Structural and Molecular Mechanisms of Cytokine-Mediated Endocrine Resistance in Human Breast Cancer Cells.

Human breast cancers that exhibit high proportions of immune cells and elevated levels of pro-inflammatory cytokines predict poor prognosis. Here, we demonstrate that treatment of human MCF-7 breast cancer cells with pro-inflammatory cytokines results in ERα-dependent activation of gene expression and proliferation, in the absence of ligand or presence of 4OH-tamoxifen (TOT). Cytokine activation of ERα and endocrine resistance is dependent on phosphorylation of ERα at S305 in the hinge domain. Phosphorylation of S305 by IKKß establishes an ERα cistrome that substantially overlaps with the estradiol (E2)-dependent ERα cistrome. Structural analyses suggest that S305-P forms a charge-linked bridge with the C-terminal F domain of ERα that enables inter-domain communication and constitutive activity from the N-terminal coactivator-binding site, revealing the structural basis of endocrine resistance. ERα therefore functions as a transcriptional effector of cytokine-induced IKKß signaling, suggesting a mechanism through which the tumor microenvironment controls tumor progression and endocrine resistance.

Dual-mechanism estrogen receptor inhibitors.

Efforts to improve estrogen receptor-α (ER)-targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, noncanonical structural perturbations of the receptor ligand-binding domain (LBD) to antagonize proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Our structural analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access nonconsensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity.

Predictive features of ligand-specific signaling through the estrogen receptor.

Some estrogen receptor-α (ERα)-targeted breast cancer therapies such as tamoxifen have tissue-selective or cell-specific activities, while others have similar activities in different cell types. To identify biophysical determinants of cell-specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X-ray crystallography. Ligands that regulate the dynamics and stability of the coactivator-binding site in the C-terminal ligand-binding domain, called activation function-2 (AF-2), showed similar activity profiles in different cell types. Such ligands induced breast cancer cell proliferation in a manner that was predicted by the canonical recruitment of the coactivators NCOA1/2/3 and induction of the GREB1 proliferative gene. For some ligand series, a single inter-atomic distance in the ligand-binding domain predicted their proliferative effects. In contrast, the N-terminal coactivator-binding site, activation function-1 (AF-1), determined cell-specific signaling induced by ligands that used alternate mechanisms to control cell proliferation. Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ERα.

Ligand Accessibility and Bioactivity of a Hormone-Dendrimer Conjugate Depend on pH and pH History.

Estrogen conjugates with a polyamidoamine (PAMAM) dendrimer have shown remarkably selective regulation of the nongenomic actions of estrogens in target cells. In response to pH changes, however, these estrogen-dendrimer conjugates (EDCs) display a major morphological transition that alters the accessibility of the estrogen ligands that compromises the bioactivity of the EDC. A sharp break in dynamic behavior near pH 7 occurs for three different ligands on the surface of a PAMAM-G6 dendrimer: a fluorophore (tetramethylrhodamine [TMR]) and two estrogens (17α-ethynylestradiol and diphenolic acid). Collisional quenching and time-resolved fluorescence anisotropy experiments with TMR-PAMAM revealed high ligand shielding above pH 7 and low shielding below pH 7. Furthermore, when the pH was cycled from 8.5 (conditions of ligand-PAMAM conjugation) to 4.5 (e.g., endosome/lysosome) and through 6.5 (e.g., hypoxic environment) back to pH 8.5, the 17α-ethynylestradiol- and diphenolic acid-PAMAM conjugates experienced a dramatic, irreversible loss in cell stimulatory activity; dynamic NMR studies indicated that the hormonal ligands had become occluded within the more hydrophobic core of the PAMAM dendrimer. Thus, the active state of these estrogen-dendrimer conjugates appears to be metastable. This pH-dependent irreversible masking of activity is of considerable relevance to the design of drug conjugates with amine-bearing PAMAM dendrimers.

