While the roles of ERα and ERβ in hormone-dependent diseases such as breast and prostate cancers are becoming increasingly elucidated, with ERα having a proliferative and ERβ having an anti-proliferative role, the mechanism by which these two receptors interact with each other in both normal and diseased states has remained elusive. Because the co-expression of ERβ along with ERα dampens the proliferative action of ERα, direct interaction of ERα and ERβ is thought to convey growth inhibitory effects, and the ERα/β heterodimer has been proposed to activate target genes mediating these anti-proliferative effects 
. However, the heterogeneous population of dimer pairs present when ERα and ERβ are co-expressed and the lack of full length heterodimerized ER structures prevent a clear understanding of their biological function. Thus, in order to shed light upon the biological action of these ERα/β heterodimers, we sought to identify small molecule ligands capable of specifically inducing heterodimers while not inducing ERα/α homodimers or ERβ/β homodimers with the rationale that these ligands could be used to decipher the biological action of ERα/β heterodimers.
The BRET technology developed in our lab 
allowed the examination of each ER dimer pair (ERα/α homodimers, ERβ/β homodimers, and ERα/β heterodimers) in isolation. This segregation was especially essential in the case of the ERα/β heterodimer, as the co-expression of ERα and ERβ leads to the formation of all three dimer forms and prevents separation of the action of each individual dimer pair as they function in concert in vivo
. However, the BRET assay allows the examination of ERα/β without observing homodimer formation. Specifically, we have previously shown that two phytoestrogens, genistein and liquiritigenin, preferentially induce different ER dimers 
. Liquiritigenin selectively induced formation of ERβ/β homodimers and ERα/β heterodimers but not ERα/α homodimers at 1 µM 
, which provides proof-of-principle that small molecule compounds which preferentially induce ERα/β heterodimers over ERα/α homodimers do indeed exist. We had further shown that BRET assays can be optimized for HTS 
. The goal of secondary HTS BRET screening in this study was to find a compound with similar characteristics to liquiritigenin but with greater ERα/β heterodimer selectivity. If a library compound was able to induce ERα/β heterodimerization while not inducing ER homodimerization, the ligand could be used in biological systems to determine the function of these heterodimers with minimal interference from either homodimer.
Two lead compounds were successfully identified in BRET screening. The two lead compounds are flavonoids, a group of potentially chemoprotective compounds widely distributed in fruit, vegetables, and beverages of plant origin including tea and wine, and have similar structures that consist of two phenolic benzene rings linked to a heterocyclic pyre or pyrone 
. Isoflavones represent an important group of phytoestrogens and are found mainly in plants belonging to the Leguminosae family. Angolensin (Trifolium pretense
, 2′,4′-dihydroxy-4″-methoxy-α-methyldeoxybenzoin, 1-(2,4-dihydroxyphenyl)-2-(4-methoxyphenyl)propan-1-one; CAS 642-39-7), is an isoflavone that was first isolated from the wood of Pterocarpus angolensis
and later from the wood and bark of Pterocarpus indicus
. Angolensin is a metabolite of Biochanin A and formononetin, which are present in red clover 
. Dietary supplements manufactured from red clover are widely marketed to provide beneficial health effects of isoflavones without dietary changes. Specifically, red clover supplements are often consumed for the purported alleviation of post-menopausal symptoms. Cosmosiin (apigenin 7-O-beta-glucoside; apigenin-7-D-glucoside; apigenin-7-O-beta-D-glucopyranoside; apigenin-7-glucoside; cosmetin, Cosmosiine, Apigetrin,5-hydroxy-2-(4-hydroxyphenyl)-7-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one; CAS 578-74-5) is a flavonoid present in chamomile flowers which are used pharmaceutically and cosmetically for their anti-spasmodic, anti-inflammatory and antimicrobial properties and also as a natural hair dye and fragrance. Cosmosiin has also been isolated from Veratrum grandiflorum
(white hellebore) and Kummerowia striata
(Korean clover). Cosmosiin has been shown to exhibit anti-inflammatory properties 
and has been shown to exhibit HIV anti-viral properties 
, although it has not received FDA approval for these purposes. The direct binding of angolensin and cosmosiin to the E2-binding pocket of ERs are observed (). To our knowledge, this is the first demonstration of cosmosiin as an estrogenic compound. Furthermore, we validated ERα/β-heterodimer specificity using BRET and reporter assays and showed that 1 µM cosmosiin and 10 µM angolensin are specific to ERα/β-heterodimers ().
