Sensitivity of Cell Transformation Induced by Expression of Mutant <i>EGFR</i> Characterized by Missense Mutation or Exon 19 Deletion, but not Exon 20 Insertion, to Gefitinib and Erlotinib

<div><p>(A) Anchorage-independent growth of clonal NIH-3T3 cells transformed with mutant <i>EGFR</i> or EGF-stimulated wild-type <i>EGFR</i> treated with the indicated concentrations of erlotinib immediately prior to suspension in soft agar. Transformation induced by expression of L858R, G719S, and L747_E749del A750P <i>EGFR,</i> but not EGF-stimulated wild-type <i>EGFR</i> or D770_N771insNPG <i>EGFR,</i> was inhibited by 0.1 μM erlotinib. Representative photographs are shown.</p> <p>(B) Number of colonies formed in soft agar by clonal NIH-3T3 cells expressing L858R <i>EGFR</i> and D770_N771insNPG <i>EGFR</i> treated with the indicated concentrations of gefitinib or erlotinib immediately prior to suspension in soft agar. Transformation by cells expressing the L858R <i>EGFR</i> was inhibited by 0.1 μM gefitinib or erlotinib, whereas transformation by cells expressing the insertion mutant was resistant to low concentrations of these inhibitors. Colonies were quantitated by counting ten fields each of triplicate wells photographed with a 10× objective; mean ± standard deviation is shown. Ins, D770_N771insNPG <i>EGFR</i>.</p> <p>(C) Transformation induced by expression of D770_N771insNPG <i>EGFR</i> is inhibited 10-fold more efficiently by the irreversible EGFR inhibitor CL-387,785 [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0020313#pmed-0020313-b35" target="_blank">35</a>]. Clonal NIH-3T3 cells expressing the insertion mutant were treated with the indicated concentrations of gefitinib, erlotinib, or CL-387,785 immediately prior to suspension in soft agar. This assay was not done in triplicate, but the results are representative of two independent experiments. The number of colonies was normalized to maximum colony formation for each treatment, and sigmoidal dose response curves were fitted to the data using Prism Graphpad software to determine IC50s.</p></div

Ligand-Independent Activation of the Mutant EGFR

<div><p>(A) Cells expressing the wild-type or mutant <i>EGFR</i> were lysed and immunoblotted with antibodies to total EGFR or antibodies that recognize specific phosphorylation sites in the EGFR C-terminal tail as labeled. All four mutant EGFR proteins, representative of the four classes of <i>EGFR</i> mutations observed in lung adenocarcinoma tumor DNA, exhibited constitutive phosphorylation on the indicated C-terminal autophosphorylation sites. Note that the nomenclature for the anti-phospho-EGFR antibodies reflects elimination of the 24-amino acid signal peptide. Due to difficulties in isolating clonal cell lines with the same levels of mutant EGFR expression, G719S is expressed at higher levels and D770_N771ins NPG at lower levels than the other mutant EGFR. del, L747_E749del A750P; ins, D770_N771insNPG; pBp, pBabe-Puro vector control; wt, wild-type EGFR.</p> <p>(B) Cells expressing the wild-type or L858R <i>EGFR</i> were placed in media containing 0.5% CS for 24 h. A combination of three neutralizing antibodies (anti-EGF, anti-TGFα, and anti-EGFR) was added 3 h prior to EGF stimulation and lysis. Upper row of blots show the anti-phospho-EGFR Y1068 immunoblots. The lower row shows anti-EGFR immunoblots. No inhibition of L858R EGFR autophosphorylation was observed upon treatment with a combination of three neutralizing antibodies (“neutr Ab”) sufficient to prevent EGF stimulation of autophosphorylation of the wild-type EGFR.</p></div

Sensitivity of Mutant EGFR Autophosphorylation to EGFR Inhibitors Reflects Inhibition of Anchorage-Independent Growth

<p>Cells expressing wild-type, L858R, or D770_N771insNPG <i>EGFR</i> were treated for 2 h with the indicated concentrations of gefitinib or CL-387,785. Cells expressing the wild-type EGFR were then stimulated for 10 min with 7 ng/ml EGF, and all plates were lysed. Whole-cell lysates were immunoblotted for phospho-EGFR Y1068 (upper row of blots), total EGFR (middle row), and actin as a loading control (lower row). Although compound concentrations necessary for inhibition of autophosphorylation do not exactly correspond to inhibition of anchorage-independent growth, the relative sensitivity of autophosphorylation of the wild-type and mutant EGFR to gefitinib or CL-387,785 mirrors the relative sensitivity of colony formation to these inhibitors.</p

Mammalian Cells Expressing the Lung Cancer-Derived Mutant EGFR Grow in an Anchorage-Independent Manner