Neural-Network Scoring Functions Identify Structurally Novel Estrogen-Receptor Ligands.

The magnitude of the investment required to bring a drug to the market hinders medical progress, requiring hundreds of millions of dollars and years of research and development. Any innovation that improves the efficiency of the drug-discovery process has the potential to accelerate the delivery of new treatments to countless patients in need. "Virtual screening," wherein molecules are first tested in silico in order to prioritize compounds for subsequent experimental testing, is one such innovation. Although the traditional scoring functions used in virtual screens have proven useful, improved accuracy requires novel approaches. In the current work, we use the estrogen receptor to demonstrate that neural networks are adept at identifying structurally novel small molecules that bind to a selected drug target, ultimately allowing experimentalists to test fewer compounds in the earliest stages of lead identification while obtaining higher hit rates. We describe 39 novel estrogen-receptor ligands identified in silico with experimentally determined Ki values ranging from 460 nM to 20 µM, presented here for the first time.

VAV3 mediates resistance to breast cancer endocrine therapy.

INTRODUCTION: Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process. METHODS: A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA-mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression. RESULTS: The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10-4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy. CONCLUSIONS: This study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer.

Synthesis and receptor binding in trans-CD ring-fused A-CD estrogens: comparison with the cis-fused isomers.

Ligands which selectively activate only one of the estrogen receptors, ERα or ERß, are current pharmaceutical targets. Previously, we have reported on substituted cis A-CD ligands in which the B-ring of the steroidal structure has been removed and cis refers the stereochemistry of the CD ring junction as compared to trans in estradiol. These compounds often showed good potency and selectivity for ERß. Here we report the synthesis and binding affinities for a similar series of trans A-CD ligands, and compare them to the cis-series. Counterintuitively, trans A-CD ligands, which are structurally more closely related to the natural ligand estradiol, show weaker binding and less ß-selectivity than their cis-counterparts.

Somatic estrogen receptor α mutations that induce dimerization promote receptor activity and breast cancer proliferation.

Physiologic activation of estrogen receptor α (ERα) is mediated by estradiol (E2) binding in the ligand-binding pocket of the receptor, repositioning helix 12 (H12) to facilitate binding of coactivator proteins in the unoccupied coactivator binding groove. In breast cancer, activation of ERα is often observed through point mutations that lead to the same H12 repositioning in the absence of E2. Through expanded genetic sequencing of breast cancer patients, we identified a collection of mutations located far from H12 but nonetheless capable of promoting E2-independent transcription and breast cancer cell growth. Using machine learning and computational structure analyses, this set of mutants was inferred to act distinctly from the H12-repositioning mutants and instead was associated with conformational changes across the ERα dimer interface. Through both in vitro and in-cell assays of full-length ERα protein and isolated ligand-binding domain, we found that these mutants promoted ERα dimerization, stability, and nuclear localization. Point mutations that selectively disrupted dimerization abrogated E2-independent transcriptional activity of these dimer-promoting mutants. The results reveal a distinct mechanism for activation of ERα function through enforced receptor dimerization and suggest dimer disruption as a potential therapeutic strategy to treat ER-dependent cancers.

Exploration of dimensions of estrogen potency: parsing ligand binding and coactivator binding affinities.

The estrogen receptors, ERα and ERß, 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 ligand-binding domain followed by coactivator recruitment to the ER·ligand complex; this ligand·receptor·coactivator 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 ligand·receptor·coactivator complex. Thus, to better understand the molecular basis of estrogen potency, we developed a completely in vitro system (using radiometric and time-resolved FRET assays) to quantify independently three parameters: (a) the affinity of ligand binding to ER, (b) the affinity of coactivator binding to the ER·ligand 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 ERα and ERß. 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 ERß 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.

BC-spiro-estradiols. Synthesis and estrogen receptor binding affinity of four new estradiol isomers.

