Suzanne A. W. Fuqua

International Union of Pharmacology. LXIV. Estrogen Receptors

Estrogen receptors (ERs[1][1]) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. In the late 1950s, the existence of a receptor molecule that could bind 17β-estradiol was demonstrated by Jensen and Jacobsen ([Jensen and Jordan, 2003][2]). The first

Selective Estrogen Receptor Modulators: Structure, Function, and Clinical Use

The sex hormone estrogen is important for many physiologic processes. Prolonged stimulation of breast ductal epithelium by estrogen, however, can contribute to the development and progression of breast cancer, and treatments designed to block estrogens effects are important options in the clinic. Tamoxifen and other similar drugs are effective in breast cancer prevention and treatment by inhibiting the proliferative effects of estrogen that are mediated through the estrogen receptor (ER). However, these drugs also have many estrogenic effects depending on the tissue and gene, and they are more appropriately called selective estrogen receptor modulators (SERMs). SERMs bind ER, alter receptor conformation, and facilitate binding of coregulatory proteins that activate or repress transcriptional activation of estrogen target genes. Theoretically, SERMs could be synthesized that would exhibit nearly complete agonist activity on the one hand or pure antiestrogenic activity on the other. Depending on their functional activities, SERMs could then be developed for a variety of clinical uses, including prevention and treatment of osteoporosis, treatment and prevention of estrogen-regulated malignancies, and even for hormone replacement therapy. Tamoxifen is effective in patients with ER-positive metastatic breast cancer and in the adjuvant setting. The promising role for tamoxifen in ductal carcinoma-in-situ or for breast cancer prevention is evolving, and its use can be considered in certain patient groups. Other SERMs are in development, with the goal of reducing toxicity and/or improving efficacy, and future agents have the potential of providing a new paradigm for maintaining the health of women.

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.

Comprehensive Genomic Analysis Identifies Novel Subtypes and Targets of Triple-Negative Breast Cancer

Purpose: Genomic profiling studies suggest that triple-negative breast cancer (TNBC) is a heterogeneous disease. In this study, we sought to define TNBC subtypes and identify subtype-specific markers and targets. Experimental Design: RNA and DNA profiling analyses were conducted on 198 TNBC tumors [estrogen receptor (ER) negativity defined as Allred scale value ≤ 2] with >50% cellularity (discovery set: n = 84; validation set: n = 114) collected at Baylor College of Medicine (Houston, TX). An external dataset of seven publically accessible TNBC studies was used to confirm results. DNA copy number, disease-free survival (DFS), and disease-specific survival (DSS) were analyzed independently using these datasets. Results: We identified and confirmed four distinct TNBC subtypes: (i) luminal androgen receptor (AR; LAR), (ii) mesenchymal (MES), (iii) basal-like immunosuppressed (BLIS), and (iv) basal-like immune-activated (BLIA). Of these, prognosis is worst for BLIS tumors and best for BLIA tumors for both DFS (log-rank test: P = 0.042 and 0.041, respectively) and DSS (log-rank test: P = 0.039 and 0.029, respectively). DNA copy number analysis produced two major groups (LAR and MES/BLIS/BLIA) and suggested that gene amplification drives gene expression in some cases [FGFR2 (BLIS)]. Putative subtype-specific targets were identified: (i) LAR: androgen receptor and the cell surface mucin MUC1, (ii) MES: growth factor receptors [platelet-derived growth factor (PDGF) receptor A; c-Kit], (iii) BLIS: an immunosuppressing molecule (VTCN1), and (iv) BLIA: Stat signal transduction molecules and cytokines. Conclusion: There are four stable TNBC subtypes characterized by the expression of distinct molecular profiles that have distinct prognoses. These studies identify novel subtype-specific targets that can be targeted in the future for the effective treatment of TNBCs. Clin Cancer Res; 21(7); 1688–98. ©2014 AACR. See related commentary by Vidula and Rugo, p. 1511

