其他EGFR结合配体。尽管如此，该突变受体仍可发生组成型磷酸化，并能够低度激活下游信号通路。为全面解析EGFRvIII的下游信号传导效应，本研究整合了表达梯度水平该突变受体的U87MG胶质母细胞瘤细胞的磷酸化蛋白质组学、转录表达谱及DNase测序（DNase-Seq）数据。整体实验设计：从经工程改造以表达不同水平EGFRvIII的U87MG细胞中提取总RNA，涵盖中等表达组（U87M；每细胞含150万份EGFRvIII受体拷贝）、高表达组（U87H；每细胞含200万份拷贝）、超高表达组（U87SH；每细胞含250万份拷贝），以及激酶失活型EGFRvIII组（U87DK；每细胞含200万份激酶失活型EGFRvIII拷贝）。将提取的RNA与Affymetrix基因芯片进行杂交。