The estrogen receptor alpha (ERa) signaling plays an essential role in breast cancer progression and endocrine therapy. Mitogen-activated protein kinase (MAPK/ Erk1/2) has been implicated in ligand-independent activation of ER, resulting in the cross-talk between growth factor and ER mediated signaling. In this study, we examined the e ect of the cross-talk on estradiol (E 2 )-mediated signaling, tumor growth and its e ect on anti-estrogen therapy. Our ®ndings demonstrate that expression of constitutively activated mitogen activated kinase kinase (MEK1), an immediate upstream activator of MAPK in estrogen receptor positive MCF-7 breast cancer cells (MEK/MCF-7), showed an increase in ERadriven transcriptional activation. In MEK/MCF-7 cells maximal transactivation levels were achieved in response to treatment with much lower E 2 concentrations (10 710 M E 2 ) when compared to MCF-7 control cells (10 78 M E 2 ). Furthermore, we have seen an increased association between ERa and its nuclear coactivators AIB1 or TIF-2, in MEK/MCF-7 cells relative to those seen in MCF-7 control cells. In addition, in vivo studies show that MEK/ MCF-7 cell tumors are *threefold larger than those of MCF-7 cell, in the presence of E 2 . Immunohistochemical staining demonstrates that progesterone receptor (PR) and pS2, two E 2 -regulated gene products, are signi®-cantly increased in MEK/MCF-7 cell tumors compared to those of MCF-7 control tumors, suggesting that activation of ERa by MAPK enhances the expression of E 2 -regulated genes and accelerates tumor growth. Remarkably, the antiestrogens tamoxifen and ICI 182,780, were shown both in vitro and in vivo studies to e ciently antagonize the stimulatory e ects of E 2 on ER regulated transactivation and tumor growth in MEK/ MCF-7 as well as MCF-7 cell lines. Taken together, these data suggest that MAPK/ER cross-talk enhances ERa-mediated signaling and accelerates E 2 -dependent tumor growth without diminishing sensitivity to the inhibitory e ects of anti-estrogens.

The human estrogen receptor-␣, a member of the nuclear receptor superfamily, is a ligand-regulated transcriptional modulator. Because comparatively little is known about the extreme carboxyl-terminal region of the estrogen receptor (F domain), we used secondary structure prediction to design mutations that delete the F domain (S554stop), disrupt a possible turn (G556L/ G557L), and alter a predicted helix (S559A/E562A, Q565P), and we evaluated the effects of these mutations on hormone binding and transcription activation in response to estradiol and the mixed agonist/antagonist 4-hydroxytamoxifen. Mutations that deleted the F domain (S554stop) or targeted the predicted helix (S559A/ E562A, Q565P) greatly reduced or eliminated the agonist activity of 4-hydroxytamoxifen. Deleting the F domain increased the affinity of the receptor for estradiol and decreased the antagonist activity of 4-hydroxytamoxifen. The Q565P mutant exhibited a non-cooperative hormone-binding mechanism, as well as an impaired response to estradiol and increased antagonist activity of 4-hydroxytamoxifen. Our results show that mutations in the F domain alter not only the response to estradiol, the affinity for hormone, and the interaction between receptor subunits but can uncouple the agonist and antagonist activities of 4-hydroxytamoxifen. These results suggest that the F domain modulates the activity of the estrogen receptor-␣ by multiple mechanisms.

The estrogen receptor (ER), of which there are two forms, ER␣ and ER␤, is a ligand-modulated transcription factor important in both normal biology and as a target for agents to prevent and treat breast cancer. Crystallographic studies of the ER␣ ligand-binding domain suggest that Leu-536 may be involved in hydrophobic interactions at the start of a helix, "helix 12," that is crucial in the agonist-stimulated activity of ER␣, as well as in the ability of antagonists to block the activity of ER␣. We found that certain mutations of Leu-536 increased the ligand-independent activity of ER␣ although greatly reducing or eliminating the agonist activity of 17␤-estradiol (E 2 ) and 4-hydroxytamoxifen (4OHT), on an estrogen response element-driven and an AP-1-driven reporter. The mutations impaired the interaction of the ER ligand-binding domain with the SRC1 receptor-interacting domain in a mammalian two-hybrid system. When tested in the yeast two-hybrid system, mutation of Leu-536 increased the basal reactivity of ER␣ to probes that recognize the agonist-bound conformation but did not significantly alter its reactivity to these probes in the presence of E 2 . Most interestingly, mutation of Leu-536 reduced the interaction of the 4OHT-bound ER␣ and increased the reactivity of the raloxifene-or ICI 182,780-bound ER␣, with probes that recognize the 4OHT-bound ER␣ conformation in a yeast two-hybrid system. These results show that Leu-536 is critical in coupling the binding of ligand to the modulation of the conformation and activity of ER␣.