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Most prostate cancers express the androgen receptor (AR), and tumor growth and progression are facilitated by exceptionally low levels of systemic or intratumorally produced androgens. Thus, absolute inhibition of the androgen signaling axis remains the goal of current therapeutic approaches to treat prostate cancer (PCa). Paradoxically, high dose androgens also exhibit considerable efficacy as a treatment modality in patients with late-stage metastatic PCa. Here we show that low levels of androgens, functioning through an AR monomer, facilitate a non-genomic activation of the mTOR signaling pathway to drive proliferation. Conversely, high dose androgens facilitate the formation of AR dimers/oligomers to suppress c-MYC expression, inhibit proliferation and drive a transcriptional program associated with a differentiated phenotype. These findings highlight the inherent liabilities in current approaches used to inhibit AR action in PCa and are instructive as to strategies that can be used to develop new therapeutics for this disease and other androgenopathies., (© 2024. The Author(s).)

Purpose: HOXB13 is an androgen receptor (AR) coregulator specifically expressed in cells of prostatic lineage. We sought to associate circulating tumor cell (CTC) HOXB13 expression with outcomes in men with mCRPC treated with abiraterone or enzalutamide., Experimental Design: We conducted a retrospective analysis of the multicenter prospective PROPHECY trial of mCRPC men (NCT02269982, n = 118) treated with abiraterone/enzalutamide. CTC detection and HOXB13 complementary DNA (cDNA) expression was measured using a modified Adnatest, grouping patients into 3 categories: CTC 0 (undetectable); CTC+ HOXB13 CTC low (<4 copies); or CTC+ HOXB13 CTC high. The HOXB13 threshold was determined by maximally selected rank statistics for prognostic associations with overall survival (OS) and progression-free survival (PFS)., Results: We included 102 men with sufficient CTC HOXB13 cDNA, identifying 25%, 31%, and 44% of patients who were CTC 0, CTC+ HOXB13 low, and CTC+ HOXB13 high, respectively. Median OS were 25.7, 27.8, and 12.1 months whereas the median PFS were 9.0, 7.7, and 3.8 months, respectively. In subgroup analysis among men with CellSearch CTCs ≥5 copies/mL and adjusting for prior abi/enza treatment and Halabi clinical risk score, the multivariate HR for HOXB13 CTC detection was 2.39 (95% CI, 1.06-5.40) for OS and 2.78 (95% CI, 1.38-5.59) for PFS, respectively. Low HOXB13 CTC detection was associated with lower CTC PSA, PSMA, AR-FL, and AR-V7 detection, and more liver/lung metastases (41% vs. 25%)., Conclusions: Higher CTC HOXB13 expression is associated with AR-dependent biomarkers in CTCs and is adversely prognostic in the context of potent AR inhibition in men with mCRPC., (©2024 American Association for Cancer Research.)

This study aimed to examine the demographic characteristics of pregnant women in a Healthy Start program who are presumed eligible for the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC), but who have not yet applied for WIC benefits. We used a cross sectional evaluation of data collected from pregnant women (n=203) participating in a Healthy Start program. Data came from surveys administered at enrollment in the Healthy Start program from July 15th, 2019 until January 14th, 2022. The primary outcome was WIC application status, which was determined by whether the woman had applied or was receiving benefits at the time of enrollment. Covariates included race/ethnicity, marital status, insurance, education, income, age, employment, and having previous children/pregnancies. Fisher exact tests and logistic regression were used to examine associations. Approximately 65% of women had not yet applied for WIC benefits. Marshallese women (80.9%) and other NHPI women (80.0%) had the highest need for assistance. In adjusted analyses, White women (p = 0.040) and Hispanic women (p = 0.005) had lower rates of needing assistance applying for WIC than Marshallese women. There were higher rates of needing assistance in applying for women with private insurance or with no insurance and for those with higher incomes. Nearly two out of every three pregnant women who were eligible for WIC had not yet applied for benefits. The findings highlight the need for outreach for all populations that may be eligible, particularly among racial/ethnic minorities and those with higher incomes., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Despite continued interest in the development of nonsteroidal estrogens and antiestrogens, there are only a few chemotypes of estrogen receptor ligands. Using targeted screening in a ligand sensing assay, we identified a phenolic thieno[2,3- d ]pyrimidine with affinity for estrogen receptor α. An efficient three-step synthesis of the heterocyclic core and structure-guided optimization of the substituents resulted in a series of potent nonsteroidal estrogens. The chemical tractability of the thieno[2,3- d ]pyrimidine chemotype will support the design of new estrogen receptor ligands as therapeutic hormones and antihormones., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

Competing Interests: Donald P. McDonnellEmployment: Duke UniversityStock and Other Ownership Interests: Zentalis, G1 Therapeutics, Viba Therapeutics, Rappta Therapeutics, X-RAD TherapeuticsHonoraria: NovartisConsulting or Advisory Role: Zentalis, G1 therapeutics, Bristol-Myers Squibb, Rappta TherapeuticsResearch Funding: Bristol-Myers Squibb, Novartis, ZentalisPatents, Royalties, Other Intellectual Property: Inventor on two patents (assigned to Duke) licensed to Radius Health covering the use of Rad1901 for Breast cancer. I am an inventor on two patents (assigned to Duke) that covers the use of lasofoxifene for ESR1-mutant breast cancers. Licensed to SermonixTravel, Accommodations, Expenses: Bristol-Myers Squibb Suzanne E. WardellConsulting or Advisory Role: ZentalisResearch Funding: Zentalis, Bristol-Myers SquibbPatents, Royalties, Other Intellectual Property: I am listed as an inventor on a patent for the use of RAD1901 in metastatic breast cancer Ching-Yi ChangResearch Funding: Novartis Institutes for BioMedical Research, Bristol-Myers SquibbPatents, Royalties, Other Intellectual Property: Sermonix. Patent application for the use of lasofoxifene as treatment for breast cancer John D. NorrisConsulting or Advisory Role: G1 Pharmaceuticals, CelgeneResearch Funding: G1 Therapeutics, CelgeneNo other potential conflicts of interest were reported.

