Unliganded and liganded estrogen receptors protect against cancer invasion via different mechanisms

While estrogens are mitogenic in breast cancer cells, the presence of estrogen receptor alpha (ER alpha) clinically indicates a favorable prognosis in breast carcinoma. To improve our understanding of ER alpha action in breast cancer, we used an original in vitro method, which combines transient transfection and Matrigel invasion assays to examine its effects on cell invasiveness. ER alpha expression in MDA-MB-231 breast cancer cells reduced their invasiveness by 3-fold in the absence of hormone and by 7-fold in its presence. Integrity of hormone and DNA-binding domains and activating function 2 were required for estradiol-induced inhibition, suggesting that transcriptional activation of estrogen target genes was involved. In contrast, these domains were dispensable for hormone-independent inhibition. Analysis of deletion mutants of ER alpha indicated that amino acids 179-215, containing the N-terminal zinc finger of the DNA-binding domain, were required for ligand-independent receptor action. Among different members of the nuclear receptor family, only unliganded ER alpha and ER beta reduced invasion. Calreticulin, a Ca2+-binding protein that could interact with amino acids 206-211 of ER alpha, reversed hormone-independent ER alpha inhibition of invasion. However, since calreticulin alone also inhibited invasion, we propose that this protein probably prevents ER alpha interaction with another unidentified invasion-regulating factor. The inhibitor role of the unliganded ER was also suggested in three ER alpha-positive cell lines, where ER alpha content was inversely correlated with cell migration. We conclude that ER alpha protects against cancer invasion in its unliganded form, probably by protein-protein interactions with the N-terminal zinc finger region, and after hormone binding by activation of specific gene transcription.