Loss of activating EGFR mutant gene contributes to acquired resistance to EGFR tyrosine kinase inhibitors in lung cancer cells.

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance

Comparison of sensitivity to various drugs between erlotinib- and gefitinib- resistant cell lines and their drug sensitive parental counterparts, PC9 and 11–18 cells.

a)<p>IC50 values are calulated from logit regression line from triplicate assays. IC50 values (µM) for erlotinib, gefitinib, lapatinib, SU11274, BIBW2992 and cisplatin are 0.04, 0.03, 3.16, 3.97, 0.00021 and 2.07 in PC9, 9.96, 5.04, 14.54, 3.97, 0.41 and 2.89 in PC9/ER1, 0.87, 0.32, 2.57, 4.22, 0.35 and 5.37 in 11–18, 20.88, 8.32, 2.83, 4.64, 0.49 and 1.61 in 11–18/ER1-7, 95.7, 20.48, 4.11, 5.06, 0.74 and 9.13 in 11–18/ER2-1, >29.58, 11.52, 13.36, 3.38 and 2.41 in 11–18/GEF10-1, and >29.58, 10.24, 14.14, 3.80 and 1.61 in 11–18/GEF20-1 respectively. The relative resistance is defined as the IC50 value divided by the IC50 value of the parental PC9 or 11–18.</p>b)<p>nt, not tested.</p

The effect of erlotinib or knockdown of EGFR, HER2, or HER3 by their siRNAs on PC9/ER1 cells.

<p>A, Exponentially growing PC9 and PC9/ER1 cells were exposed to various doses of erlotinib for 5 h, and followed by western blot analysis. B, C, D, PC9 and PC9/ER1 cells were treated for 48 hr with 10 nM scramble (sc) siRNA, 2 nM or 10 nM EGFR siRNA, HER2 siRNA or HER3 siRNA respectively, and followed by Western blot analysis.</p

Immunostaining images for EGFR expression in both histological and cytological samples in human NSCLC.

<p>Total EGFR antibody stained all four histological and cytological samples. Anti-delE746-A750 antibody stained only cancer cells with the delE746-A750 mutation (A, case 1) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041017#pone-0041017-t002" target="_blank">Table 2</a>), and the EGFR L858R antibody stained only cancer cells with the L858R mutations (B, case 9) in both histological and cytological samples. No staining was evident by both EGFR delE746-A750 and EGFR L858R antibodies in cancer cells without the activating EGFR mutations in cytological samples (C: case 6 and D: case 11).</p

The effect of erlotinib, lapatinib and BIBW2992 on phosphorylation of Akt and EGFR family proteins in PC9/ER1 cells.

<p>A, PC9/ER1 cells were treated with or without 1 µM erlotinib, and 5 µM lapatinib for 5 hrs, and followed Western blot analysis. B, PC9/ER1 cells were treated with 10 nM of siRNAs of scrumble and EGFR family genes, and exposed to erlotinib (1 µM) or BIBW2992 (1 µM) for 5 hrs, and followed Western blot analysis.</p

Summary of EGFR mutation status in cell samples of refractory cancer patients^a).

a)<p>EGFR mutation status including wild-type (WT), E746-A750 del (del), L858R and T790M was determined by both IHC and RNA-LNA PCR clamp assays with 11 clinical samples of cancer patients refractory to gefitinib treatment.</p>b)<p>EGFR mutation status determined by IHC analysis is presented by scoring (0, 1+, 2+, 3+) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041017#pone-0041017-g008" target="_blank">Figure 8</a>) of immunostaining intensity in cancer cells in primary tumor and disseminated samples of 11 patients (Primary tumor sample/Disseminated and/or metastatic sample). nd, not determined.</p

Our hypothetic model how drug resistance to erlotinib is acquired in lung cancer cells harboring activated mutant EGFR (mEGFR).

<p>Cell proliferation and survival of human lung cancer cells harboring activated mutant EGFR (PC9 and 11–18 cells) closely depend upon EGFR-driven PI3K/Akt pathway, and this proliferation/survival is highly susceptible to erlotinib and other EGFR TKIs. First, there is partial or complete loss of mEGFR gene allele in drug-resistant cell lines, and then gain of addiction to HER2/HER3 and PI3K/Akt signaling (PC9/ER1 cells). However, more definitive analysis on resistant cell lines of 11–18 is required because 11–18 resistant cell lines show only partial loss of mEGFR.</p

Comparison of protein expression of EGFR family proteins and the down-stream molecules in erlotinib-resistant cell lines in the absence or presence of erlotinib.

<p>A, Western blot analysis showing the expression of pEGFR, EGFR, pHER2, HER2, pHER3, HER3, pc-Met, c-Met, PTEN, pAkt, Akt, pERK1/2, and ERK1/2 proteins, and α-tubulin as a loading control. B, Exponentially growing PC9 and PC9/ER1 cells were exposed to various doses of erlotinib for 5 hr, and followed by Western blot analysis. C, Exponentially growing 11–18, 11–18/ER1-7, and 11–18/ER2-1 cells were exposed to various doses of erlotinib for 5 hr, and followed by Western blot analysis.</p