A dominant-negative FGF1 mutant (the R50E mutant) suppresses tumorigenesis and angiogenesis.

Fibroblast growth factor-1 (FGF1) and FGF2 play a critical role in angiogenesis, a formation of new blood vessels from existing blood vessels. Integrins are critically involved in FGF signaling through crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and induces FGF receptor-1 (FGFR1)-FGF1-integrin αvβ3 ternary complex. We previously generated an integrin binding defective FGF1 mutant (Arg-50 to Glu, R50E). R50E is defective in inducing ternary complex formation, cell proliferation, and cell migration, and suppresses FGF signaling induced by WT FGF1 (a dominant-negative effect) in vitro. These findings suggest that FGFR and αvβ3 crosstalk through direct integrin binding to FGF, and that R50E acts as an antagonist to FGFR. We studied if R50E suppresses tumorigenesis and angiogenesis. Here we describe that R50E suppressed tumor growth in vivo while WT FGF1 enhanced it using cancer cells that stably express WT FGF1 or R50E. Since R50E did not affect proliferation of cancer cells in vitro, we hypothesized that R50E suppressed tumorigenesis indirectly through suppressing angiogenesis. We thus studied the effect of R50E on angiogenesis in several angiogenesis models. We found that excess R50E suppressed FGF1-induced migration and tube formation of endothelial cells, FGF1-induced angiogenesis in matrigel plug assays, and the outgrowth of cells in aorta ring assays. Excess R50E suppressed FGF1-induced angiogenesis in chick embryo chorioallantoic membrane (CAM) assays. Interestingly, excess R50E suppressed FGF2-induced angiogenesis in CAM assays as well, suggesting that R50E may uniquely suppress signaling from other members of the FGF family. Taken together, our results suggest that R50E suppresses angiogenesis induced by FGF1 or FGF2, and thereby indirectly suppresses tumorigenesis, in addition to its possible direct effect on tumor cell proliferation in vivo. We propose that R50E has potential as an anti-cancer and anti-angiogenesis therapeutic agent ("FGF1 decoy")

A dominant-negative FGF1 mutant (the R50E mutant) suppresses tumorigenesis and angiogenesis.

Fibroblast growth factor-1 (FGF1) and FGF2 play a critical role in angiogenesis, a formation of new blood vessels from existing blood vessels. Integrins are critically involved in FGF signaling through crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and induces FGF receptor-1 (FGFR1)-FGF1-integrin αvβ3 ternary complex. We previously generated an integrin binding defective FGF1 mutant (Arg-50 to Glu, R50E). R50E is defective in inducing ternary complex formation, cell proliferation, and cell migration, and suppresses FGF signaling induced by WT FGF1 (a dominant-negative effect) in vitro. These findings suggest that FGFR and αvβ3 crosstalk through direct integrin binding to FGF, and that R50E acts as an antagonist to FGFR. We studied if R50E suppresses tumorigenesis and angiogenesis. Here we describe that R50E suppressed tumor growth in vivo while WT FGF1 enhanced it using cancer cells that stably express WT FGF1 or R50E. Since R50E did not affect proliferation of cancer cells in vitro, we hypothesized that R50E suppressed tumorigenesis indirectly through suppressing angiogenesis. We thus studied the effect of R50E on angiogenesis in several angiogenesis models. We found that excess R50E suppressed FGF1-induced migration and tube formation of endothelial cells, FGF1-induced angiogenesis in matrigel plug assays, and the outgrowth of cells in aorta ring assays. Excess R50E suppressed FGF1-induced angiogenesis in chick embryo chorioallantoic membrane (CAM) assays. Interestingly, excess R50E suppressed FGF2-induced angiogenesis in CAM assays as well, suggesting that R50E may uniquely suppress signaling from other members of the FGF family. Taken together, our results suggest that R50E suppresses angiogenesis induced by FGF1 or FGF2, and thereby indirectly suppresses tumorigenesis, in addition to its possible direct effect on tumor cell proliferation in vivo. We propose that R50E has potential as an anti-cancer and anti-angiogenesis therapeutic agent ("FGF1 decoy")

R50E suppresses WT FGF1- induced tube formation of endothelial cells in vitro.

<p>Serum starved HUVECs were plated on Matrigel-coated plates, and incubated in WT FGF1 (5 ng/ml) or the mixture of WT FGF1 (5 ng/ml) and R50E (250 ng/ml) for 8 h. a. Representative tube formation images are shown. Scale bar = 200 µm. b. The number of branch points was counted per field from the digital images. Data is shown as means +/− SE. Statistical analysis was done by one-way ANOVA plus Tukey analysis.</p

R50E suppresses WT FGF1-induced angiogenesis in rat aortic ring.

