(593d) A Mechanism Leading to Imbalanced EGFR Oncogenic Signaling Common to Lung and Brain Cancers with Distinct EGFR-Activating Mutations

Elevated expression of the epidermal growth factor receptor (EGFR) is observed in many cancers, but efforts to treat such cancers with EGFR-targeted therapeutics have resulted in limited success.  Surprisingly, EGFR mutations resulting in constitutive receptor activity tend to promote cellular sensitivity to EGFR-targeted inhibitors, an effect which has been suggested to result from a general augmentation of both pro- and anti-apoptotic signaling activity downstream of the receptor in which pro-apoptotic pathways are more sustained in response to receptor inhibition.  Previous work in our lab demonstrated that the EGFR-activating mutations in exons 19 and 21 which arise in non-small cell lung cancer (NSCLC) lead to enhanced cellular sensitivity to the EGFR kinase inhibitors gefitinib and erlotinib in part because of an impairment in the activation of the extracellular regulated kinase (ERK) due to altered function of the protein tyrosine phosphatase SHP2.  This mechanism appears to involve the sequestration of biochemically active SHP2 with the endocytosis-impaired EGFR mutants at the plasma membrane in a manner which prevents the participation of SHP2 in the activation of ERK.  In the present study, we sought to determine the extent to which a similar mechanism might exist in the context of elevated expression of the structurally distinct, constitutively active EGFRvIII mutant (which results from an in-frame deletion of exons 2-7) in glioblastoma multiforme (GBM) cells.  Using a panel of GBM cell lines expressing three different levels of EGFRvIII or a kinase-dead counterpart, we found that the magnitude of EGF-mediated activation of ERK decreased sharply with increasing EGFRvIII expression levels.  In fact, for cells with the highest expression of EGFRvIII (two million receptors per cell), treatment with 10 ng/mL EGF failed to induce detectable ERK phosphorylation.  Also consistent with our findings in NSCLC cells, we found that increasing expression of EGFRvIII reduced the rate of ligand-mediated receptor internalization.  Immunofluorescence imaging revealed that ligand-induced redistribution of SHP2 from the cell center outward was least pronounced for cells with the highest EGFRvIII expression levels, where SHP2 was basally distributed throughout the cell.  Immunoprecipitation of SHP2 from cell lysates revealed that basal association of SHP2 with the adapter protein GAB1 and EGFR increased with increasing EGFRvIII expression. Taken together, the preceding data suggest that sufficiently high EGFRvIII expression may result in sequestration of SHP2 with adapter proteins and the receptor at the cell periphery in a complex which may lead to an impaired ability to promote ERK activity in response to EGFR ligands.  We further explored this model in the panel of GBM cell lines through stable knockdown of SHP2 using RNA interference.  SHP2 knockdown led to decreased ERK phosphorylation in all cell lines, but the magnitude of this effect decreased with increasing EGFRvIII expression.  Consistent with this observation, we also found that SHP2 knockdown inhibited rates of cell proliferation least significantly in cells with the highest EGFRvIII expression.  Thus, our data confirm the hypothesis that as EGFRvIII expression increases the extent to which SHP2 participates in ERK activation is diminished in a manner that reduces overall ERK activity levels and alters cellular proliferation rates.  Our findings further demonstrate that structurally distinct EGFR-activating mutations may result in qualitatively similar perturbations to downstream signaling leading to the surprising impairment of receptor-mediated ERK activation.  Given that elevated expression of these mutant receptors generally promotes cellular response to EGFR inhibitors, this work identifies SHP2 and ERK as potentially critical nodes in the signaling network downstream of EGFR for co-inhibition in settings where EGFR inhibitors are not effective as single agents.