Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma

Abstract

Glioblastoma multiforme (GBM) is the most aggressive brain tumor in adults and remains incurable despite multimodal intensive treatment regimens. EGFRvIII is a truncated extracellular mutant of the EGF receptor (EGFR) commonly found in GBMs that confers enhanced tumorigenic behavior. To gain a molecular understanding of the mechanisms by which EGFRvIII acts, we have performed a large-scale analysis of EGFRvIII-activated phosphotyrosine-mediated signaling pathways and thereby have identified and quantified 99 phosphorylation sites on 69 proteins. Distinct signaling responses were observed as a function of titrated EGFRvIII receptor levels with the phosphatidylinositol 3-kinase pathway being dominant over the MAPK and STAT3 pathways at a high level of EGFRvIII expression. Within this data set, the activating phosphorylation site on the c-Met receptor was found to be highly responsive to EGFRvIII levels, indicating cross-activation of the c-Met receptor tyrosine kinase by EGFRvIII. To determine the significance of this finding, we devised a combined treatment regimen that used a c-Met kinase inhibitor and either an EGFR kinase inhibitor or cisplatin. This regimen resulted in enhanced cytotoxicity of EGFRvIII-expressing cells compared with treatment with either compound alone. These results suggest that the clinical use of c-Met kinase inhibitors in combination with either EGFR inhibitors or standard chemotherapeutics might represent a previously undescribed therapeutic approach to overcome the observed chemoresistance in patients with GBMs expressing EGFRvIII.

Fig. 1.

Cell lines and experimental strategy. (A) EGFRvIII expression levels in retrovirally transfected U87MG cell lines. (B) Western blot of U87MG cell lines expressing titrated levels of EGFRvIII. Cells were serum-starved for 36 h, lysed, and probed for EGFRvIII or phosphotyrosine levels. (C) Outline of MS-based experimental strategy.

Fig. 2.

Effect of EGFRvIII receptor levels on downstream signaling networks. (A) Relative quantification of EGFRvIII phosphorylation sites across the four cell lines. Phosphorylation levels are normalized relative to that of the DK cell line. (B) Visualization of the fold change in phosphorylation levels in the canonical EGFR signaling cascades as a function of titrated EGFRvIII levels. (C) Clustering analysis of phosphotyrosine protein networks using self-organizing maps (SOMs). Each column within the matrix components represent the relative phosphorylation level in the -DK, -M, -H, and -SH U87MG cell lines normalized against the U87H cell line. Optimal SOM architecture was a 3 × 3 matrix, because smaller matrices tended to cluster dissimilar phosphorylation profiles. (D) Protein phosphorylation sites found within the highly responsive cluster.

Fig. 3.

c-Met receptor activation and kinase inhibition. (A) Western blot of specific phosphorylation sites on the c-Met receptor (Y1230/Y1234/Y1235) across the four different cell lines in vitro after 24-h serum starvation. (B) Western blot of c-Met receptor phosphorylation levels of in vivo parental (P), DK, or EGFRvIII high-expressing U87MG-derived xenografts. (C) Western blot of U87-H cell line subjected to 1 h AG1478 dose escalation after 24-h serum starvation. (D) Comparison of the quantification of the phosphorylation levels for c-Met Y1234 upon treatment with either DMSO (control) or 10 μM c-Met kinase inhibitor SU11274 for 1 h after 24-h serum starvation. Two biological replicates were performed and peak areas for iTRAQ marker ions enable quantification of phosphorylation for each condition.

Fig. 4.

Dose–response of U87-H cell line upon treatment with kinase inhibitors or cisplatin. (A) Dose–response of U87-H cells to AG1478, SU1127, or a combination of SU11274 and 5 μM AG1478 over 72 h after 24-h serum starvation. Viability was measured by using the metabolic dye WST-1. Combination treatment significantly enhanced cytotoxicity at 10 μM SU11274 (P < 0.001). (B) Apoptosis measured by caspase 3/7 cleavage upon drug treatment over 24 h after 24-h serum starvation. Concentration of drugs used was 10 μM SU11274, 10 μM AG1478, or a combination of 10 μM SU11274 and 5 μM AG1478. Combination treatment significantly enhanced apoptosis (P < 0.01). (C) Dose–response of U87-H to AG1478, PHA665752, or a combination of PHA665752 and 5 μM AG1478 over 72 h after 24-h serum starvation. Combination treatment significantly enhanced cytotoxicity at 10 μM PHA665752 (P < 0.0001). (D) Viability of U87-H cells in response to a combination treatment of 10 μg/ml cisplatin with either AG1478 or SU11274.