Molecular mechanism of action at estrogen receptor alpha of a new clinically relevant antiestrogen (GW7604) related to tamoxifen

Tamoxifen is the endocrine treatment of choice for all stages of estrogen r eceptor (ER)-positive breast cancer, and it is the first drug approved to r educe the incidence of breast cancer in high-risk women. Unfortunately, tam oxifen also possesses some estrogen-like effects in the uterus that cause a modest increase in the risk of endometrial cancer. GW5638 is a tamoxifen d erivative with a novel carboxylic acid side chain with no uterotropic activ ity in the rat (Willson et al., J Med Chem, 1994, 37:1550-1552). We have compared and contrasted the actions of 4-hydroxytamoxifen (4-OHT, t he active metabolite of tamoxifen) with GW7604 [the presumed metabolite of GW5638 in breast (MCF-7) and endometrial (ECC-1) cell lines in, vitro]. GW7 604 did not cause the growth of ECC-1 cells at any concentration (10(-11)-1 0(-6) M), but 4-OHT was weakly estrogen-like at low concentrations (10(-11) -10(-10) M). Compounds (10(-7) M) blocked the growth promoting action of es tradiol (10(-10) M) in both ECC-1 and MCF-7 cells. Western blotting was use d to show that GW7604 and raloxifene did not affect ER levels significantly , compared with controls, in MCF-7 cells; whereas the pure antiestrogen ICI 182,780 decreased ER levels (P < 0.05). An assay system was used that can classify compounds into tamoxifen-like, r aloxifene-like, or pure antiestrogens. The assay depends on the activation of the transforming growth factor <alpha> (TGF alpha) gene in situ by wild- type or D351Y mutant ER stably transfected into MDA-MB-231 cells (MacGregor -Schafer et al., Cancer Res, 1999, 59: 4308-4313). GW7604 inhibited both es tradiol(10(-9) hi) and 4-OHT (10(-8), 10(-7) M) induction of TGF alpha in a concentration related manner (10(-9)-10(-6) M). GW7604 and raloxifene stim ulated TGF alpha with the D351YER. In contrast, ICI 182,780 (10(-6) M) did not initiate TGF alpha and blocked the induction of TGF alpha with GW7604, raloxifene, and 4-OHT in D351Y-transfected cells. Using computer-assisted m olecular models of ER complexes, we found that the antiestrogenic side chai n of 4-OHT weakly interacted with the surface amino acid 351 (aspartate), b ut the carboxylic acid of GW7604 caused a strong repulsion of aspartate 351 . We propose that GW7604 is less estrogen-like than 4-OHT, because it disru pts the surface charge around aa351 required for coactivator docking in the 4-OHT:ER complex. This charge is restored in the D351Y ER, thus converting GW7604 from an antiestrogen to an estrogen-like molecule.