Table 1.
Comparison of sensitivity to various drugs between erlotinib- and gefitinib- resistant cell lines and their drug sensitive parental counterparts, PC9 and 11–18 cells.
Figure 1.
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.
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.
Figure 2.
Comparison of EGFR gene and protein expression between PC9 and PC9/ER1 cells.
A, Western blots showing expression of delE746-A750 EGFR and total EGFR in PC9 and PC9/ER1 cells. B, Detection of wild-type and mutation sequences using specific primers. PC9 cells show both wild-type and mutant-specific bands, while PC9/ER1, 11–18, and QG56 cells show only the wild-type-specific band. Mut, mutant exon19, and WT, wild-type exon19. C, PCR analysis shows heteroduplex (Mut/WT) and homoduplex (WT/WT and Mut/Mut) in PC9 cells, and only homoduplex (WT/WT) in PC9/ER1 cells, 11–18 cells harboring L858R mutation, and QG56 cells harboring wild-type EGFR.
Figure 3.
Comparison of EGFR gene and protein expression in 11–18, 11–18/ER1-7, and 11–18/ER2-1.
A, Western blot analysis using L858R-specific antibody and total EGFR antibody recognizing both wild-type and mutant EGFR. Expression levels of mutant EGFR (L858R), total EGFR, and L858R versus total EGFR (L858R/total EGFR) are normalized by their expression levels in 11–18 cells. Western blot analysis with primary human endothelial cell (HUVEC) shows expression of total EGFR, but not mutant L858R EGFR. B, DNA sequences reads of 15 bases around the L858R mutation in 11–18, 11–18/ER1-7, and 11–18/ER2-1 cells are compared. Arrows indicate base at 2573. Note that the nucleotide of 11–18 at 2573 was G/T heterozygote. C, PLACE-SSCP analysis of DNA samples of 11–18, 11–18/ER1-7, and 11–18/ER2-1 cells in the absence or presence of equal amounts of DNA from human vascular endothelial cells (HUVECs) are shown. Two peaks show wild-type (WT) and mutant (Mut) EGFR gene (a). Based on the equations shown in Materials and Methods, copy number changes of the wild-type and mutant EGFR genes are estimated (b).
Figure 4.
The effect of PI3K inhibitors and PIK3CA knockdown on PC9 and PC9/ER1 cells.
A, B, Exponentially growing PC9 and PC9/ER1 cells were exposed to various doses of wortmannin (A) and LY294002 (B) for 5 hr, and followed by Western blot analysis. C. PC9 and PC9/ER1 cells were treated with PIK3CA siRNA or scramble (SC) siRNA for 48 h, and followed by western blot analysis.
Figure 5.
The effect of erlotinib or knockdown of EGFR, HER2, or HER3 by their siRNAs on PC9/ER1 cells.
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.
Figure 6.
The effect of erlotinib, lapatinib and BIBW2992 on phosphorylation of Akt and EGFR family proteins in PC9/ER1 cells.
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.
Figure 7.
Restoration of sensitivity to erlotinib by expression of activating mutant EGFR in PC9/ER1 and 11–18/ER1-7.
A, PC9/ER1 cells were transfected with del EGFR (E746-A750) cDNA, and followed incubation for various days. Western blot analysis was performed with antibodies recognizing del EGFR and total EGFR. B, Dose response curves of mock- and activated mutant EGFR cDNA transfectants of PC9/ER1 were determined by cytotoxicity assay. Each value is average of triplicate dishes ± SD. C, 11–18/ER1-7 cells were transfected with L858R EGFR cDNA, and followed by Western blot analysis with a specific antibody of L858R EGFR. D, Dose response curves of mock- and activated mutant EGFR cDNA transfectants of 11–18/ER1-7. Each values is average of triplicate dishes ± SD.
Figure 8.
Immunostaining images for EGFR expression in both histological and cytological samples in human NSCLC.
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 Table 2), 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).
Table 2.
Summary of EGFR mutation status in cell samples of refractory cancer patientsa).
Figure 9.
Our hypothetic model how drug resistance to erlotinib is acquired in lung cancer cells harboring activated mutant EGFR (mEGFR).
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.