{'fraction_non_alphanumeric': 0.018497109826589597, 'fraction_numerical': 0.006551059730250481, 'mean_word_length': 5.409876543209877, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Quantitative phosphoproteomic analysis of EGFRvIII cellular signaling networks in glioblastoma

2529 EGFRvIII is a truncated mutant of the epidermal growth factor receptor (EGFR) which is implicated in the progression of glioblastoma multiforme. While much work has been done to elucidate the pathways initiated by EGFRvIII, the global map of its signaling network is still incomplete, making it difficult to assess downstream components involved in tumor progression. As a model system, we have used the U87MG glioblastoma cell line retrovirally transfected to express three different levels of EGFRvIII. Employing a mass spectrometric strategy previously developed in our laboratory, we have quantitatively measured global tyrosine phosphorylation events in these cell lines. This has allowed us to obtain a systems view of signaling networks initiated by the EGFRvIII receptor. We have identified many critical signaling proteins which are differentially tyrosine phosphorylated as a function of increasing EGFRvIII levels, in particular, PI-3 kinase and the cellular migration/invasion machinery. In addition, pathways normally activated by wildtype EGFR, such as the MAP kinase cascades are not responsive to EGFRvIII. K-means clustering of the phosphoproteomic data revealed a cluster of phosphorylation sites which are highly responsive to the expression levels of EGFRvIII. These include tyrosine phosphorylation sites in the activation loop of the c-Met receptor, PLC-γ and pyruvate kinase 3. EGFRvIII-positive tumors have previously been shown to be resistant to EGFR kinase inhibitors and alternative therapeutic candidates from our phosphoproteomic screen may represent a means by which to overcome this chemoresistance. We chose the c-Met receptor as an example for target validation. Analysis of c-Met phosphorylation in the presence of an Anti-HGF antibody indicates that the activation of c-Met is ligand independent. Temporal treatment of EGFRvIII expressing cells with AG1478, an EGFRvIII tyrphostin inhibitor, showed that c-Met activation is a result of direct crosstalk with the EGFRvIII receptor. Treatment of EGFRvIII expressing cells with SU11274, a c-Met specific inhibitor, led to a dose-dependent decrease in cell growth and increase in apoptosis. In addition, siRNA-mediated knockdown of the c-Met receptor resulted in a decrease in cell proliferation. Our data suggests that the c-Met receptor may be an alternative target in the treatment of EGFRvIII positive tumors and establishes phosphoproteomic analysis by mass spectrometry as a means of drug target discovery.