Estrogen receptor cofactors expression in breast and endometrial human cancer cells

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Abstract

In order to approach the molecular basis of the tissue-specific agonistic activity of antioestrogens, we have compared, at the mRNA level, the expression of various transcriptional cofactors (activators or repressors) of estrogen receptors in different breast (MCF7, ZR75-1, T47D, MDA-MB231) and endometrial (Ishikawa, RL-95-2 and HEC1A) human cancer cell lines. We showed that for SRC-1, CBP, TIF1α, RIP140, N-CoR, and SMRT, no significant differences in the expression levels were observed between breast and endometrial cells. For TIF1α mRNA, both isoforms were also detected at similar levels in all the cells tested. By contrast, over-expression of AIB1 mRNA was observed in MCF7 cells, but not in other breast or endometrial cells, irrespective of their ER-status. We then used protein-protein interaction assay (far-Western blot) to confirm the increased expression of at least one of the p160 proteins in MCF7 cells. Finally, we demonstrated that RIP140 mRNA is directly induced by estrogens in ER-positive MCF7 breast cancer cell lines but not in Ishikawa endometrial cells. Together these results indicate that some differences exist between breast and endometrial cancer cell lines at the level of estrogen receptor transcription cofactor expression.

Introduction

Estrogens are important for the growth and development of normal mammary gland (Topper and Freeman 1980) and both epidemiological and cell biology studies have highlighted their role in the promotion of breast cancers (Dickson and Lippman 1988). Endocrine therapy using antagonists that block estrogen action has been widely developed in the last decades and the most commonly used drug is the nonsteroidal triphenylethylene-related compound tamoxifen (Jordan, 1990). However, this molecule exhibits mixed estrogenic and antiestrogenic activities depending on the species, tissue, cell, and promoter context (Berry et al., 1990, Tzukerman et al., 1994). Agonistic effects of tamoxifen have been observed in human endometrial cancer cells both at the level of gene expression (Jamil et al., 1991, Simard et al., 1997, Barsalou et al., 1998) and cell proliferation (Satyaswaroop et al., 1984, Gottardis et al., 1988, Anzai et al., 1989, Jamil et al., 1991) which may explain why its use in hormone therapy of breast cancer has been associated with an increased risk of endometrial carcinoma (Fornander et al., 1989, Fisher et al., 1994, Kedar et al., 1994, van Leeuwen et al., 1994, Assikis et al., 1996).

The effects of estrogens and antiestrogens are mediated by specific nuclear estrogen receptors (ER) α and β (Katzenellenbogen and Korach 1997) which belong to a superfamily of nuclear receptors that function as ligand-dependent transcription factors (Parker, 1993, Beato et al., 1995). Transcription is mediated by means of two activation regions, activating function (AF-1) located in the N-terminal domain and AF-2 located in the C-terminal hormone binding domain. The AF1 function is constitutively active whereas AF2 requires the presence of hormone and is inactivated by antiestrogen. Determination of the crystal structure of the ER ligand-binding domain with either estradiol or an antiestrogen (Brzozowski et al. 1997) has revealed a ligand-induced swing of the α-helix H12 in the presence of hormone which is not properly achieved in the presence of the antihormone. This agonist-induced conformational transition then allows interaction with transcriptional intermediary factors (TIFs) or coactivators (Shibata et al. 1997b) recently identified using in vitro protein interaction assays or yeast two-hybrid systems. These include different related proteins (p160s) from the steroid receptor coactivator (SRC-1) family (Onate et al., 1995, Hong et al., 1996, Takeshita et al., 1996, Voegel et al., 1996, Anzick et al., 1997, Li et al., 1997, Torchia et al., 1997), CREB binding protein (CBP)/p300 (Chakravarti et al., 1996, Kamei et al., 1996, Smith et al., 1996) and p/CAF (Yang et al. 1996) which are able to form multimeric complexes with activated nuclear receptors. Both p/CAF, CBP/p300, and members of the SRC-1 family have been shown to possess intrinsic acetyltransferase activities capable of acetylating histones, histone acetyl transferase (HAT) (Bannister and Kouzarides, 1996, Ogryzko et al., 1996, Chen et al., 1997, Spencer et al., 1997), as well as other proteins such as transcription factors (Gu and Roeder, 1997, Imhof et al., 1997). Others proteins such as RIP140 (Cavailles et al. 1995) or TIF1α (Le Douarin et al., 1995, Thenot et al., 1997), also bind hormone-activated nuclear receptors. However, the role of these proteins is less clear because in transient transfection experiments, their over-expression leads to a repression of the hormone-dependent transcriptional activity of ER (Cavailles et al., 1995, Le Douarin et al., 1995)