Using ERα/β-heterodimer selective compounds at specific concentrations, we are able to show that the ERα/β-heterodimer is growth inhibitory. These compounds inhibit cell proliferation in HC11 and PC3 cells which co-express ERα and ERβ. Inhibition of cell growth () and migration () due to 1 µM cosmosiin and 10 µM angolensin is ablated with treatment of ICI 182,780 or the silencing of either ERα or ERβ in PC3-shERα and PC3-shERβ, respectively. These compounds, however, did not have an effect on ER-negative MDA-MB-231 and ERα-negative/ERβ-positive DU-145 cells, further supporting that the growth inhibitory effects observed with these compounds were dependent on expression of both ERα and ERβ. While these compounds appear to have little or no effect on ERα/α homodimerization and transcriptional activation in HEK293 BRET and ERE-luciferase assays employing exogenous ERs (), treatment of breast and prostate cancer cells expressing ERα at a much higher level than ERβ (PC3-shERβ) results in ERα-dependent growth increases (). This result is in agreement with the common theme that ERα is a major growth driver, and it also implicates the dependence of these compounds' growth effects on the relative expression ratio of ERα
ERβ, as these compounds ablate growth increases in PC3 and HC11, in which ERα and ERβ expression levels are relatively similar and heterodimerization may be favored 
. Taken together, these data suggest that the ratio of ERα
ERβ in the same tumor cells is extremely important for physiological effects of these compounds. While the data presented herein provide initial evidence for a growth-inhibitory function of the ERα/β heterodimer, identification of higher affinity compounds with greater ERα/β heterodimer selectivity will be needed to validate our findings since both compounds are weak agonists, and cosmosiin at 10 µM appears to have off-target effects.
Compounds exhibiting ERα/β heterodimer-selectivity may have therapeutic or preventive efficacy in hormone-dependent diseases. A recent study shows that the tamoxifen metabolite endoxifen is capable of degrading ERα 
, stabilizing ERβ, and inducing ERα/β heterodimerization in a concentration dependent manner 
. Tamoxifen is a widely-utilized FDA-approved breast cancer treatment and prevention drug. This finding suggests that tamoxifen's cancer preventive effects may be mediated by stimulation of ERα/β heterodimer formation. The possibility is supported by the fact that both ERs are expressed in normal mammary epithelial cells 
. Similarly, naturally-occurring estrogen-like compounds such as phytoestrogens, a group of plant-derived compounds with estrogenic and/or antiestrogenic activities hold promise for action as preventive or therapeutic ER-regulators via their abilities to mediate estrogenic responses tissue-specifically. Indeed, consumption of soy phytoestrogens has been correlated with decreased breast cancer risk 
, although these data remain somewhat controversial 
. Furthermore, consumption of genistein 
, resveratrol 
, and soy 
has been inversely correlated with prostate cancer risk. Although these compounds may stimulate the proliferative action of ERα when ERβ is lost in tumors, they may have preventative effects under normal physiological conditions when both ERs are expressed.
Furthermore, our examination of nuclear co-localization of ERα and ERβ within the same tumor cell using the AQUA® technology () support that ERα/β heterodimerization could potentially occur within tumor cells. Prior to these studies, the co-localization of ERα and ERβ within the same cell had not been examined. The punctate staining pattern suggests that ERα and ERβ are co-localized on DNA, and therefore may be transcriptionally active in these cells as ERα/β heterodimers. Furthermore, AQUA® analysis showed that the ERα
ERβ ratio is higher in malignant states compared to benign tissue samples, in agreement with the finding that ERβ levels often decrease in malignant breast cancers 
. The growth inhibitory effects of ERα/β heterodimers might due to their activation of different target genes from their respective homodimers. Recently, global ChIP-Seq analyses of ERα and ERβ target genes show that perfectly or imperfectly palindromic EREs are preferential binding sites for ERα/β heterodimers as compared to ERα/α or ERβ/β homodimers which are more flexible in DNA recognition 
. This is consistent with other reports that ERα/β heterodimers might regulate distinct genes 
. The ERα/β heterodimer-selective ligands identified in this study will allow identification of heterodimer target genes in cells co-expressing ERα and ERβ (e.g. PC3). While our findings implicate the ERα/β heterodimer as a putative preventative and therapeutic target for hormone-responsive cancers, this example highlights the imminent need to decipher the role these heterodimers in breast and prostate cancers.
In conclusion, these data provide a proof-of-principle that ERα/β heterodimer-selective ligands can inhibit cell growth and migration in ERα/ERβ-positive cells such as PC3 and HC11 when ERα and ERβ are expressed at similar levels. We also found that the compounds' growth effects depend on the relative expression levels of ERα and ERβ. Upon knockdown of ERβ in PC3 cells, cosmosiin increases PC3 cell growth in an ERα-dependent manner. Thus, more heterodimer selective ligands need to be identified to clarify whether the heterodimer-selective ligands become growth stimulatory when ERβ expression is lost in human tumors. Although more studies are needed to demonstrate the ERα/β heterodimer as a therapeutic target, the concept of inducing ERβ to pair with ERα, thus antagonizing ERα's proliferative function, is distinct from existing breast cancer therapeutic strategies of targeting ERα alone. We also suggest that the relative ERα and ERβ expression levels in patient tumors should be carefully evaluated to better understand the ER-targeted drugs' therapeutic performance, as many of these drugs have not been evaluated for their dimer selectivity, and ERβ expression in patient tumors is not routinely evaluated.