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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 tumorigenesis in vivo.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).
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pone-0057927-g001: R50E suppresses tumorigenesis in vivo.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).

Mentions: To test if R50E may act as an antagonist to FGF signaling in vivo, we stably expressed R50E or WT FGF1 in a secretion vector in DLD-1 colon carcinoma cells, and tested if R50E affects tumor growth in vivo. These cells secreted 6His-tagged R50E or WT FGF1 into culture medium (Fig. 1a). The expression of WT FGF1 or R50E had little or no effect on cell survival in vitro in the presence of FCS (Fig. 1b). The expression of WT FGF1 significantly enhanced cell survival in the absence of serum, but the expression of R50E did not (Fig. 1c). When the population of DLD-1 colon cancer cells that stably express WT FGF1 or R50E were injected subcutaneously into nude mice (1 million cells/site, two sites per mouse), cells that secrete WT FGF1 generated bigger tumors (n = 8) but cells that secrete R50E generated smaller tumors (n = 8) than mock-transfected cells (n = 7) (Fig. 1d and 1e). These results suggest that R50E suppressed tumorigenesis in vivo while WT FGF1 markedly enhanced it. Since R50E did not affect tumor cell proliferation or survival in vitro, it is likely that R50E suppressed tumorigenesis in vivo indirectly through blocking FGF signaling in endothelial cells (angiogenesis) or stromal cells. We thus tested the effect of R50E on angiogenesis.


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 tumorigenesis in vivo.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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585250&req=5

pone-0057927-g001: R50E suppresses tumorigenesis in vivo.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).
Mentions: To test if R50E may act as an antagonist to FGF signaling in vivo, we stably expressed R50E or WT FGF1 in a secretion vector in DLD-1 colon carcinoma cells, and tested if R50E affects tumor growth in vivo. These cells secreted 6His-tagged R50E or WT FGF1 into culture medium (Fig. 1a). The expression of WT FGF1 or R50E had little or no effect on cell survival in vitro in the presence of FCS (Fig. 1b). The expression of WT FGF1 significantly enhanced cell survival in the absence of serum, but the expression of R50E did not (Fig. 1c). When the population of DLD-1 colon cancer cells that stably express WT FGF1 or R50E were injected subcutaneously into nude mice (1 million cells/site, two sites per mouse), cells that secrete WT FGF1 generated bigger tumors (n = 8) but cells that secrete R50E generated smaller tumors (n = 8) than mock-transfected cells (n = 7) (Fig. 1d and 1e). These results suggest that R50E suppressed tumorigenesis in vivo while WT FGF1 markedly enhanced it. Since R50E did not affect tumor cell proliferation or survival in vitro, it is likely that R50E suppressed tumorigenesis in vivo indirectly through blocking FGF signaling in endothelial cells (angiogenesis) or stromal cells. We thus tested the effect of R50E on angiogenesis.

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