The estrogen receptor (ER/ ESR1 ) is expressed in a majority of breast cancers and drugs that inhibit ER signaling are the cornerstone of breast cancer pharmacotherapy. Currently, aromatase inhibitors are the frontline endocrine interventions of choice although their durability in metastatic disease is limited by activating point mutations within the ligand-binding domain of ESR1 that permit ligand-independent activation of the receptor. It has been suggested that the most commonly occurring ESR1 mutations would likely compromise the clinical activity of selective estrogen receptor downregulators and selective estrogen receptor modulators (SERMs) when used as second-line therapies. It was unclear, however, how these mutations, which are likely coexpressed in cells with ER WT , may impact response to ER ligands in a clinically meaningful manner. To address this issue, we dissected the molecular mechanism(s) underlying ESR1 -mutant pharmacology in models relevant to metastatic disease. These studies revealed that the response of ESR1 mutations to ligands was dictated primarily by the relative coexpression of ER WT in cells. Specifically, dysregulated pharmacology was only evident in cells in which the mutants were overexpressed relative to ligand-activated ER WT ; a finding that highlights the role of allelism in determining ER-mutant pharmacology. Importantly, we demonstrated that the antagonist activity of the SERM, lasofoxifene, was not impacted by mutant status; a finding that has led to its clinical evaluation as a treatment for patients with advanced ER-positive breast cancer whose tumors harbor ESR1 mutations., (©2020 American Association for Cancer Research.)

Purpose: The combination of targeting the CDK4/6 and estrogen receptor (ER) signaling pathways with palbociclib and fulvestrant is a proven therapeutic strategy for the treatment of ER-positive breast cancer. However, the poor physicochemical properties of fulvestrant require monthly intramuscular injections to patients, which limit the pharmacokinetic and pharmacodynamic activity of the compound. Therefore, an orally available compound that more rapidly reaches steady state may lead to a better clinical response in patients. Here, we report the identification of G1T48, a novel orally bioavailable, non-steroidal small molecule antagonist of ER., Methods: The pharmacological effects and the antineoplastic mechanism of action of G1T48 on tumors was evaluated using human breast cancer cells (in vitro) and xenograft efficacy models (in vivo)., Results: G1T48 is a potent and efficacious inhibitor of estrogen-mediated transcription and proliferation in ER-positive breast cancer cells, similar to the pure antiestrogen fulvestrant. In addition, G1T48 can effectively suppress ER activity in multiple models of endocrine therapy resistance including those harboring ER mutations and growth factor activation. In vivo, G1T48 has robust antitumor activity in a model of estrogen-dependent breast cancer (MCF7) and significantly inhibited the growth of tamoxifen-resistant (TamR), long-term estrogen-deprived (LTED) and patient-derived xenograft tumors with an increased response being observed with the combination of G1T48 and the CDK4/6 inhibitor lerociclib., Conclusions: These data show that G1T48 has the potential to be an efficacious oral antineoplastic agent in ER-positive breast cancer.

Before Europeans arrived to Eastern North America, prehistoric, indigenous peoples experienced a number of changes that culminated in the development of sedentary, maize agricultural lifeways of varying complexity. Inherent to these lifeways were several triggers of social stress including population nucleation and increase, intergroup conflict (warfare), and increased territoriality. Here, we examine whether this period of social stress co-varied with deadlier weaponry, specifically, the design of the most commonly found prehistoric archery component in late pre-contact North America: triangular stone arrow tips (TSAT). The examination of modern metal or carbon projectiles, arrows, and arrowheads has demonstrated that smaller arrow tips penetrate deeper into a target than do larger ones. We first experimentally confirm that this relationship applies to arrow tips made from stone hafted onto shafts made from wood. We then statistically assess a large sample (n = 742) of late pre-contact TSAT and show that these specimens are extraordinarily small. Thus, by miniaturizing their arrow tips, prehistoric people in Eastern North America optimized their projectile weaponry for maximum penetration and killing power in warfare and hunting. Finally, we verify that these functional advantages were selected across environmental and cultural boundaries. Thus, while we cannot and should not rule out stochastic, production economizing, or non-adaptive cultural processes as an explanation for TSAT, overall our results are consistent with the hypothesis that broad, socially stressful demographic changes in late pre-contact Eastern North America resulted in the miniaturization-and augmented lethality-of stone tools across the region., Competing Interests: The authors have declared that no competing interests exist.

The article Pharmacokinetic and pharmacodynamic analysis of fulvestrant in preclinical models of breast cancer to assess the importance of its estrogen receptor-α degrader activity in antitumor efficacy, written by Suzanne E. Wardell, Alexander P. Yllanes, Christina A. Chao, Yeeun Bae, Kaitlyn J. Andreano, Taylor K. Desautels, Kendall A. Heetderks, Jeremy T. Blitzer, John D. Norris, Donald P. McDonnell, was originally published electronically on the publisher's internet portal on September 27, 2019 without open access. With the author(s)' decision to opt for Open Choice the copyright of the article changed on November 16, 2019 to © The Author(s) 2019 and the article is forthwith distributed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The original article has been corrected.

Purpose: Fulvestrant is a selective estrogen receptor downregulator (SERD) that is approved for first- or second-line use as a single agent or in combination with cyclin dependent kinase or phosphatidylinositol 3-kinase inhibitors for the treatment of metastatic breast cancer. Fulvestrant exhibits exceptionally effective antitumor activity in preclinical models of breast cancer, a success that has been attributed to its robust SERD activity despite modest receptor downregulation in patient tumors. By modeling human exposures in animal models we probe the absolute need for SERD activity., Methods: Three xenograft models of endocrine therapy-resistant breast cancer were used to evaluate the efficacy of fulvestrant administered in doses historically used in preclinical studies in the field or by using a dose regimen intended to model clinical exposure levels. Pharmacokinetic and pharmacodynamic analyses were conducted to evaluate plasma exposure and intratumoral ER downregulation., Results: A clinically relevant 25 mg/kg dose of fulvestrant exhibited antitumor efficacy comparable to the historically used 200 mg/kg dose, but at this lower dose it did not result in robust ER downregulation. Further, the antitumor efficacy of the lower dose of fulvestrant was comparable to that observed for other oral SERDs currently in development., Conclusion: The use of clinically unachievable exposure levels of fulvestrant as a benchmark in preclinical development of SERDs may negatively impact the selection of those molecules that are advanced for clinical development. Further, these studies suggest that antagonist efficacy, as opposed to SERD activity, is likely to be the primary driver of clinical response.

