Overexpression of basic fibroblast growth factor in MCF-7 human breast cancer cells: Lack of correlation between inhibition of cell growth and MAP kinase activation

Basic fibroblast growth factor (bFGF, FGF-2) is progressively lost from mammary epithelial cells as they become malignant. To investigate the effects of restoring the expression of bFGF in breast cancer cells, we constructed MCF-7 cells that permanently overexpress 18-kD cytoplasm-localizing bFGF (MCF-7/ΔA_FGF(18) cells) and cells that express both the 18-kD along with the 22- and 24-kD nucleus-localizing bFGF peptides (MCF-7/NCF_{FGF(18,22,24)} cells), using retroviral transduction. These stable cell constructs grew more slowly and had a larger fraction of their populations in the G₀/G₁ phase of the cell cycle than control cells. All forms of bFGF were eluted from MCF-7/NCF_{FGF(18,22,24)} cell monolayers with 2 M NaCl, in contrast to fibroblasts that were demonstrated to secrete only the 18-kD bFGF isoform. High-affinity binding of 18-kD ¹²⁵I-bFGF to these cells was significantly decreased, probably because of competitive binding by the autocrine-secreted bFGF. Recombinant 18-kD bFGF that was previously demonstrated in our laboratory to inhibit proliferation, activate MAP kinase, and induce the cyclin-dependent kinase inhibitor p21^WAF1/CIP1 in MCF-7 cells, further inhibited MCF-7/ΔA_FGF(18) cells but had no effect on MCF-7/NCF_{FGF(18,22,24)} cells. The total cellular content of the high-affinity FGF receptors 1–3 was unchanged, but FGF receptor 4 was decreased in MCF-7/NCF_{FGF(18,22,24)} cells. Both cell types overexpressing bFGF isoforms had elevated levels of the cyclin-dependent kinase inhibitor p27^Kip1 but not that of p21^WAF1/CIP1. In MCF-7/ΔA_FGF(18) cells, FGFR1 and MAP kinase were constitutively phosphorylated. Exogenous recombinant 18-kD bFGF did not accentuate these effects but did induce an increase in the levels of p21^WAF1/CIP1 corresponding to the further inhibition induced by exogenous bFGF in these cells. In MCF-7/NCF_{FGF(18,22,24)} cells, FGFR1 and MAP kinase were not phosphorylated at baseline nor upon stimulation with recombinant bFGF, and exogenous bFGF only had a minimal effect on low steady-state p21^WAF1/CIP1 levels. However, stimulation of these cells with phorbol ester or insulin did result in MAP kinase phosphorylation. While growth-inhibited in the G₁ phase of the cell cycle, MCF-7/NCF_{FGF(18,22,24)} cells retained active isoforms of cdk2 and the hyperphosphorylated form of Rb. These data suggest that high molecular weight forms of bFGF overexpressed in MCF-7 cells do not activate the receptor-mediated MAP kinase pathway, and do not induce p21^WAF1/CIP1 in an autocrine manner, but inhibit proliferation through other, possibly direct nuclear signalling mechanisms. J. Cell. Physiol. 177:411–425, 1998. © 1998 Wiley-Liss, Inc.