Molecular characterization of EGFR and EGFRvIII signaling networks in human glioblastoma tumor xenografts

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.

Fig. 1.

Characterization of human GBM xenografts differentially expressing wtEGFR and EGFRvIII. A, GBM xenografts were generated from 100–200 μl of tumor homogenate, derived from primary tumors from different patients undergoing surgical treatment and serially passaged in mice before this study, mixed 1:1 with matrigel and subcutaneously injected into the flanks of nude mice. Tumors were grown for 30–60 days at which time mice were sacrificed and subcutaneous tumors were harvested. B and C, For all blots the upper panel shows either EGFR or phosphotyrosine and the lower panel indicates the GAPDH loading control blot. B, EGFR immunoblotting across eight human GBM xenograft lines (four biological replicates). Tumor lines GBM6, 8, 10, 12, 15, 26, 39, and 59 are indicated by numbers. There are two biological replicates for GBM6. Biological replicate 1 (6¹) and biological replicate 4 (6⁴) were analyzed in the place of biological replicate 2 and 3 respectively. For all analyses there are four biological replicates for the seven remaining GBM xenograft lines. wtEGFR is indicated by the higher MW band and EGFRvIII is indicated by the lower MW band. Bottom panel; immunoblot band intensities were quantified across all four biological replicates, EGFR expression was normalized to GAPDH, then normalized to GBM8. C, Total phosphotyrosine immunoblots of 8 human GBM xenograft lines (four biological replicates).

Fig. 2.

Schematic diagram of the experimental mass spectrometric workflow. Eight GBM xenograft lines grown subcutaneously in mice were homogenized, reduced, alkylated, and digested with trypsin and then peptides were differentially labeled with iTRAQ8plex. Phosphotyrosine peptide enrichment was carried out by immunoprecipitation using anti-phosphotyrosine antibodies and peptides were further enriched using an IMAC step. Phosphotyrosine peptides were then analyzed by LC-MS/MS using an Orbitrap XL instrument. Peptides were identified and quantified using CID and HCD scans. Protein expression profiling was carried out using an IEF workflow in which peptides were separated into five fractions based on their pI. Each fraction underwent LC-MS/MS using a 5600 triple ToF instrument and peptides were identified by CID.

Fig. 3.

Summary of phosphotyrosine and protein expression profiles identified and quantified across eight human GBM xenograft lines. A, Venn diagram showing the number of proteins identified and quantified across four biological replicates (1, 2, 3, and 4). A total of 1588 proteins were identified and quantified across two or more replicates. Proteins that were only identified in one replicate are shown in gray and were not included in further analyses. B, Venn diagram showing the number of phosphotyrosine peptides quantified across four biological replicates (1, 2, 3, and 4).

Fig. 4.

Proteins quantified across eight GBM xenograft lines differentially overexpressing wtEGFR and EGFR vIII. A, Heat map of the 1588 proteins quantified across eight GBM xenograft lines expressing wtEGFR, overexpressing wtEGFR (wtEGFR+) or overexpressing EGFRvIII (EGFRvIII+) indicated at the top of the heat map. The heat map is a representation of iTRAQ8plex fold changes normalized to the mean of all eight channels and Log 2 transformed. Proteins were clustered using affinity propagation to reveal groups of proteins that show similar expression profiles (see experimental procedures). B, Panther GO biological processes annotation analyses of all 1588 proteins identified and quantified. C, Panther GO protein classes' annotation analyses of all 1588 proteins identified and quantified. D, Compilation of the 44 EGFR peptides identified and quantified across the eight GBM xenografts. The amino acid position in the full length is indicated on the x axis. The amino acid sequence that is specific to the wtEGFR is indicated alongside the amino acid sequence specific to both wtEGFR and the truncated EGFRvIII. wtEGFR (wt) tumors are shown in gray, wtEGFR overexpressing tumors (wt+) are shown in blue, and EGFRvIII overexpressing tumors (vIII+) are shown in red.

Fig. 5.

Proteins significantly increased in EGFRvIII expressing tumors. A, Heat map of the 63 protein significantly differentially expressed in the EGFRvIII tumors when compared with the wtEGFR tumors. The heat map is a representation of iTRAQ8plex fold changes normalized to the mean of all eight channels and Log 2 transformed. * indicates the proteins depicted in section C. B, GO protein class annotation for the 63 proteins differentially expressed in the EGFRvIII tumors. C, Kaplan meier survival plots of the four proteins with the most significant effect on survival across all gliomas in REMBRANDT. n refers to the number of human patients and the p value shown is associated with the survival curves for the specified up-regulated gene expression (>2 fold) compared with intermediate expression calculated with a log rank (Mantel-Cox) test. D, Kaplan meier survival curve for the simultaneous up-regulation of all five of the most significant genes shown in panel C. E, The bar charts shown are representative of quantification of immunoblotting images shown in supplemental Fig. S2. The wtEGFR, wtEGFR+, and EGFRvIII+ band intensities were averaged together and the mean value across all four biological replicates are shown in the bar charts for S100A10, MVP, CAIII, and GBP1. The standard error across four biological replicates is shown.

Fig. 6.

Phosphotyrosine sites quantified across GBM xenografts differentially overexpressing wtEGFR and EGFR vIII. A, Heat map of the 225 phosphotyrosine sites quantified across eight GBM xenograft lines expressing wtEGFR (wt), overexpressing wtEGFR (wtEGFR+), or overexpressing EGFRvIII (vIII+) as indicated at the top of the heat map. The heat map is a representation of iTRAQ8plex fold changes normalized to the mean of all 8 channels and Log 2 transformed. B, GO protein classes' annotation for the 168 proteins identified and quantified in the phosphotyrosine analyses. C, Graphical representation of the 10 phosphotyrosine site clusters. Phosphotyrosine sites were clustered using affinity propagation.

Fig. 7.

Quantification of EGFR phosphorylation sites and known downstream signaling molecules. A, Seven EGFR phosphorylation sites are depicted along with their fold change relative to the mean. Specific downstream signaling molecules SHC, PLC-γ, STAT3, Gab1, and Cbl are shown. The y axis on all graphs indicates fold changes relative to the mean, and x axis indicates the tumor in which the signal was identified and quantified. Error bars represent standard deviations. B, Validation Western blotting of phosphotyrosine sites on EGFR and directly downstream. EGFR protein expression, EGFR pY1173 and pY1045, STAT3 protein expression and STAT3 pY705, PLC-γ protein expression and Gab1 protein expression and Gab1 py627 across all four biological replicates β-tubulin is the loading control.

Fig. 8.

Proteins that are significantly differentially tyrosine phosphorylated in EGFRvIII expressing tumors. A, Eight tyrosine phosphorylation sites were found to be significantly (p < 0.05) differentially phosphorylated in the five wtEGFR tumors relative to the three EGFRvIII tumors. Average phosphorylation levels in the wtEGFR, wtEGFR+, and EGFRvIII expressing tumors are represented. ** indicates proteins where p < 0.01 and * indicates p < 0.05. Error bars indicate standard deviations. B, Western blotting analyses to identify protein expression of Cbl, GEFH1, Hrs, and Shp2, and β-tubulin is the loading control across all four biological replicates.

Fig. 9.

Increased expression of wtEGFR or EGFRvIII leads to differential effects on protein expression and tyrosine phosphorylation signaling networks. Quantification of protein expression and tyrosine phosphorylation profiles in the eight human GBM tumor xenografts identified patterns of expression and phosphorylation that were specific to each tumor. Despite the inter-tumor heterogeneity, it was still possible to identify proteins and phosphorylation sites that were differentially expressed in the EGFRvIII tumors.