Cell type- and estrogen receptor-subtype specific regulation of selective estrogen receptor modulator regulatory elements

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Abstract

Selective estrogen receptor modulators (SERMs), such as tamoxifen and raloxifene can act as estrogen receptor (ER) antagonists or agonists depending on the cell type. The antagonistic action of tamoxifen has been invaluable for treating breast cancer, whereas the agonist activity of SERMs also has important clinical applications as demonstrated by the use of raloxifene for osteoporosis. Whereas the mechanism whereby SERMs function as antagonists has been studied extensively very little is known about how SERMs produce agonist effects in different tissues with the two ER types; ERα and ERβ. We examined the regulation of 32 SERM-responsive regions with ERα and ERβ in transiently transfected MCF-7 breast, Ishikawa endometrial, HeLa cervical and WAR-5 prostate cancer cells. The regions were regulated by tamoxifen and raloxifene in some cell types, but not in all cell lines. Tamoxifen activated similar number of regions with ERα and ERβ in the cell lines, whereas raloxifene activated over twice as many regions with ERβ compared to ERα. In Ishikawa endometrial cancer cells, tamoxifen activated 17 regions with ERα, whereas raloxifene activated only 2 regions, which might explain their different effects on the endometrium. Microarray studies also found that raloxifene regulated fewer genes than tamoxifen in U2OS bone cancer cells expressing ERα, whereas tamoxifen was equally effective at regulating genes with ERα and ERβ. Our studies indicate that tamoxifen is a non-selective agonist, whereas raloxifene is a relative ERβ-selective agonist, and suggest that ERβ-selective SERMs might be safer for treating clinical conditions that are dependent on the agonist property of SERMs.

Introduction

Two major classes of estrogenic drugs are used clinically to target the two estrogen receptor (ER) subtypes, ERα and ERβ (Dahlman-Wright et al., 2006, Heldring et al., 2007, Koehler et al., 2005). The first class of drugs that interact with ERs are a family of mammalian estrogens that include estradiol, estrone, and equilin, and synthetic estrogens such as ethinyl estradiol. The mammalian estrogens have been used extensively to treat menopausal symptoms and osteoporosis, whereas synthetic estrogens are commonly found in oral contraceptive pills. This class of estrogens function as agonists in tissues and regulate genes by recruiting coactivators to the activation function (AF)-2 surface of ERs (Smith and O’Malley, 2004). The second class of drugs used clinically to target the ERs is the selective ER modulators (SERMs). These compounds are distinguished from estrogens by their capacity to function as both agonists and antagonists in different cell types (Jordan, 2004). The main SERMs approved for clinical use are tamoxifen and raloxifene. The antagonistic action of tamoxifen on ER signaling in breast cells has been exploited for treating and preventing breast cancer (MacGregor and Jordan, 1998, O’Regan and Jordan, 2002). Raloxifene also acts as an antagonist in breast cells and decreases the risk of ER positive breast tumors (Cummings et al., 1999, Vogel et al., 2006). A major clinical difference in the antagonistic action of the two SERMs is that tamoxifen prevents both invasive and non-invasive breast tumors, whereas raloxifene only prevents invasive tumors (Grady et al., 2008). The molecular mechanism of the antagonistic action of SERMs has been studied extensively (MacGregor and Jordan, 1998). SERMs act as antagonists by at least three mechanisms. First, the SERMs bind to ERs with high affinity and competitively block the binding of estradiol. Second, the SERMs disrupt the movement of helix 12, which prevents the binding of coactivators to AF-2 in the ligand binding domain (Nettles and Greene, 2005, Shiau et al., 1998). Third, the SERMs induce the recruitment of corepressor proteins, such as N-CoR to ERs (Shang et al., 2000), which causes the repression of gene transcription by histone deacetylation and chromatin remodeling (Torchia et al., 1998).

