Estrogen receptor β inhibits 17β-estradiol-stimulated proliferation of the breast cancer cell line T47D

Anders Ström

Johan Hartman

James Foster

Silke Kietz

Jay Wimalasena

Jan Gustafsson

^{Department of Biosciences, Karolinska Institutet, Novum, S-14157 Huddinge, Sweden;}^{Department of Obstetrics and Gynecology, Graduate School of Medicine, University of Tennessee Medical Center, Knoxville, TN 37920; and}^{Department of Medical Nutrition and Biosciences, Karolinska Institutet, Novum, 14186 Huddinge, Sweden}

^{To whom correspondence should be addressed. E-mail:}es.ik.tundem@nossfatsug.eka-naj.

Contributed by Jan-Åke Gustafsson, December 15, 2003

Abstract

Estrogen receptor (ER) β counteracts the activity of ERα in many systems. In agreement with this, we show in this study that induced expression of ERβ in the breast cancer cell line T47D reduces 17β-estradiol-stimulated proliferation when expression of ERβ mRNA equals that of ERα. Induction of ERβ reduces growth of exponentially proliferating cells with a concomitant decrease in components of the cell cycle associated with proliferation, namely cyclin E, Cdc25A (a key regulator of Cdk2), p45^{(a key regulator of p27}^{proteolysis), and an increase in the Cdk inhibitor p27}^{. We also observed a reduced Cdk2 activity. These findings suggest a possible role for ERβ in breast cancer and imply that ERβ-specific ligands may reduce proliferation of ER-positive breast cancer cells through actions on the G}₁ phase cell-cycle machinery.

Keywords: cell cycle, p27, cyclin E, Cdc25A, Cdk2

Abstract

The proliferative actions of 17β-estradiol (E2) mediated via estrogen receptor (ER) α can be opposed by ERβ. The mechanism behind this antagonism seems to be related to the different actions of E2–ERα and E2–ERβ complexes at AP-1 sites, respectively (1). This antiproliferative effect of ERβ might explain protection by estrogen against development of colon cancer, which has been seen in women on hormone replacement therapy (2) and may be of significance in the progression of colon cancer, where it has been shown that the malignant cells in the colon lose expression of ERβ (1). The ventral prostate and the uterus are other tissues where ERβ exerts antiproliferative actions. In the absence of ERβ (in ERβ-/- mice), there is hyperplasia of the prostate epithelium (2) and hypersensitivity of the uterus to the growth effects of E2 (3).

Breast cancer cell lines are extensively used as model systems to study aberrant E2-dependent and E2-independent growth. One commonly used cell line whose growth is stimulated by E2 is the ERα-positive cell line T47D (3, 4). E2 regulates expression of key cell-cycle genes such as c-Myc, cyclin D1, cyclin E, A, Cdc25A, p45^{, and p27}⁽5–10). The cyclin D promoter is one site where ERβ opposes ERα-mediated activation and the ERβ–antiestrogen complex can stimulate transcription (1). Increased transcription of the cyclin D genes occurs 3–4 h after E2 exposure of cells (10). Its transcription is reduced by antiestrogens, and this reduction in levels of cyclin D1 contributes to reduced cell proliferation (11). Another indication of the importance of cyclin D1 in E2 signaling is that its overexpression in breast cancer cells leads to resistance to antiestrogens (12).

Cyclins E and A are important later in the G₁ phase of the cell cycle when they participate in activation of Cdk2, a crucial step in moving the cell into the S phase of the cell cycle (13). Another factor that is essential for activation of Cdk2 activity is the phosphatase Cdc25A, which is itself induced by E2 (10). Inhibitors of Cdk2 also play an important role in cell-cycle regulation. p27^{is one such inhibitor. In its turn, level of p27}^{is regulated by ubiquitin-induced degradation, which is mediated by p45}⁽14); when p45^{is overexpressed, levels of the cell-cycle inhibitor p27}^{are reduced and the cell becomes resistant to antiestrogens.}

In a recent report, Omoto et al. (15) expressed ERβ stably in MCF-7 cells under the control of a cytomegalovirus promoter and found that the receptor had a negative effect on proliferation of these cells and reduced the number of colonies in an anchorage–independence assay.

In the present study, we have investigated how ERβ affects cellular proliferation in response to E2 in T47D cells stably transfected with tetracycline-regulated ERβ expression plasmid. We have investigated the specific effects of ERβ expression on the components of the cell cycle machinery Cdk2, cyclin D1, Cdc25A, cyclin E, and p27^{in these cells.}

Acknowledgments

This work was supported by the Swedish Cancer Fund, KaroBio, and the National Institutes of Health.

Acknowledgments

Notes

Abbreviations: ER, estrogen receptor; E2, 17β-estradiol; DCCFBS, Dextran-coated charcoal-treated FBS.

Notes

Abbreviations: ER, estrogen receptor; E2, 17β-estradiol; DCCFBS, Dextran-coated charcoal-treated FBS.

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