Notwithstanding the positive clinical impact of endocrine therapies in estrogen receptor-alpha (ERα)-positive breast cancer, de novo and acquired resistance limits the therapeutic lifespan of existing drugs. Taking the position that resistance is nearly inevitable, we undertook a study to identify and exploit targetable vulnerabilities that were manifest in endocrine therapy-resistant disease. Using cellular and mouse models of endocrine therapy-sensitive and endocrine therapy-resistant breast cancer, together with contemporary discovery platforms, we identified a targetable pathway that is composed of the transcription factors FOXA1 and GRHL2, a coregulated target gene, the membrane receptor LYPD3, and the LYPD3 ligand, AGR2. Inhibition of the activity of this pathway using blocking antibodies directed against LYPD3 or AGR2 inhibits the growth of endocrine therapy-resistant tumors in mice, providing the rationale for near-term clinical development of humanized antibodies directed against these proteins., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The recurrent p.Gly84Glu germline mutation (G84E) in HOXB13 is consistently associated with prostate cancer (PCa), although the mechanisms underlying such linkage remain elusive. The majority of the PCa-associated HOXB13 mutations identified are localized to two conserved domains in HOXB13 that have been shown to mediate the interaction with MEIS cofactors belonging to the TALE family of homeodomain transcription factors. In this study, we sought to interrogate the biochemical and functional interactions between HOXB13 and MEIS in prostatic cells with a goal of defining how the HOXB13-MEIS complex impacts PCa pathobiology and define the extent to which the oncogenic activity of G84E is related to its effect on HOXB13-MEIS interaction/function., Methods: HOXB13 and MEIS paralog expression in prostate epithelial cells and PCa cell lines was characterized by qPCR and immunoblot analyses. HOXB13 and MEIS1 co-expression in human prostate tissue was confirmed by IHC, followed by co-IP mapping of HOXB13-MEIS1 interactions. Proliferation of the PCa cell line LAPC4 following shRNA-mediated knockdown of each gene or both genes was assessed using DNA- and metabolic-based assays. Transcriptional targets of HOXB13 and MEIS1 were identified by gene expression profiling and qPCR. Finally, protein stability of HOXB13 in the context of MEIS1 was determined using pulse-chase assays., Results: HOXB13 and MEIS1 are co-expressed and interact in prostate cells. Both of the putative MEIS interacting domains (MID) within HOXB13 were shown to be capable of mediating the interaction between HOXB13 and MEIS1 independently and such interactions were not influenced by the G84E mutation. The inhibitory effect of either HOXB13 or MEIS1 knockdown on cellular proliferation was augmented by knockdown of both genes, and MEIS1 knockdown abolished HOXB13-driven regulation of BCHE and TNFSF10 mRNA expression. Notably, we demonstrated that MEIS1 stabilized the HOXB13 protein in LAPC4 cells., Conclusions: Our study provides evidence for functional HOXB13-MEIS1 interactions in PCa. MEIS1 may contribute to the cancer-promoting actions of HOXB13 in cellular proliferation and gene regulation by prolonging HOXB13 half-life. Our data demonstrates that G84E is not a loss-of-function mutation that interferes with HOXB13 stability or ability to interact with MEIS1., (© 2018 Wiley Periodicals, Inc.)

A drug used in hormone replacement therapy can target estrogen receptors that have become resistant to breast cancer treatments., Competing Interests: SW, JN, DM No competing interests declared, (© 2019, Wardell et al.)

In breast cancer, estrogen receptor alpha (ERα) positive cancer accounts for approximately 74% of all diagnoses, and in these settings, it is a primary driver of cell proliferation. Treatment of ERα positive breast cancer has long relied on endocrine therapies such as selective estrogen receptor modulators, aromatase inhibitors, and selective estrogen receptor degraders (SERDs). The steroid-based anti-estrogen fulvestrant (5), the only approved SERD, is effective in patients who have not previously been treated with endocrine therapy as well as in patients who have progressed after receiving other endocrine therapies. Its efficacy, however, may be limited due to its poor physicochemical properties. We describe the design and synthesis of a series of potent benzothiophene-containing compounds that exhibit oral bioavailability and preclinical activity as SERDs. This article culminates in the identification of LSZ102 (10), a compound in clinical development for the treatment of ERα positive breast cancer.

In this issue of Cancer Cell, Jeselsohn et al. dissect the function of several of the most clinically important estrogen receptor alpha mutants associated with endocrine therapy resistance in breast cancer and demonstrate that they manifest disease-relevant neomorphic activities that likely contribute to tumor pathogenesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Resistance to second-generation androgen receptor (AR) antagonists and CYP17 inhibitors in patients with castration-resistant prostate cancer (CRPC) develops rapidly through reactivation of the androgen signaling axis and has been attributed to AR overexpression, production of constitutively active AR splice variants, or the selection for AR mutants with altered ligand-binding specificity. It has been established that androgens induce cell-cycle progression, in part, through upregulation of cyclin D1 (CCND1) expression and subsequent activation of cyclin-dependent kinases 4 and 6 (CDK4/6). Thus, the efficacy of the newly described CDK4/6 inhibitors (G1T28 and G1T38), docetaxel and enzalutamide, was evaluated as single agents in clinically relevant in vitro and in vivo models of hormone-sensitive and treatment-resistant prostate cancer. CDK4/6 inhibition (CDK4/6i) was as effective as docetaxel in animal models of treatment-resistant CRPC but exhibited significantly less toxicity. The in vivo effects were durable and importantly were observed in prostate cancer cells expressing wild-type AR, AR mutants, and those that have lost AR expression. CDK4/6i was also effective in prostate tumor models expressing the AR-V7 variant or the AR F876L mutation, both of which are associated with treatment resistance. Furthermore, CDK4/6i was effective in prostate cancer models where AR expression was lost. It is concluded that CDK4/6 inhibitors are a viable alternative to taxanes as therapeutic interventions in endocrine therapy-refractory CRPC. Implications: The preclinical efficacy of CDK4/6 monotherapy observed here suggests the need for near-term clinical studies of these agents in advanced prostate cancer. Mol Cancer Res; 15(6); 660-9. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The clinical utility of inhibiting cytochrome P450 17A1 (CYP17), a cytochrome p450 enzyme that is required for the production of androgens, has been exemplified by the approval of abiraterone for the treatment of castration-resistant prostate cancer (CRPC). Recently, however, it has been reported that CYP17 inhibitors can interact directly with the androgen receptor (AR). A phase I study recently reported that seviteronel, a CYP17 lyase-selective inhibitor, ædemonstrated a sustained reduction in prostate-specific antigen in a patient with CRPC, and another study showed seviteronel's direct effects on AR function. This suggested that seviteronel may have therapeutically relevant activities in addition to its ability to inhibit androgen production. Here, we have demonstrated that CYP17 inhibitors, with the exception of orteronel, can function as competitive AR antagonists. Conformational profiling revealed that the CYP17 inhibitor-bound AR adopted a conformation that resembled the unliganded AR (apo-AR), precluding nuclear localization and DNA binding. Further, we observed that seviteronel and abiraterone inhibited the growth of tumor xenografts expressing the clinically relevant mutation AR-F876L and that this activity could be attributed entirely to competitive AR antagonism. The results of this study suggest that the ability of CYP17 inhibitors to directly antagonize the AR may contribute to their clinical efficacy in CRPC.