On the other hand, two factors, namely nuclear receptor corepressor (N-CoR) and silencing mediator for retinoid and thyroid receptor (SMRT) have been isolated on the basis of their interaction with unliganded thyroid hormone and retinoic acid receptors. They have been shown to mediate transcriptional repression by recruiting a complex of proteins, including mSIN3 and HDAC1 histone deacetylase (HD) (Pazin and Kadonaga 1997). These corepressors also play a role in the effects of steroid hormone receptors (estrogen or progesterone) in the presence of antihormone (Jackson et al., 1997, Smith et al., 1997, Lavinsky et al., 1998, Zhang et al., 1998) and it has been suggested that the balance between coactivators (SRC-1 or L7/SPA) and these corepressors could regulate the partial agonistic activity of antihormones.

In order to assess the potential role of these transcriptional cofactors in the tissue-specific activity of ER ligands, we have analyzed by Northern blot the expression and regulation of several TIFs in breast and endometrial human cancer cell lines.

Section snippets

Cell culture and transient transfection

Breast (MCF7, ZR75-1, T47D, and MDA-MB231) and endometrial cells (Ishikawa, RL95-2 and HEC1-A) were derived from stocks routinely maintained in the laboratory. Monolayer cultures were grown in Dulbecco’s modified Eagle’s medium (DMEM) or in Ham’s F-12/DMEM (1:1) supplemented with 10% fetal calf serum (GIBCO BRL, Cergy-Pontoise, France) and antibiotics. Before hormonal treatments, MCF7 and Ishikawa cells were stripped of endogenous steroids by passage in medium without phenol red containing 5%

Expression of estrogen receptor transcriptional cofactors

Based on microinjection experiments (Lavinsky et al. 1998) or transient transfection assays (Jackson et al. 1997; Smith et al. 1997), it has recently been suggested that the balance between nuclear receptor transcriptional coactivators (SRC-1 for instance) and corepressors (N-CoR and SMRT) should define in a given cell the agonist/antagonist behaviour of antihormones such as 4-hydroxytamoxifen or RU486.

In an attempt to precise the molecular basis of the agonistic activity of antioestrogens

Discussion

Breast and endometrium are two estrogen target tissues which exhibit differences in their response to ER ligands. This tissue-specific sensitivity is of therapeutical importance in the case of antiestrogens such as tamoxifen that are used clinically to treat breast cancer patients. Several recent reports have suggested that the relative agonist activity of this drug in a given cell could depend on the particular concentration of the different cofactors, either coactivators or corepressors, that

Acknowledgements

We thank J.-M. Rey for the AIB1 probe, P. Balaguer, M.G. Parker, R. Evans and R. Goodman for plasmids. We are also grateful to J.Y. Cance for illustrations and to C.A. Royer, P. Augereau, D. Chalbos and F. Vignon for critical reading of the manuscript. This work was supported by the ‘Institut National de la Santé et de la Recherche Médicale’, the University of Montpellier I, the ‘Ligue Nationale contre le Cancer’ and the ‘Association pour la Recherche sur le Cancer’ and by funds from the

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