Targeting the heparin-binding domain of fibroblast growth factor receptor 1 as a potential cancer therapy

Abstract

Background: Aberrant activation of fibroblast growth factor receptors (FGFRs) deregulates cell proliferation and promotes cell survival, and may predispose to tumorigenesis. Therefore, selective inactivation of FGFRs is an important strategy for cancer therapy. Here as a proof-of-concept study, we developed a FGFR1 neutralizing antisera, IMB-R1, employing a novel strategy aimed at preventing the access of essential heparan sulfate (HS) co-receptors to the heparin-binding domain on FGFR1.

Methods: The mRNA and protein expression level of FGFR1 and other FGFRs were examined in several lines of breast cancer and osteosarcoma cells and corresponding normal cells using Taqman real-time quantitative PCR and Western blot analysis. The specificity of IMB-R1 against FGFR1 was assessed with various ELISA-based approaches and Receptor Tyrosine Kinase array. Proliferation assay and apoptosis analysis were performed to assess the effect of IMB-R1 on cancer cell growth and apoptosis, respectively, in comparison with known FGFR1 inhibitors. The IMB-R1 induced alteration of intracellular signaling and gene expression were analysed using Western blot and microarray approaches. Immunohistochemical staining of FGFR1 using IMB-R1 were carried out in different cancer tissues from clinical patients. Throughout the study, statistical differences were determined by Student's t test where appropriate and reported when a p value was less than 0.05.

Results: We demonstrate that IMB-R1 is minimally cross-reactive for other FGFRs, and that it potently and specifically inhibits binding of heparin to FGFR1. Furthermore, IMB-R1 blocks the interaction of FGF2 with FGFR1, the kinase activity of FGFR1 and activation of intracellular FGFR signaling. Cancer cells treated with IMB-R1 displayed impaired FGF2 signaling, were unable to grow and instead underwent apoptosis. IMB-R1-induced cell death correlated with a disruption of antioxidative defense networks and increased expression of several tumor suppressors and apoptotic proteins, including p53. Immunostaining with IMB-R1 was stronger in human cancer tissues in which the FGFR1 gene is amplified.

Conclusion: Our study suggests that blocking HS interaction with the heparin-binding domains of FGFR1 inhibited cancer cell growth, which can be an attractive strategy to inactivate cancer-related heparin-binding proteins.

Fig. 1

Expression of FGFR1 in cells from breast and bone tissue. a, FGFR mRNA transcript levels in normal and cancer cells. b, Fold change in FGFR mRNA based on expression in cancers cells relative to normal cells. c, FGFR protein levels in normal versus cancer cells. Results are from triplicate experiments and the Western blot is representative of the triplicates. (*p < 0.05)

Fig. 2

Specificity of IMB-R1 for FGFR1. a, Binding of IMB-R1 and MAB765 to FGFR1 isoforms (representative blot from triplicate experiments). b, Affinity of IMB-R1 for FGFR isoforms (results are from triplicate experiments). c, Binding of FGFR to heparin in the presence or absence of IMB-R1 (results are from triplicate experiments). d, Binding of FGF2 to FGFR in the presence or absence of IMB-R1 (results are from triplicate experiments). e, FGFR phosphorylation stimulated by FGF2 in the presence or absence of IMB-R1 including the fold change of FGFR phosphorylation as determined by a comparison of means

Fig. 3

Effects of FGFR1 inhibitors on cell growth. a, Fold change in cell number 48 h after treatment with IMB-R1. b, Temporal change in MG63 cell number following treatment with IMB-R1 (1:250). c, Cell number following FGF2 treatment (20 ng/ml for MG63, 5 ng/ml for MDAMB468 and T47D) for 48 h. Cells were pre-treatment with IMB-R1 for 1 h before FGF2 treatment. d, Cell number (MG63) following 48 h treatment with IMB-R1 or antigen-purified IMB-R1. e, Cell numbers following 48 h treatment with varying doses of FGFR inhibitors. f, Cell numbers following 48 h treatment with the FGFR1 antibody (MAB765) at varying doses. Unless otherwise stated, IMB-R1 was applied at a dilution of 1:250. All experiments were performed in triplicate

Fig. 4

IMB-R1 induced cell apoptosis with varying potency. a, Caspase 3 activity was used as an assay for apoptosis in MG63 cells and the apoptotic activity of IMB-R1 (1:250) compared with Staurosporine (15 nM). b, Cells were treated with either IMB-R1 or SU5402 for 24 h and stained with a combination of Annexin V-FITC and PI to detect early apoptosis (positive for Annexin V-FITC, AV+/PI-) and late apoptosis (positive for both Annexin V-FITC and PI, AV+/PI+). Total apoptotic level represents AV+/PI- plus AV+/PI+. FACS plots are representative of triplicate experiments, and bar graphs show percentage cells in each population from triplicate experiments

Fig. 5

Downstream signaling targets of IMB-R1. Western blot analysis of signaling targets in serum-starved (SS) cells (48 h) released into FGF2 in the absence (a) or presence (b) of IMB-R1 for the time points indicated. a, Following SS, cells were stimulated with FGF2 for 10 min and protein expression determined. b, Following SS, cells were pre-treated with IMB-R1 (1:250 dilution) for 1, 2, 6 or 24 h then stimulated with 20 ng/ml (MG63) or 5 ng/ml (T47D) of FGF2 for 10 min and protein expression determined. c, Cells were dosed with U0126 for 1 h and phosphorylated-ERKs detected by immunoblotting. d, Cells were treated with U0126 for 24 h and stained with a combination of Annexin V-FITC and PI and presented as per Figure 4 b. e, Cells were treated with IMB-R1 (1:250 dilution) for 48 h and the protein expression determined by immunoblotting

Fig. 6

The effect of IMB-R1 on gene expression. Cells were treated with IMB-R1 (1:250 dilution) for 48 h and RNA extracted for microarray analysis. a, The heatmaps: Red, up-regulation; Green, down-regulation. b, Top 10 affected cellular functions by IMB-R1 as determined by Ingenuity Pathway Analysis. c, The number of common genes affected across the different cells. d, The antioxidant genes significantly regulated by IMB-R1. e, The proposed signaling mechanisms disrupted by IMB-R1 during FGF2/FGFR1 dependent cell growth and survival in cancer cells

Fig. 7

IMB-R1 targets FGFR1 in multiple human cancers. a, IMB-R1 histochemical staining from a human cancer tissue array. Right panel, enlarged image of boxed area highlighting increased FGFR1 expression (detected by IMB-R1) in breast cancer tissues from twenty separate donors compared with adjacent healthy breast tissue. b, The intensity of FGFR1 expression (detected by IMB-R1) was scored and the average scores for the various cancer tissues compared with those from adjacent healthy tissues