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Molecular Mechanism of Action at Estrogen Receptor of a New Clinically Relevant Antiestrogen (GW7604) Related to Tamoxifen¹

Department of Surgery (D.J.B.), The Robert H. Lurie Comprehensive Cancer Center (R.C.D., H.L., J.M.S., V.C.J.), Northwestern University Medical School, Chicago, Illinois 60611; and Signal Pharmaceutical (J.W.Z.), San Diego, California 92121

Address all correspondence and requests for reprints to: V. Craig Jordan, The Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Olson Pavilion 8258, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: vcjordan{at}northwestern.edu

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 (TGF) 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 TGF in a concentration related manner (10^-9–10^-6 M). GW7604 and raloxifene stimulated TGF with the D351Y ER. In contrast, ICI 182,780 (10^-6 M) did not initiate TGF and blocked the induction of TGF 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.

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