The synthesis of four new isomers of estradiol in which the ring A to ring C planes are perpendicular to each other as a result of a spiro BC ring junction is described. Heterocyclic analogs and carbocyclic homologs of these compounds are also reported. Estrogen receptor binding studies show that the spiro compounds with the natural stereochemistry at C9 bind almost as strongly as estradiol but with greater ß to α selectivity. These studies show that the estrogen receptors can readily accommodate isomers of estrogen with substantially different fixed shapes than the native ligand.

Bicyclic core estrogens as full antagonists: synthesis, biological evaluation and structure-activity relationships of estrogen receptor ligands based on bridged oxabicyclic core arylsulfonamides.

Compounds that block estrogen action through the estrogen receptor (ER) or downregulate ER levels are useful for the treatment of breast cancer and endocrine disorders. In our search for structurally novel estrogens having three-dimensional core scaffolds, we found some compounds with a 7-oxabicyclo[2.2.1]heptene core that bound well to the ERs. The best of these compounds, a phenyl sulfonate ester (termed OBHS for oxabicycloheptene sulfonate), was a partial antagonist on both ERα and ERß. Although OBHS bears no structural resemblance to other estrogen antagonists, it appears to achieve its partial antagonist character by stabilizing a novel conformation of the ER that involves a significant distortion of helix-11. To enhance the antagonist properties of these oxabicyclo[2.2.1]heptane core ligands, we expanded the functional diversity of OBHS by replacing the sulfonate with secondary or tertiary sulfonamides (-SO(2)NR-), isoelectronic and potentially isostructural molecular replacements. An array of 16 OBHS sulfonamide analogues were prepared through a Diels-Alder reaction of a 3,4-diarylfuran using various N-aryl vinyl sulfonamide dienophiles. While the more polar secondary sulphonamides were weak ligands, certain of the tertiary sulfonamides had very good ER binding affinity. In HepG2 cell reporter gene assays, the sulphonamides had moderate potency, but they showed lower intrinsic transcriptional activity on ERα than the selective estrogen receptor modulator (SERM) hydroxytamoxifen or OBHS, and they were inverse agonists on ERß. Thus, the behaviour of these OBH-sulfonamides more closely mirrors the activity of full antagonists like the drug fulvestrant (ICI 182 780), and their greater antagonist biocharacter appears to arise from an accentuated distortion of helix-11.

Defining the Energetic Basis for a Conformational Switch Mediating Ligand-Independent Activation of Mutant Estrogen Receptors in Breast Cancer.

Although most primary estrogen receptor (ER)-positive breast cancers respond well to endocrine therapies, many relapse later as metastatic disease due to endocrine therapy resistance. Over one third of these are associated with mutations in the ligand-binding domain (LBD) that activate the receptor independent of ligand. We have used an array of advanced computational techniques rooted in molecular dynamics simulations, in concert with and validated by experiments, to characterize the molecular mechanisms by which specific acquired somatic point mutations give rise to ER constitutive activation. By comparing structural and energetic features of constitutively active mutants and ligand-bound forms of ER-LBD with unliganded wild-type (WT) ER, we characterize a spring force originating from strain in the Helix 11-12 loop of WT-ER, opposing folding of Helix 12 into the active conformation and keeping WT-ER off and disordered, with the ligand-binding pocket open for rapid ligand binding. We quantify ways in which this spring force is abrogated by activating mutations that latch (Y537S) or relax (D538G) the folded form of the loop, enabling formation of the active conformation without ligand binding. We also identify a new ligand-mediated hydrogen-bonding network that stabilizes the active, ligand-bound conformation of WT-ER LBD, and similarly stabilizes the active conformation of the ER mutants in the hormone-free state. IMPLICATIONS: Our investigations provide deep insight into the energetic basis for the structural mechanisms of receptor activation through mutation, exemplified here with ER in endocrine-resistant metastatic breast cancers, with potential application to other dysregulated receptor signaling due to driver mutations.

Development of [F-18]fluorine-substituted Tanaproget as a progesterone receptor imaging agent for positron emission tomography.

