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Originally published In Press as doi:10.1074/jbc.M412707200 on January 4, 2005

J. Biol. Chem., Vol. 280, Issue 9, 7829-7835, March 4, 2005
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Limiting Effects of RIP140 in Estrogen Signaling

POTENTIAL MEDIATION OF ANTI-ESTROGENIC EFFECTS OF RETINOIC ACID*

Kristina A. White{ddagger}, Mark M. Yore, Dexin Deng, and Michael J. Spinella§

From the Department of Pharmacology and Toxicology, Dartmouth Medical School, and the Norris Cotton Cancer Center, Dartmouth Hitchcock Medical Center, Hanover, New Hampshire 03755

Received for publication, November 10, 2004 , and in revised form, December 21, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The receptor interacting protein 140 (RIP140) belongs to a unique subclass of nuclear receptor coregulators with the ability to bind and repress the action of a number of agonist-bound hormone receptors. We have previously demonstrated that all-trans-retinoic acid (RA) induction of RIP140 constitutes a rate-limiting step in the regulation of retinoid receptor signaling. Here we demonstrate that RIP140 is also a limiting regulator of estrogen receptor signaling. Overexpression of RIP140 dose dependently inhibits estrogen-dependent reporter activity in human breast cancer cells. Furthermore, small interfering RNA to RIP140 enhances estrogen-dependent signaling. Our previous studies indicate that RIP140 is a direct target of RA. We report here that RA can abrogate estrogen-mediated cell cycle re-entry. In addition, RA treatment of estrogen-dependent breast cancer cells opposes estrogen receptor-dependent reporter activity, implying that a proportion of RA effects are anti-estrogenic. We provide evidence for a role for RIP140 in mediating anti-estrogenic effects of RA. RIP140 small interfering RNA blocks RA-mediated repression of estrogen receptor activity and provides a growth advantage to estrogen-dependent cells. Together these data implicate a regulatory role for RIP140 in mediating anti-estrogenic effects of RA in estrogendependent breast cancer cells and suggest that acute regulation of coregulator expression may be a general mechanism to integrate diverse hormone signals.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear receptors are a superfamily of hormone-activated transcription factors, which include retinoic acid receptors (RARs)1 and estrogen receptors (ERs). Organized in a modular fashion, nuclear receptors contain discrete domains that mediate distinct aspects of receptor function, such as DNA binding, ligand binding, dimerization, and transactivation (1–4). RAR and ER regulate gene transcription through binding to consensus retinoic acid responsive elements (RAREs) and estrogen responsive elements (EREs) in proximal promoter regions of their respective target genes (1–4). Nuclear receptor-mediated gene expression is regulated by complex and precisely choreographed recruitment of large multiprotein complexes whose activity depends on mutual interactions and post-translational modifications (5–7). The conformation of nuclear receptors depend on the presence of ligand, which determines binding of coregulators, components of basal transcription, and other cofactors, resulting in changes in target gene expression (5–7). Over the past decade, much progress has been made in elucidating the identity and order of recruitment of coregulatory proteins (5–7). However, relatively little attention has focused on mechanisms driving regulation of specific coregulators themselves.

Co-repressors N-CoR and SMART, through direct interactions with the AF-2 domain of nuclear receptors, actively repress transcription in the absence of ligand by recruitment of histone deacetylase-associated complexes (8, 9). In the presence of ligand, corepressor complexes dissociate allowing interaction of histone acetyltransferase-associated coactivators of the p160 family (SRC1, SCR2, and SRC3) with the AF-2 domain of receptors (5–7). LXXLL motifs present in SRC1–3 are essential for hormone-dependent interaction with nuclear receptors (10). The coregulator RIP140 contains 10 LXXLL motifs and like a classic coactivator interacts preferentially with ligand-bound nuclear receptors (11). However, RIP140 inhibits the transactivation function of ligand-bound nuclear receptors in reporter assays (12–22). RIP140 is now recognized as one of the first proteins of a unique class of coregulators that are able to repress ligand-bound nuclear receptors (23). Our previous studies have shown that RIP140 not only represses ligandactivated RAR signaling but is itself an immediate target of RA (24–26). RA induction of RIP140 mRNA occurs within 3 h and does not require de novo protein synthesis consistent with the RIP140 gene being a direct target of RARs (24–26).