Tetrahydroisoquinoline 40 has been identified as a potent ERα antagonist and selective estrogen receptor degrader (SERD), exhibiting good oral bioavailability, antitumor efficacy, and SERD activity in vivo. We outline the discovery and chemical optimization of the THIQ scaffold leading to THIQ 40 and showcase the racemization of the scaffold, pharmacokinetic studies in preclinical species, and the in vivo efficacy of THIQ 40 in a MCF-7 human breast cancer xenograft model.

Triple-negative breast cancer (TNBC) has a faster rate of metastasis compared to other breast cancer subtypes, and no effective targeted therapies are currently FDA-approved. Recent data indicate that the androgen receptor (AR) promotes tumor survival and may serve as a potential therapeutic target in TNBC. Studies of AR in disease progression and the systemic effects of anti-androgens have been hindered by the lack of an AR-positive (AR+) immunocompetent preclinical model. In this study, we identified the transgenic MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mouse mammary gland carcinoma model of breast cancer and Met-1 cells derived from this model as tools to study the role of AR in breast cancer progression. AR protein expression was examined in late-stage primary tumors and lung metastases from MMTV-PyMT mice as well as in Met-1 cells by immunohistochemistry (IHC). Sensitivity of Met-1 cells to the AR agonist dihydrotestosterone (DHT) and anti-androgen therapy was examined using cell viability, migration/invasion, and anchorage-independent growth assays. Late-stage primary tumors and lung metastases from MMTV-PyMT mice and Met-1 cells expressed abundant nuclear AR protein, while negative for estrogen and progesterone receptors. Met-1 sensitivity to DHT and AR antagonists demonstrated a reliance on AR for survival, and AR antagonists inhibited invasion and anchorage-independent growth. These data suggest that the MMTV-PyMT model and Met-1 cells may serve as valuable tools for mechanistic studies of the role of AR in disease progression and how anti-androgens affect the tumor microenvironment.

Clinical resistance to the second-generation antiandrogen enzalutamide in castration-resistant prostate cancer (CRPC), despite persistent androgen receptor (AR) activity in tumors, highlights an unmet medical need for next-generation antagonists. We have identified and characterized tetra-aryl cyclobutanes (CBs) as a new class of competitive AR antagonists that exhibit a unique mechanism of action. These CBs are structurally distinct from current antiandrogens (hydroxyflutamide, bicalutamide, and enzalutamide) and inhibit AR-mediated gene expression, cell proliferation, and tumor growth in several models of CRPC. Conformational profiling revealed that CBs stabilize an AR conformation resembling an unliganded receptor. Using a variety of techniques, it was determined that the AR-CB complex was not recruited to AR-regulated promoters and, like apo AR, remains sequestered in the cytoplasm, bound to heat shock proteins. Thus, we have identified third-generation AR antagonists whose unique mechanism of action suggests that they may have therapeutic potential in CRPC., Competing Interests: Statement. A patent covering this work has been published (Publication No. WO 2015/048246).

Purpose: Endocrine therapy, using tamoxifen or an aromatase inhibitor, remains first-line therapy for the management of estrogen receptor (ESR1)-positive breast cancer. However, ESR1 mutations or other ligand-independent ESR1 activation mechanisms limit the duration of response. The clinical efficacy of fulvestrant, a selective estrogen receptor downregulator (SERD) that competitively inhibits agonist binding to ESR1 and triggers receptor downregulation, has confirmed that ESR1 frequently remains engaged in endocrine therapy-resistant cancers. We evaluated the activity of a new class of selective estrogen receptor modulators (SERM)/SERD hybrids (SSH) that downregulate ESR1 in relevant models of endocrine-resistant breast cancer. Building on the observation that concurrent inhibition of ESR1 and the cyclin-dependent kinases 4 and 6 (CDK4/6) significantly increased progression-free survival in advanced patients, we explored the activity of different SERD- or SSH-CDK4/6 inhibitor combinations in models of endocrine therapy-resistant ESR1(+) breast cancer., Experimental Design: SERDs, SSHs, and the CDK4/6 inhibitor palbociclib were evaluated as single agents or in combination in established cellular and animal models of endocrine therapy-resistant ESR1(+) breast cancer., Results: The combination of palbociclib with a SERD or an SSH was shown to effectively inhibit the growth of MCF7 cell or ESR1-mutant patient-derived tumor xenografts. In tamoxifen-resistant MCF7 xenografts, the palbociclib/SERD or SSH combination resulted in an increased duration of response as compared with either drug alone., Conclusions: A SERD- or SSH-palbociclib combination has therapeutic potential in breast tumors resistant to endocrine therapies or those expressing ESR1 mutations. See related commentary by DeMichele and Chodosh, p. 4999., (©2015 American Association for Cancer Research.)

Androgen receptor (AR)-dependent transcription is a major driver of prostate tumor cell proliferation. Consequently, it is the target of several antitumor chemotherapeutic agents, including the AR antagonist MDV3100/enzalutamide. Recent studies have shown that a single AR mutation (F876L) converts MDV3100 action from an antagonist to an agonist. Here we describe the generation of a novel class of selective androgen receptor degraders (SARDs) to address this resistance mechanism. Molecules containing hydrophobic degrons linked to small-molecule AR ligands induce AR degradation, reduce expression of AR target genes and inhibit proliferation in androgen-dependent prostate cancer cell lines. These results suggest that selective AR degradation may be an effective therapeutic prostate tumor strategy in the context of AR mutations that confer resistance to second-generation AR antagonists., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Drugs that inhibit estrogen receptor alpha (ERα) or that block the production of estrogens remain frontline interventions in the treatment and management of breast cancer at all stages. However, resistance to endocrine therapies, especially in the setting of advanced disease, remains an impediment to durable clinical responses. Although the mechanisms underlying resistance to existing agents are complex, preclinical studies suggest that selective estrogen receptor downregulators (SERDs), molecules which eliminate ERα expression, may have particular utility in the treatment of breast cancers that have progressed on tamoxifen and/or aromatase inhibitors. The discovery and development of orally bioavailable SERDs provide the opportunity to evaluate the utility of eliminating ERα expression in advanced metastatic breast cancers.

