Key elements for discriminating selective estrogen receptor modulators and selective receptor estrogen down regulators: a molecular approach to classify antiestrogens.

Abstract

Abstract Abstract #3036 Breast cancer is the most common type of malignancy among women in the world. About 60% of breast tumours express the oestrogen receptor alpha (ERα) and are considered hormone-responsive. Thus endocrine therapies have been the treatment of choice. However, the estrogen-like agonist effect of the available selective estrogen receptor modulators such as tamoxifen and the development of resistances require the development of new treatments that act through different mechanisms.
 The objective of our study is to design new anti-estrogens and tools that can help to understand the molecular mechanisms involved in ligand dependent modulation or degradation of ERα. It has been proposed that ligand dependent ERα degradation of antagonist may result from the presence of an aliphatic side chain on a steroidal body. Our aim is to exploit the structural characteristics of anti-estrogens to create new compounds with different lateral side chains for the study of the relationship between structure and activity of antiestrogens.
 We selected the following compounds: RU 39411, RU 58668, which have similar structures of the steroid body, but different side-chain motifs: RU 39411 has a side chain similar to that of Tamoxifen, while RU 58668 has one similar to Faslodex. The third compound, EM 652, has a body of intermediate structure between stilbestrol and oestradiol while the side chain is similar to that of Raloxifen.
 First, we analysed the degradation of ERα in the presence of the different ligands: Fulvestrant and RU 58668 induce degradation of ERα,in a protasome dependent manner, while RU 39411 and EM 652 have the same effect as Tamoxifen and stabilize ERα. To further analyze the molecular mechanism we generated three mutants of ERα; L540Q (in H12 of ligand binding domain), K362A (interaction with co-activators,) and A350V-D351Y (interaction with side chain of antiestrogens).
 Using transient transfection of the different mutant constructs of MDA-MB231(ER-negative) cells, we tested the effect of the ligands on gene expression of a luciferase reporter gene under the control of an estrogen inducible promoter and ligand-induced degradation of ER. Our results demonstrate that antiestrogens can be classified into two groups based on structure and function: Tamoxifen and RU39411 fall into a first group, and Faslodex and RU58668 into a second group. However EM 652 has an intermediate behavior. Preliminary results on stable cell lines show that results from transient transfections can be reproduced.and confirm the importance of the integrity of H12 for antagonist activity. Molecular modelisation demonstrates that variations in the structure of the ER ligand binding domain in the presence of different antiestrogens correlate with functional differences.
 We have used molecular studies to screen and classify antiestrogen compounds into SERM or SERD with the aim to develop novel molecules for endocrine treatment of breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3036.