Because little is known about the actions of botanical estrogens (BEs), widely consumed by menopausal women, we investigated the mechanistic and cellular activities of some major BEs. We examined the interactions of genistein, daidzein, equol, and liquiritigenin with estrogen receptors ERα and ERβ, with key coregulators (SRC3 and RIP140) and chromatin binding sites, and the regulation of gene expression and proliferation in MCF-7 breast cancer cells containing ERα and/or ERβ. Unlike the endogenous estrogen, estradiol (E2), BEs preferentially bind to ERβ, but their ERβ-potency selectivity in gene stimulation (340- to 830-fold vs. E2) is enhanced at several levels (coregulator recruitment, chromatin binding); nevertheless, at high (0.1 or 1 μM) concentrations, BEs also fully activate ERα. Because ERα drives breast cancer cell proliferation and ERβ dampens this, the relative levels of these two ERs in target cells and the BE dose greatly affect gene expression and proliferative response and will be crucial determinants of the potential benefits vs. risks of BEs. Our findings reveal key and novel mechanistic differences in the estrogenic activities of BEs vs. E2, with BEs displaying patterns of activity distinctly different from those seen with E2 and provide valuable information to inform future studies.

Estrogen receptor-α (ERα) and progesterone receptor (PR) are expressed in most human breast cancers and are important predictive factors for directing therapy. Because of de novo and acquired resistance to endocrine therapy, there remains a need to identify which ERα-positive (ERα+)/PR-positive (PR+) tumors are most likely to respond. The purpose of this study was to use estrogen- and progestin-based radiopharmaceuticals to image ERα and PR in mouse mammary tumors at baseline and after hormonal therapy and to determine whether changes in these imaging biomarkers can serve as an early predictive indicator of therapeutic response. Methods Mammary adenocarcinomas that spontaneously develop in aged female mice deficient in signal transducer and activator of transcription-1 (STAT1) were used. Imaging of ERα and PR in primary tumor–bearing mice and mice implanted with mammary cell lines (SSM1, SSM2, and SSM3) derived from primary STAT1-deficient (STAT1−/−) tumors was performed. Hormonal treatments consisted of estradiol, an ER agonist; letrozole, an aromatase inhibitor; and fulvestrant, a pure ER antagonist. Small-animal PET/CT was performed using 18F-fluoroestradiol (18F-FES) for ER, 18F-fluoro furanyl norprogesterone (18F-FFNP) for PR, and 18F-FDG for glucose uptake. Tracer uptake in the tumor was quantified and compared with receptor concentration determined by in vitro assays of resected tumors. Results Primary STAT1−/− mammary tumors and implanted SSM2 and SSM3 tumors showed high 18F-FES and 18F-FFNP uptake and were confirmed to be ERα+/PR+. Classic estrogen-induced regulation of the progesterone receptor gene was demonstrated by increased 18F-FFNP uptake of estradiol-treated SSM3 tumors. Treatment with fulvestrant decreased 18F-FFNP, 18F-FES, and 18F-FDG uptake and inhibited growth of SSM3 tumors but decreased only 18F-FES uptake in SSM2 tumors, with no effect on growth, despite both tumors being ERα+/PR+. Decreased 18F-FFNP uptake by SSM3 tumors occurred early after initiation of treatment, before measurable tumor growth inhibition. Conclusion Using small-animal PET, a profile was identified that distinguished fulvestrant-sensitive from fulvestrant-resistant ERα+/PR+ tumors before changes in tumor size. This work demonstrates that imaging baseline tumoral 18F-FES uptake and initial changes in 18F-FFNP uptake in a non-invasive manner is a potentially useful strategy to identify responders and nonresponders to endocrine therapy at an early stage.

