Figure 1.
Flow scheme of high throughput screening and characterization of compounds with selectivity for ERα/ERβ heterodimers.
A library of >5200 small molecules was screened ER transcriptional activity using T47D-KBLuc cells. Molecules with transcriptional activity were then screened for ERα/α, ERα/β, or ER β/β dimerization potential using BRET assays. Two phytoestrogens, angolensin and cosmosiin, were identified as ER dimer selective ligands. These molecules were validated using in vitro binding assays and BRET and ERE-luciferase reporter assays. Heterodimer selective concentrations were identified as 10 µM angolensin and 1 µM cosmosiin. The cellular effects of these two heterodimer-selective concentrations were characterized using cell migration and proliferation assays.
Figure 2.
Fluorescence polarization competition binding assays for ERα and ERβ.
Cosmosiin (A) and angolensin (B) bind to recombinant ERα and ERβ with µM affinities.
Figure 3.
The dimer selectivity for cosmosiin and angolensin was demonstrated in dose-response BRET assays (A and B) and reporter assays (C and D) in HEK293 cells.
ER dimer-specific BRET assays were performed over a range of compound concentrations of cosmosiin (A) and angolensin (B). HEK293T ERE-luciferase transcriptional assays reveal each compound's ability to transcriptionally activate various dimer pairs (C and D). ERα alone, ERβ alone, or ERα+ERβ was transfected along with an ERE-luciferase element in order to test the ability of cosmosiin (C) and angolensin (D) to transcriptionally activate these various ER dimer pairs. RLU, relative luciferase units. Error bars represent standard deviations from the mean of triplicate samples. In BRET (A), p values indicate all pairs with statistical significance by the Student's T-Test.
Figure 4.
Determination of relative expression levels of ERα and ERβ in various cell lines.
Western blotting analyses of ERα and ERβ expression in HC11 (lane 1), PC3 (lane 2), PC3shERα (lane 3), PC3shERβ (lane 4), MDA-MB-231 (lane 5), and DU145 (lane 6).
Figure 5.
Cosmosiin and angolensin inhibit PC3 cell motility and migration.
(A) Wound healing assays showing the effect of 1 µM cosmosiin and 10 µM angolensin on ERα,β-positive PC3 cells. Vehicle (DMSO) treatment resulted in cell motility to fill the wound (top panels) that was inhibited by 1 µM cosmosiin (middle panels) and 10 µM angolensin (bottom panels). (B) Transwell migration assays measured the ability of cosmosiin (B) and angolensin (C) to inhibit cellular migration of PC3 cells toward a chemoattractant. Cosmosiin (B) and angolensin (C) decreased the ability of PC3 cells to migrate through the pore, and this decreased ability was ablated by the antagonist ICI 182,780 at 100 nM.
Figure 6.
ER dimer-selective compounds influence cell growth in an ER-dependent and dose-dependent manner.
Cosmosiin (A) and angolensin (B) decrease the growth of PC3 prostate cancer cells in a dose-dependent manner. These decreases are ER-specific for 1 µM cosmosiin and 10 µM angolensin, since the growth decreases are ablated by the ER antagonist ICI 182,780. These ER-specific effects by 1 µM cosmosiin (C) and 10 µM angolensin (D) are lost with the silencing of ERα in PC3-shERα cells, while ER non-specific effects due to 10 µM cosmosiin are retained. Silencing ERβ in PC3-shERβ results in cosmosiin-dependent increases in cell growth (E) that are ablated in the presence of the antagonist, and furthermore, when ERs are antagonized, ER non-specific growth inhibition in PC3-shERβ is retained. Angolensin has no statistically significant effects in PC3-shERβ (F). Error bars represent standard deviations from the mean of triplicate samples. * indicates statistical significance by the Student's T-Test.
Figure 7.
Automated quantitative measurement of ERα and ERβ expression in breast cancer tissue microarrays.
(A) Tissue microarray analysis with the AQUA® technology shows that the ERα∶ERβ ratio increases from benign samples throughout various stages of malignancy. Pairwise two-sample t-tests between the benign and malignant samples showed a statistically significant difference (p-values<0,01). (B) AQUA® analysis indicates that ERα and ERβ colocalize to the nucleus within the same cell in human breast tumors. Figures are shown at 200× magnification, and scale bars are present in the lower left corners of each image. The blow-up picture is an amplified section of AQUA staining showing co-localization of ERα and ERβ to the nucleus. * indicates statistical significance by the Student's T-Test. NAT = cancer adjacent normal breast tissue.