Retinoids are known to be important in development, differentiation, and cell growth. Retinoids at pharmacological doses exhibit a variety of activities associated with cancer prevention. They suppress transformation of cells in vitro, inhibit carcinogenesis in various organs in animal models, reduce premalignant human epithelial lesions, and prevent second primary tumors (as reviewed in Refs. 27–30). Several studies have suggested that retinoids predominately inhibit the growth of estrogen-dependent but not hormone refractory breast cancers (31–38). Although the underlying mechanism remains elusive, there is evidence to suggest that the growth suppressive effects of retinoids may be, in part, anti-estrogenic in nature (33, 34, 36, 39, 40). The current study was designed to assess the role of RIP140 as a mechanism of cross-talk between RAR and ER signaling.

Under retinoid dominant conditions, we demonstrated that RA induction of RIP140 constitutes a functional negative feedback signal limiting RA activity at the level of nuclear receptor coregulation in human embryonal carcinoma (25). Furthermore, we demonstrated that RIP140 has potent rate-limiting effects both in modulating retinoid receptor activity and on the efficacy of retinoids to signal growth suppression and differentiation (25). Specifically, silencing RIP140 in human embryonal carcinoma enhances and accelerates retinoid receptor transactivation and RA-mediated maturation (25). Acute regulation of coregulators, such as RIP140, may be a general regulatory mechanism in hormonal signaling. RIP140 is predicted to inhibit several, if not all, nuclear receptors (23). Therefore, we investigated the potential role of RIP140 in mediating crosstalk between RA and ER signaling in a system where both receptors have been characterized, namely retinoid-responsive and estrogen-dependent human breast cancer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines, Hormones, Induction Protocols, and Northern Analysis—ER positive, MCF-7 and T47D, and ER negative, MDA-MB-231, human breast cancer cell lines were purchased from American Type Culture Collection (Manassas, VA). All cells were cultured under humidified 5% CO2 at 37 °C in high glucose Dulbecco's modified Eagle's medium/F-12 (50:50) with 10% fetal bovine serum supplemented with penicillin, streptomycin, and glutamine. Charcoal-absorbed serum was employed for all-trans-retinoic acid (RA) and 17 {beta}-estradiol (E2) inductions to deplete endogenous serum retinoids and estrogens unless otherwise indicated. E2 and RA were purchased from Sigma. RA was stored protected from light under liquid N2 asa10mM stock solution dissolved in dimethyl sulfoxide (Me2SO). E2 was stored at –20 °C as a 100 µM stock solution dissolved in ethanol (EtOH). The RA induction protocol was as follows. On day –1 cells were fed with complete medium containing charcoal-absorbed sera. On day 0, the medium was replaced with fresh charcoal-absorbed medium containing specific dosages of RA or the vehicle control, Me2SO. The estrogen induction protocol was as follows. On day –3 cells were seeded into phenol-free media with 10% charcoal-absorbed fetal bovine serum supplemented with penicillin, streptomycin, and glutamine. On day 0, medium was replaced with fresh phenol-free medium containing specific dosages of E2 or the vehicle control, EtOH. For Northern analysis, total RNA was isolated using TriReagent (Invitrogen). Northern hybridizations were performed as described previously (24). The RIP140 probe was a radiolabeled BglII/XhoI 1-kb fragment from plasmid pSG5-HAhRIP140 (22).

