In Situ Analysis of Mutant EGFRs Prevalent in Glioblastoma Multiforme Reveals Aberrant Dimerization, Activation, and Differential Response to Anti-EGFR Targeted Therapy

Abstract

Aberrations in epidermal growth factor receptor (EGFR/ErbB1) are the most common oncogenic alterations in glioblastoma multiforme (GBM), the most common primary brain tumor. Interactions between wild-type (wt) and mutant EGFRs and their subsequent activation are of biologic and potential therapeutic importance in GBMs. We recently showed that in situ proximity ligation assay (PLA) allows for quantitative evaluation of EGFR dimerization and activation in intact cells. Using this in situ platform, we show the aberrant homo-/heterodimeric properties of EGFRvIII and EGFRc958 mutants, the two most common EGFR mutants in GBMs. In addition, dimer phosphoactivation status could be detected by PLA with superior signal-noise ratio (>17-fold) and sensitivity (>16-fold) than immunofluorescence-based phospho-EGFR measurements. Dimer activation analysis indicated quantitative activation differences of mutant dimers. These aberrant features were not overexpression dependent but appeared independent of cellular expression levels, suggesting inherent properties of the mutant receptors. Moreover, we observed in situ detection of EGFRwt-EGFRvIII heterodimerization in GBM specimens, supporting our cell line observations. Notably, currently used anti-EGFR therapeutics, such as cetuximab, matuzumab, and panitumumab, could effectively block EGFRwt dimerization and activation but did not equally impair EGFRvIII homodimers, EGFRwt-EGFRvIII, or EGFRvIII-EGFRc958 heterodimers. EGFRvIII appears to have intrinsic phosphoactivation independent of dimerization as matuzumab blockade of homodimerization had no effect on receptor phosphorylation levels. These data suggest differences in the dimerization-blocking efficacy of EGFR monoclonal antibodies as mutant EGFR dimer configurations prevalent in GBMs can evade blockade by anti-EGFR treatments. Further studies are warranted to evaluate whether this evasion contributes to poor therapeutic response or resistance. Mol Cancer Res; 10(3); 428-40. Ó2012 AACR.

Targeting a Glioblastoma Cancer Stem-Cell Population Defined by EGF Receptor Variant III

“…Antibodies and small molecule inhibitors against wtEGFR also bind to EGFRvIII, but are less potent against this variant (43–45). Thus, if EGFRvIII + cells represent the tumor initiating population there may be a CSC basis for why anti-EGFR therapy has not been effective against tumors expressing EGFRvIII (46).…”

Early signaling dynamics of the epidermal growth factor receptor

“…One candidate mechanism for the slower phosphorylation kinetics of Gab1 versus Shc is a difference in recruitment to the receptor. To test this hypothesis, proximity ligation assays (PLAs) were used to verify selected immediate early phosphorylation events in situ and quantify receptor-adaptor interactions with low-nanometer resolution in intact cells (20,21). To measure early association events, cells seeded on glass coverslips were stimulated with 20 nM EGF for 0, 10, or 60 s; frozen on liquid nitrogen; and fixed in 4% paraformaldehyde for analysis.…”

First in human nanotechnology doxorubicin delivery system to target epidermal growth factor receptors in recurrent glioblastoma

“…First, despite EGFR representing a clear target for GBM, multiple clinical trials with EGFR inhibitors [8,9] have been disappointing. The molecular basis for these disappointing results remains unclear, although it may include inactivation of a conformational change, extracellular domain mutations, as well as interactions between wild-type and mutant EGFR [28,29]. These potential issues may be less important for the proposed drug delivery system, compared with the extent of distribution and the degree of drug internalization.…”