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. 2013 Mar 25;2(3):e39.
doi: 10.1038/oncsis.2013.4.

A mechanism of resistance to gefitinib mediated by cellular reprogramming and the acquisition of an FGF2-FGFR1 autocrine growth loop

K E Ware  1 , T K Hinz, E Kleczko, K R Singleton, L A Marek, B A Helfrich, C T Cummings, D K Graham, D Astling, A-C Tan, L E Heasley
Affiliations

A mechanism of resistance to gefitinib mediated by cellular reprogramming and the acquisition of an FGF2-FGFR1 autocrine growth loop

K E Ware et al. Oncogenesis. .

Abstract

Despite initial and often dramatic responses of epidermal growth factor receptor (EGFR)-addicted lung tumors to the EGFR-specific tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, nearly all develop resistance and relapse. To explore novel mechanisms mediating acquired resistance, we employed non-small-cell lung cancer (NSCLC) cell lines bearing activating mutations in EGFR and rendered them resistant to EGFR-specific TKIs through chronic adaptation in tissue culture. In addition to previously observed resistance mechanisms including EGFR-T790M 'gate-keeper' mutations and MET amplification, a subset of the seven chronically adapted NSCLC cell lines including HCC4006, HCC2279 and H1650 cells exhibited marked induction of fibroblast growth factor (FGF) 2 and FGF receptor 1 (FGFR1) mRNA and protein. Also, adaptation to EGFR-specific TKIs was accompanied by an epithelial to mesenchymal transition (EMT) as assessed by changes in CDH1, VIM, ZEB1 and ZEB2 expression and altered growth properties in Matrigel. In adapted cell lines exhibiting increased FGF2 and FGFR1 expression, measures of growth and signaling, but not EMT, were blocked by FGFR-specific TKIs, an FGF-ligand trap and FGFR1 silencing with RNAi. In parental HCC4006 cells, cell growth was strongly inhibited by gefitinib, although drug-resistant clones progress within 10 days. Combined treatment with gefitinib and AZD4547, an FGFR-specific TKI, prevented the outgrowth of drug-resistant clones. Thus, induction of FGF2 and FGFR1 following chronic adaptation to EGFR-specific TKIs provides a novel autocrine receptor tyrosine kinase-driven bypass pathway in a subset of lung cancer cell lines that are initially sensitive to EGFR-specific TKIs. The findings support FGFR-specific TKIs as potentially valuable additions to existing targeted therapeutic strategies with EGFR-specific TKIs to prevent or delay acquired resistance in EGFR-driven NSCLC.

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Figures

Figure 1
Figure 1
FGFR1 and FGF2 are induced following adaption of EGFR-dependent NSCLC cell lines to EGFR-specific TKIs. (a, b) Total RNA was purified from the indicated cell lines following chronic adaption to a reversible EGFR inhibitor (gefitinib 3 μℳ) or irreversible EGFR inhibitor (BIBW2992 2 μℳ) that inhibits EGFR-T790M and submitted to quantitative RT–PCR analysis of (a) FGFR1 or (b) FGF2. The data are presented as relative expression following normalization for GAPDH expression. Statistical analysis by t-test revealed significant increases in mRNA expression where *indicates P<0.05, **indicates P<0.01 and ***indicates P<0.001. (c) Cell lysates from the indicated NSCLC cell lines resistant to 3 μℳ gefitinib or 2 μℳ BIBW2292 (R) or DMSO-treated passage control (d) were immunoblotted for FGFR1, phospho-EGFR-Y1046, EGFR and the α subunit of the NaK-ATPase as a loading control. (d) The cell extracts from panel C were submitted to measurement of FGF2 protein by ELISA using a commercially available kit (R&D Systems, Minneapolis, MN, USA). The levels of FGF2 are presented as picograms per μg of cellular protein.
Figure 2
Figure 2
Effect of TKIs on ERK and AKT phosphorylation in control and gefitinib-resistant HCC4006 and H1650 cells. (a, b) HCC4006 and H1650 control (DMSO) or gefitinib-resistant cells were treated for 2 h with inhibitors: Reversible EGFR inhibitor (lapatinib/gefitinib, 1 μℳ), irreversible EGFR inhibitor (BIBW2992, 0.1 μℳ), FGFR inhibitors (AZD8010, 0.3 μℳ, RO4383596, 1 μℳ) or Met inhibitor (crizotinib, 0.2 μℳ). Cell lysates were immunoblotted for phospho-ERK and phospho-AKT-T308 and phospho-AKT-S473 as indicated. The filters were stripped and reprobed for total ERK1/2 and AKT as loading controls. The phospho-AKT-T308 blot was submitted to densitometry and a graphical presentation is shown in Supplementary Figure S4.
Figure 3
Figure 3
Gefitinib-resistant HCC4006, H1650 and HCC2279 cell cultures are dependent on FGFR signaling for cell growth. (a) HCC4006 control and gefitinib-resistant cells were analyzed for anchorage-independent growth. Cells were treated with increasing concentrations of the EGFR reversible inhibitor, lapatinib (0, 0.03, 0.05, 0.1, 0.3, 1 μℳ), the irreversible EGFR inhibitor, BIBW2992 (0, 0.03, 0.05, 0.1, 0.3, 0.5 μℳ), and the FGFR inhibitor AZ8010 (0, 0.03, 0.05, 0.1, 0.3, 0.5 μℳ). (b) HCC4006 control and gefitinib-resistant cells were analyzed for anchorage-independent growth with 0.1 μg/ml FGF-ligand trap (FP-1039). (c) HCC2279 control (DMSO) and gefitinib-resistant cells were analyzed for cell proliferation by cell counts after 7 days of treatment with DMSO or inhibitors: (1 μℳ, lapatinib, 0.1 μℳ BIBW2992 or 0.3 μℳ AZ8010. D) H1650 control (DMSO) and gefitinib-resistant cells were analyzed for proliferation as above except with the addition of EGFR inhibitor AG1478 (0.1 μℳ). Statistical analysis by two-way ANOVA revealed significant decreases in cell growth where *indicates P<0.05, **indicates P<0.005 and ***indicates P<0.001.
Figure 4
Figure 4
Induction of EMT in EGFR TKI-resistant NSCLC cell lines is associated with induction of FGFR1. (a) Cell lysates from the indicated NSCLC cell lines resistant to 3 μℳ gefitinib or 2 μℳ BIBW2292 (R) or DMSO-treated passage control (d) were immunoblotted for E-cadherin, vimentin and the α subunit of the NaK-ATPase as a loading control. (b) HCC4006 DMS0 and gefitinib-resistant cells were plated in media containing 2% Matrigel and allowed to form colonies over 7 days. Colonies were imaged at × 100 magnification using a Nikon Eclipse TS100 camera. (c) HCC4006 control and gefitinib-resistant cells were treated 1, 3 or 6 days with DMSO or 0.3 μℳ AZ8010. Cell lysates were immunoblotted for E-cadherin and the α-subunit of NaK-ATPase as a loading control.
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