Cell Proliferation Assays—On day –2, MCF-7, T47D, and 231 cell lines were seeded 1:3 into phenol red containing medium with 10% normal sera. On day –1, cell lines were seeded in triplicate into 6-well dishes at 1.75 x 105 cells per well in the above media. 24 h later (on day 0), this medium was replaced with fresh medium containing various doses of RA or vehicle control (Me2SO). Cells were re-fed at 48-h intervals with fresh medium and drug. Cells were harvested via trypsinization and trypan blue-excluded cells were counted using a hemocytometer. Each experimental condition was cultured in triplicate. Average counts were determined from three independent samplings from each triplicate well. Error bars were determined as standard deviations.

Expression and Reporter Constructs—The HA-tagged RIP140 expression vector in pSG5, pSG5-HAhRIP140, was kindly provided by Dr. Jan-Ake Gustafsson (Karolinska Institute, Sweden) (22). The reporter gene, containing tandem RAREs fused to the minimal herpes simplex virus-thymidine kinase promoter and luciferase (RARE-TK-Luc), has been described previously (41). The reporter gene containing tandem EREs fused to the minimal herpes simplex virus-thymidine kinase promoter and luciferase (ERE2-TK-Luc) were a kind gift from Dr. James DiRenzo (Dartmouth Medical School, Hanover, NH) and have also been described previously (42).

Transient Transfection Reporter Assays—Transient transfection of MCF-7, MDA-MB-231, and NT2/D1 cells has been described previously (43). Briefly, 1.75 x 105 cells per 6-well plate were transfected with 2.75 µg of DNA including 0.75 µg of reporter plasmid, 1.5 µg of expression plasmid, and 0.5 µg of a cytomegalovirus {beta}-galactosidase expression plasmid using Polyfect (Qiagen). In all cases, insertless pSG5 expression plasmid was used to normalize DNA content. All plates transfected with a given plasmid combination received a portion of the same liposomal mixture. Cells were exposed to precipitates for 14–16 h and then washed and cultured with or without RA or E2 for an additional 24 or 48 h before harvest. For siRNA, 0.75 µg of RARE-TK-Luc, 0.5 µg of {beta}-galactosidase expression plasmid, and 1.0 µg of siRNA were co-transfected using Polyfect. Luciferase activities were measured as described (44) and normalized to {beta}-galactosidase activity. Data points represent the average of triplicate transfection with similar results obtained in at least three independent experiments.

siRNA Design and Transfection—The sequence of the custom siRNA duplex for RIP140 (5'-GAAGGAAGCUUUGCUAGCU-3') corresponds to the human RIP140 cDNA starting 331 bp downstream of the ATG start codon as described previously (25) and was purchased from Dharmacon. The siRNA duplex control used was the Scramble II sequence from Dharmacon. Transfection of siRNA was performed with OligofectAMINE reagent (Invitrogen) according to the manufacturer's instructions. The final concentration of siRNA used in each experiment was ~150 nM. In independent experiments, transfection efficiency was assessed to be greater than 90% using fluorescein-labeled siRNA (Dharmacon). For select experiments a second siRNA for RIP140 (5'-CAAACAGGAUAGCACAUUA-3') corresponding to human RIP140 cDNA starting 1986 bp downstream of the ATG start codon was employed.

Cell Cycle Analysis—For cell cycle phase analysis, 1 x 106 cells were fixed with 70% ethanol and stained with propidium iodide for 16 h. The percentages of cells in G1, S, and G2/M were then determined on a BD Biosciences FACscan flow cytometer using Modfit LT software and established techniques (43).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Overexpression of RIP140 Represses ER Activity—In prior studies it was shown that RA acutely up-regulates the nuclear receptor coregulator, RIP140 (24–26). Because it has been suggested that RIP140 can inhibit nuclear receptor activation, including that mediated by ER, the potential role of RIP140 as a rate-limiting regulator of ER was investigated. Overexpression of RIP140 greatly reduced estrogen-dependent ERE-TK-Luc activity in MCF-7 cells (Fig. 1A). Even relatively low amounts of transfected RIP140 was able to potently suppress estrogen-dependent promoter activity in a dose-dependent manner (Fig. 1B). RIP140 transfection had no effect on a SV40-driven control reporter or a TK-control reporter (data not shown). RIP140 overexpression had little effect on basal ERE-dependent reporter activity, which is in support of previous findings that RIP140 most effectively targets estrogen-bound ER.