The SERMs also have agonist activity in specific tissues. Some agonist actions of SERMs lead to beneficial effects, such as increasing bone mineral density (Love et al., 1988) and the prevention of fractures (Ettinger et al., 1999). This important property has led to the approval of raloxifene for the prevention and treatment of osteoporosis. However, the agonist properties can produce some adverse effects, including the stimulation of blood clots (Vogel et al., 2006). While much is known about the mechanism of the antagonist action of SERMs, little is known about how they produce agonist effects. It is also important to understand the mechanism whereby diverse SERMs produce different agonist effects from each other. For example, it is unclear why tamoxifen increases the risk of endometrial cancer, whereas raloxifene does not (Vogel et al., 2006). Studies indicate that the agonist effect is mediated by coactivators (Shang and Brown, 2002) that bind to and potentiate the constitutive activity of AF-1 in the A/B domain of ERs (Dutertre and Smith, 2003, Webb et al., 1998). This is in contrast to the agonist action of estrogens, which is mediated by the recruitment of coactivators to AF-2 in the ligand binding domain (Feng et al., 1998, Shiau et al., 1998). A more complete understanding of the molecular mechanism for the agonist actions of SERMs could lead to the development of safer and more selective SERMs.

One of the major problems that impede investigating the agonist action of SERMs at the genomic level is the lack of regulatory regions from native genes. Therefore, most studies have focused on the activation of an AP-1 site by the SERMs (Paech et al., 1997, Webb et al., 1995). While this site might be responsible for mediating the SERM's effects on several genes, it is clearly not the only SERM-responsive regulatory region. To identify a broad range of regulatory regions from native target genes, we used a chromatin immunoprecipitation-cloning and sequencing strategy. We isolated multiple regions from different genes that were regulated by SERMs in U2OS osteosarcoma cells (Levy et al., 2008). Using some of these regulatory regions, the goal of this study was to determine if the SERM-responsive regions are activated by tamoxifen and raloxifene in ER subtype specific manner in different cell lines. Our findings suggest that tamoxifen is a non-selective ER subtype SERM, whereas raloxifene is a relative ERβ-selective SERM. These results provide a potential mechanism whereby tamoxifen and raloxifene produce different clinical effects.

Section snippets

Materials

Estradiol, tamoxifen and raloxifene were obtained from Sigma–Aldrich. All other compounds were obtained as previously described (Levy et al., 2008).

Cell lines and cell culture

Tetracycline-inducible U2OS-ERα and U2OS-ERβ cells were characterized and maintained as previously described (Kian Tee et al., 2004). U2OS, MCF-7, HeLa, and Ishikawa cells were obtained from the UCSF cell culture facility and maintained as previously described (Kian Tee et al., 2004, Levy et al., 2008). WAR-5 prostate cancer cells were prepared as

Cell type-specific regulation of SERM regulatory regions

Using a chromatin immunoprecipitation-cloning and sequencing strategy, we previously isolated 173 regulatory regions from ERα target genes in U2OS cells (Levy et al., 2008). Most of these regions were regulated by the SERMs, tamoxifen and raloxifene in U2OS cells. In the current study, we examined whether regulatory regions are regulated by SERMs in a cell type-specific manner with ERα and ERβ. Of the 173 original regulatory regions, 32 were studied. These regions were selected because they

Discussion

The SERMs are a unique class of drugs that bind to ERs, and are distinguished from estrogens by their capacity to act as an antagonist or agonist in various tissues (MacGregor and Jordan, 1998). While the antagonist property of SERMs have been exploited for breast cancer treatment for decades the value of the agonist property of SERMs has only recently been realized in clinical practice by use of raloxifene for osteoporosis prevention and treatment (Ettinger et al., 1999). However, the agonist

Disclosure

N.L., C.G. and W.A.R. have nothing to declare. X.Z., M.T., and I.C., are employees of Bionovo, Inc. T.P.S., G.L.F. and D.C.L. are on the Scientific Advisory Board of Bionovo, Inc. D.C.L. has received financial support for research from Bionovo, Inc.

Acknowledgements

We thank Pierre Chambon and Jan-Åke Gustafsson for providing plasmids. We also thank Zhijin Wu and Yunxia Sui for providing us the dbRMA package for microarray analysis. This work was supported by a grant from the American Cancer Society to D.C.L.

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