<p>Isolated rat aortic ring was embedded in collagen gels in DMEM containing WT FGF1 (50 ng/ml), R50E (50 ng/ml) or the mixture of WT FGF1 (50 ng/ml) and R50E (2500 ng/ml) and cultured for 10 days. Representative phase contrast images of 3 independent experiments are shown. Scale bars, 100 µm.</p

R50E suppresses tumorigenesis in vivo.

<p>a. Transfected DLD-1 cells secrete WT FGF1 or R50E into culture medium. DLD-1 cells that stably express WT FGF1 or R50E were generated. The WT FGF1 and R50E have a 6His-tag at the N-terminus. To detect FGF1 secreted from the transfected cells, we analyzed the culture media by Western blotting with anti-6His antibodies. Mock-transfected cells were used as a control. As a loading control, we ran the same samples in gel in parallel and stained the gel with Coomassie Brilliant Blue (CBB). b. Proliferation of DLD-1 cells in the presence of 10% FCS. DLD-1 cells that secrete R50E grew in the medium that contains FCS in vitro at levels comparable to those of WT-FGF1 expressing cells or mock transfected cells. Statistical analysis was done by one-way ANOVA plus Tukey analysis. c. Proliferation of DLD-1 cells in the absence of FCS. DLD-1 cells that secrete R50E grew in vitro in the medium without FCS at levels comparable to that of mock-transfected cells. Cells that express WT FGF1 grew faster than mock-transfected and R50E expressing cells. Statistical analysis was done by one-way ANOVA plus Tukey analysis. d. The growth curve of DLD-1 cells in vivo. WT FGF1 enhanced tumor growth in vivo, while R50E suppressed it (as shown by the growth curve and the sizes of DLD-1 tumors removed at day 31). We injected the DLD-1 cells that secrete WT FGF1or R50E into nude mice (1 million cells/site) at right and left inguinal regions (4 mice per group, 2 tumors/mouse). Mock-transfected cells were used as a control. Statistical analysis of tumor sizes at Day 31 was done by t-test (n = 8 for mock and wt FGF, n = 7 for R50E). e. The sizes of tumors at Day 31. DLD-1 cells secreting wt FGF1 grew faster, and cells secreting R50E slower, than mock-transfected cells (n = 8 for mock and wt FGF, n = 7 for R50E).</p

R50E suppresses WT FGF1-induced endothelial cell migration.

<p>Lower side of the filter in the modified Boyden chamber was coated with fibronectin (10 µg/ml). The lower chamber was filled with serum-free medium with WT FGF1 (5 ng/ml) or the mixture of WT FGF1 and excess R50E (5 and 250 ng/ml, respectively). HUVECs were plated on the filter and incubated for 6 h. Chemotaxed cells were counted from the digital images of the stained cells. Data is shown as means +/− SE per field. Statistical analysis was done by one-way ANOVA plus Tukey analysis.</p

R50E suppresses angiogenesis in Matrigel plug assays in rat.

<p>Matrigel plug containing WT FGF1 (1 µg/ml), R50E (1 µg/ml) or the mixture of WT FGF1 (1 µg/ml) and excess R50E (50 µg/ml) were injected subcutaneously into the back of rat, respectively. The plugs (n = 4−5) were removed 10 days after injection and tissue sections were stained for von Willebrand factor, a blood vessel marker. a. Representative images are shown. Scale bar = 50 µm. b. The number of extended blood vessels were counted under a light microscope. Data is shown as means +/− SE. Statistical analysis was done by one-way ANOVA plus Tukey analysis.</p

R50E suppresses FGF1- and FGF2-induced angiogenesis (branching formation) in CAM models.

<p>Saline- or FGF- impregnated filter disks are placed on blood vessels in otherwise avascular sections of CAM (day 11) to induce angiogenesis. The disks and underlying CAM tissue (day 13) are then harvested. Neovascularization was then scored by counting vessel branches present in the CAM tissue below the filter from digital images. a) and b) Quantification of dose response. Five ng/ml is optimum, c) Suppression of FGF1-induced angiogenesis by excess R50E. d) Suppression of FGF2-induced angiogenesis by excess R50E. The data suggest that R50E suppresses FGF1- and FGF2-induced angiogenesis in the CAM model. Statistical analysis was done by one-way ANOVA plus Tukey analysis.</p