Regulation of insulin‐like growth factor‐binding‐protein‐1, 2, 3, 4, 5, and 6: Synthesis, secretion, and gene expression in estrogen receptor‐negative human breast carcinoma cells

Insulin‐like growth‐factors I and II (IGF‐I, II) are potent mitogens for breast carcinoma proliferation. In extracellular fluids, most of the IGF‐I and II is associated with specific IGF‐binding proteins (IGFBPs). The role of these IGFBPs in IGF action is still not clear, but it has been demonstrated that these proteins may either enhance or inhibit IGF‐mediated cellular effects. Synthesis and secretion of IGFBPs have been demonstrated in breast carcinoma cells. In this study, we examined retinoic acid (RA) and IGF‐I modulation of IGFBP mRNA and IGFBP levels in two ER‐negative human breast carcinoma cell lines. Treatment of MDAMB‐231 and MDA‐MB‐468 cells with RA increased the levels in conditioned media of a Mr 42‐46‐kDa IGFBP, which was immunoprecipitated by an IGFBP‐3 antibody. IGF‐I also increased the accumulated levels of IGFBP‐3 in the conditioned media of both cell lines. Both cell lines expressed high basal levels of IGFBP‐3 mRNA; the addition of RA increased IGFBP‐3 mRNA levels by 1.5‐fold, whereas the addition of IGFI had no effect on IGFBP‐3 mRNA levels in either cell line. The difference in the magnitude of the RA enhancement of IGFBP‐3 mRNA levels (1.5‐fold) and RA stimulation of IGFBP‐3 levels in conditioned media (3.5–4‐fold) suggests that some of the effect of RA is at a posttranscriptional level. IGF‐I increased the levels of IGFBP‐2 and IGFBP‐5 in conditioned media by greater than tenfold but had no effect on IGFBP‐2 and IGFBP‐5 mRNA levels, again suggesting the involvement of posttranscriptional controls. Pretreatment of MDA‐MB‐468 and MDA‐MB‐231 cells with IGF‐I receptor antibody (αIR3) blocked the IGF‐I effect on IGFBP‐3 levels in the media in both cell lines and IGFBP‐2 and IGFBP‐5 secreted levels in MDA‐MB‐468 cell conditioned media. The addition of RA also blocked IGF‐I stimulation of IGFBP2 and IGFBP‐5 levels. Cycloheximide treatment completely blocked the RA and/or IGF‐I‐mediated modulation of these binding proteins, suggesting that these agents enhance IGFBP‐3, IGFBP‐2, and IGFBP‐5 synthesis and consequent secretion. MDA‐MB‐468 cells expressed IGFBP‐5 mRNA, whereas both MDA‐MB‐231 and MDA‐MB‐468 expressed IGFBP‐6 mRNA. RA enhanced IGFBP‐6 gene expression by threefold in MDA‐MB‐231 cells, whereas IGF‐1 had no effect on IGFBP‐6 gene expression in either cell line. These results demonstrate that RA and IGF‐I modulate IGFBP levels in a number of breast carcinoma cell lines. Such modulation of IGFBPs may in turn affect IGF mediated cellular responses and thus the biologic behavior of these malignant cells.