Target specificity of selective estrogen receptor modulators within human endometrial cancer cells

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Abstract

Selective estrogen receptor modulators (SERMs) are estrogen receptor (ER) ligands that function as antagonists in some tissues, but have either partial or full agonist activity in others. SERMs often display variable partial agonist activity in uterine tissues and this activity can be displayed in uterine cell lines such as the human Ishikawa endometrial adenocarcinoma cell line. In this study, we compared the effects of several ER ligands including some SERMs on alkaline phosphatase (AP) activity and the expression of an ER target gene, the progesterone receptor (PR), in Ishikawa cells. As expected, estradiol (E2) was a potent and efficacious activator of both AP activity and PR mRNA expression. 4-Hydroxytamoxifen (4OHT) stimulated AP activity to a level 47% of that of E2 (100 nM), while CP 336156 (lasofoxifene) increased AP activity 18%. A benzothiophene, such as LY 117018, a raloxifene analog, stimulated AP even less with values approximately 11% of E2-stimulated levels. A pure antiestrogen, ICI 182,780 did not stimulate AP activity. Interestingly, when we examined the ability of these compounds to increase the expression of the ER target gene, PR, a different rank order of efficacy was detected. After E2, CP 336156 was the most efficacious in increasing PR mRNA with a maximal stimulation of 20% of E2 levels, while 4OHT stimulated only 17%. LY 117018 increased PR mRNA expression 8% while ICI 182,780 did not increase PR mRNA expression at all. These data illustrate the target specificity that a SERM is able to display within a single cell type independent of “tissue specificity” and differential levels of expression of various cofactors. While 4OHT is 160% more active than CP 336156 in terms of inducing AP activity in the Ishikawa cells, CP 336156 has equivalent activity as 4OHT when one examines the ability of these SERMs to induce PR mRNA expression. Since the stimulation of Ishikawa cells by ER ligands is often used to assess the potential in vivo uterotrophic activity, these data indicate that examination of several endpoints in these cells may be necessary in order to fully characterize the activity of SERMs.

Introduction

The estrogen receptor (ER) is a ligand-dependent transcription factor that belongs to the nuclear hormone receptor superfamily [1]. ER plays an essential role in mediation of the action of estrogens in a variety of biological processes including differentiation, homeostasis and reproduction. However, additional actions of estrogens have been characterized in the skeletal, cardiovascular, and nervous systems. Estrogens act by binding directly to ER and inducing a conformational change, which allows for receptor dimerization followed by recognition of specific DNA sequences (estrogen response elements; EREs) in the regulatory regions of estrogen regulated genes within target cell nuclei. Once activated and positioned on the ERE, ER can modulate the transcription of the target gene. Until 1996, it was believed that the actions of estrogens were mediated by a sole receptor; however, an additional ER isoform was identified, termed ERβ, with a distinct pattern of expression compared to ERα that added a degree of complexity to ER pharmacology [2], [3].

Clinically, ER ligands are utilized for a variety of indications including contraception, hormone replacement therapy (HRT), and the prevention and treatment of breast cancer [4]. Several of the ER ligands used in the clinic are selective estrogen receptor modulators (SERMs). SERMs are ER ligands that function as antagonists in some tissues, but have either partial or full agonist activity in others. One target tissue of estrogens of particular physiological and clinical relevance is the uterus. Estrogens cause an increase in proliferation of the uterine endometrial cells, which may limit the chronic use of ER agonists due to increased risk of endometrial cancer. However, SERMs display a wide range of partial agonist activity with some exhibiting negligible uterine agonist activity allowing for chronic therapy for indications such as the prevention and treatment of osteoporosis as is the case for raloxifene [5].

The mechanism by which SERMs display their tissue selectivity is unclear. In cotransfections, SERMs display differential selectivity based on the promoter and cell context [6]. However, this specificity does not appear to be mediated by ER isoform selectivity since SERMs do not possess significant receptor isoform selectivity and the expression profile of ERα versus ERβ is inconsistent with the tissue/cell selectivity profile of the SERMs [7]. The promoter or cell type specific requirement for activation domain 1 and/or 2 (AF-1 and/or AF-2) of ER has been demonstrated to be a determinate of a particular SERMs pharmacological function [8]. Crystallographic evidence demonstrates that SERMs, such as tamoxifen and raloxifene, induce a conformational structure in the ligand binding domain of ER that is distinct from that induced by estrogen agonists [9], [10], [11], which is consistent with previous data using the degree of protease resistance of ligand-bound ER to predict differences in the conformational state of the receptor [12], [13], [14]. Differential recruitment of receptor cofactors may also play a role in tissue selective action of SERMs, which may be governed by the selective patterns of expression of these cofactors and/or selective receptor interactions with various cofactors due to the unique conformations induced by the distinct ligands [6], [15]. Direct protein–protein interaction with and modulation of the activity of other factors such as alkaline phosphatase (AP)-1 by ER, independent of its own DNA binding activity, may also play a role in selectivity since various SERMs appear to differentially influence these signaling pathways [16].

In the current study, we examined the effects of a series of SERMs on two endpoints within a human endometrial adenocarcinoma cell line (Ishikawa). We compared the ability of various SERMs to increase alkaline phosphatase activity and progesterone receptor (PR) mRNA expression. AP activity was selected to be monitored because of the significant amount of historical data that has been published characterizing estrogen receptor ligands in this system (Section 4). PR mRNA was chosen as a second endpoint to measure since PR is a target gene for ER [17] and is of particular physiological importance in the uterus due to the requirement for estrogens to prime the uterus for the increase in progesterone that occurs during the luteal phase of the ovarian cycle and which is essential for preparing the secretory phase uterus for implantation of the conceptus [18]. Interestingly, some SERMs displayed differential activity in the Ishikawa cell line depending on the endpoint examined (AP or PR).

Section snippets

Compounds

Estradiol (E2), 4-hydroxytamoxifen (4OHT), clomiphene, and droloxifene were obtained from Sigma. ICI 182,780 was obtained from Tocris (Ballwin, MO). LY 117018 and CP 336156 were synthesized using standard organic synthesis methods. Compounds were diluted first in 100% DMSO to yield a 10 mM stock solution followed by dilution in cell medium. Final DMSO concentrations within the cell medium was 0.1%.

Cell culture and experimental design

The human Ishikawa cell line derived from a well-differentiated endometrial adenocarcinoma was

Results

Fig. 1 illustrates the chemical structures of the ER ligands utilized in this study. 17β-estradiol and ICI 182,780 were used as the standard agonist and antagonist, respectively. Three triphenylethylene SERMs were evaluated in this study including 4OHT, chlomiphene, and droloxifene. A benzothiophene SERM was tested: LY 117018 (a raloxifene analogue). In addition, a napthalene SERM, CP 336156 (lasofoxifene), was examined.

We first assessed the agonist activity of the ER ligands with respect to

Discussion

Several SERMs are currently approved for clinical use; however, each displays a unique tissue specificity profile [5]. The intrinsic tissue specificity profile of a particular SERM often specifies the clinical indication for the compound. For example, both tamoxifen and raloxifene act as ER agonists in bone suggesting that they would be efficacious in the prevention and treatment of osteoporosis, but tamoxifen also retains significant uterine agonist activity precluding its use due to increased

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