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. 2008 Oct;7(10):3408-19.
doi: 10.1158/1535-7163.MCT-08-0444.

Fibroblast growth factor receptor-mediated signals contribute to the malignant phenotype of non-small cell lung cancer cells: therapeutic implications and synergism with epidermal growth factor receptor inhibition

Hendrik Fischer  1 , Ninon Taylor, Sigrid Allerstorfer, Michael Grusch, Gudrun Sonvilla, Klaus Holzmann, Ulrike Setinek, Leonilla Elbling, Heidelinde Cantonati, Bettina Grasl-Kraupp, Christine Gauglhofer, Brigitte Marian, Michael Micksche, Walter Berger
Affiliations

Fibroblast growth factor receptor-mediated signals contribute to the malignant phenotype of non-small cell lung cancer cells: therapeutic implications and synergism with epidermal growth factor receptor inhibition

Hendrik Fischer et al. Mol Cancer Ther. 2008 Oct.

Abstract

Fibroblast growth factors (FGF) and their high-affinity receptors (FGFR) represent an extensive cellular growth and survival system. Aim of this study was to evaluate the contribution of FGF/FGFR-mediated signals to the malignant growth of non-small cell lung cancer (NSCLC) and to assess their potential as targets for therapeutic interventions. Multiple FGFR mRNA splice variants were coexpressed in NSCLC cells (n = 16) with predominance of FGFR1. Accordingly, both expression of a dominant-negative FGFR1 (dnFGFR1) IIIc-green fluorescent protein fusion protein and application of FGFR small-molecule inhibitors (SU5402 and PD166866) significantly reduced growth, survival, clonogenicity, and migratory potential of the majority of NSCLC cell lines. Moreover, dnFGFR1 expression completely blocked or at least significantly attenuated s.c. tumor formation of NSCLC cells in severe combined immunodeficient mice. Xenograft tumors expressing dnFGFR1 exhibited significantly reduced size and mitosis rate, enhanced cell death, and decreased tissue invasion. When FGFR inhibitors were combined with chemotherapy, antagonistic to synergistic in vitro anticancer activities were obtained depending on the application schedule. In contrast, simultaneous blockage of FGFR- and epidermal growth factor receptor-mediated signals exerted synergistic effects. In summary, FGFR-mediated signals in cooperation with those transmitted by epidermal growth factor receptor are involved in growth and survival of human NSCLC cells and should be considered as targets for combined therapeutic approaches.

