Benita S. Katzenellenbogen

Selective Estrogen Receptor Modulators: Discrimination of Agonistic versus Antagonistic Activities by Gene Expression Profiling in Breast Cancer Cells

Selective estrogen receptor modulators (SERMs) such as tamoxifen are effective in the treatment of many estrogen receptor-positive breast cancers and have also proven to be effective in the prevention of breast cancer in women at high risk for the disease. The comparative abilities of tamoxifen versus raloxifene in breast cancer prevention are currently being compared in the Study of Tamoxifen and Raloxifene trial. To better understand the actions of these compounds in breast cancer, we have examined their effects on the expression of ∼12,000 genes, using Affymetrix GeneChip microarrays, with quantitative PCR verification in many cases, categorizing their actions as agonist, antagonist, or partial agonist/antagonist. Analysis of gene stimulation and inhibition by the SERMs trans-hydroxytamoxifen (TOT) and raloxifene (Ral) or ICI 182,780 (ICI) and by estradiol (E2) in estrogen receptor-containing MCF-7 human breast cancer cells revealed that (a) TOT was the most E2-like of the three compounds, (b) all three compounds either partially or fully antagonized the action of E2 on most genes, with the order of antagonist activity being ICI > Ral > TOT, (c) TOT and Ral, but not ICI, displayed partial agonist/partial antagonist activity on a number of E2-regulated genes, (d) several stimulatory cell cycle-related genes were down-regulated exclusively by ICI, (e) the estrogen-like activity of Ral nearly always overlapped with that of TOT, indicating that Ral has little unique agonist activity different from that of TOT, and (f) some genes were specifically up-regulated by TOT but not Ral, ICI, or E2. Hence, gene expression profiling can discern fundamental differences among SERMs and provides insight into the distinct biologies of TOT, Ral, and ICI in breast cancer.

Direct acetylation of the estrogen receptor alpha hinge region by p300 regulates transactivation and hormone sensitivity.

Regulation of nuclear receptor gene expression involves dynamic and coordinated interactions with histone acetyl transferase (HAT) and deacetylase complexes. The estrogen receptor (ERα) contains two transactivation domains regulating ligand-independent and -dependent gene transcription (AF-1 and AF-2 (activation functions 1 and 2)). ERα-regulated gene expression involves interactions with cointegrators (e.g.p300/CBP, P/CAF) that have the capacity to modify core histone acetyl groups. Here we show that the ERα is acetylated in vivo.p300, but not P/CAF, selectively and directly acetylated the ERα at lysine residues within the ERα hinge/ligand binding domain. Substitution of these residues with charged or polar residues dramatically enhanced ERα hormone sensitivity without affecting induction by MAPK signaling, suggesting that direct ERα acetylation normally suppresses ligand sensitivity. These ERα lysine residues also regulated transcriptional activation by histone deacetylase inhibitors and p300. The conservation of the ERα acetylation motif in a phylogenetic subset of nuclear receptors suggests that direct acetylation of nuclear receptors may contribute to additional signaling pathways involved in metabolism and development.

Novel Ligands that Function as Selective Estrogens or Antiestrogens for Estrogen Receptor-α or Estrogen Receptor-β1

We report on the identification of novel, nonsteroidal ligands that show pronounced subtype-selective differences in ligand binding and transcriptional potency or efficacy for the two estrogen receptor (ER) subtypes, ER alpha and ER beta. An aryl-substituted pyrazole is an ER alpha potency-selective agonist, showing higher binding affinity for ER alpha and 120-fold higher potency in stimulation of ER alpha vs. ER beta in transactivation assays in cells. A tetrahydrochrysene (THC) has a 4-fold preferential binding affinity for ER beta; it is an agonist on ER alpha, but a complete antagonist on ER beta. Intriguingly, the antagonist activity of THC is associated with the R,R-enantiomer (R,R-THC). The S,S-enantiomer (S,S-THC) is an agonist on both ER alpha and ER beta but has a 20-fold lower affinity for ER beta than R,R-THC. This difference in binding affinity accounts for the full ER beta antagonist activity of the THC racemate (a 1:1 mixture of R,R-THC and S,S-THC). These compounds should be useful in probing the conformational changes in these two ERs that are evoked by agonists and antagonists, and in evaluating the distinct roles that ER beta and ER alpha may play in the diverse target tissues in which estrogens act.