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  		FIG. 1.
Repression of ligand-dependent ER transactivation by RIP140. A, the effect of RIP140 on estrogen-induced luciferase activity of a heterologous ERE-TK-Luc promoter. Estrogen-depleted MCF-7 cells were transfected with an ERE-TK-Luc reporter plasmid in the absence or presence of 1.0 µg of RIP140 expression plasmid. Cells were treated for 24 h with the indicated concentrations of 17 {beta}-estradiol (E) prior to assessing luciferase activity. Each transfectant also contained 2an equivalent quantity of a cytomegalovirus {beta}-galactosidase expression plasmid and results are corrected for {beta}-galactosidase activity. Each point is the average of three independent determinations. Error bars are S.D. This is representative of two independent determinations. B, RIP140 suppresses estrogen induction of ER-dependent reporter activity in a dose-dependent fashion. ERE-TK-Luc reporter assays were performed as above in the presence of the indicated amounts of HA-RIP140 expression plasmid. 17 {beta}-estradiol (E2) treatment was 100 nM for 48 h. Each point is the average of three independent determinations. Error bars are S.D. This is representative of three independent determinations.

 
Repression of RIP140 Enhances ER Activity—Because RIP140 contains 10 LXXLL motifs and binds receptors in a ligand-dependent fashion, both features of classic coactivators, it has been questioned whether the repressor effects of overexpressed RIP140 are because of a non-physiologic squelchingtype mechanism. To further address this question we designed siRNA to RIP140 to assess the effects of suppressing endogenous RIP140 expression on ER activity. siRNA specific to RIP140, but not a control siRNA, robustly inhibited both basal and RA-induced expression of RIP140 in MCF-7 cells (Fig. 2A). As shown in Fig. 2B, RIP140 siRNA enhanced ERE-TK Luc reporter activity. Control, scrambled siRNA had no effect on RARE reporter activity and RIP140 siRNA had no effect on SV40 {beta}-galactosidaseor p53-dependent reporter activities (data not shown). This finding supports the hypothesis that endogenous RIP140 functions to inhibit ligand-activated ER. The low level of enhancement seen with siRNA to RIP140 in the Me2SO control may be because of the presence of low levels of endogenous estrogen in charcoal-absorbed sera. RIP140 siRNA also stimulated ERE activity in ER-independent NT2/D1 cells co-transfected with an expression plasmid for ER (Fig. 2C).



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  		FIG. 2.
RIP140 silencing enhances ER activity. A, Northern analysis depicting silencing of basal and RA-induced expression of RIP140. MCF-7 cells were transfected with the indicated siRNA and treated with 1 µM RA for the indicated times points prior to RNA isolation. B, RIP140 siRNA enhances estrogen induction of ERE reporter activity in MCF-7 cells. Liposome-based transfections were performed in estrogen-depleted MCF-7 or MDA-MB-231 cells with a ERE-TK-Luc promoter in the presence or absence of RIP140 siRNA or control siRNA. 17 {beta}-estradiol (E2) treatment was 100 nM for 48 h. Error bars are S.D. This is representative of three independent experiments. C, RIP140 siRNA enhances estrogen-dependent ER signaling in human embryonal carcinoma cells. Estrogen-depleted NT2/D1 cells were transfected with ERE-TK-Luc reporter plasmid and an ER expression plasmid in the presence of the indicted siRNA. 17 {beta}-estradiol (E2) treatment was 100 nM for 48 h. Error bars are S.D. and representative of two independent experiments.

