Analysis of Phosphotyrosine Signaling in Glioblastoma Identifies STAT5 as a Novel Downstream Target of ΔEGFR

Abstract

An in-frame deletion mutation in Epidermal Growth Receptor (EGFR), ΔEGFR is a common and potent oncogene in glioblastoma (GBM), promoting growth and survival of cancer cells. This mutated receptor is ligand independent and constitutively active. Its activity is low in intensity and thought to be qualitatively different from acutely ligand stimulated wild type receptor implying that the preferred downstream targets of ΔEGFR play a significant role in malignancy. To understand the ΔEGFR signal we compared it to that of a kinase-inactivated mutant of ΔEGFR and wild-type EGFR with shotgun phosphoproteomics using an electron-transfer dissociation (ETD) enabled ion trap mass spectrometer. We identified and quantified 354 phosphopeptides corresponding to 249 proteins. Among the ΔEGFR-associated phosphorylations were the previously described Gab1, c-Met and Mig-6, and also novel phosphorylations including that of STAT5 on Y694/9. We have confirmed the most prominent phosphorylation events in cultured cells and in murine xenograft models of glioblastoma. Pathway analysis of these proteins suggests a preference for an alternative signal transduction pathway by ΔEGFR compared to wild type EGFR. This understanding will potentially benefit the search for new therapeutic targets for ΔEGFR expressing tumors.

Soluble Urokinase Receptor Is Released Selectively by Glioblastoma Cells That Express Epidermal Growth Factor Receptor Variant III and Promotes Tumor Cell Migration and Invasion*

“…As we have shown previously, this modest decrease in uPAR is accompanied by a major increase in uPA expression, which has the net effect of activating uPAR signaling (42,53). This shift in GBM cell signaling has been implicated in promoting survival of tumor cells treated with EGFR-targeting drugs and in promoting GBM cell migration (42,50,52).…”

“…Nevertheless, tumors in which EGFRvIII is identified tend to be highly aggressive (43,51). This may reflect novel co-receptor interactions in the cells that express EGFRvIII or activation of downstream signaling pathways that are unique to EGFRvIII (52). We previously showed that in EGFRvIII-expressing GBM cells, membrane-anchored uPAR functions as an important co-receptor promoting activation of STAT5b (42).…”

Molecular Characterization of EGFR and EGFRvIII Signaling Networks in Human Glioblastoma Tumor Xenografts

“…Although EGFRvIII signaling has been extensively studied in GBM cell lines, the molecular mechanisms of increased tumorigenesis driven by EGFRvIII overexpression in human tumors have not been fully elucidated (20,21). In addition, tissue culture conditions dramatically change the genetic and molecular characteristics found in primary human tumors.…”

Cucurbitacin-I inhibits Aurora kinase A, Aurora kinase B and survivin, induces defects in cell cycle progression and promotes ABT-737-induced cell death in a caspase-independent manner in malignant human glioma cells

“…48 Chumbalkar et al performed a tyrosine-directed search for signaling events downstream of DEGFR (EGFRviii) and identified STAT5 phosphorylation at Y699 as a key event. 56 Later, Latha et al demonstrated that EGFRviii cooperated with STAT5 to regulate the Bcl -xL promoter. 57 This prompted us to investigate the role of JAK/STAT inhibitor cucurbitacin-I and the Bcl -2/Bcl -xL antagonist ABT-737 in glioma cell lines.…”