Insulin-like growth factor (IGF)-independent action of IGF-binding protein-3 in Hs578T human breast cancer cells. Cell surface binding and growth inhibition

Estrogen receptor-negative Hs578T human breast cancer cells secrete insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-4 as major binding protein (BP) species. Our previous immunohistochemical studies (Oh, Y., Muller, H. L., Pham, H., Lamson, G., and Rosenfeld, R. G. (1992) Endocrinology 131, 3123-3125) have demonstrated the existence of cell surface-associated IGFBP-3 and release of cell surface-associated IGFBP-3 into conditioned media by addition of IGF peptide in Hs578T cells. In this study, we have demonstrated that IGFBP-3 binding on the cell surface is specific and receptor-mediated, by showing: 1) a dose-dependent increase of IGFBP-3 binding by addition of divalent cations (CaCl₂ and MnCl₂); 2) dose- dependent competition of ¹²⁵I-IGFBP-3(E. coli) by unlabeled IGFBP-3(E. coli) (>80% competition at 100 nM), but not by IGFBP-1 or fibronectin. In addition, exogenous IGFBP-3 treatment resulted in a significant inhibitory effect on monolayer growth of Hs578T cells. This inhibitory effect of IGFBP- 3 was shown to be specific and IGF-independent by demonstrating: 1) dose- dependent inhibition on cell growth (60% inhibition at 20 nM) and inhibition on DNA synthesis (10 nM; p < 0.05, 20 nM; p < 0.005) by exogenous IGFBP-3(E. coli), but not by IGFBP-1; 2) absence of stimulatory effects on monolayer cell growth by either native IGFs or IGF analogs which have significantly decreased affinity for IGFBPs, but retain full affinity for type 1 and 2 IGF receptors; 3) significant diminution of the IGFBP-3 inhibitory effects on monolayer growth by coincubation with native IGFs, but not by coincubation with IGF analogs with decreased affinity for IGFBP-3. In conclusion, exogenous IGFBP-3 shows specific binding on the cell surface and can inhibit Hs578T cell monolayer growth by itself, suggesting the existence of specific membrane-associated proteins or receptors for IGFBP-3. Furthermore, IGF-I and -II can attenuate inhibitory effect of IGFBP-3 by forming IGF·IGFBP-3 complexes, thereby preventing cell surface binding of IGFBP-3.