 
Repression of RIP140 Sensitizes MCF-7 Cells to Estrogen— The effects of perturbing RIP140 expression on estrogen-dependent growth of MCF-7 cells was then explored. As depicted in Fig. 3A, RIP140 siRNA increased the growth of MFC-7 cells under estrogen-replete growth conditions. To explore this effect further the effect of RIP140 depletion under defined estrogen conditions was assessed. Estrogen starvation of MCF-7 cells resulted in growth suppression as evidenced by a decrease of ~30% of cells in S-phase (Fig. 3B). As compared with control siRNA, RIP140 siRNA had little effect on the ability of MCF7 cells to growth arrest with estrogen depletion. This is consistent with the inability of RIP140 to bind ER in the absence of ligand (11, 45). However, RIP140 siRNA enhanced estrogenmediated cell cycle re-entry (Fig. 3B). The enhanced sensitivity of MCF-7 RIP140 siRNA cells to estrogen could be demonstrated at estrogen levels as low as 1 pM (Fig. 3B). Thus, RIP140 silencing provided MCF-7 cells with a growth advantage under defined estrogen exposure. Similar to the situation for RA signaling in human embyronal carcinoma (24, 25), this data supports a role for RIP140 in limiting ER signaling in human breast cancer cells.



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  		FIG. 3.
RIP140 silencing sensitizes MCF-7 cells to estrogen. A, silencing of RIP140 accelerates proliferation of MCF-7 cells grown under estrogen replete conditions. MCF-7 cells were transfected with control siRNA or two independent RIP140 siRNAs. Transfected cells were plated at a density of 3 x 105 cells per 10-cm dish in complete phenol red containing media. Cell numbers were determined 5 days later by hemocytometer count of viable trypan blue-excluded cells. Average of triplicate treatments. Error bars are S.D. This is representative of two independent experiments. B, RIP140 siRNA enhances estrogen-mediated cell cycle re-entry of MCF-7 cells. MCF-7 cells were transfected with control or RIP140 siRNA and replated in estrogendepleted conditions (charcoal absorbed sera, phenol minus growth media) for 48 h. Cells were then treated for an additional 12 h with the indicated dosages of 17 {beta}-estradiol (E2) prior to harvesting for cell cycle analysis. Data were plotted as percentage of cells in S-phase relative to estrogen replete controls. Two independent experiments are shown representative of three independent experiments.

 
RA Inhibits Growth of ER-positive Breast Cancer Cells and Represses Expression of ER Target Genes—Because RA induction of RIP140 could potentially be a mechanism of repressional cross-talk between RA and estrogen signaling pathways, the extent to which RA is anti-estrogenic was investigated. Under estrogen-replete conditions, RA dose-dependently inhibited the growth of ER-positive MCF-7 and T47D breast cancer cells but not ER-negative MDA-MB-231 cells (Fig. 4A). Under these conditions RA was able to inhibit the transcription of the known direct ER-target genes pS2, Cathespin D, and Cyclin D1 in MCF-7 and T47D cells (Fig. 4A, inset, and data not shown). This has been shown previously in the case of pS2 (33, 34, 36). Interestingly, despite the lack of RA-mediated growth suppression in MDA-MB-231 cells compared with MCF-7 and T47D cells, both MCF-7 and MDA-MB-231 cells supported RA-mediated activation of an RARE-TK-Luc reporter (Fig. 4B). The induction in MDA-MB-231 cells was reduced, supporting some previous findings (40, 46). Furthermore, the transcript levels of established RA target genes RIP140, CRABP2, and HAS2 were induced by RA under estrogen-replete conditions in both MCF-7 and MDA-MB-231 cells (Fig. 4C, and data not shown). These findings suggest that RA is at least partially anti-estrogenic in MCF-7 and T47D cells, whereas MDA-MB-231 cells are resistant to RA despite partially intact RAR signaling.



