Abstract
Glioblastoma multiforme is a highly invasive and aggressive brain tumor with an invariably poor prognosis. The overexpression of epidermal growth factor receptor (EGFR) is a primary influencer of invasion and proliferation in tumor cells and the constitutively active EGFRvIII mutant, found in 30-65% of Glioblastoma multiforme, confers more aggressive invasion. To better understand how EGFR contributes to tumor aggressiveness, we investigated the effect of EGFR on the secreted levels of 65 rationally selected proteins involved in invasion. We employed selected reaction monitoring targeted mass spectrometry using stable isotope labeled internal peptide standards to quantity proteins in the secretome from five GBM (U87) isogenic cell lines in which EGFR, EGFRvIII, and/or PTEN were expressed. Our results show that cell lines with EGFR overexpression and constitutive EGFRvIII expression differ remarkably in the expression profiles for both secreted and intracellular signaling proteins, and alterations in EGFR signaling result in reproducible changes in concentrations of secreted proteins. Furthermore, the EGFRvIII-expressing mutant cell line secretes the majority of the selected invasion-promoting proteins at higher levels than other cell lines tested. Additionally, the intracellular and extracellular protein measurements indicate elevated oxidative stress in the EGFRvIII-expressing cell line. In conclusion, the results of our study demonstrate that EGFR signaling has a significant effect on the levels of secreted invasion-promoting proteins, likely contributing to the aggressiveness of Glioblastoma multiforme. Further characterization of these proteins may provide candidates for new therapeutic strategies and targets as well as biomarkers for this aggressive disease.
Highlights
Glioblastoma multiforme (GBM)1 is the most frequently occurring glioma in adults [1, 2]
Quantitative Analysis of Secreted Proteins from GBM Cells Reveals Differential Expression According to Alterations in epidermal growth factor receptor (EGFR) Signaling—To investigate the effect of EGFR signaling on the secreted levels of proteins implicated in aggressive invasiveness and proliferation of GBM cells, we performed quantitative selected reaction monitoring (SRM) analysis of selected proteins based on the following stepwise criteria: 1) Literature support for a role in invasion and regulation by EGFR; a protein was selected if it has been reported to be implicated in tumor invasion and/or to be influenced by EGFR pathway components in the published literature
Given that components of the EGFR signaling pathway play a critical role in the aggressiveness of GBM cells, we used a quantitative mass spectrometry-based proteomic analysis approach to identify the effects of alterations in the EGFR pathway on intracellular signaling proteins and the secreted levels of proteins that contribute to the invasiveness of GBM
Summary
Glioblastoma multiforme (GBM) is the most frequently occurring glioma in adults [1, 2]. The most common and best-studied mutation is the type III variant (EGFRvIII) with a deletion of exons 2 to 7 [25] This in-frame deletion of 267 amino acids removes the ligandbinding domain of the EGFR protein, encoding a mutant protein with ligand-independent constitutive activity [27]. EGFR and EGFRvIII activate the PI3K/Akt pathway in GBM, and in the absence of PTEN activity, the balance is shifted toward cell survival, angiogenesis, migration, and proliferation, leading to aggressive tumor growth. The EGFR signaling pathway has been shown to play a major role in the invasiveness of glioma cells by regulating the phosphorylation levels and expression of downstream intracellular signaling proteins [50]. We present a first comprehensive quantitative study of the effects of EGFR signaling on the levels of secreted proteins involved in invasion in GBM using selected reaction monitoring (SRM), a targeted quantitative mass spectrometry technique
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