Abstract
Glioblastoma multiforme (GBM) is a malignant primary brain tumor with a mean survival of 15 months with the current standard of care. Genetic profiling efforts have identified the amplification, overexpression, and mutation of the wild-type (wt) epidermal growth factor receptor tyrosine kinase (EGFR) in ≈ 50% of GBM patients. The genetic aberration of wtEGFR is frequently accompanied by the overexpression of a mutant EGFR known as EGFR variant III (EGFRvIII, de2-7EGFR, ΔEGFR), which is expressed in 30% of GBM tumors. The molecular mechanisms of tumorigenesis driven by EGFRvIII overexpression in human tumors have not been fully elucidated. To identify specific therapeutic targets for EGFRvIII driven tumors, it is important to gather a broad understanding of EGFRvIII specific signaling. Here, we have characterized signaling through the quantitative analysis of protein expression and tyrosine phosphorylation across a panel of glioblastoma tumor xenografts established from patient surgical specimens expressing wtEGFR or overexpressing wtEGFR (wtEGFR+) or EGFRvIII (EGFRvIII+). S100A10 (p11), major vault protein, guanylate-binding protein 1(GBP1), and carbonic anhydrase III (CAIII) were identified to have significantly increased expression in EGFRvIII expressing xenograft tumors relative to wtEGFR xenograft tumors. Increased expression of these four individual proteins was found to be correlated with poor survival in patients with GBM; the combination of these four proteins represents a prognostic signature for poor survival in gliomas. Integration of protein expression and phosphorylation data has uncovered significant heterogeneity among the various tumors and has highlighted several novel pathways, related to EGFR trafficking, activated in glioblastoma. The pathways and proteins identified in these tumor xenografts represent potential therapeutic targets for this disease.
Highlights
From the ‡Department of Bioengineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139, USA; §Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota 55905, USA; ¶Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
We propose that an improved understanding of the system-wide changes in protein expression and signaling caused by EGFRvIII expression should provide insight into specific therapeutic targets for EGFRvIII driven tumors
Characterization of Glioblastoma multiforme (GBM) Xenograft Tumors—Initial characterization of the four tumor xenografts generated for each GBM tumor was performed, with the goal of providing an estimate of the variance across different mice and different tumors (Fig. 1)
Summary
Generation of GBM Xenografts—Human GBM xenografts were established with the ectopic injection of 100 –200 l of tumor homogenate mixed 1:1 with matrigel into the flanks of nude mice (Fig. 1A). Antibody conjugated protein G was rinsed and iTRAQ 8plex labeled peptides re-suspended in the IP buffer, added to the conjugated protein G and incubated overnight at 4 °C with rotation. Phosphotyrosine peptide iTRAQ ratios were normalized based on the mean relative protein quantification ratios obtained from the total protein (i.e. protein expression analyses). Based on the biological variation, observed proteins with iTRAQ ratios below 0.75 were considered to be reduced in expression and proteins with iTRAQ ratios above 1.25 were considered to be increased in expression These values were applied to the phosphotyrosine analyses (the number of proteins shown to be changing based on this criteria can be found in supplemental Table S5). Gene Ontology Annotation—Gene ontology (GO) annotations were identified by uploading gene lists to the Protein Analysis Through Evolutionary Relationships (PANTHER) classification system (http:// www.pantherdb.org/)
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