Measurement of steroid hormone receptors in breast cancer patients on tamoxifen

SummaryEstrogen (ER) and progesterone receptor (PgR) positive breast tumors often respond to tamoxifen, but ultimately progress as they become tamoxifen resistant. An accurate assessment of receptor status in specimens from tamoxifen-resistant patients could help to understand potential mechanisms of resistance and to predict response to second line hormonal therapies. However, since tamoxifen itself can affect ER and PgR determinations, assay results can be misleading. We measured ER and PgR by both ligand binding (LBA) and immunohistochemical (IHC) assays in 34 tumors from patients on tamoxifen, 30 of whom were displaying resistance to the drug. These tumors were classified into several receptor phenotypes. Eleven patients, 8 of whom were clearly progressing, expressed both receptors while on tamoxifen. ER was significantly less often negative when measured by IHC, suggesting that ER status by LBA was falsely negative in this group due to receptor occupancy by tamoxifen. Six patients had no detectable ER by LBA or IHC but still expressed PgR. The presence of PgR suggests that ER could still be functional, though undetectable, in these tumors, or that PgR is constitutively expressed by them. Finally, 12 patients were ER and PgR-negative by both assays, suggesting hormonal independence as the mechanism for resistance in this group. In a subset of patients with receptor assays both prior to tamoxifen and at the time of progression while taking the drug, we found that most ER-positive tumors converted to an apparent ER-negative status when assayed by LBA, while PgR status frequently remained unchanged. The continued expression of ER and/or PgR in many patients with tumor progression on tamoxifen indicates that mechanisms for resistance other than receptor loss are common in breast cancer.

Low Levels of Estrogen Receptor β Protein Predict Resistance to Tamoxifen Therapy in Breast Cancer

Purpose: Breast cancer is a hormone-dependent cancer, and the presence of estrogen receptor α (ER-α) in tumors is used clinically to predict the likelihood of response to hormonal therapies. The clinical value of the second recently identified ER isoform, called ER-β, is less clear, and there is currently conflicting data concerning its potential role as a prognostic or predictive factor. Experimental Design: To assess whether ER-β expression is associated with clinical outcome, protein levels were measured by immunoblot analysis of a retrospective bank of tumor cell lysates from 305 axillary node-positive patients. A total of 119 received no adjuvant therapy, and 186 were treated with tamoxifen only. The median follow-up time was 65 months. Univariate and multivariate Cox regression modeling was done to assess the prognostic and predictive significance of ER-β expression. Results: Expression of ER-β protein did not correlate significantly with any other clinical variables, including ER and progesterone levels (as measured ligand binding assay), tumor size, age, or axillary nodal status. In the untreated population, those patients whose tumors who expressed both receptor isoforms exhibited the most favorable outcome as compared with those patients who had lost ER-α expression. However, there was no association between ER-β levels alone and either disease-free or overall survival in the untreated patient population. In contrast, in both univariate and multivariate analyses, high levels of ER-β predicted an improved disease-free and overall survival in patients treated with adjuvant tamoxifen therapy. Conclusions: These findings provide evidence that ER-β may be an independent predictor of response to tamoxifen in breast cancer. Furthermore, these results suggest that ER-β may influence tumor progression in ways different from those mediated by the ER-α isoform.

Androgen Receptor Acetylation Governs trans Activation and MEKK1-Induced Apoptosis without Affecting In Vitro Sumoylation and trans-Repression Function

ABSTRACT The androgen receptor (AR) is a nuclear hormone receptor superfamily member that conveys both trans repression and ligand-dependent trans-activation function. Activation of the AR by dihydrotestosterone (DHT) regulates diverse physiological functions including secondary sexual differentiation in the male and the induction of apoptosis by the JNK kinase, MEKK1. The AR is posttranslationally modified on lysine residues by acetylation and sumoylation. The histone acetylases p300 and P/CAF directly acetylate the AR in vitro at a conserved KLKK motif. To determine the functional properties governed by AR acetylation, point mutations of the KLKK motif that abrogated acetylation were engineered and examined in vitro and in vivo. The AR acetylation site point mutants showed wild-type trans repression of NF-κB, AP-1, and Sp1 activity; wild-type sumoylation in vitro; wild-type ligand binding; and ligand-induced conformational changes. However, acetylation-deficient AR mutants were selectively defective in DHT-induced trans activation of androgen-responsive reporter genes and coactivation by SRC1, Ubc9, TIP60, and p300. The AR acetylation site mutant showed 10-fold increased binding of the N-CoR corepressor compared with the AR wild type in the presence of ligand. Furthermore, histone deacetylase 1 (HDAC1) bound the AR both in vivo and in cultured cells and HDAC1 binding to the AR was disengaged in a DHT-dependent manner. MEKK1 induced AR-dependent apoptosis in prostate cancer cells. The AR acetylation mutant was defective in MEKK1-induced apoptosis, suggesting that the conserved AR acetylation site contributes to a pathway governing prostate cancer cellular survival. As AR lysine residue mutations that abrogate acetylation correlate with enhanced binding of the N-CoR repressor in cultured cells, the conserved AR motif may directly or indirectly regulate ligand-dependent corepressor disengagement and, thereby, ligand-dependent trans activation.

