Rapidly acquired resistance to EGFR tyrosine kinase inhibitors in NSCLC cell lines through de-repression of FGFR2 and FGFR3 expression

Abstract

Despite initial and sometimes dramatic responses of specific NSCLC tumors to EGFR TKIs, nearly all will develop resistance and relapse. Gene expression analysis of NSCLC cell lines treated with the EGFR TKI, gefitinib, revealed increased levels of FGFR2 and FGFR3 mRNA. Analysis of gefitinib action on a larger panel of NSCLC cell lines verified that FGFR2 and FGFR3 expression is increased at the mRNA and protein level in NSCLC cell lines in which the EGFR is dominant for growth signaling, but not in cell lines where EGFR signaling is absent. A luciferase reporter containing 2.5 kilobases of fgfr2 5' flanking sequence was activated after gefitinib treatment, indicating transcriptional regulation as a contributing mechanism controlling increased FGFR2 expression. Induction of FGFR2 and FGFR3 protein as well as fgfr2-luc activity was also observed with Erbitux, an EGFR-specific monoclonal antibody. Moreover, inhibitors of c-Src and MEK stimulated fgfr2-luc activity to a similar degree as gefitinib, suggesting that these pathways may mediate EGFR-dependent repression of FGFR2 and FGFR3. Importantly, our studies demonstrate that EGFR TKI-induced FGFR2 and FGFR3 are capable of mediating FGF2 and FGF7 stimulated ERK activation as well as FGF-stimulated transformed growth in the setting of EGFR TKIs. In conclusion, this study highlights EGFR TKI-induced FGFR2 and FGFR3 signaling as a novel and rapid mechanism of acquired resistance to EGFR TKIs and suggests that treatment of NSCLC patients with combinations of EGFR and FGFR specific TKIs may be a strategy to enhance efficacy of single EGFR inhibitors.

Figure 1. Induction of FGFR2 and FGFR3 mRNA and protein in EGFR inhibitor treated NSCLC cells.

A. Quantitative RT-PCR assay for FGFR2 and FGFR3 mRNAs. Total RNA was purified from the indicated cell lines following a 3-day treatment with 1 µM gefitinib and submitted to quantitative RT-PCR analysis of FGFR2, FGFR3 and GAPDH. (Cell lines with EGFR autocrine signaling or gain-of-function EGFR mutations: open bars; cell lines lacking EGFR signaling: grey bars) The data are presented as fold expression over DMSO treated cells following normalization for GAPDH mRNA. B–C. Cell lysates from the indicated NSCLC cell lines treated 3 days with or without 1 µM gefitinib or 2 µg/mL Erbitux were immunoblotted for FGFR2, FGFR3, EGFR and the α-subunit of the NaK-ATPase as a loading control. Densitometry of FGFR2, FGFR3 and EGFR expression was normalized relative to NaK-ATPase expression and is indicated under each immunoblot.

Figure 2. Transcriptional regulation of FGFR2 following EGFR inhibitor treatment.

A. H322c, H1650 or H520 cells transfected with pGL3-basic empty control vector or FGFR2-luc reporter (see Material and Methods) and TK-renilla to estimate transfection efficiency were treated with 1 µM gefitinib for 48 hrs. Cells were then lysed and luciferase activity measured and normalized to renilla activity. The data are the mean and SEM of 3 independent experiments. B. H322c cells transfected as above were treated with gefitinib (0.5 µM) or 2 µg/mL Erbitux for 48 hrs. The data are the mean and SEM of 4 independent experiments. C. FGFR2-luc transfected H322c cells were treated with the indicated inhibitors (5 µM PD98059, 1 µM saracatinib, 5 µM SB239063, or 10 µM LY294002) for 48 hrs. Luciferase expression was then measured as described above. Data are the mean and SEM of 3 independent experiments. D. H322c cells co-transfected with FGFR2-luc and empty LXSN, constitutively active MEK1 or constitutively active c-Src were treated for 48 hrs with 0.5 µM gefitinib. Luciferase expression was measured as described above. Data are the mean and SEM of 4 independent experiments. Statistical analysis by two-tailed t-test revealed significant increases in luciferase activity where ns indicates not significant, * indicates p<0.05, ** indicates p<0.005 and *** indicates p<0.001.

Figure 3. FGF-stimulated ERK and FRS2-α activation following EGFR TKI treatment.

A. NSCLC cell lines were incubated with the EGFR inhibitor, AG1478 (0.1 µM) or DMSO, for 3 days in full media. Cells were switched to HITES for 2 hrs and subsequently incubated with the FGFR inhibitor, RO4383596 (1 µM) or DMSO for 1 hour followed by FGF2 or FGF7 stimulation at 10 ng/mL for 15 minutes. Extracts were prepared, resolved on SDS-PAGE and immunoblotted for phospho-ERK. The filters were subsequently stripped and reprobed for total ERK1 and ERK2 to verify equal loading. Densitometry of phospho-ERK2 relative to total ERK2 for the designated experiment is indicated. The mean and SEM of replicate experiments is indicated in the text of the Results section. B. NSCLC cell lines were incubated with the EGFR inhibitor, gefitinib (1 µM), gefitinib in combination with FGFR inhibitor AZ12908010, or DMSO for 3 days in full media. Cells were then stimulated with PBS or FGF2 at 10 ng/mL for 15 minutes. Extracts were prepared, resolved on SDS-PAGE and immunoblotted for phopho-FRS2. The filters were subsequently stripped and reprobed for NaK-ATPase α-subunit to verify equal loading. Densitometry of phospho-FRS2 relative to NaK-ATPase is indicated under each immunoblot.

Figure 4. FGF2 and FGF7 rescue EGFR TKI dependent growth inhibition.

A. H322c and H1650 cells were analyzed for anchorage-independent growth as described in the Materials and Methods. B. HCC4006 cells were seeded at 100 cells per 35 mm dish in full media containing the indicated treatments and cultured for 2 weeks. Colony area was then quantified and shown as a percentage of control. The data are the mean and SEM of 3 independent experiments. Statistical analysis by 2way ANOVA revealed significant increases in growth where * indicates p<0.05, ** indicates p<0.005 and *** indicates p<0.001.

Figure 5. Co-culture with human fibroblasts prevents EGFR TKI dependent growth inhibition.

A. H322c cells were analyzed for anchorage-independent growth in the presence or absence of HGF-1 fibroblasts as described in the Materials and Methods. The data are the mean and SEM of 3 independent experiments. Statistical analysis by 2way ANOVA revealed significant increases in growth where * indicates p<0.05.