The FGF1 mutant (R50E) is defective in integrin binding but still binds to heparin and FGFR. R50E is defective in inducing DNA synthesis, cell proliferation, cell migration, and chemotaxis, suggesting that the direct integrin binding to FGF1 is critical for FGF signaling [12]. WT FGF1 induces ternary complex formation (integrin-FGF1-FGFR1) in NIH3T3 cells and human umbilical endothelial cells (HUVECs), selleck chemical Nutlin-3a but R50E is defective in these functions. WT FGF1 induces sustained activation of ERK1/2, but R50E is defective in this function. Notably excess R50E suppresses signals induced by WT FGF1 in vitro. Our results suggest that 1) R50E is a dominant-negative mutant, 2) ternary complex formation is involved in FGF signaling, and 3) the defect of R50E to bind to integrin may be directly related to the antagonistic action of R50E.

Taken together, these results suggest that R50E has potential as a therapeutic in cancer [13]. 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. Figure 1 R50E suppresses tumorigenesis in vivo. R50E Suppresses WT FGF-1 Induced Endothelial Cell Migration Endothelial cell migration is a critical feature of tumor angiogenesis.

We tested the effect of R50E on migration of HUVECs. Lower side of the filter in the modified Boyden chamber was coated with fibronectin (10 ��g/ml). The lower chamber was filled with serum-free EBM-2 medium with WT FGF1 (5 ng/ml) and/or R50E (5 and 250 ng/ml, respectively). HUVECs were plated on the filter and incubated for 6 h, and cells were stained with crystal violet. Carfilzomib Chemotaxed cells were counted from the digital images of the stained cells.