Obesity and altered lipid metabolism are risk factors for breast cancer in pre- and post-menopausal women. These pathologic relationships have been attributed in part to the impact of cholesterol on the biophysical properties of cell membranes and to the influence of these changes on signaling events initiated at the membrane. However, more recent studies have indicated that the oxysterol 27-hydroxycholesterol (27HC), and not cholesterol per se, may be the primary biochemical link between lipid metabolism and cancer. The enzyme responsible for production of 27HC from cholesterol, CYP27A1, is expressed primarily in the liver and in macrophages. In addition, significantly elevated expression of this enzyme within breast tumors has also been observed. It is believed that 27HC, acting through the liver X receptor in macrophages and possibly other cells, is involved in maintaining organismal cholesterol homeostasis. It has also been shown recently that 27HC is an estrogen receptor agonist in breast cancer cells and that it stimulates the growth and metastasis of tumors in several models of breast cancer. These findings provide the rationale for the clinical evaluation of pharmaceutical approaches that interfere with cholesterol/27HC synthesis as a means to mitigate the impact of cholesterol on breast cancer pathogenesis. Cancer Res; 74(18); 4976-82. ©2014 AACR., (©2014 American Association for Cancer Research.)

The androgen receptor (AR) has a critical role in the development and progression of prostate cancer (PC) and is a major therapeutic target in this disease. The transcriptional activity of AR is modulated by the coregulators with which it interacts, and consequently deregulation of cofactor expression and/or activity impacts the expression of genes whose products can have a role in PC pathogenesis. Here we report that E74-like factor 3 (ELF3), a member of the ETS family of transcription factors, is a repressor of AR transcriptional activity. Exogenous expression of ELF3 represses AR transcriptional activity when assessed using reporter-based transfection assays or when evaluated on endogenous AR target genes. Conversely, ELF3 knock down increases the AR transcriptional activity. Biochemical dissection of this activity indicates that it results from the physical interaction between ELF3 and AR and that this interaction inhibits the recruitment of AR to specific androgen response elements within target gene promoters. Significantly, we observed that depletion of ELF3 expression in LNCaP cells promotes cell migration, whereas increased ELF3 expression severely inhibits tumor growth in vitro and in a mouse xenograft model. Taken together, these results suggest that modulation of ELF3 expression and/or AR/ELF3 interaction may have utility in the treatment of PC.

The presence of heterogenous mitochondria from the host ooplast affects the acceptance of offspring obtained by somatic cell nuclear transfer. This might be avoided by obtaining oocytes from selected females, but is then complicated by low numbers of available oocytes. We examined the efficiency of equine somatic cell nuclear transfer using oocytes recovered by transvaginal aspiration of immature follicles from 11 mares. Use of metaphase I oocytes as cytoplasts and of scriptaid (a histone deacetylase inhibitor) treatment during oocyte activation were evaluated to determine if these approaches would increase blastocyst production. In experiment 1, blastocyst development was 0/14 for metaphase I oocytes and 4/103 (4%) for metaphase II oocytes. Three blastocysts were transferred to recipient mares, resulting in two pregnancies and one live foal, which died shortly after birth. In experiment 2, blastocyst development was 2/47 (4%) for control oocytes and 1/83 (1%) for scriptaid-treated oocytes. No foals were born from two blastocysts transferred in the control group. The blastocyst from the scriptaid treatment resulted in birth of a live foal. In conclusion, this is apparently the first report of production of a viable cloned foal from oocytes collected from immature follicles of live mares, supporting the possibility of cloning using oocytes from selected mares., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Reasons for Performing Study: Transvaginal ultrasound-guided follicle aspiration (TVA) is performed clinically but there is little information available on complications associated with this procedure., Objectives: It is possible that TVA is associated with damage to the ovary and may induce peritonitis or peritoneal adhesions. This study was conducted to determine the effect of repeated TVA on mare health and ovarian status., Methods: Thirty-two mares were used for oocyte recovery via repeated TVA over a 3 year period; different mares were used each year. In Year 1, ovarian status was monitored in 11 mares by transrectal palpation and ultrasonography. In Year 2, 6 of 11 mares underwent abdominocentesis and were examined by laparoscopy after one TVA and again after multiple TVAs. In Year 3, 10 mares underwent multiple TVAs with either a 15 or a 12 gauge needle and the ovaries were removed for examination., Results: Four hundred and twenty-seven aspiration sessions (390 via TVA and 37 via needle placement through the flank) and 3202 follicle punctures (3161 TVA and 41 flank) were performed. One mare developed an ovarian abscess. Transient rectal bleeding was evident after 16% of TVA sessions. No adhesions were found on laparoscopic or gross examination of ovaries and there were minimal changes on histological evaluation., Conclusions: Follicle aspiration carries a small possibility (< 0.5%) of ovarian abscess formation. There is a possibility of rectal abrasion or puncture but little gross or histological damage to the ovary., Potential Relevance: These results provide a basis for using prophylactic administration of antibiotics after TVA and for advising mare owners of the rare but potential complications associated with the procedure.

Objective: To evaluate the efficiency of foal production following intracytoplasmic sperm injection (ICSI) and blastocyst culture of oocytes from mares that died or were euthanized under field conditions., Design: Prospective case series., Animals: 16 mares (age, 3 to 19 years) that died or were euthanized for various causes., Procedures: Ovaries were collected immediately before euthanasia (n = 10) or after death (6). Ovaries were transported to the laboratory for oocyte recovery (15 mares), or oocytes were recovered at a remote location and shipped to the laboratory (1). Oocytes underwent ICSI, and presumptive zygotes were cultured for 7 to 10 days. Blastocysts were shipped to embryo transfer facilities for transcervical transfer to recipient mares., Results: Ovaries were processed 30 minutes to 12 hours (mean ± SD, 4.6 ± 3.3 hours) after mares' deaths. A mean of 14.1 ± 8.6 oocytes/mare were cultured, and 110 of 225 (49%) matured. Twenty-one blastocysts developed after ICSI and were transferred to recipient mares. Thirteen pregnancies were established; 10 healthy foals were produced from 6 donor mares. The number of blastocysts produced per mare and number of live foals produced per mare were significantly correlated with the number of oocytes recovered., Conclusions and Clinical Relevance: Foals were produced from mares after death or euthanasia under field conditions. Proportions of foals born overall (10 foals/16 mares) and mares from which ≥ 1 foal was produced (6/16) were greater than those reported following recovery and oviductal transfer of oocytes to inseminated recipients after death of donor mares under field conditions.

