The retinoic acid receptor antagonist, BMS453, inhibits normal breast cell growth by inducing active TGFbeta and causing cell cycle arrest.

Abstract

We have previously shown that a retinoic acid receptor (RAR) antagonist BMS453, which does not activate RAR-dependent gene transcription in breast cells, inhibits normal breast cell growth. In this study we have investigated the mechanisms by which this retinoid receptor antagonist inhibits cell growth. Both all trans retinoic acid (atRA) and BMS453 inhibited the proliferation of normal breast cell growth without significantly inducing apoptosis. Both retinoids caused a G1 block in the cell cycle with an increase in the proportion of cells in G0/G1 and a decrease in the proportion of cells in S phase. We then investigated the effects of the retinoids on molecules that regulate the G1 to S transition. These studies demonstrated that both atRA and BMS453 induce Rb hypophosphorylation and decrease CDK2 kinase activity. We then studied the effect of the retinoids on the expression of CDK inhibitors. atRA and BMS453 increased total p21 protein levels and CDK2-bound p21 protein, but did not change CDK4-bound p21. These results suggest that atRA and BMS453 increase p21, decrease CDK2 kinase activity, which in turn leads to hypophosphorylation of Rb and G1 arrest. Because transforming growth factor beta (TGFbeta) has been proposed as a mediator of retinoid-induced growth inhibition, we next investigated whether TGFbeta mediates the anti-proliferative effect of atRA and BMS453 in normal breast cells. These studies showed that atRA and BMS453 increased total TGFbeta activity by 3-5-fold. However, BMS453 increased active TGFbeta activity by 33-fold while atRA increased active TGFbeta activity by only threefold. These results suggest that BMS453 treatment induces conversion of latent TGFbeta to active TGFbeta. To investigate whether this increase in active TGFbeta mediates the anti-proliferative effects of these retinoids, a TGFbeta-blocking antibody was used in an attempt to prevent retinoid-induced growth inhibition. Results from these experiments showed that the anti-TGFbeta antibody prevented the inhibition of cell proliferation induced by BMS453, but did not prevent the inhibition of cell proliferation induced by atRA. These results demonstrate that BMS453 inhibits breast cell growth predominantly through the induction of active TGFbeta, while atRA inhibits growth through other mechanisms. These results suggest that retinoid analogs that increase active TGFbeta may be promising agents for the prevention of breast cancer.