Activities of estrogen receptor alpha- and beta-selective ligands at diverse estrogen responsive gene sites mediating transactivation or transrepression

Estrogens exert their regulatory transcriptional effects, which can be stimulatory or repressive, at diverse gene sites via two estrogen receptors, ERalpha and ERbeta. Since these two ERs have different tissue distributions, ligands that have the capacity to selectively activate or inhibit these two ERs would be useful in elucidating the biology of these two receptors and might assist in the development of estrogen pharmaceuticals with improved tissue selectivity. We have developed several ligands that showed ERalpha or ERbeta selectivity at promoter-gene sites containing consensus estrogen response elements (EREs): ERalpha-selective agonist (propyl-pyrazole-triol (PPT)), ERalpha-selective antagonist (methyl-piperidino-pyrazole (MPP)), ERbeta-potency selective agonist (diarylpropionitrile (DPN)) and ERbeta-selective antagonist/ERalpha-agonist (R,R-tetrahydrochrysene (R,R-THC)). In this study, we have examined the activity of these compounds at a range of gene sites where ER stimulates gene expression through non-consensus EREs (complement C3), or multiple half-EREs (NHE-RF/EBP50), or by tethering to DNA via other proteins (TGF beta3 and progesterone receptor A/AP-1), and at gene sites where ER represses gene transcription (interleukin-6). At all of these genes, PPT showed full stimulation through ERalpha while displaying no agonism through ERbeta. MPP antagonized estradiol actions on gene transactivation and transrepression through ERalpha, with little or no effect on transcription mediated through ERbeta. DPN displayed subtype-selective agonism, being ca. 30-fold more potent through ERbeta. R,R-THC was a complete antagonist through ERbeta and displayed agonism through ERalpha, the level of which was promoter dependent. Because these ligands maintain their agonist or antagonist character and ER subtype-selectivity at gene sites of diverse nature, where estradiol is either stimulatory or inhibitory, these compounds should prove useful in elucidating the biological functions of ERalpha and ERbeta.

Estrogen receptor transcription and transactivation: Estrogen receptor alpha and estrogen receptor beta: regulation by selective estrogen receptor modulators and importance in breast cancer.

Estrogens display intriguing tissue-selective action that is of great biomedical importance in the development of optimal therapeutics for the prevention and treatment of breast cancer, for menopausal hormone replacement, and for fertility regulation. Certain compounds that act through the estrogen receptor (ER), now referred to as selective estrogen receptor modulators (SERMs), can demonstrate remarkable differences in activity in the various estrogen target tissues, functioning as agonists in some tissues but as antagonists in others. Recent advances elucidating the tripartite nature of the biochemical and molecular actions of estrogens provide a good basis for understanding these tissue-selective actions. As discussed in this thematic review, the development of optimal SERMs should now be viewed in the context of two estrogen receptor subtypes, ERα and ERβ, that have differing affinities and responsiveness to various SERMs, and differing tissue distribution and effectiveness at various gene regulatory sites. Cellular, biochemical, and structural approaches have also shown that the nature of the ligand affects the conformation assumed by the ER-ligand complex, thereby regulating its state of phosphorylation and the recruitment of different coregulator proteins. Growth factors and protein kinases that control the phosphorylation state of the complex also regulate the bioactivity of the ER. These interactions and changes determine the magnitude of the transcriptional response and the potency of different SERMs. As these critical components are becoming increasingly well defined, they provide a sound basis for the development of novel SERMs with optimal profiles of tissue selectivity as medical therapeutic agents.

Conformational Changes and Coactivator Recruitment by Novel Ligands for Estrogen Receptor-α and Estrogen Receptor-β: Correlations with Biological Character and Distinct Differences among SRC Coactivator Family Members1

Ligands for the estrogen receptor (ER) that have the capacity to selectively bind to or activate the ER subtypes ERα or ERβ would be useful in elucidating the biology of these two receptors and might assist in the development of estrogen pharmaceuticals with improved tissue selectivity. In this study, we examine three compounds of novel structure that act as ER subtype-selective ligands. These are a propyl pyrazole triol (PPT), which is a potent agonist on ERα but is inactive on ERβ, and a pair of substituted tetrahydrochrysenes (THC), one enantiomer of which (S,S-THC) is an agonist on both ERα and ERβ, the other (R,R-THC) being an agonist on ERα but an antagonist on ERβ. To investigate the molecular mechanisms underlying the ER subtype-selective actions of these compounds, we have determined the conformational changes induced in ERα and ERβ by these ligands using protease digestion sensitivity, and we have tested the ability of these ligands to promote the recruitment of representatives of the three SRC/...

Antagonists Selective for Estrogen Receptor α

To develop compounds that are antagonists on ERα, but not ERβ, we have added basic side-chains typically found in nonsteroidal antiestrogens to pyrazole compounds that bind with much higher affinity to ERα than to ERβ. In this way we have developed basic side-chain pyrazoles (BSC-pyrazoles) that are high affinity, potent, selective antagonists on ERα. These BSC-pyrazoles are themselves inactive on ERα and ERβ, and they antagonize E2 stimulation by ERα only. We investigated seven basic side-chain substituents on various alkyl-triaryl-substituted pyrazoles, and the most ERα-selective compound was methyl-piperidino-pyrazole (MPP). ERα-selective antagonism was observed on diverse reporter-promoter gene constructs containing estrogen response elements that are consensus, nonconsensus (pS2), or comprised of multiple half-estrogen response elements (NHERF/EBP50) and on genes in which ER works indirectly by tethering to other DNA-bound proteins (TGFβ3). In contrast to these BSC-pyrazoles, the antiestrogens trans-...

