title: Molecular mechanism of action at estrogen receptor alpha of a new clinically relevant antiestrogen (GW7604) related to tamoxifen.
abstract: Tamoxifen is the endocrine treatment of choice for all stages of estrogen receptor (ER)-positive breast cancer, and it is the first drug approved to reduce the incidence of breast cancer in high-risk women. Unfortunately, tamoxifen also possesses some estrogen-like effects in the uterus that cause a modest increase in the risk of endometrial cancer. GW5638 is a tamoxifen derivative with a novel carboxylic acid side chain with no uterotropic activity 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, the active metabolite of tamoxifen) with GW7604 [the presumed metabolite of GW5638 in breast (MCF-7) and endometrial (ECC-1) cell lines in vitro]. GW7604 did not cause the growth of ECC-1 cells at any concentration (10(-11)-10(-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 estradiol (10(-10) M) in both ECC-1 and MCF-7 cells. Western blotting was used to show that GW7604 and raloxifene did not affect ER levels significantly, compared with controls, in MCF-7 cells; whereas the pure antiestrogen ICI182,780 decreased ER levels (P < 0.05). An assay system was used that can classify compounds into tamoxifen-like, raloxifene-like, or pure antiestrogens. The assay depends on the activation of the transforming growth factor alpha (TGFalpha) 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 estradiol (10(-9) M) and 4-OHT (10(-8), 10(-7) M) induction of TGFalpha in a concentration related manner (10(-9)-10(-6) M). GW7604 and raloxifene stimulated TGFalpha with the D351Y ER. In contrast, ICI 182,780 (10(-6) M) did not initiate TGFalpha and blocked the induction of TGFalpha with GW7604, raloxifene, and 4-OHT in D351Y-transfected cells. Using computer-assisted molecular models of ER complexes, we found that the antiestrogenic side chain of 4-OHT weakly interacted with the surface amino acid 351 (aspartate), but 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 disrupts 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.
authors.affiliation: Department of Surgery, The Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611, USA.
authors.forename: D
authors.initials: D
authors.lastname: Bentrem
authors.forename: R
authors.initials: R
authors.lastname: Dardes
authors.forename: H
authors.initials: H
authors.lastname: Liu
authors.forename: J
authors.initials: J
authors.lastname: MacGregor-Schafer
authors.forename: J
authors.initials: J
authors.lastname: Zapf
authors.forename: V
authors.initials: V
authors.lastname: Jordan
mesh_terms: D002277:Carcinoma / Q000473:pathology; D002455:Cell Division / Q000187:drug effects; D002934:Cinnamates / Q000737:chemistry / Q000494:pharmacology*; D016889:Endometrial Neoplasms / Q000473:pathology; D020847:Estrogen Receptor Modulators / Q000494:pharmacology*; D047628:Estrogen Receptor alpha; D004967:Estrogens / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D008958:Models, Molecular; D012333:RNA, Messenger / Q000378:metabolism; D011960:Receptors, Estrogen / Q000187:drug effects* / Q000378:metabolism; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology; D013267:Stilbenes / Q000737:chemistry / Q000494:pharmacology*; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology; D016211:Transforming Growth Factor alpha / Q000235:genetics; D014407:Tumor Cells, Cultured
chemical_list: D002934:Cinnamates; D020847:Estrogen Receptor Modulators; D047628:Estrogen Receptor alpha; D004967:Estrogens; C401858:GW 7604; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D020845:Selective Estrogen Receptor Modulators; D013267:Stilbenes; D016211:Transforming Growth Factor alpha; D013629:Tamoxifen; C016601:afimoxifene