Ribozyme targeting demonstrates that the nuclear receptor coactivator AIB1 is a rate-limiting factor for estrogen-dependent growth of human MCF-7 breast cancer cells

Human breast tumorigenesis is promoted by the estrogen receptor pathway, and nuclear receptor coactivators are thought to participate in this process. Here we studied whether one of these coactivators, AIB1 (amplified in breast cancer 1), was rate-limiting for hormone-dependent growth of human MCF-7 breast cancer cells. We developed MCF-7 breast cancer cell lines in which the expression of AIB1 can be modulated by regulatable ribozymes directed against AIB1 mRNA. We found that depletion of endogenous AIB1 levels reduced steroid hormone signaling via the estrogen receptor alpha or progesterone receptor beta on transiently transfected reporter templates. Down-regulation of AIB1 levels in MCF-7 cells did not affect estrogen-stimulated cell cycle progression but reduced estrogen-mediated inhibition of apoptosis and cell growth. Finally, upon reduction of endogenous AIB1 expression, estrogen-dependent colony formation in soft agar and tumor growth of MCF-7 cells in nude mice was decreased. From these findings we conclude that, despite the presence of different estrogen receptor coactivators in breast cancer cells, AIB1 exerts a rate-limiting role for hormone-dependent human breast tumor growth.     

A life-long search for the molecular pathways of steroid hormone action

The O'Malley laboratory first showed that estrogen and progesterone act in the nucleus to stimulate synthesis of specific mRNAs (ovalbumin and avidin), coding for their respective inducible proteins. The overall molecular pathway of steroid-receptor-DNA-mRNA-protein-function was then established and provided a coherent foundation for future studies of the impact of estrogen and progesterone receptors on endocrine tissue development, adult function, and in pathologies such as cancer. The lab group went on to: biochemically demonstrate ligand-induced conformational activation of progesterone and estrogen receptors, discover the concept of ligand-independent activation of steroid receptors, discover key steroid receptor coactivator intermediary coactivators for receptor function, and define the role of coactivators/corepressors in selective receptor modulator drug action and in cell homeostasis. This body of work advanced our molecular understanding of the critical role of steroid hormones in normal and abnormal physiology and also generated a base of scientific knowledge that served to further modern hormonal therapy and disease management.     

A structural and in vitro characterization of asoprisnil: a selective progesterone receptor modulator

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.     

Three amino acids specify coactivator choice by retinoid X receptors

Binding of agonists to nuclear receptors results in a conformational change in receptor structure that promotes interaction between activated receptors and coactivators. Receptor-coactivator interactions are mediated by the agonist-dependent formation of a hydrophobic pocket on the part of receptors, and short leucine-rich sequences termed LxxLL motifs or nuclear receptor boxes present in coactivators. RXR-PPARgamma (retinoid X receptor-peroxisome proliferator-activated receptor-gamma) heterodimers play important roles in adipocyte and macrophage differentiation and have been implicated as therapeutic targets in diabetes, atherosclerosis, and cancer. Analysis of interactions between RXR-PPARgamma heterodimers and coactivator nuclear receptor boxes suggests that RXR and PPARgamma can distinguish among coactivators by recognizing distinct structural features of nuclear receptor boxes. The results also indicate that coactivator choice by RXR is mediated by three nonconserved amino acids of the nuclear receptor box. The ability of an optimized seven-amino acid nuclear receptor box to specifically interact with RXR and function as a selective inhibitor suggests the coactivator-binding pocket may serve as a new target for drug discovery.