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

Mori S, Tran V, Nishikawa K, Kaneda T, Hamada Y, Kawaguchi N, Fujita M, Saegusa J, Takada YK, Matsuura N, Zhao M, Takada Y - PLoS ONE (2013)

Bottom Line: 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.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").

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pathology, Osaka University Graduate School of Medicine, Division of Health Sciences, Suita, Osaka, Japan.

ABSTRACT
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").

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R50E suppresses WT FGF1- induced tube formation of endothelial cells in vitro.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.
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pone-0057927-g003: R50E suppresses WT FGF1- induced tube formation of endothelial cells in vitro.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.

Mentions: One of the most specific tests for angiogenesis is the measurement of the ability of endothelial cells to form three-dimensional structures (tube formation) [20]. Endothelial cells of all origins appear to be able to form tubules in vitro on extracellular matrix components. We examined the effect of R50E on the tube formation of HUVECs in vitro. We plated serum-starved HUVECs on reconstituted extracellular matrix (Matrigel, growth factor reduced)-coated plates, and incubated with WT FGF1 and/or R50E (5 and 250 ng/ml, respectively) for 8 h. We counted the number of branching points per field from the digital images. We found that WT FGF1 markedly enhanced tube formation and R50E (5 ng/ml) did not induce tube formation. High dose R50E weakly induced tube formation. Excess R50E (250 ng/ml) significantly suppressed tube formation induced by WT FGF1 (Fig. 3). This suggests that R50E directly affects endothelial cell and competes with WT FGF1 for its binding to integrin to generate tube-like structure.


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

Mori S, Tran V, Nishikawa K, Kaneda T, Hamada Y, Kawaguchi N, Fujita M, Saegusa J, Takada YK, Matsuura N, Zhao M, Takada Y - PLoS ONE (2013)

R50E suppresses WT FGF1- induced tube formation of endothelial cells in vitro.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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585250&req=5

pone-0057927-g003: R50E suppresses WT FGF1- induced tube formation of endothelial cells in vitro.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.
Mentions: One of the most specific tests for angiogenesis is the measurement of the ability of endothelial cells to form three-dimensional structures (tube formation) [20]. Endothelial cells of all origins appear to be able to form tubules in vitro on extracellular matrix components. We examined the effect of R50E on the tube formation of HUVECs in vitro. We plated serum-starved HUVECs on reconstituted extracellular matrix (Matrigel, growth factor reduced)-coated plates, and incubated with WT FGF1 and/or R50E (5 and 250 ng/ml, respectively) for 8 h. We counted the number of branching points per field from the digital images. We found that WT FGF1 markedly enhanced tube formation and R50E (5 ng/ml) did not induce tube formation. High dose R50E weakly induced tube formation. Excess R50E (250 ng/ml) significantly suppressed tube formation induced by WT FGF1 (Fig. 3). This suggests that R50E directly affects endothelial cell and competes with WT FGF1 for its binding to integrin to generate tube-like structure.

Bottom Line: 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.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").

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pathology, Osaka University Graduate School of Medicine, Division of Health Sciences, Suita, Osaka, Japan.

ABSTRACT
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").

Show MeSH
Related in: MedlinePlus