The level of progesterone receptors (PRs) in breast tumors can be used to guide the selection of endocrine therapies for breast cancer patients. To this end, we have prepared a fluorine-18 labeled analogue of Tanaproget, a nonsteroidal progestin with very high PR binding affinity and low affinity for androgen and glucocorticoid receptors, and have studied its tissue distribution in estrogen-primed rats to evaluate its potential for imaging PR levels by positron emission tomography. 4-[(18)F]Fluoropropyl-Tanaproget ([(18)F]9, FPTP) was prepared in three steps, within 140 min at an overall decay-corrected yield of 5% and effective specific activity of >550 Ci/mmol. In biodistribution studies, [(18)F]9 uptake was high in target tissues at both 1 and 3 h (uterus, 4.55 and 5.26%ID/g; ovary, 2.32 and 2.20%ID/g, respectively) and was cleanly blocked by coinjection of excess unlabeled compound. Uterus to blood and muscle activity ratios were 9.2 and 5.2 at 1 h and 32 and 26 at 3 h, respectively. The biodistribution of [(18)F]9 compares favorably to that of previously prepared F-18 labeled steroidal progestins, FENP and FFNP. Its high target tissue uptake efficiency and selectivity, and prolonged retention, suggest that it has excellent promise as a PET imaging agent for PR-positive breast tumors.

A mutant form of ERα associated with estrogen insensitivity affects the coupling between ligand binding and coactivator recruitment.

A homozygous missense mutation in the gene encoding the estrogen receptor α (ERα) was previously identified in a female patient with estrogen insensitivity syndrome. We investigated the molecular features underlying the impaired transcriptional response of this mutant (ERα-Q375H) and four other missense mutations at this position designed to query alternative mechanisms. The identity of residue 375 greatly affected the sensitivity of the receptor to agonists without changing the ligand binding affinity. Instead, the mutations caused changes in the affinity of coactivator binding and alterations in the balance of coactivator and corepressor recruitment. Comparisons among the transcriptional regulatory responses of these six ERα genotypes to a set of ER agonists showed that both steric and electrostatic factors contributed to the functional deficits in gene regulatory activity of the mutant ERα proteins. ERα-coregulator peptide binding in vitro and RIME (rapid immunoprecipitation mass spectrometry of endogenous) analysis in cells showed that the degree of functional impairment paralleled changes in receptor-coregulator binding interactions. These findings uncover coupling between ligand binding and coregulator recruitment that affects the potency rather than the efficacy of the receptor response without substantially altering ligand binding affinity. This highlights a molecular mechanism for estrogen insensitivity syndrome involving mutations that perturb a bidirectional allosteric coupling between ligand binding and coregulator binding that determines receptor transcriptional output.

Analogs of methyl-piperidinopyrazole (MPP): antiestrogens with estrogen receptor alpha selective activity.

Methyl-piperidino-pyrazole (MPP), an estrogen receptor alpha (ERalpha)-selective antagonist we developed, has a basic side chain (BSC) attached to an ERalpha-selective agonist ligand, methyl-pyrazole-triol (MPT) through an ether linkage. To remove the possibility that metabolic cleavage of the BSC in MPP would regenerate MPT, we have replaced the N-piperidinylethoxy moiety with an N-piperidinylpropyl group, giving MPrP. This new analog retains the high ERalpha-selective binding affinity and antagonist potency of MPP.

Deconstructing estradiol: removal of B-ring generates compounds which are potent and subtype-selective estrogen receptor agonists.

Estradiol and related estrogens have been widely used as supplements to relieve menopausal symptoms, but they lead to an increased risk of breast and endometrial cancer. Here we report the synthesis of a new family of compounds where we have removed the B-ring from the steroid ABCD structure, and functionalized the A-ring. These A-CD compounds show a preferential affinity for the estrogen receptor subtype ERbeta. Some show binding affinities which are greater than estradiol. The presence of electron-withdrawing substituents on the A-ring should reduce the tendency of these compounds to form carcinogenic metabolites, so they might lead to a safer approach to hormone replacement therapy.

