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Identification of estrogen receptor dimer selective ligands reveals growth-inhibitory effects on cells that co-express ER? and ER?.

ABSTRACT: Estrogens play essential roles in the progression of mammary and prostatic diseases. The transcriptional effects of estrogens are transduced by two estrogen receptors, ER? and ER?, which elicit opposing roles in regulating proliferation: ER? is proliferative while ER? is anti-proliferative. Exogenous expression of ER? in ER?-positive cancer cell lines inhibits cell proliferation in response to estrogen and reduces xenografted tumor growth in vivo, suggesting that ER? might oppose ER?'s proliferative effects via formation of ER?/? heterodimers. Despite biochemical and cellular evidence of ER?/? heterodimer formation in cells co-expressing both receptors, the biological roles of the ER?/? heterodimer remain to be elucidated. Here we report the identification of two phytoestrogens that selectively activate ER?/? heterodimers at specific concentrations using a cell-based, two-step high throughput small molecule screen for ER transcriptional activity and ER dimer selectivity. Using ER?/? heterodimer-selective ligands at defined concentrations, we demonstrate that ER?/? heterodimers are growth inhibitory in breast and prostate cells which co-express the two ER isoforms. Furthermore, using Automated Quantitative Analysis (AQUA) to examine nuclear expression of ER? and ER? in human breast tissue microarrays, we demonstrate that ER? and ER? are co-expressed in the same cells in breast tumors. The co-expression of ER? and ER? in the same cells supports the possibility of ER?/? heterodimer formation at physio- and pathological conditions, further suggesting that targeting ER?/? heterodimers might be a novel therapeutic approach to the treatment of cancers which co-express ER? and ER?.

SUBMITTER: Powell E

PROVIDER: S-EPMC3274540 | biostudies-other | 2012

REPOSITORIES: biostudies-other

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Identification of estrogen receptor dimer selective ligands reveals growth-inhibitory effects on cells that co-express ERα and ERβ.

Powell Emily E Shanle Erin E Brinkman Ashley A Li Jun J Keles Sunduz S Wisinski Kari B KB Huang Wei W Xu Wei W

PloS one 20120207 2

Estrogens play essential roles in the progression of mammary and prostatic diseases. The transcriptional effects of estrogens are transduced by two estrogen receptors, ERα and ERβ, which elicit opposing roles in regulating proliferation: ERα is proliferative while ERβ is anti-proliferative. Exogenous expression of ERβ in ERα-positive cancer cell lines inhibits cell proliferation in response to estrogen and reduces xenografted tumor growth in vivo, suggesting that ERβ might oppose ERα's prolifera ...[more]

PMID: 22347418

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Project description:AimTo discover novel ligands of estrogen receptor (ER) β using pharmacophore mapping and structure-based screening.MethodsA computer-aided strategy combining pharmacophore mapping and structure-based screening was used to screen the Maybridge and Enamine databases. Yeast two-hybrid (Y2H) assay was used to detect the activity and selectivity of the chosen compounds. The transcriptional activities of the chosen compounds were demonstrated with luciferase reporter assays. The anti-proliferative effects of ER antagonists against MCF-7 and MDA-MB-231 breast cancer cells were examined using MTT assay, and the mechanisms of action were analyzed with flow cytometry analysis and Western blotting.ResultsThrough in silico screen, 95 compounds were chosen for testing in Y2H assay, which led to 20 potent ligands, including 10 agonists, 8 antagonists and 2 partial agonists with EC50 or IC50 values at μmol/L. Furthermore, 6 agonists exhibited absolute selectivity for ERβ, and 3 agonists showed higher selectivity for ERβ. The agonists 1g and 1h (10, 25, and 50 μmol/L) dose-dependently increased ER transcriptional activities, whereas the antagonists 2a and 2d (10, 25, and 50 μmol/L) caused dose-dependent inhibition on the activities. The antagonists and partial agonists at 100 μmol/L suppressed the proliferation of ERα positive MCF-7 cells and ERβ positive MDA-MB-231 cells, but were more effective against MDA-MB-231 cells. Treatment of MDA-MB-231 cells with antagonists 2a and 2d (25 and 50 μmol/L) dose-dependently increased the population of cells in the S phase. Both 2a and 2d treatment dose-dependently decreased the expression levels of cyclin A and CDK2. Meanwhile, the downregulation of cyclin E was only caused by 2d, while 2a treatment did not cause significant changes in the protein levels of cyclin E.ConclusionThe selective ligands discovered in this study are promising drug candidates to be used as molecular probes to explore the differences between ERα and ERβ.

Project description:UnlabelledOur understanding of estrogen (17β-estradiol, E2) receptor biology has evolved in recent years with the discovery and characterization of a 7-transmembrane-spanning G protein-coupled estrogen receptor (GPER/GPR30) and the development of GPER-selective functional chemical probes. GPER is highly expressed in certain breast, endometrial, and ovarian cancers, establishing the importance of noninvasive methods to evaluate GPER expression in vivo. Here, we developed (99m)Tc-labeled GPER ligands to demonstrate the in vivo status of GPER as an estrogen receptor (ER) and for GPER visualization in whole animals. A series of (99m)Tc(I)-labeled nonsteroidal tetrahydro-3H-cyclopenta[c]quinolone derivatives was synthesized utilizing pyridin-2-yl hydrazine and picolylamine chelates. Radioligand receptor binding studies revealed binding affinities in the 10 to 30 nmol/L range. Cell signaling assays previously demonstrated that derivatives retaining a ketone functionality displayed agonist properties, whereas those lacking such a hydrogen bond acceptor were antagonists. In vivo biodistribution and imaging studies performed on mice bearing human endometrial and breast cancer cell xenografts yielded significant tumor uptake (0.4-1.1%ID/g). Blocking studies revealed specific uptake in multiple organs (adrenals, uterus, and mammary tissue), as well as tumor uptake with similar levels of competition by E2 and G-1, a GPER-selective agonist. In conclusion, we synthesized and evaluated a series of first-generation (99m)Tc-labeled GPER-specific radioligands, demonstrating GPER as an estrogen-binding receptor for the first time in vivo using competitive binding principles, and establishing the utility of such ligands as tumor imaging agents. These results warrant further investigation into the role of GPER in estrogen-mediated carcinogenesis and as a target for diagnostic/therapeutic/image-guided drug delivery.ImplicationsThese studies provide a molecular basis to evaluate GPER expression and function as an ER through in vivo imaging.

Dataset Information

Identification of estrogen receptor dimer selective ligands reveals growth-inhibitory effects on cells that co-express ER? and ER?.

Publications

Identification of estrogen receptor dimer selective ligands reveals growth-inhibitory effects on cells that co-express ERα and ERβ.

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