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.

Mechanisms enforcing the estrogen receptor ß selectivity of botanical estrogens.

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 ERß, 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 ERß. Unlike the endogenous estrogen, estradiol (E2), BEs preferentially bind to ERß, but their ERß-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 ERß 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.

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.

Enhancing Diagnostic Follow-up and Care Coordination for Children with Autism in a Busy Resident Continuity Clinic: Leveraging the Electronic Health Record.

PURPOSE: A high-quality primary care clinic should provide clear action points and important care coordination for a child receiving a new diagnosis of autism spectrum disorder (ASD). Unfortunately, a substantial proportion of caregivers report little-to-no post-diagnosis support from their home clinics and primary care providers often report lack of training and resources in providing these supports. METHODS: We implemented an intervention package to investigate the impact on the frequency and quality of follow-up care for children with ASD in a busy, high-volume resident continuity clinic. The package consisted of a care coordination scheduling pathway and a standardized clinical template-embedded in the electronic health record (EHR)-that guided providers through best-practice recommendations and patient resources. RESULTS: As a result of these interventions, 74% of patients had ASD-specific follow-up, a more than threefold increase from baseline with a majority of providers using the EHR-embedded template to guide their visit. Providers also indicated a high degree of usability for the system and that it aided them in following best-practice guidelines for ASD care. CONCLUSION: Through explicit scheduling pathways and a novel EHR template, we saw a significant increase in ASD-specific follow-up visits and implementation of best practices for ASD care, demonstrating a new process for training and engaging primary care providers in clear action steps for post-diagnostic care without having to rely on tertiary referrals.

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.

Direct-to-patient telehealth equity: Reaching diverse pediatric populations in primary care.

INTRODUCTION: Telehealth is the use of electronic information and technology for long-distance clinical care. In direct-to-patient (DTP) telehealth, the patient initiates care from a personal computer or mobile device to a medical provider. While information on standard clinic-to-clinic telehealth exists, less is known about DTP telehealth in pediatric populations. Using quantitative and qualitative data, we examined DTP telehealth for low-income pediatric patient-families and compared the experience of English and non-English speakers. METHOD: Telehealth visits for acute and preventive care took place from April 2020 to May 2020 at a pediatric primary care clinic (80% Medicaid-insured, 40% non-English-speaking). Patients and primary care providers conducted the visit through the clinic's portal or other platforms (WhatsApp, FaceTime, Zoom). Providers completed an electronic survey with patient feedback about the telehealth experience and their own observations. An iterative inductive/deductive approach informed a coding scheme for free-text survey responses consisting of five domains. RESULTS: REDCap surveys were completed for 258 (52%) of telehealth visits. There was an overrepresentation of English visits compared to the overall clinic population and the majority of visits were via mobile phone. Visits with English speakers utilized the patient portal and had positive process ease ratings more often than those with non-English speakers. Providers rated most telehealth visits as satisfactory, with contributing elements including family call environment, technology process and experience, value added, and barriers. DISCUSSION: Expanding telehealth in pediatrics without worsening health disparities requires building digital health that is user-friendly on mobile technology, facilitating patient preferred language, and simplifying logistical processes. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

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.

Fidelity Protocol Development for a Telehealth Type 1 Diabetes Occupation-Based Coaching Intervention.

Background. Preserving fidelity ascertains that the intervention is delivered as intended in occupational therapy (OT) contexts. The process of conceptualizing and developing fidelity standards, however, is seldom documented in the existing literature. Purpose. The purpose of this methodological description paper was to (a) describe the process of generating a comprehensive fidelity plan based on the National Institutes of Health Behavioral Change Consortium's five-domain fidelity framework and (b) evaluate the development process and utility of the end product, the Occupation-Based Coaching (OBC) Fidelity Protocol. Key Issues. There is no known research that documents the process of developing fidelity standards and tools to support the OBC intervention. Implications. The OBC Fidelity Protocol proposes an example of how a comprehensive fidelity plan and tools can be developed from a well-established scientific framework. This can also inform OT practitioners and researchers to deliver OBC sessions with consistency across clients, providers, and interventions/studies.

Pediatric emergency department to primary care transfer protocol: Transforming access for patients' needs.

BACKGROUND: Previous interventions to reduce emergency department (ED) overutilization from non-urgent visits have shown little success. At our hospital, we created an ED to primary care clinic (PCC) transfer protocol for non-urgent ED visits of established patients. Our study analyzed the impact of this protocol on patient encounters. METHODS: Chart reviews were conducted for a retrospective cohort of transfers from the ED to PCC from 9/01/17-8/31/18. Primary outcomes included length of stay (LOS), cost, and need for return to the ED. Cost savings were calculated by comparing encounters with identical primary diagnoses in the ED with internal technical and professional financial data. Secondary outcomes were final diagnoses and primary care services provided. RESULTS: 374 patient encounters were transferred from ED to PCC. The five most common diagnoses were viral upper respiratory infection (n=80, 21.4%), dermatologic diagnoses (n=37, 9.9%), acute otitis media (n=35, 9.4%), pharyngitis (n=34, 9.1%), and influenza (n=34, 9.1%). Overall, total cost savings equaled approximately $100,000. For the top 10 diagnoses, costs were reduced from $29-$46 per $100 of ED costs and LOS was reduced by a mean of 49 min/encounter. For 9 of these 10 conditions, costs exceeded reimbursement in both settings; however, evaluation in PCC versus ED reduced the loss of revenue by 10-68%. Sixty-four encounters (17.1%) received additional primary care services. There were no safety events or inappropriate transfers. CONCLUSIONS: This protocol provided a safe, efficient method for patients to be evaluated in their medical home while reducing non-urgent emergency visits in the ED. LEVEL OF EVIDENCE: VI.

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.