Fibroblast growth factor (FGF) and FGF receptor-mediated autocrine signaling in non-small-cell lung cancer cells

Abstract

Despite widespread expression of epidermal growth factor (EGF) receptors (EGFRs) and EGF family ligands in non-small-cell lung cancer (NSCLC), EGFR-specific tyrosine kinase inhibitors (TKIs) such as gefitinib exhibit limited activity in this cancer. We propose that autocrine growth signaling pathways distinct from EGFR are active in NSCLC cells. To this end, gene expression profiling revealed frequent coexpression of specific fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in NSCLC cell lines. It is noteworthy that FGF2 and FGF9 as well as FGFR1 IIIc and/or FGFR2 IIIc mRNA and protein are frequently coexpressed in NSCLC cell lines, especially those that are insensitive to gefitinib. Specific silencing of FGF2 reduced anchorage-independent growth of two independent NSCLC cell lines that secrete FGF2 and coexpress FGFR1 IIIc and/or FGFR2 IIIc. Moreover, a TKI [(+/-)-1-(anti-3-hydroxy-cyclopentyl)-3-(4-methoxy-phenyl)-7-phenylamino-3,4-dihydro-1H-pyrimido-[4,5-d]pyrimidin-2-one (RO4383596)] that targets FGFRs inhibited basal FRS2 and extracellular signal-regulated kinase phosphorylation, two measures of FGFR activity, as well as proliferation and anchorage-independent growth of NSCLC cell lines that coexpress FGF2 or FGF9 and FGFRs. By contrast, RO4383596 influenced neither signal transduction nor growth of NSCLC cell lines lacking FGF2, FGF9, FGFR1, or FGFR2 expression. Thus, FGF2, FGF9 and their respective high-affinity FGFRs comprise a growth factor autocrine loop that is active in a subset of gefitinib-insensitive NSCLC cell lines.

Fig. 1.

Expression of FGFs and FGFRs in NSCLC cell lines. Total RNA prepared from a panel of 33 NSCLC cell lines was reverse-transcribed and submitted to quantitative RT-PCR with primers (see Table 1) specific for FGF2, FGF7, or FGF9 (A) or FGFR1 or FGFR2 (B). The values are normalized to GAPDH mRNA measured in replicate samples and presented as relative expression. The horizontal bars denote the median expression among the 33 NSCLC cell lines.

Fig. 2.

Expression of FGF2, FGF7 and FGF9 mRNA and FGF2 secretion by NSCLC cell lines. A, quantitative RT-PCR assay for FGF2 and FGF9 mRNAs was performed on the indicated NSCLC cell lines and normalized for GAPDH mRNA levels. B, NSCLC cell lines were cultured for 2 days in serum-free HITES medium. FGF2 secreted into the conditioned medium was measured by ELISA (R&D Systems) and normalized to protein measured in the attached cells.

Fig. 3.

FGFR1 and FGFR2 protein expression in NSCLC cell lines. A, quantitative RT-PCR assay for FGFR1 and FGFR2 mRNAs was performed on the indicated NSCLC cell lines and normalized for GAPDH mRNA levels. B, membrane preparations from the indicated NSCLC cell lines were resolved by SDS-PAGE and immunoblotted for FGFR1 and FGFR2. The filters were stripped and reprobed for EGFR or the α-subunit of NaK-ATPase as a loading control.

Fig. 4.

Expression of FGFR1 and FGFR2 IIIb and IIIc splice variants in NSCLC cells. Total RNA from the indicated NSCLC cell lines was reverse-transcribed and submitted to PCR with published primers (Kwabi-Addo et al., 2001) flanking the cDNA sequences encoding the alternatively spliced third Ig-like extracellular domains from FGFR1 or FGFR2. The amplified FGFR1 PCR products were incubated with AflII (A) that digests the FGFR1 IIIc isoform or Bsm1 (B) that digests the FGFR1 IIIb isoform or incubated with neither enzyme (uc). Similar analysis was performed on the FGFR2 PCR products where Ava 1 (A) cuts the FGFR2 IIIb isoform and Hinc II (H) cuts the FGFR2 IIIc isoform; uc is uncut product. The reactions were submitted to electrophoresis on 1.5% agarose gels and stained with ethidium bromide.

Fig. 5.