Steroidal bivalent ligands for the estrogen receptor: design, synthesis, characterization and binding affinities.

Steroidal bivalent ligands for the estrogen receptor (ER) were designed using crystal structures of ERalpha dimers as a template. The syntheses of several 17alpha-ethynylestradiol-based bivalent ligands with varying linker compositions and lengths are described. The binding affinities of these bivalent ligands for ERalpha and ERbeta were determined. In the two series of bivalent ligands that we synthesized, there is a clear correlation between linker length and binding affinity, both of which reach a maximum at the same tether length. Further studies are underway to explore aspects of bivalent ligand and control compound binding to the ERs and their effects on ER dimer formation; these results will be reported in a subsequent publication.

Synthesis and evaluation of aryl-substituted diarylpropionitriles, selective ligands for estrogen receptor beta, as positron-emission tomographic imaging agents.

We have investigated halogen-substituted non-steroidal estrogens with selective binding affinity for the estrogen receptor beta (ERbeta that might be used for imaging the levels of this ER-subtype in breast tumors by positron emission tomography (PET). Based on diarylpropionitrile (DPN, 1a), a compound previously reported that has a 72-fold binding selectivity for ERbeta, we developed a series of DPN analogs having methyl-, hydroxyl-, and halogen substituents, including fluoroethyl and fluoropropyl groups. In competitive radiometric binding assays with [(3)H]estradiol, all of these DPN analogs showed high ERbeta/ERalpha selectivity; while the selectivity varied, in some cases it reached nearly 300-fold (RBA: ERalpha, 0.023%; ERbeta, 6.25%). The absolute ERbeta binding affinities, however, were not sufficient to merit further consideration for developing these ligands as PET imaging agents.

Monoaryl-substituted salicylaldoximes as ligands for estrogen receptor beta.

Salicylaldoximes possess a hydrogen-bonded pseudocyclic A' ring in place of the typical phenolic A ring that is characteristic of most estrogen receptor (ER) ligands. Monoaryl-substituted salicylaldoximes were obtained by replacing the phenol moiety (ring A) of the ERbeta pharmacophore with the pseudocycle A' ring, which has previously been shown to behave as a bioequivalent of phenols in nonselective ER ligands. In this series, small substituents (CH 3, CN, Cl) were introduced into the central phenyl scaffold. An efficient sequential halogen-selective double cross-coupling reaction was developed for the synthesis of the methyl-substituted ER ligand. The measured ERbeta affinity proved to be very sensitive to the effect of central core substituents. The binding affinities of the compounds herein reported were in good agreement with the results of computational docking analysis. The chloro-substituted derivative showed the highest beta affinity and selectivity, and it also proved to be an ERbeta partial agonist with an EC 50 of 11 nM.

Elemental isomerism: a boron-nitrogen surrogate for a carbon-carbon double bond increases the chemical diversity of estrogen receptor ligands.

To increase the chemical diversity of bioactive molecules by incorporating unusual elements, we have examined the replacement of a C=C double bond with the isoelectronic, isostructural B-N bond in the context of nonsteroidal estrogen receptor (ER) ligands. While the B-N bond was hydrolytically labile in the unhindered cyclofenil system, the more hindered anilino dimesitylboranes, analogs of triarylethylene estrogens, were easily prepared, hydrolytically stable, and demonstrated substantial affinity for ERs. X-ray analysis of one ERalpha-ligand complex revealed steric clashes with the para methyl groups distorting the receptor; removal of these groups resulted in an increase in affinity, potency, and transcriptional efficacy. These studies define the structural determinants of stability and cellular bioactivity of a B-N for C=C substitution in nonsteroidal estrogens and provide a framework for further exploration of "elemental isomerism" for diversification of drug-like molecules.