A naturally occurring estrogen receptor mutation results in increased estrogenicity of a tamoxifen analog.

We previously identified a codon 351 (Asp-->Tyr) mutant estrogen receptor (ER) in a tamoxifen-stimulated human breast tumor line. To examine its biological activity, we have constructed cell lines from the ER-negative human breast cancer cell line MDA-MB-231 that stably express either the wild type (S30) or mutant ER (BC-2). ER expression was confirmed by Western blot, ligand-binding studies, and ER-enzyme immunoassay. The growth characteristics of the S30 and BC-2 cell lines were compared when treated with estradiol, fixed-ring 4-hydroxytamoxifen [(fr) 4-OH TAM], or ICI 182,780. (fr) 4-OH TAM is a stable, high affinity tamoxifen analog. Many investigators have recognized that growth of ER-negative cell lines stably transfected with ER is inhibited by estradiol. Similarly, both S30 and BC-2 cell lines are inhibited by estradiol in a concentration-dependent manner. (fr) 4-OH TAM has no effect on S30 proliferation but inhibits the growth of BC-2 cells. The pure antiestrogen ICI 182,780 can block the growth-inhibitory effect of estradiol in both cell lines and the growth-inhibitory effect of (fr) 4-OH TAM in the BC-2 cells. In transient transfection analyses using a luciferase reporter plasmid containing two copies of the Xenopus vitellogenin A2 estrogen response element, estradiol stimulated luciferase transcription through both the wild type and mutant estrogen receptors, while (fr) 4-OH TAM stimulated transcription to a greater extent through the mutant receptor. These results demonstrate that the estrogenicity of (fr) 4-OH TAM is increased by binding to the codon 351 mutant ER, and that ER activation and growth inhibition are associated.