A MODEL TO DESCRIBE HOW A POINT MUTATION OF THE ESTROGEN-RECEPTOR ALTERS THE STRUCTURE-FUNCTION RELATIONSHIP OF ANTIESTROGENS

The antiestrogen tamoxifen [(Z)-1(p-beta-dimethylamino-ethoxyphenyl)-1,2-diphenylbut-1-ene] is an effective anticancer agent for the treatment of hormone responsive breast cancer. Previous studies have demonstrated that a point mutation in the estrogen receptor (ER) resulted in an alteration of the pharmacology of 4-hydroxytamoxifen, the active metabolite of tamoxifen (Jiang et al, Mol Endocrinol 6:2167-2174, 1992). We have extended our studies to evaluate the effect of a point mutation, a Val substitution for Gly at amino acid 400 in the ligand binding domain of ER, on the pharmacology of other antiestrogens in ER stable transfectants derived from the ER-negative breast cancer cell line MDA-MB-231 CL10A. The compounds were tested with or without estradiol-17beta (E2) for their effects on cell growth in cells expressing the wild type ER (S30) or the mutant ER (MLalpha2H) or in control antisense ER transfectant AS23 which does not express ER protein. MCF-7 cells, which express the wild type ER, were also used as a control. The growth of AS23 cells was not affected by any of the compounds at a concentration of 1 muM. E2 stimulated the growth of MCF-7 cells but inhibited the growth of ER transfectants S30 and MLalpha2H. The MLalpha2H cells were about 10 to 100-fold less sensitive to E2 and antiestrogens than S30 and MCF-7 cells. Keoxifene, an antiestrogen with a high affinity for the ER, maintained antiestrogenic activities in both ER transfectants and MCF-7 cells. However, the pharmacology of the steroidal antiestrogen RU 39411 was altered in the MLalpha2H cells. The compound did not block the effects of E2 but acted as an estrogen. Overall, this and previous studies from this laboratory demonstrate that it is possible to predict that the mutation will enhance the estrogenic activity of antiestrogens which have a side chain projecting in a position analogous to that of 4-hydroxytamoxifen. In contrast, compounds that do not have a side chain that occupies the same area, e.g. keoxifene and ICI 164,384, are unaffected by the mutation. We have extended our previous model (Lieberman et al, J Biol Chem 258:4741-4745, 1983) to incorporate our new observations.