EGF-independent activation of cell-surface EGF receptors harboring mutations found in gefitinib-sensitive lung cancer

Abstract

Several somatic mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) have been identified that predict clinical response of non-smallcell lung carcinoma (NSCLC) patients to gefitinib. To test the hypothesis that these mutations cause constitutive EGF receptor signaling, and to investigate its mechanistic basis, we expressed representative examples in a null background and analysed their biochemical properties. Each mutation caused significant EGF-independent tyrosine phosphorylation of EGFR, and allowed the receptor to promote Ba/F3 cell mitogenesis in the absence of EGF, arguing that these are oncogenic mutations. Active mutated receptors are present at the cell surface and are fully competent to bind EGF. Recent structural studies show that the inactive EGFR tyrosine kinase domain is autoinhibited by intramolecular interactions between its activation loop and aC helix. We find that mutations predicted to disrupt this autoinhibitory interaction (including several that have not been described in NSCLC) elevate EGF-independent tyrosine kinase activity, thus providing new insight into how somatic mutations activate EGFR and other ErbB family members.

Epidermal growth factor receptor activation in prostate cancer by three novel missense mutations

“…Most EGFR mutations in lung and head/neck cancer reside in the activation loop or alpha helix C region (Lynch et al, 2004;Pao et al, 2004;Lee et al, 2005;Nagahara et al, 2005). These mutations disrupt the autoinhibition of interaction between intracellular kinase domains and render the mutated kinase constitutively active (Greulich et al, 2005;Zhang et al, 2006;Choi et al, 2007). Interestingly, while they occur in the kinase domain of EGFR in prostate cancer, none of the four novel somatic EGFR mutations are located in the activation loop or alpha helix C region.…”

“…In lung cancer, cells whose growth depends on EGFR with mutations in exons 19 and 21 are sensitive to tyrosine kinase inhibitors of EGFR (Lynch et al, 2004;Paez et al, 2004;Pao et al, 2004;Bell et al, 2005;Choi et al, 2007). Glioblastoma cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors (Lee et al, 2006).…”

“…The L858R EGFR mutation, also alternatively identified as L834R for the human EGFR sequence occuring in lung and head neck cancer, has been extensively studied and serves as a prototype for EGFR kinase domain mutational studies in many other cancers. L858R is located in the activation loop encoded by exon 21 (Pao et al, 2004;Arteaga, 2006;Zhang et al, 2006;Choi et al, 2007).…”

Combined Treatment with Silibinin and Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors Overcomes Drug Resistance Caused by T790M Mutation

“…Silibinin can inhibit EGFR activity through alternative mechanisms in cells with EGFR mutations because kinase domain mutations in the EGFR gene lead to constitutive ligand-independent stimulation of the tyrosine kinase activity (24). To determine how silibinin inhibits the activity of EGFR family in cells with EGFR mutations, the affinity of silibinin to the ATP-binding pocket of the EGFR tyrosine kinase domain was measured by using a direct binding assay (3).…”

A Multiscale Computational Approach to Dissect Early Events in the Erb Family Receptor Mediated Activation, Differential Signaling, and Relevance to Oncogenic Transformations

“…Experimental observation of constitutive activation in the gefitinib-sensitive EGFR mutants has been recorded independently in several studies, which report significantly elevated basal phosphorylation (in the absence of the stimulating ligand) of the mutant systems in comparison to the wildtype. 10,11,48,61 Ligand and Substrate Binding Affinities for EGFRTK We determine the ATP and erlotinib binding modes and binding affinities based on our multiple conformations docking strategy (Section S1.2) and analyze several low-energy clusters of ligand conformations. For both wildtype and L834R mutant EGFRTK systems, one lead erlotinib binding conformation is found to be closely aligned with that in the crystal structure: the hydrogen bonds between the N1 of the quinazoline and Met769 are present and so is the interaction between Thr766 and the quinazoline nitrogen atom (N3).…”