<div><p>(A) NIH-3T3 cells infected with retroviruses encoding the indicated wild-type or mutant <i>EGFR</i> were selected in the presence of 2.5 μg /ml puromycin for 4 d. In the top photomicrographs, 1 × 10⁵ cells were suspended in soft agar for a colony formation assay and photographed after 3 wk incubation at 37 °C. Expression of lung cancer-derived missense <i>EGFR</i> mutants, but not wild-type or kinase-inactive D837A <i>EGFR,</i> induces colony formation in soft agar. In the bottom photomicrographs, samples were identical, but 20 ng/ml EGF was added to the top agar. Representative photomicrographs are shown.</p> <p>(B) Anti-EGFR immunoblot analysis of pooled stable NIH-3T3 cells infected as described in (A). All <i>EGFR</i> constructs are expressed at similar levels. pBp, pBabe-Puro vector; wt, wild-type EGFR.</p> <p>(C) Lysates from 4 × 10⁴ cells from the human lung adenocarcinoma cell line H3255, harboring the L858R mutation in <i>EGFR,</i> or the wild-type or L858R <i>EGFR</i>-overexpressing NIH-3T3 cells, were immunoblotted for total <i>EGFR</i> levels. Although total protein levels per cell are lower for the H3255 than the NIH-3T3 cells, EGFR expression levels are slightly higher in the H3255s.</p> <p>(D) NIH-3T3 cells infected with retroviruses encoding the mutant <i>EGFR</i> were selected in the presence of 2 μg/ml puromycin for 9 d. Selected cells (1 × 10⁵) were suspended in soft agar for a colony formation assay and photographed after 3 wk incubation at 37 °C. Expression of the deletion and insertion <i>EGFR</i> mutants induced formation of colonies in soft agar with higher efficiency than expression of L858R. Representative photos are shown. Polyoma mT, NIH-3T3 cells infected with positive control pBabe-Puro retrovirus encoding the polyoma middle T antigen.</p> <p>(E) hTBE cells expressing the SV40 early region and hTERT were infected with control virus pBabe-Puro (pBp) or with viruses encoding the indicated <i>EGFR</i> alleles. Cells were plated in 0.4% Noble agar, and colonies were counted with an automated imager at 6 wk. Mean ± standard deviation is shown for three independent determinations. Control cells (pBp) formed many microscopic colonies, but colonies formed by cells expressing <i>EGFR</i> mutants were more numerous and larger. del, L747_E749del A750P mutated EGFR; ins, D770_N771insNPG mutated EGFR; pBp, pBabe-Puro vector; RasV12, V12 H-Ras; wt, wild-type EGFR.</p> <p>(F) Anti-EGFR immunoblot analysis of hTBE cells infected as described in (E). All <i>EGFR</i> constructs were expressed at similar levels. pBp, pBabe-Puro vector; wt, wild-type EGFR.</p></div

EGFR Missense Mutations Are Transforming and Tumorigenic

<div><p>(A) Anchorage-independent growth of NIH-3T3 cells expressing various <i>EGFR</i> alleles as mean number of colonies ± standard deviation (bar graph, above). The lanes (below) show EGFR and actin immunoblots of whole cell lysates from NIH-3T3 subclones plated in soft agar. EGF (10 ng/ml) was added to the top agar where indicated.</p> <p>(B) Tumorigenicity of NIH-3T3 cells stably expressing the indicated <i>EGFR</i> alleles in nude mice. Mean tumor size ± standard deviation was determined 3–4 wk after subcutaneous inoculation into nude mice (<i>n</i> = 6 per cell line).</p></div

<i>EGFR</i> Missense Mutations Sensitize Cells to EGFR Kinase Inhibitors

<div><p>(A) Effect of increasing concentrations of the EGFR inhibitor erlotinib (0–10 μM) on the viability of IL-3 independent Ba/F3 subclones expressing EGFR ectodomain mutants (R108K, T263P, A289V, G598V, and EGFRvIII), the EGFR kinase domain mutants (L858R and L861Q), or the erlotinib-resistant EGFR double mutant L858R-T790M (LTM). Parental Ba/F3 cells and Ba/F3 cells expressing wild-type EGFR are not IL-3 independent and were included as controls. Viability (a mean percent of control ± standard deviation) was determined after exposure to erlotinib for 48 h.</p> <p>(B) Oncogenic EGFR ectodomain mutations map to interdomain interfaces. Shown are ribbon and surface diagrams of the EGFR [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030485#pmed-0030485-b046" target="_blank">46</a>] with sites of amino acid substitutions highlighted. Blue, domain I; green, domain II; red, domain III; and yellow, domain IV. Sites of the most prevalent amino acid substitutions are shown in red. Images were created with PyMOL (<a href="http://pymol.sourceforge.net/" target="_blank">http://pymol.sourceforge.net/</a>). P596 is not visible in this view.</p></div

EGFR Missense Mutations in Glioblastoma Cluster in the Extracellular Domain and Are Associated with Increased <i>EGFR</i> Gene Dose

<div><p>(A) Location of missense mutations within the EGFR protein in a panel of 151 gliomas (132 glioblastomas, 11 WHO grade III gliomas, and eight glioblastoma cell lines). Each diamond represents one sample harboring the indicated mutation. Amino acid (AA) numbers are based on the new convention for EGFR numbering, which starts at the initiator methionine of pro-EGFR. Ligand-binding domains (I and III), cysteine-rich domains (II and IV), kinase domain (kinase), and the extracellular deletion mutant EGFRvIII [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030485#pmed-0030485-b045" target="_blank">45</a>] are indicated as reference.</p> <p>(B) Increased <i>EGFR</i> gene dose in tumors harboring <i>EGFR</i> missense mutations. The array (left) shows a high-resolution view of Affymetrix 100K SNP array at the <i>EGFR</i> gene locus for ten glioblastoma tumors and three normal controls (sample numbers are indicated above each column). <i>EGFR</i> mutation and log₂ ratio (see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030485#st2" target="_blank">Methods</a>) are indicated below each column. The plot (left) shows a comparison of <i>EGFR</i> gene copy number determination by SNP array (y-axis, EGFR log₂ ratios) and FISH (x-axis). AMP, amplified; NON-AMP, non amplified.</p> <p>(C) RT-PCR for <i>EGFRvIII</i> and full-length <i>EGFR</i> in 14 fresh-frozen glioblastoma tumors (see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030485#st2" target="_blank">Methods</a>). The upper band represents full-length <i>EGFR</i> (1,044 bp), the lower band <i>EGFRvIII</i> (243 bp), and the inset shows glyceraldehyde-3-phosphate dehydrogenase <i>(GAPDH)</i> RT-PCR results.</p></div