INSULIN-LIKE GROWTH-FACTOR BINDING-PROTEIN 3 MEDIATES RETINOIC ACID-NDUCED AND TRANSFORMING GROWTH-FACTOR BETA-2-INDUCED GROWTH-INHIBITION IN HUMAN BREAST-CANCER CELLS

Retinoic acid (RA) is a potent in vitro inhibitor of cell proliferatio n in various malignant cell lines. The exact mechanisms of its actions , however, are not fully understood. To further elucidate the nature o f this inhibition, we investigated the effects of RA in an estrogen re ceptor-negative human breast cancer cell line, MDA-MB-231. RA (0.01-5 mu M) significantly inhibited MDA-MB-231 cell growth by 35-40% as comp ared with untreated controls, Similar growth inhibitory actions were o bserved when cells were treated with transforming growth factor beta 2 (TGF-beta 2), another factor with antiproliferative actions in breast cancer cells. Both RA and TGF-beta 2 increased the levels of insulin- like growth factor binding protein (IGFBP) 3 (2-3-fold) and mRNA (1.5- 2-fold), whereas IGFBP-4 levels remained essentially unchanged. The di rect involvement of IGFBP-3 in cell growth inhibition was further conf irmed by its action on cell growth: exogenous IGFBP-3 directly and sig nificantly inhibited MDA-MB-231 cell number by 40%. These results prov ided circumstantial evidence that IGFBP-3 may mediate RA and TGF-beta 2 growth inhibitory actions in human breast cancer cells. To test this hypothesis, we used an antisense IGFBP-3 oligodeoxynucleotide (ODN) w hich specifically inhibits IGFBP-3 expression, The antisense IGFBP-3 O DN dramatically blocked both RA- and TGF-beta 2-induced increases in I GFBP-3 protein (90%) and mRNA levels (90%). This effect was not observ ed when RA- or TGF-beta 2-exposed cells were treated with sense IGFBP- 3 ODN. Moreover, antisense ODN did not significantly affect IGFBP-4 pr otein or mRNA levels, strongly supporting the specificity of the antis ense IGFBP-3 ODN effect on IGFBP-3 mRNA. This specific effect of antis ense IGFBP-3 ODN on IGFBP-3 protein and mRNA levels was accompanied by significant attenuation of the inhibition of cell proliferation attai ned with RA or TGF-beta 2 (approximately 40% of either RA- or TGF beta 2-induced inhibition). The control sense IGFBP-3 ODN did not reduce t he growth inhibition observed with either RA or TGF-beta 2. These resu lts indicate that IGFBP-3 is an important mediator of RA-and TGF-beta 2-induced cell growth inhibition in human breast cancer cells.