Autocrine TGFβ signaling mediates vitamin D3 analog-induced growth inhibition in breast cells

In this study, we address whether TGF beta signaling mediates vitamin D3 analog-induced growth inhibition in nonmalignant and malignant breast cells. Normal mammary epithelial cells (184), immortalized nonmalignant mammary epithelial cells (184A1 and MCF10A), and breast cancer cells (early passage MCF7: MCF7E) were sensitive to the inhibitory effects of vitamin D3 analogs (EB1089 and MC1288) while late passage MCF7 breast cancer (MCF7L) cells were relatively resistant. A similar pattern of sensitivity to TGF beta was observed with these cells. Thus, the sensitivity to the vitamin D3 analogs correlated with the sensitivity to TGF beta. MCF7L TGF beta RII-transfected cells, which have autocrine TGF beta activity, were more sensitive to EB1089 than MCF7L cells. TGF beta neutralizing antibody was found to block the inhibitory effects of these analogs. These results are consistent with the idea that autocrine TGF beta signaling mediates the anti-proliferative effects of the vitamin D3 analogs in these cells. The expression of TGF beta isoforms and/or TGF beta receptors was induced by the analogs in the vitamin D3 and TGF beta sensitive cells. Vitamin D3 analogs did not induce TGF beta or TGF beta receptor expression in the resistant MCF7L cel Is. Therefore, EB1089 induces autocrine TGF beta activity through increasing expression of TGF beta isoforms and/or TGF beta receptors. In addition, EB1089 induced nuclear VDR protein levels in the sensitive 184A1 cells but not in the resistant MCF7L cells. 184A1 cells were more sensitive to EB1089-induced VDR-dependent transactivation than MCF7L cells as measured by a luciferase reporter construct containing the VDRE, indicating a defect of VDR signaling in MCF7L cells. Smad3, a TGF beta signaling mediator, coactivated VDR-dependent transactivation in 184A1 cells but not in MCF7L cells. These results indicate that Smad3 coactivates VDR to further enhance TGF beta signaling and vitamin D3 signaling in the sensitive 184A1 cells. The results also indicate that Smad3 is not of itself sufficient to coactivate VDR in TGF beta /vitamin D3 resistant MCF7L cells and other factors are required. We found that the PI 3-kinase pathway inhibitor LY29004 inhibited the synergy of TGF beta and EB1089 on VDR-dependent transactivation activity. This indicates that the crosstalk between TGF beta and vitamin D signaling is also PI 3-kinase pathway dependent.