HOXB13 is a member of the homeodomain family of sequence-specific transcription factors and, together with the androgen receptor (AR), plays a critical role in the normal development of the prostate gland. We demonstrate here that, in prostate cancer cells, HOXB13 is a key determinant of the response to androgens. Specifically, it was determined that HOXB13 interacts with the DNA-binding domain of AR and inhibits the transcription of genes that contain an androgen-response element (ARE). In contrast, the AR:HOXB13 complex confers androgen responsiveness to promoters that contain a specific HOXB13-response element. Further, HOXB13 and AR synergize to enhance the transcription of genes that contain a HOX element juxtaposed to an ARE. The profound effects of HOXB13 knockdown on androgen-regulated proliferation, migration, and lipogenesis in prostate cancer cells highlight the importance of the observed changes in gene expression.

Advanced prostate cancers preferentially metastasize to bone, suggesting that this tissue produces factors that provide a suitable microenvironment for prostate cancer cells. Recently, it has become clear that even in antiandrogen-resistant cancers, the androgen receptor (AR)-signaling axis is required for prostate cancer progression. Therefore, we hypothesized that AR may be involved in the regulation of pathways that are responsible for the homing of prostate cancer cells to select microenvironments. In support of this hypothesis, we have determined that chemokine (C-X-C motif) receptor 4 (CXCR4), the receptor for the chemokine CXCL12, is up-regulated in prostate cancer cells in response to androgens. Given that the levels of CXCL12 are elevated at sites of known prostate cancer metastases such as bone, these results suggest that androgens may influence prostate cancer metastasis. Specifically, we demonstrate that androgens increase the levels of both CXCR4 mRNA and functional protein in LNCaP prostate cancer cells. Importantly, androgens enhanced the migration of LNCaP cells toward a CXCL12 gradient, an effect that could be blocked by the specific CXCR4 antagonist AMD3100. Interestingly, CXCR4 is not directly regulated by androgens but rather is positively up-regulated by Krüppel-like factor 5 (KLF5), a transcription factor that we have shown to be an early, direct target of AR. Further, KLF5 is both required and sufficient for androgen-mediated CXCR4 expression and migration toward CXCL12. Taken together, these findings demonstrate that AR can utilize the CXCL12/CXCR4 axis through induction of KLF5 expression to promote prostate cancer progression and highlight the potential utility of CXCR4 antagonists as prostate cancer therapeutics.

The impact of ligand binding on nuclear receptor (NR) structure and the ability of target cells to distinguish between different receptor-ligand complexes are key determinants of the pharmacological activity of NR ligands. However, until relatively recently, these mechanistic insights have not been used in a prospective manner to develop screens for NR modulators with specific therapeutic activities. Driven by the need for unique androgen receptor (AR) antagonists that retain activity in hormone-refractory prostate cancer, we developed and applied a conformation-based screen to identify AR antagonists that were mechanistically distinct from existing drugs of this class. Two molecules were identified by using this approach, D36 and D80, which interact with AR in a unique manner and allosterically inhibit AR agonist activity. Unlike the clinically important antiandrogens, casodex and hydroxyflutamide, both D36 and D80 block androgen action in cellular models of hormone-refractory prostate cancer. Mechanistically, these compounds further distinguish themselves from classical AR antagonists in that they do not promote AR nuclear translocation and quantitatively inhibit the association of AR with DNA even under conditions of overexpression. Although the therapeutic potential of these antiandrogens is apparent, it is the demonstration that it is possible, to modulate the interaction of cofactors with agonist-activated AR, using second-site modulators, that has the greatest potential with respect to the therapeutic exploitation of AR and other NRs.

The pharmacological activity of different nuclear receptor ligands is reflected by their impact on receptor structure. Thus, we asked whether differential presentation of protein-protein interaction surfaces on the androgen receptor (AR), a surrogate assay of receptor conformation, could be used in a prospective manner to define the pharmacological activity of bound ligands. To this end, we identified over 150 proteins/polypeptides whose ability to interact with AR is influenced in a differential manner by ligand binding. The most discriminatory of these protein-AR interactions were used to develop a robust compound-profiling tool that enabled the separation of ligands into functionally distinguishable classes. Importantly, the ligands within each class exhibited similar pharmacological activities, a result that highlights the relationship between receptor structure and activity and provides direction for the discovery of novel AR modulators.

Androgens, through their actions on the androgen receptor (AR), are required for the development of the prostate and contribute to the pathologic growth dysregulation observed in prostate cancers. Consequently, androgen ablation has become an essential component of the pharmacotherapy of prostate cancer. In this study, we explored the utility of targeting processes downstream of AR as an alternate approach for therapy. Specifically, we show that the serum and glucocorticoid-regulated kinase 1 (SGK1) gene is an androgen-regulated target gene in cellular models of prostate cancer. Furthermore, functional serum- and glucocorticoid-regulated kinase 1 (SGK1) protein, as determined by the phosphorylation of its target Nedd4-2, was also increased with androgen treatment. Importantly, we determined that RNA interference-mediated knockdown of SGK1 expression attenuates the androgen-mediated growth of the prostate cancer cell line LNCaP. Given these findings, we explored the utility of SGK1 as a therapeutic target in prostate cancer by developing and evaluating a small-molecule inhibitor of this enzyme. From these studies emerged GSK650394, a competitive inhibitor that quantitatively blocks the effect of androgens on LNCaP cell growth. Thus, in addition to androgen ablation, inhibition of pathways downstream of AR is likely to have therapeutic utility in prostate cancer.

We have previously identified a family of novel androgen receptor (AR) ligands that, upon binding, enable AR to adopt structures distinct from that observed in the presence of canonical agonists. In this report, we describe the use of these compounds to establish a relationship between AR structure and biological activity with a view to defining a rational approach with which to identify useful selective AR modulators. To this end, we used combinatorial peptide phage display coupled with molecular dynamic structure analysis to identify the surfaces on AR that are exposed specifically in the presence of selected AR ligands. Subsequently, we used a DNA microarray analysis to demonstrate that differently conformed receptors facilitate distinct patterns of gene expression in LNCaP cells. Interestingly, we observed a complete overlap in the identity of genes expressed after treatment with mechanistically distinct AR ligands. However, it was differences in the kinetics of gene regulation that distinguished these compounds. Follow-up studies, in cell-based assays of AR action, confirmed the importance of these alterations in gene expression. Together, these studies demonstrate an important link between AR structure, gene expression, and biological outcome. This relationship provides a firm underpinning for mechanism-based screens aimed at identifying SARMs with useful clinical profiles.