Non-nuclear estrogen receptor α signaling promotes cardiovascular protection but not uterine or breast cancer growth in mice

Steroid hormone receptors function classically in the nucleus as transcription factors. However, recent data indicate that there are also non-nuclear subpopulations of steroid hormone receptors, including estrogen receptors (ERs), that mediate membrane-initiated signaling of unclear basis and significance. Here we have shown that an estrogen-dendrimer conjugate (EDC) that is excluded from the nucleus stimulates endothelial cell proliferation and migration via ERalpha, direct ERalpha-Galphai interaction, and endothelial NOS (eNOS) activation. Analysis of mice carrying an estrogen response element luciferase reporter, ER-regulated genes in the mouse uterus, and eNOS enzyme activation further indicated that EDC specifically targets non-nuclear processes in vivo. In mice, estradiol and EDC equally stimulated carotid artery reendothelialization in an ERalpha- and G protein-dependent manner, and both agents attenuated the development of neointimal hyperplasia following endothelial injury. In contrast, endometrial carcinoma cell growth in vitro and uterine enlargement and MCF-7 cell breast cancer xenograft growth in vivo were stimulated by estradiol but not EDC. Thus, EDC is a non-nuclear selective ER modulator (SERM) in vivo, and in mice, non-nuclear ER signaling promotes cardiovascular protection. These processes potentially could be harnessed to provide vascular benefit without increasing the risk of uterine or breast cancer.

Estrogen action and male fertility: Roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function

Estrogen receptor α (ERα) is essential for male fertility. Its activity is responsible for maintaining epithelial cytoarchitecture in efferent ductules and the reabsorption of fluid for concentrating sperm in the head of the epididymis. These discoveries and others have helped to establish estrogens bisexual role in reproductive importance. Reported here is the molecular mechanism to explain estrogens role in fluid reabsorption in the male reproductive tract. It is shown that estrogen regulates expression of the Na+/H+ exchanger-3 (NHE3) and the rate of 22Na+ transport, sensitive to an NHE3 inhibitor. Immunohistochemical staining for NHE3, carbonic anhydrase II (CAII), and aquaporin-I (AQP1) was decreased in ERα knockout (αERKO) efferent ductules. Targeted gene-deficient mice were compared with αERKO, and the NHE3 knockout and CAII-deficient mice showed αERKO-like fluid accumulation, but only the NHE3 knockout and αERKO mice were infertile. Northern blot analysis showed decreases in mRNA for NHE3 in αERKO and antiestrogen-treated mice. The changes in AQP1 and CAII in αERKO seemed to be secondary because of the disruption of apical cytoarchitecture. Ductal epithelial ultrastructure was abnormal only in αERKO mice. Thus, in the male, estrogen regulates one of the most important epithelial ion transporters and maintains epithelial morphological differentiation in efferent ductules of the male, independent of its regulation of Na+ transport. Finally, these data raise the possibility of targeting ERα in developing a contraceptive for the male.

Antiestrogens: Mechanisms of action and resistance in breast cancer

Antiestrogens have proven to be highly effective in the treatment of hormone-responsive breast cancer. However, resistance to antiestrogen therapy often develops. In addition, although tamoxifen-like antiestrogens are largely inhibitory and function as estrogen antagonists in breast cancer cells, they also have some estrogen-like activity in other cells of the body. Thus, recent efforts are being directed toward the development of even more tissue-selective antiestrogens, i.e. compounds that are antiestrogenic on breast and uterus while maintaining the beneficial estrogen-like actions on bone and the cardiovascular system. Efforts are also being directed toward understanding ligand structure-estrogen receptor (ER) activity relationships and characterizing the molecular changes that underlie alterations in parallel signal transduction pathways that impact on the ER. Recent findings show that antiestrogens, which are known to exert most of their effects through the ER of breast cancer cells, contact a different set of amino acids in the hormone binding domain of the ER than those contacted by estrogen, and evoke a different receptor conformation that results in reduced or no transcriptional activity on most genes.Resistance to antiestrogen therapy may develop due to changes at the level of the ER itself, and at pre- and post-receptor points in the estrogen receptor-response pathway. Resistance could arise in at least four ways: (1) ER loss or mutation; (2) Post-receptor alterations including changes in cAMP and phosphorylation pathways, or changes in coregulator and transcription factor interactions that affect the transcriptional activity of the ER; (3) Changes in growth factor production/sensitivity or paracrine cell-cell interactions; or (4) Pharmacological changes in the antiestrogen itself, including altered uptake and retention or metabolism of the antiestrogen. Model cell systems have been developed to study changes that accompany and define the antiestrogen resistant versus sensitive breast cancer phenotype. This information should lead to the development of antiestrogens with optimized tissue selectivity and agents to which resistance may develop more slowly. In addition, antiestrogens which work through somewhat different mechanisms of interaction with the ER should prove useful in treatment of some breast cancers that become resistant to a different category of antiestrogens.