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Fibroblast growth factor (FGF) signaling can bypass a requirement for estrogen receptor (ER) activation in the growth of ER positive breast cancer cells. Although it is constitutively bound through its N-terminal phosphotyrosine binding domain (PTB) to FGF receptors (FGFRs), FGF stimulation leads to phosphorylation of the adaptor protein SNT-1 on C-terminal tyrosine residues which are docking sites for molecules that activate downstream pathways. By expressing the PTB domain of SNT-1 alone (SNT-1 PTB) in a doxycycline-inducible manner in MCF-7 cells, a ER positive breast carcinoma line, we asked whether we could uncouple FGFR activation from its downstream signaling components. As we previously reported, the induction of SNT-1 PTB expression resulted in a significant decrease of FGF-dependent tyrosine phosphorylation of endogenous wild-type SNT-1 and a strong inhibition of complex formation between SNT-1, Gab-1 and Sos-1. This resulted in the inhibition of Ras and MAP kinase activation as well as in the inhibition of FGF dependent colony formation in the presence of pure antiestrogen ICI 182,780. Since we demonstrated a significant decrease in the involvement of Gab1 in the SNT-1 associated multiprotein complex by SNT-1 PTB overexpression, it would be reasonable to expect a dominant negative effect of this molecule in the PI3 kinase pathway. Inducible expression of SNT-1 PTB in MCF-7 cells significantly inhibited phosphorylation of Akt Ser 473 but phosphorylation of this site remained unchanged in cells treated with heregulin β1. We also analysed the effect of the SNT-1 PTB domain on the activity of the important mediator of the ERK and PI3-kinase pathways, p70S6K. We observed inhibition of the phosphorylation of sites Thr 421/Ser 424 and Ser 411 of p70S6K by SNT-1 PTB overexpression which may result from the strong inhibition of MAP kinase activity. On the other hand, inhibition of phosphorylation of the MAPK independent site Thr 389 of p70S6K may reflect both inhibition of PI-3 kinase pathways and mTOR dependent signaling, since the FGF-1 induced phosphorylation of the Thr 389 site was sensitive to the treatment with the PI3-K and mTOR inhibitors LY294002 and rapamycin. Overexpression of the SNT-1 PTB domain did not affect the phosphorylation of p70S6K induced by heregulin β1 in estrogen depleted conditions. Collectively these results suggest that SNT-1 selectively plays a pivotal role in FGF-dependent Ras-MAP kinase activation, as well as in the PI-3 kinase and mTOR pathways in these cells.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]