Tamoxifen is effective for the prevention and treatment of estrogen-dependent breast cancers, but is associated with an increased incidence of endometrial tumors. We report the crystal structure of the estrogen receptor alpha (ERalpha) ligand binding domain (LBD) bound to the structurally similar compound GW5638, which has therapeutic potential and does not stimulate the uterus. Like tamoxifen, GW5638 relocates the carboxy-terminal helix (H12) to the known coactivator-docking site in the ERalpha LBD. However, GW5638 repositions residues in H12 through specific contacts with the N terminus of this helix. In contrast to tamoxifen, the resulting increase in exposed hydrophobic surface of ERalpha LBD correlates with a significant destabilization of ERalpha in MCF-7 cells. Thus, the GW5638-ERalpha LBD structure reveals an unexpected mode of SERM-mediated ER antagonism, in which the stability of ERalpha is decreased through an altered position of H12. This dual mechanism of antagonism may explain why GW5638 can inhibit tamoxifen-resistant breast tumors.

The full-length human androgen receptor with an N-terminal biotin acceptor peptide tag was overexpressed in Spodoptera frugiperda cells in the presence of 1 microM dihydrotestosterone. Site-specific biotinylation of BAP was achieved in vivo by co-expression of E. coli biotin holoenzyme synthetase. The androgen receptor was purified by single-step affinity chromatography using Streptavidin Mutein Matrix under native conditions. The resultant protein was active, stable, 95% homogeneous, and we obtained sufficient yield for use in functional and structural studies.

Several lines of evidence have indicated that the estrogen receptor (ER) can recruit the corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid receptors (SMRT), to target genes in the presence of tamoxifen, suggesting a possible role for NCoR/SMRT in regulating ER pharmacology. However, a tamoxifen-dependent, direct interaction between NCoR/SMRT and ER in vitro has not been demonstrated. To investigate the possible involvement of different corepressors in the actions of antiestrogen-bound ER, we have constructed a phage display library that expresses 23-amino acid peptides containing the canonical CoRNR box motif in an otherwise random background. Screening of the CoRNR box library with apo-ER or ER treated with tamoxifen or ICI 182,780 led to the isolation of peptides whose ability to interact with ER was influenced by the nature of the bound ligand. Using a series of ERalpha mutants, we found that helix 12 was not required for the binding of CoRNR box peptides, whereas disruption of helixes 3 and 5 had a marked effect on peptide binding. One mutant, ER-L372R, lost the ability to interact with CoRNR box-containing peptides without affecting its binding to LXXLL motif-containing peptides. The estradiol- and tamoxifen-mediated transcriptional activity of ER-L372R was dramatically increased by 11- and 3-fold, respectively, compared with that of wild-type ERalpha. The ICI 182,780-mediated repressional activity of this mutant was also reduced by 4-fold compared with that of wild-type ERalpha. These results suggest that leucine 372 may be an important part of the interaction surface on ER that is responsible for corepressor binding. In addition, our data suggest that corepressors, other than NCoR/SMRT, may be involved in ER signaling.

The term selective estrogen receptor modulator (SERM) describes a group of pharmaceuticals that manifest estrogen receptor (ER) agonist activity in some tissues but opposes estrogen action in others. Although the name describing this class of drugs is new, the concept is not, as compounds exhibiting tissue-selective ER agonist/antagonist properties have been available for nearly 40 years. What is new is the idea that it may be possible to capitalize on the paradoxical activities of SERMs and develop them as target organ-selective ER agonists for the treatment of osteoporosis and other estrogenopathies. This realization has provided the impetus for research in this area, the progress of which is described in this review.

Estrogen regulates a plethora of functionally dissimilar processes in a broad range of tissues. Recent progress in the study of the molecular mechanism of action of estrogen(s) has revealed why different cells can respond to the same hormone in a different manner. Three of these findings are of particular importance: (i) There are two genetically and functionally distinct estrogen receptors that have distinct expression patterns in vivo; (ii) the positive and negative transcriptional activities of these receptors require them to engage transcription cofactors (coactivators or corepressors) in target cells; and (iii) not all cofactors are functionally equivalent, nor are they expressed in the same manner in all cells. Thus, although the estrogen receptor is required for a cell to respond to an estrogenic stimulus, the nature and extent of that response are determined by the proteins, pathways, and processes with which the receptor interacts.

ERalpha is a ligand-activated transcription factor and a key regulator of the processes involved in cellular proliferation and differentiation. In addition, aberrant ERalpha activity is linked to several pathological conditions including breast cancer. A complex network of coregulatory proteins is largely believed to determine the transcriptional activity of ERalpha. We report here the isolation of a protein, denoted RTA for repressor of tamoxifen transcriptional activity, which contains an RNA recognition motif and interacts with the receptor N-terminal activation domain. RTA interacts with RNA in vitro, and its overexpression inhibits the partial agonist activity manifest by the antiestrogen tamoxifen while minimally affecting E2-activated transcription. Mutation of the RNA recognition motif alters RNA binding specificity and results in a dominant negative form of RTA that leads to derepression of ERalpha transcriptional activity, allowing all classes of antiestrogens to manifest partial agonist activity and enhancing agonist efficacy. These findings suggest a role for RNA binding proteins as coregulatory factors of the nuclear receptor family and reveal a novel mechanism by which antiestrogens can manifest agonist activities in some tissues.

The term selective estrogen receptor modulators describes a group of pharmaceuticals that function as estrogen receptor (ER) agonists in some tissues but that oppose estrogen action in others. Although the name for this class of drugs has been adopted only recently, the concept is not new, as compounds exhibiting tissue-selective ER agonist/antagonist properties have been around for nearly 40 years. What is new is the idea that it may be possible to capitalize on the paradoxical activities of these drugs and develop them as target organ-selective ER agonists for the treatment of osteoporosis and other estrogenopathies. This realization has provided the impetus for research in this area, the progress of which is discussed in this review.

