Fibroblast and Epidermal Growth Factors Utilize Different Signaling Pathways to Induce Anchorage-independent Cell Transformation in JB6 Cl41 Mouse Skin Epidermal Cells

BACKGROUND Extracellular stimulation of cells with growth factors such as epidermal growth factor (EGF) induces cell proliferation and cell transformation. Although fibroblast growth factor (FGF) is a well-known family member of growth factors and acts as a ligand of FGF receptor (FGFR), a receptor tyrosine kinase, in cytoplasmic membrane, the tumor promoter potential of FGF has not been clearly understood. METHODS The role of FGF as a tumor promoter was determined measuring its effects of cell proliferation and transformation by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and anchorage-independent cell transformation assays, respectively. The antibody specificity of phospho-RSK2 Tyr529 was determined by Western blotting using a purified FGFR kinase domain in vitro and the membrane fraction of JB6 Cl41 cells ex vivo. The signaling pathways mediated by FGF or EGF were determined by the comparisons of phosphorylation inhibitory efficacy using signaling inhibitors including kaempferol. RESULTS FGF acted as a tumor promoter. FGF induced cell proliferation by stimulation of G1/S cell cycle transition, and anchorage-independent cell transformation in JB6 Cl41 cells. FGF-induced FGFR phosphorylation was suppressed by kaempferol treatment in a dose dependent manner. Interestingly, FGF stimulation utilized a non-canonical signaling pathway to activate RSK2 and activating transcription factor (ATF)-1, which was not transduced by EGF stimulation. Importantly, kaempferol inhibited tyrosine phosphorylation of FGFR by FGF stimulation and nuclear accumulation of phospho-ATF-1 at Ser63. Moreover, although kaempferol, 4'-N-benzoyl staurosporine (PKC412), 2-(2'-amino-3'-methoxyphenyl)oxanaphthalen-4-one (PD98059) and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)buta-diene (U0126) inhibited EGF-induced anchorage-independent cell transformation in JB6 Cl41 cells, FGF-induced cell transformation in soft agar was only inhibited by PKC412 and kaempferol, but not by PD98059 and U0126. CONCLUSIONS FGF acts as a tumor promoter and dual inhibition of kaempferol on the kinase activities of FGFR3 and RSK2 suppresses the FGF-induced neoplastic cell transformation through a non-canonical signaling pathway which is not utilized by EGF stimulation.