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Figures

Figure 1
Figure 1
Expression of FGFR mRNA variants in NSCLC cells (A) and blockage of FGF-induced phosphorylation of downstream signaling molecules by dnFGFR1 (B) and small-molecule inhibitor PD166866 (C). A, expression of IgIII mRNA splice variants of FGFR1, FGFR2, and FGFR3 was analyzed by reverse transcription-PCR in 16 NSCLC cell lines. The percentage of positive cell lines separated into histologic sub-groups adenocarcinoma-derived, SCC-derived, and large cell carcinoma (LC)-derived cell lines is shown. FGFR primer pairs were designed to be variant specific with respect to the third IgG loop (variant FGFR-IIIb or FGFR-IIIc). B, GFP and dnFGFR1 (representing a GFP fusion protein) expression and effect on phosphorylation of ERK were analyzed after adenoviral transduction by Western blotting in VL-10 cells with respective antibodies. After infection, cells were serum starved for 48 h and stimulated with the indicated FGF for 15 min. Blots (left) were quantified densitometrically (mean and SD of three experiments; right). Values are expressed as pERK/ERK ratios normalized to the serum-free controls set arbitrarily as 1. C, effects of the indicated concentrations of PD166866 on phosphorylation of ERK and S6 were analyzed by Western blotting. VL-10 cells were serum starved for 48 h, incubated with PD166866 for 1 h, and stimulated with FGF2 and FGF7 (20 ng/mL) for 10 min. Blots (left) were quantified densitometrically. Values are expressed as pERK/ ERK and pS6/S6 ratios normalized to the serum-free controls set arbitrarily as 1 (right). One of three experiments delivering comparable results.
Figure 2
Figure 2
Effects of dnFGFR1 expression on NSCLC cells. Cells were transfected with GFP or dnFGFR1 vectors by electroporation (A-C). A, photomicrographs were taken of A549 cells 24 h (note the similar proportion of green fluorescent cells showing comparable transfection rates) and 7 d after transfection as indicated. B, proportion of GFP- or dnFGFR1-expressing, viable A549 cells was analyzed by fluorescence-activated cell sorting at the indicated time points after transfection. One of three experiments yielding comparable results. C, after transfection of the indicated NSCLC cell lines and selection with G418 for about 3 wk, numbers of green clones were determined. Due to the high variation in the cloning potential of the analyzed cell lines, the data are given relatively to the number of controlGFP clones. D, A427 cells were transduced with GFP or dnFGFR1 adenoviruses and photomicrographs taken at the indicated time points.
Figure 3
Figure 3
Expression of dnFGFR1 in NSCLC cells leads to apoptotic cell death. The indicated NSCLC cell lines were either transduced with GFP (open columns) or dnFGFR1 adenoviruses (hatched columns; A-C) or treated with small-molecule FGFR inhibitors (D). At the indicated time points after infection, the number of cells alive (A) and the percentage of dead cells (B) were assessed by trypan blue exclusion assay. Percentage of apoptotic cells (C) was analyzed by 4′,6-diamidino-2-phenylindole staining. D, NSCLC cell lines and nonmalignant bronchial epithelial cells BEAS-2B were treated with the indicated concentrations of the FGFR inhibitors SU5402 (top) and PD166866 (bottom) in growth medium containing 1% FCS. Following a 96 h exposure, cell number was analyzed by MTT assay. Representative results for each cell line are shown normalized to controls without drugs. Columns, mean; bars, SD. Significant differences to controls were determined by Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.
Figure 4
Figure 4
Blockade of FGFR leads to inhibition of NSCLC cell migration. After transduction with GFP or dnFGFR1 adenoviruses as indicated, cell migration was analyzed by scratch assays as described in Materials and Methods (A and B). Representative photomicrographs of scratch wounds in a VL-8 monolayer taken at the indicated time points are shown in A and quantitative evaluations for three NSCLC cell lines are shown in B. C, VL-8 cells were seeded on transmigration filters and treated with PD166866 (10 μmol/L) and SU5402 (15 μmol/L) for 72 h in medium with 10% FCS. Thereafter, cells on the lower surface of the filters were fixed, stained with crystalviolet, and counted. Left, columns, mean of three experiments; bars, SD. Significant difference to the controlwas determined by Student’s t test. *, P < 0.05; **, P < 0.01.
Figure 5
Figure 5
Inhibition of in vivo NSCLC growth by expression of dnFGFR1. Tumor formation of SCC-derived VL-8 and adenocarcinoma-derived A427 cells transduced with GFP or dnFGFR1 adenoviruses were analyzed by s.c. implantation in SCID BALB/c mice. Forty-nine days after s.c. injection, the mice were killed and the tumors were isolated and further analyzed. Photographs of representative tumors are shown (A, top). Tumor volumes (A, bottom) were calculated as described in Materials and Methods. Each experimental group contained three to six animals. Mean ± SD. Significant differences to controls were determined by two-way ANOVA with Bonferroni post-test. Formalin-fixed A427-derived tumor specimens were routinely processed for paraffin embedding, and sections were stained with pan-cytokeratin (B) or H&E and Ki-67 (D) as indicated. B, controltumors grew in an infiltrative manner, with a jagged and fissured margin and a high frequency of isolated tumor islets invading the surrounding stroma. In contrast, dnFGFR1-expressing tumors grew in a more encapsulated fashion and exhibited stronger compression of the surrounding stromal tissue (arrows). Boxed regions (top) are shown at higher magnifications (bottom). C, mitotic (left) and apoptotic (right) cells were counted in viable boundary regions of H&E-stained tumor sections. D, apoptotic cells were counted in at least 10 small prenecrotic (top, H/E stain) and Ki-67-negative (bottom) regions as representatively shown (right). Bar, 50 μm (B, bottom) and 100 μm (B, top, and D). Columns, mean of at least 15 optical fields (C) or 10 regions (D) analyzed; bars, SD. Significant differences to controls were determined by Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.
Figure 6
Figure 6
Combination of small-molecule FGFR inhibitors with chemotherapeutic drugs and EGFR inhibitors. Concentrations of the drugs [vinblastine (VBL), erlotinib, and lapatinib] used for combination experiments (A and B, right) were selected from single substance dose-response curves (data not shown) and covered the range from ineffective to around 50% growth inhibition. A, A549 cells were simultaneously treated for 96 h with the indicated VBL + PD166866 combinations (left), 72 h with PD166866 followed by 72 h VBL (middle), or 72 h with VBL followed by 72 h with PD166866 (right) and cell viability was analyzed by MTT assays. B, NSCLC cell lines were simultaneously treated with the indicated combinations of the FGFR inhibitor PD166866 with the EGFR inhibitor erlotinib (top) or the EGFR/HER-2 dualtyrosine kinase inhibitor lapatinib (bottom) for 96 h in growth medium containing 1% FCS and cell survival was analyzed by MTT assay. Data in A and B present CI of the respective drug combinations calculated according to Chou and Talalay (19) with CalcuSyn software. Following this evaluation, CI < 1 indicates synergism, CI = 1 stands for additive effects, and CI >1 depicts antagonistic effects. C, effect of a 2 h exposure of the indicated RTK inhibitors on the phosphorylation of the downstream signaling molecules ERK and S6 was analyzed by Western blotting. Representatively, data for SCC-derived VL-10 cells are shown. D, VL-8 cells were transduced with dnFGFR1 or dnEGFR1 adenoviruses alone or in combination whereby the viral dose was kept constant by complementing with respective amounts of control virus. After 48 h exposure, numbers of viable (open columns) and dead cells (black columns) were determined by trypan blue exclusion assay.
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