The term Selective Estrogen Receptor Modulators (SERMs) has been used of late to describe a group of pharmaceuticals that manifest estrogen receptor (ER) agonist activity in some tissues, but that oppose estrogen action in others. Whereas the name describing this class of drugs is new, the concept is not. Indeed, compounds exhibiting tissue-selective ER agonist/antagonist properties have been around for nearly 40 years. What is new is the idea that it may be possible to capitalize on the paradoxical activities of these drugs and develop them as treatments for estrogenopathies where it is desirable to direct therapy to a specific estrogen-responsive target organ. This realization has provided the impetus for research in this area and has pushed the development and clinical use of this class of drugs. The objective of this review is to describe how the medical need for SERMs arose and how recent studies of the mechanism of action of the currently available drugs are paving the way for the development of novel drugs with improved selectivity.

Tamoxifen inhibits estrogen receptor (ER) transcriptional activity by competitively inhibiting estradiol binding and inducing conformational changes in the receptor that may prevent its interaction with coactivators. In bone, the cardiovascular system, and some breast tumors, however, tamoxifen exhibits agonist activity, suggesting that the tamoxifen-ER complex is not recognized identically in all cells. We used phage display to demonstrate that the antiestrogen GW5638 induces a unique structural change in the ER. The biological significance of this conformational change was revealed in studies that demonstrated that tamoxifen-resistant breast tumor explants are not cross-resistant to GW5638. Because of these properties, this drug is currently being developed as a potential therapeutic for tamoxifen-resistant breast cancers.

We have developed a series of high-affinity peptide antagonists that inhibit the transcriptional activity of both subtypes of the human estrogen receptor (ERalpha and ERbeta). We believe that it will be possible to develop these peptides, or corresponding peptidomimetic derivatives, into pharmaceuticals for use in the treatment of breast cancer and other estrogenopathies. It is anticipated that drugs of this type could be used in combination with classical antiestrogens, such as tamoxifen, to achieve a complete blockage of ER-transcriptional activity. Although ER has been the primary target of our studies to date, it is likely that the insights gained from this work will apply to other nuclear receptors and transcription factors.

A transcriptional coactivator of the peroxisome proliferator-activated receptor-gamma (PPARgamma), PPARgamma-coactivator-1(PGC-1) interacts in a constitutive manner with the hinge domain of PPARgamma and enhances its transcriptional activity. In this study we demonstrate that PGC-1 is a coactivator of estrogen receptor-alpha (ERalpha)-dependent transcriptional activity. However the mechanism by which PGC-1 interacts with ERalpha is different from that of PPARgamma. Specifically, it was determined that the carboxyl terminus of PGC-1 interacts in a ligand-independent manner with the ERalpha hinge domain. In addition, an LXXLL motif within the amino terminus of PGC-1 was shown to interact in an agonist-dependent manner with the AF2 domain within the carboxyl terminus of ERalpha. The ability of PGC-1 to associate with and potentiate the transcriptional activity of an ERalpha-AF2 mutant that is unable to interact with the p160 class of coactivators suggests that this coactivator may have a unique role in estrogen signaling. It is concluded from these studies that PGC-1 is a bona fide ERalpha coactivator, which may serve as a convergence point between PPARgamma and ERalpha signaling.

Antiestrogens such as tamoxifen are one of the most effective methods of treating estrogen receptor (ERalpha) positive breast cancers; however, the effectiveness of this therapy is limited by the almost universal development of resistance to the drug. If antiestrogens are recognized differently by the cell as it has been suggested, then in disease conditions where tamoxifen fails to function effectively, a mechanistically different antiestrogen might yield successful results. Although many antiestrogens have been developed, a direct comparison of their mechanisms of action is lacking, thus limiting their utility. Therefore, to determine if there are mechanistic differences among available antiestrogens, we have carried out a comprehensive analysis of the molecular mechanisms of action of 4-hydroxy-tamoxifen (40HT), idoxifene, raloxifene, GW7604, and ICI 182,780. Using a novel set of peptides that recognize different surfaces on ERalpha, we have found that following binding to ERalpha, each ligand induces a distinct ERalpha-ligand conformation. Furthermore, transcriptional assays indicate that each ERalpha-ligand complex is recognized distinctly by the transcription machinery, and consequently, antiestrogens vary in their ability to inhibit estradiol- and 40HT-mediated activities. Relative binding assays have shown that the affinity of these ligands for ERalpha is not always representative of their inhibitory activity. Using this assay, we have also shown that the pharmacology of each antiestrogen is influenced differently by hormone binding proteins. Furthermore, GW7604, like ICI 182,780, but unlike the other antiestrogens evaluated, decreases the stability of the receptor. Overall, our results indicate that there are clear mechanistic distinctions among each of the antiestrogens studied. However, GW7604 and ICI 182,780 differ more significantly from tamoxifen than idoxifene and raloxifene. These data, which reveal differences among antiestrogens, should assist in the selection of compounds for the clinical regulation of ERalpha function.

Recruitment of transcriptional coactivators following ligand activation is a critical step in nuclear receptor-mediated target gene expression. Upon binding an agonist, the receptor undergoes a conformational change which facilitates the formation of a specific coactivator binding pocket within the carboxyl terminus of the receptor. This permits the alpha-helical LXXLL motif within some coactivators to interact with the nuclear receptors. Until recently, the LXXLL motif was thought to function solely as a docking module; however, it now appears that sequences flanking the core motif may play a role in determining receptor selectivity. To address this issue, we used a combinatorial phage display approach to evaluate the role of flanking sequences in influencing these interactions. We sampled more than 10(8) variations of the core LXXLL motif with estradiol-activated estrogen receptor alpha (ERalpha) as a target and found three different classes of peptides. All of these peptides interacted with ERalpha in an agonist-dependent manner and disrupted ERalpha-mediated transcriptional activity when introduced into target cells. Using a series of ERalpha-mutants, we found that these three classes of peptides showed different interaction patterns from each other, suggesting that not all LXXLL motifs are the same and that receptor binding selectivity can be achieved by altering sequences flanking the LXXLL core motif. Most notable in this regard was the discovery of a peptide which, when overexpressed in cells, selectively disrupted ERbeta- but not ERalpha-mediated reporter gene expression. This novel ERbeta-specific antagonist may be useful in identifying and characterizing the ERbeta-regulated process in estradiol-responsive cells. In conclusion, using a combinatorial approach to define cofactor-receptor interactions, we have clearly been able to demonstrate that not all LXXLL motifs are functionally equivalent, a finding which suggests that it may be possible to target receptor-LXXLL interactions to develop receptor-specific antagonists.