The basis for the differential repressive effects of antiestrogens on transactivation by estrogen receptor-alpha (ERalpha) remains incompletely understood. Here, we show that the full antiestrogen ICI182,780 and, to a lesser extent, the selective ER modulator raloxifene (Ral), induce accumulation of exogenous ERalpha in a poorly soluble fraction in transiently transfected HepG2 or stably transfected MDA-MB231 cells and of endogenous receptor in MCF7 cells. ERalpha remained nuclear in HepG2 cells treated with either compound. Replacement of selected hydrophobic residues of ERalpha ligand-binding domain helix 12 (H12) enhanced receptor solubility in the presence of ICI182,780 or Ral. These mutations also increased transcriptional activity with Ral or ICI182,780 on reporter genes or on the endogenous estrogen target gene TFF1 in a manner requiring the integrity of the N-terminal AF-1 domain. The antiestrogen-specific effects of single mutations suggest that they affect receptor function by mechanisms other than a simple decrease in hydrophobicity of H12, possibly due to relief from local steric hindrance between these residues and the antiestrogen side chains. Fluorescence anisotropy experiments indicated an enhanced regional stabilization of mutant ligand-binding domains in the presence of antiestrogens. H12 mutations also prevent the increase in bioluminescence resonance energy transfer between ERalpha monomers induced by Ral or ICI182,780 and increase intranuclear receptor mobility in correlation with transcriptional activity in the presence of these antiestrogens. Our data indicate that ICI182,780 and Ral locally alter the ERalpha ligand binding structure via specific hydrophobic residues of H12 and decrease its transcriptional activity through tighter association with an insoluble nuclear structure.

The estrogen receptors, ER␣ and ER␤, are ligand-regulated transcription factors that control gene expression programs in target tissues. The molecular events underlying estrogen action involve minimally two steps, hormone binding to the ER ligandbinding domain followed by coactivator recruitment to the ER⅐ligand complex; this ligand⅐receptor⅐coactivator triple complex then alters gene expression. Conceptually, the potency of an estrogen in activating a cellular response should reflect the affinities that characterize both steps involved in the assembly of the active ligand⅐receptor⅐coactivator complex. Thus, to better understand the molecular basis of estrogen potency, we developed a completely in vitro system (using radiometric and timeresolved FRET assays) to quantify independently three parameters: (a) the affinity of ligand binding to ER, (b) the affinity of coactivator binding to the ER⅐ligand complex, and (c) the potency of ligand recruitment of coactivator. We used this system to characterize the binding and potency of 12 estrogens with both ER␣ and ER␤. Some ligands showed good correlations between ligand binding affinity, coactivator binding affinity, and coactivator recruitment potency with both ERs, whereas others showed correlations with only one ER subtype or displayed discordant coactivator recruitment potencies. When ligands with low receptor binding affinity but high coactivator recruitment potencies to ER␤ were evaluated in cell-based assays, elevation of cellular coactivator levels significantly and selectively improved their potency. Collectively, our results indicate that some low affinity estrogens may elicit greater cellular responses in those target cells that express higher levels of specific coactivators capable of binding to their ER complexes with high affinity.Estrogens of diverse structure are used for many clinical needs and various health benefits. Ethynylestradiol is used for fertility regulation (1, 2), and the drug Premarin (3, 4), which contains a mixture of 10 structurally different equine estrogens, is widely prescribed for menopausal hormone replacement therapy. Non-steroidal estrogens, such as diethylstilbestrol, are used to suppress androgen production in the treatment of prostate cancer (5, 6), and soy isoflavone extracts that contain the phytoestrogen genistein are consumed by older women as estrogen supplement for its possible beneficial effect in relieving some of the post-menopausal symptoms (7,8). In addition, estrogens selective for one of the estrogen receptor (ER) 2 subtypes, ER␤, are under active investigation for the management of breast, prostate, and colon cancers and specific cases of cardiovascular and central nervous system disorders (9, 10). Little is known, however, about what structural features of a particular estrogen and what molecular interactions it undergoes during its course of action contribute to its potency and to its selectivity of action through the two ER subtypes, ER␣ and ER␤.ER␣ and ER␤ are members of the nuclear receptor (NR) family of li...