EGFR-T790M is a rare lung cancer susceptibility allele with enhanced kinase activity

Abstract

The use of tyrosine kinase inhibitors (TKI) has yielded great success in treatment of lung adenocarcinomas. However, patients who develop resistance to TKI treatment often acquire a somatic resistance mutation (T790M) located in the catalytic cleft of the epidermal growth factor receptor (EGFR) enzyme. Recently, a report describing EGFR-T790M as a germ-line mutation suggested that this mutation may be associated with inherited susceptibility to lung cancer. Contrary to previous reports, our analysis indicates that the T790M mutation confers increased Y992 and Y1068 phosphorylation levels. In a human bronchial epithelial cell line, overexpression of EGFR-T790M displayed a growth advantage over wild-type (WT) EGFR. We also screened 237 lung cancer family probands, in addition to 45 bronchoalveolar tumors, and found that none of them contained the EGFR-T790M mutation. Our observations show that EGFR-T790M provides a proliferative advantage with respect to WT EGFR and suggest that the enhanced kinase activity of this mutant is the basis for rare cases of inherited susceptibility to lung cancer.

Figure 1

Phosphorylation of Y1068 is enhanced in EGFR-T790M. HEK293 cells (A) and COS-7 cells (B) were transfected with the indicated DNAs and lysed 48 h after transfection. Lysates were immunoblotted with anti-EGFR and anti–EGFR-Y1068 as indicated. Phosphorylation of Y1068 is enhanced in the EGFR-T790M mutant. C, phosphorylation and activation of EGFR-T790M are evident at lower levels of protein expression compared with higher levels in COS-7 cells (lane 3 versus lane 5). D, gefitinib does not affect the phosphorylation status of T790M-EGFR. E, phosphorylation of MAPK is enhanced in EGFR-T790M–expressing (versus EGFR-WT expressing) cells. Cells were stimulated with EGF (10 ng/mL) for 5 min.

Figure 2

Colony formation growth assay for EGFR-T790M. Viral transduction and selection of HBEC cells stably expressing EGFR were done as described previously (11). A, cells (200) were plated in triplicate and grown in keratinocyte serum-free medium (with 50 μg/mL bovine pituitary extract and without EGF). After 10 d, cells were stained with methylene blue. B, stained cell colonies >3 mm in diameter were counted. *, P < 0.0001, two-tailed t test. C, Western blots for EGFR, EGFR-Y1068, EGFR-Y992, MAPK, MAPK (T202/Y204), AKT, AKT (S473), cyclin D1, and α-tubulin in the HBEC stable cell lines were done as indicated.

Figure 3

EGFR genotyping and linkage analysis for chromosome 7 with SimWalk2. A, the EGFR-T790M mutation was not observed in familial lung cancer blood DNA, BAC tumors and normals, or fresh-frozen resected tumors. B, the genetic position for EGFR is at ~73 cM (based on interpolation), between markers D7S1818 (70 cM) and D7S3046 (79 cM). Simwalk2 analysis was based on the parametric method using the genetic model reported by Moscatello et al. (18). Fifty-five pedigrees with five or more affected members were used. C and D, two representative pedigrees of GELCC collected families with predisposition to lung cancer. In (C) and (D), ●. (females) or ■ (males) represent lung, throat, or laryngeal cancer. Numbers below each individuals, sample numbers; numbers in brackets, individual ages at the time the pedigree was constructed; slashes, dead individuals.