Breast Cancer Patients with Progesterone Receptor PR-A-Rich Tumors Have Poorer Disease-Free Survival Rates

Purpose: No study has yet analyzed whether changes in relative expression levels of progesterone receptor (PR) isoforms A and B in human breast tumors have significance in predicting clinical outcome. Human PRs are ligand-activated nuclear transcription factors that mediate progesterone action. Their presence in breast tumors is used to predict functional estrogen receptors (ERs) and, therefore, also to predict the likelihood of response to endocrine therapies and disease prognosis. The two PR isoforms, PR-A and PR-B, possess different in vitro and in vivo activities, suggesting that in tumors, the ratio of their expression may control hormone responsiveness. In general, PR-B are strong transcriptional activators, whereas PR-A can act as dominant repressors of PR-B and ER. Thus their balance may affect tamoxifen response in breast cancers. Experimental Design: To determine whether differential expression of the PR isoforms is associated with clinical outcome and hormonal responsiveness, PR-A and PR-B were measured by immunoblot analysis of cell lysates from 297 axillary node-positive breast tumors. Results: Expression of the two isoforms correlated with each other, as well as with ER. Additional analyses revealed that patients with PR-positive tumors but high PR-A:PR-B ratios, which were often caused by high PR-A levels, were 2.76 times more likely to relapse than patients with lower ratios, indicating resistance to tamoxifen. Conclusions: This study suggests that knowledge of the PR-A:PR-B ratio may identify a subgroup of ER-positive/PR-positive patients with node-positive breast cancer that benefit poorly from endocrine therapy.

Phosphorylation of Estrogen Receptor α Blocks Its Acetylation and Regulates Estrogen Sensitivity

Estrogen receptor (ER) α is mutated (lysine 303 to arginine, K303R) in approximately one third of premalignant breast hyperplasias, which renders breast cancer cells expressing the mutant receptor hypersensitive for proliferation in response to low doses of estrogen. It is known that ERα is posttranslationally modified by protein acetylation and phosphorylation by a number of secondary messenger signaling cascades. The K303R ERα mutation resides at a major protein acetylation site adjacent to a potential protein kinase A (PKA) phosphorylation site at residue 305 within the hinge domain of the receptor. Mutation of this phosphorylation site to aspartic acid to mimic constitutive phosphorylation blocks acetylation of the K303 ERα site and generates an enhanced transcriptional response similar to that seen with the naturally occurring K303R mutant receptor. Activation of PKA signaling by the cell-permeable cyclic AMP (cAMP) analog 8-bromo-cAMP further enhances estrogen sensitivity of the mutant receptor, whereas a specific PKA inhibitor antagonizes this increase. We propose that the hypersensitive ERα mutant breast cancer phenotype involves an integration of coupled acetylation and phosphorylation events by upstream signaling molecules.

Cytokine Receptor CXCR4 Mediates Estrogen-Independent Tumorigenesis, Metastasis, and Resistance to Endocrine Therapy in Human Breast Cancer

Estrogen independence and progression to a metastatic phenotype are hallmarks of therapeutic resistance and mortality in breast cancer patients. Metastasis has been associated with chemokine signaling through the SDF-1-CXCR4 axis. Thus, the development of estrogen independence and endocrine therapy resistance in breast cancer patients may be driven by SDF-1-CXCR4 signaling. Here we report that CXCR4 overexpression is indeed correlated with worse prognosis and decreased patient survival irrespective of the status of the estrogen receptor (ER). Constitutive activation of CXCR4 in poorly metastatic MCF-7 cells led to enhanced tumor growth and metastases that could be reversed by CXCR4 inhibition. CXCR4 overexpression in MCF-7 cells promoted estrogen independence in vivo, whereas exogenous SDF-1 treatment negated the inhibitory effects of treatment with the anti-estrogen ICI 182,780 on CXCR4-mediated tumor growth. The effects of CXCR4 overexpression were correlated with SDF-1-mediated activation of downstream signaling via ERK1/2 and p38 MAPK (mitogen activated protein kinase) and with an enhancement of ER-mediated gene expression. Together, these results show that enhanced CXCR4 signaling is sufficient to drive ER-positive breast cancers to a metastatic and endocrine therapy-resistant phenotype via increased MAPK signaling. Our findings highlight CXCR4 signaling as a rational therapeutic target for the treatment of ER-positive, estrogen-independent breast carcinomas needing improved clinical management.