ERK pathway activation by exogenous FGF2 or EGF. The indicated NSCLC cell lines were incubated for 2 h in serum-free HITES medium and then for another 15 min with or without 10 ng/ml EGF or 10 ng/ml FGF2. Cell extracts were prepared, resolved by SDS-PAGE, and immunoblotted for phospho-ERK1/ERK2 as a measure of EGFR and FGFR activation. The filters were stripped and re-probed with total ERK antibodies to verify equal sample loading.

Fig. 6.

FGF2 silencing inhibits ERK activity and anchorage-independent growth of H226 and H1703 cells. A, the FGF2 shRNA-encoding lentiviral vectors (TRCN0000003329 or TRCN0000003332) or the pLKO.1 vector encoding a shRNA targeting GFP as a control were packaged into lentiviruses, transduced into H226 or H1703 cells, and stable transfectants were selected with puromycin. Pooled puromycin-resistant cultures were seeded in 12-well plates and aliquots of media conditioned by the cells for three days were assayed for FGF2 by ELISA. The data are presented as picograms of FGF2 per milligram of cellular protein (mean ± S.E.M.), where GFP indicates cells transduced with the control GFP shRNA and 3329 and 3332 denote cultures transduced with the TRCN0000003329 or TRCN0000003332 constructs, respectively. B, basal ERK phosphorylation in the H226 and H1703 transfectants was measured by immunoblot analysis as described under Materials and Methods. The pERK levels relative to total ERK levels in H226-3329, H226-3332, and H1703-3332 were 24, 9 and 16%, respectively, of the levels observed in the appropriate H226-GFP and H1703-GFP controls. C, the H226 and H1703 transfectants expressing the GFP or FGF2 shRNAs were assayed for anchorage-independent growth as described under Materials and Methods.

Fig. 7.

RO4383596 inhibits FGF2-stimulated, but not EGF-stimulated, ERK and FRS2 phosphorylation in H226 NSCLC cells. H226 cells in 35-mm wells were incubated for 1 h in HITES medium in the presence of the indicated concentrations of RO4383596; DMSO was added as a diluent control. Subsequently, FGF2 or EGF was added at 10 ng/ml for another 15 min, and cell extracts were prepared and immunoblotted for phospho-ERK and phospho-FRS2-Y196 as described under Materials and Methods. The filters were stripped and reprobed with antibodies to total ERK or FRS2 to verify equal protein loading. Note that the mobility of pFRS2 is decreased after FGF2 treatment, consistent with increased phosphorylation of the protein on additional sites. In addition, the mobility of pFRS2 detected in cells treated with FGF2 and 1 μM RO4383596 was greatly increased, suggesting that FRS2 is dephosphorylated after TKI treatment.

Fig. 8.

RO4383596 inhibits basal ERK and FRS2 phosphorylation in NSCLC cell lines coexpressing FGFs and FGFRs. A, NSCLC cell lines were seeded in 12-well plates and cultured in full medium to 50 to 90% confluence. The growth medium was replaced with serum-free HITES medium, and after a 2-h incubation, RO4383596 or gefitinib was added at the indicated concentrations, and incubation was continued for another 2 h. Cell-free extracts were prepared and submitted to immunoblot analysis for pERK as described under Materials and Methods. The filters were stripped and reprobed for total ERK1 and ERK2 with rabbit polyclonal antibodies from Santa Cruz Biotechnology, Inc. Representative pERK and ERK1/ERK2 blots are shown for H226, H1703, H322c, and HCC4006 cells. B, immunoblot data from the entire panel of NSCLC cell lines was submitted to densitometry analysis and is presented as the ratio of pERK2 to total ERK2. C, the indicated NSCLC cell lines were incubated in HITES medium for 16 h and then for an additional 2 h with or without 1 μM RO4383596. Extracts were prepared and immunoblotted for pFRS2-Y196 as described under Materials and Methods. The filters were subsequently stripped and reprobed for total FRS2.

Fig. 9.

Selective inhibition of NSCLC proliferation and anchorage-independent growth by RO4383596. A, the indicated NSCLC cell lines were seeded at 2000 cells/well in 96-well plates and subsequently incubated for 5 days with growth medium containing the indicated concentrations of RO4383596. Viable cells were measured with the MTT assay as described under Materials and Methods. B, anchorage-independent growth of the indicated NSCLC cell lines in the presence of RO4383596 was measured as described under Materials and Methods. The plates were incubated for three weeks, the colonies were stained with nitro blue tetrazolium, and quantified after digital photography and MetaMorph software analysis. The symbols in the figure are consistent with those used in Fig. 8.