Implication of epithelial-mesenchymal transition in IGF1R-induced resistance to EGFR-TKIs in advanced non-small cell lung cancer

Abstract

The underlying mechanisms for acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in about 30%-40% of non-small cell lung cancer (NSCLC) patients remain elusive. Recent studies have suggested that activation of epithelial-mesenchymal transition (EMT) and type 1 insulin-like growth factor receptor (IGF1R) is associated with acquired EGFR-TKIs resistance in NSCLC. Our study aims to further explore the mechanism of EMT and IGF1R in acquired EGFR-TKIs resistance in NSCLC cell lines with mutant (PC-9) or wild-type EGFR (H460). Compared to parental cells, EGFR-TKIs-resistant PC-9/GR and H460/ER cells displayed an EMT phenotype and showed overexpression of IGF1R. SiIGF1R in PC-9/GR and H460/ER cells reversed EMT-related morphologies and reversed their resistance to EGFR-TKIs. Exogenous IGF-1 alone induced EMT in EGFR-TKIs-naïve PC-9 and H460 cells and increased their resistance against EGFR-TKIs. Inducing EMT by TGF-β1 in PC-9 and H460 cells decreased their sensitivity to EGFR-TKIs, whereas reversing EMT by E-cadherin overexpression in PC-9/GR and H460/ER cells restored their sensitivity to EGFR-TKIs. These data suggest that IGF1R plays an important role in acquired drug resistance against EGFR-TKIs by inducing EMT. Targeting IGF1R and EMT may be a potential therapeutic strategy for advanced NSCLC with acquired EGFR-TKIs resistance.

Keywords: drug resistance; epidermal growth factor receptor-tyrosine kinase inhibitors; epithelial-mesenchymal transition; non-small cell lung cancer; type 1 insulin-like growth factor receptor.

Figure 1. Role of IGF1R on the sensitivity to gefitinib and erlotinib in EGFR-TKIs-resistant cells

A. The sensitivity to gefitinib and erlotinib of PC-9/GR, H460/ER, and their parental cells was assessed by MTT assays. Cells were treated with the indicated doses of gefitinib or erlotinib for 72 h. IC50 values for different conditions are provided in the table within individual figures. B. Expression of IGF1R, phosphor-IGF1R, EGFR, and phosphor-EGFR in EGFR-TKIs-resistant cells by immunoblotting analysis. C. Effect of IGF1R siRNA on expression of IGF1R, phosphor-IGF1R, EGFR, and phosphor-EGFR in EGFR-TKIs-resistant cells. β-actin was used as an internal control. D. IC50 of gefitinib/erlotinib in PC-9/GR and H460/ER cells increased significantly following IGF1R knock-down when compared with the control cells. Data represent means ± S.D. of three independent experiments.

Figure 2. EMT in EGFR-TKIs-resistant cells

A. Morphology of PC-9/GR, H460/ER, and their parental cells grown for 3 days until 90% confluence. In contrast to the parental cells, the PC-9/GR and H460/ER cells displayed long spindle-like shape with loose cell junctions. Photographs were taken at × 200 magnification. B–C. Loss of E-cadherin was seen in PC-9/GR cells, H460 cells did not express E-cadherin, and there was increased expression of Vimentin, transcription factor Snail and nuclear β-catenin in PC-9/GR and H460/ER cells shown by immunoblotting analysis. In addition, Vimentin expression at the protein level increased in a time dependent manner after the induction of drug resistance. β-actin was used as an internal control. D, E. Enhanced migratory capacity of EGFR-TKIs-resistant cells according to Scratch assay. Confluent cells were scraped by a pipette tip to generate wounds and then were cultured in serum-free culture medium for 48 h. Representative images of wounds were taken at 0 and 48 h. Cell motility was examined with a light microscope (×40) and the width of the wound was quantified. F, G. Enhanced invasiveness of EGFR-TKIs-resistant cells according to transwell assay. The cells were incubated for 24 h in modified Boyden chambers. Those cells that migrated through the filters were stained and counted under a light microscope. Quantification was done in 10 randomly chosen fields. The data are reported as means ± S.D. The photographs were taken at × 200 magnification.

Figure 3. Effect of TGF-β1 on EMT and the sensitivity to gefitinib and erlotinib in EGFR-TKIs-resistant cells

A. Morphology of PC-9 and H460 cells grown with 10 ng/mL TGF-β1 for 3 days until 90% confluence. The photographs were taken at × 200 magnification. B. TGF-β1-induced downregulation of E-cadherin and upregulation of Vimentin in PC-9 and H460 cells according to immunoblotting analysis. β-actin was used as an internal control. C. The effects of sequential treatment with the TGF-β1 on cell viability of PC-9 and H460 cells exposed to gefitinib and erlotinib by MTT uptake assays, respectively. The data represent the means ± S.D. of three independent experiments.

Figure 4. Effect of E-cadherin (CDH1) overexpression on EMT and the sensitivity to gefitinib and erlotinib in EGFR-TKIs-resistant cells

A. E-cadherin-overexpressing cell lines PC-9/GR and H460/ER after CDH1 transfection showed an epithelial-like morphology and a remarkably increased expression of E-cadherin according to immunofluorescence assay. The nuclei were stained with DAPI (blue fluorescence), and E-cadherin was stained with Cy3-conjugated antibodies (red fluorescence). B. Effect of E-cadherin overexpression on expression levels of EMT markers in EGFR-TKIs resistant cells. β-actin was used as an internal control. C. The IC50 of gefitinib/erlotinib in E-cadherin-overexpressing PC-9/GR-CDH1 and H460/ER-CDH1 cells was significantly greater than that in PC-9/GR-control and H460/ER-control cells. The data represent the means ± S.D. of three independent experiments. D, E. E-cadherin overexpression repressed migration of EGFR-TKIs-resistant cells according to Scratch assay. F, G. E-cadherin overexpression suppressed invasion of EGFR-TKIs-resistant cells according to transwell assay.

Figure 5. IGF1R activation led to EMT and decreased sensitivity against EGFR-TKIs in PC-9 and H460 cells upon IGF-1 induction

EGFR-TKIs-naïve lung cancer cells PC-9 and H460 were serum-starved overnight and then treated with fresh RPMI 1640 containing 0.5% FBS and 200 ng/ml IGF-I for 24 h. A. After IGF-1 induction, IGF1R and pIGF1R were activated. EMT phenotype, decreased expression of E-cadherin, increased Vimentin, nuclear β-catenin and Snail were observed. β-actin was used as an internal control. B. Mesenchymal phenotype of PC-9 and H460 cells after IGF-1 induction. C. β-catenin relocated from cell membrane to nucleus after IGF-1 induction as shown by immunofluorescence experiment. The photographs were taken at × 200 magnification. D. Exogenous IGF-1 application increased resistance to EGFR-TKIs in PC-9 and H460 cells. Data represent means ± S.D.of three independent experiments.

Figure 6. Effects of IGF1R on EMT and ERK/MAPK signaling of EGFR-TKIs-resistant cells

A, B. Morphologic change of PC-9/GR and H460/ER after IGF1R knockdown. Green fluorescence represented the successful transfection of IGF1R siRNA. C. Upregulation of E-cadherin. and downregulation of Vimentin. nuclear β-catenin and Snail by IGF1R silencing. D, E. Attenuated ERK/MAPK signaling in PC-9 and H460 after IGF1R activation by IGF-1 and IGF1R knockdown. β-actin was used as an internal control.* p < 0.05