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  		FIG. 4.
RA inhibits the growth of ER-positive but not ER-negative human breast cancer cell lines. A, estrogen-dependent human breast cancer cell lines (MCF-7 and T47D) are growth suppressed by retinoic acid. Cells were plated at a density of 1.75 x 105 cells per well of a 6-well plate in estrogen-replete media and the next day exposed to the indicated dosages of RA for 6 days. Cell numbers were determined by hemocytometer count of viable trypan blue-excluded cells. Data were normalized to viable cell number in the absence of RA. Average of triplicate treatments. Error bars are S.D. and representative of three independent experiments. Inset, RA represses ER target gene expression in MCF-7 cells. MCF-7 cells plated in estrogen-replete media were treated with 10 µM RA for 6 days prior to mRNA isolation. RT-PCR was performed for the estrogen target genes pS2 and Cathespin D. B, MCF-7 and MDA-MB-231 cells both support RAR-dependent transactivation. MCF-7 and MDA-MB-231 cells were transfected with an RARE-TK-Luc reporter plasmid (left) or an ERE-TK-Luc reporter plasmid (right) in estrogen-depleted media. Cells were treated for 24 h with either 1 µM RA or 100 nM 17 {beta}-estradiol (E2) prior to assessing luciferase activity. Each transfectant also contained an equivalent quantity of a cytomegalovirus {beta}-galactosidase expression plasmid and results were corrected for {beta}-galactosidase activity. Each point is the average of three independent determinations. Error bars are S.D. C, MCF-7 and MDA-MB-231 cells each support RA-dependent induction of the RA target genes, RIP140. Cells in estrogenreplete media were treated with 1 µM RA for the indicated time points prior to isolation of RNA.

 
RA Blocks Mitogenic Effects of Estrogen in MCF-7 Cells—To further establish the anti-estrogenic effects of RA on MCF-7 cells, cell cycle kinetics were investigated. Fig. 5A depicts the cell cycle profile of ER-positive MCF-7 cells either in the continued presence of estrogen or starved of estrogen for 48 h. Estrogen starvation resulted in a decline in S-phase cells and synchronization (45 to 10% S-phase cells). Replenishing the cultures with estrogen released the cells from the G1 block such that 75% of cells are in S-phase within 10 h. A 24-h pretreatment with RA completely abolished estrogen-mediated cell cycle re-entry (Fig. 5C). However, RA had no effect on cells that were continually starved of estrogen (Fig. 5B).



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  		FIG. 5.
RA abrogates estrogen-dependent cell cycle re-entry. Cell cycle distribution of MCF7 cells replete of estrogen (E2-Repl) and starved of estrogen for 48 h (E2-Strv) are shown. A, after 48 h of estrogen starvation, 10 nM estrogen was added to the cultures for the indicated time points. B, 1 µM RA was added to the culture during the last 24 h of estrogen starvation and the indicated time points were taken thereafter in the continued presence of RA. C, 1 µM RA was added to the culture during the last 24 h of estrogen starvation and the indicated time points taken thereafter in the continued presence of RA with addition of 10 nM estrogen.

 
Repression of RIP140 Reverses Anti-estrogenic Effects of RA—Because RA potently induces RIP140 expression we asked whether RIP140 could participate in cross-talk between RAR and ER signaling. Whereas RA has no effect on basal ER activity, RA dose-dependently inhibited estrogen-dependent ER activity (Fig. 6A). This result further supports that a portion of RA effects are anti-estrogenic. Importantly, silencing RIP140 in MCF-7 cells opposed the anti-estrogenic effects of RA and fully restored estrogen-dependent ER activity (Fig. 6B).



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  		FIG. 6.
RIP140 silencing reverses anti-estrogenic effects of RA. A, RA inhibits estrogen-dependent ER transactivation. The effect of RA on estrogen-induced luciferase activity of a heterologous ERE-TK-Luc promoter. Estrogen-depleted MCF-7 cells were transfected with an ERE-TK-Luc reporter. Cells were treated for 48 h with 100 nM 17 {beta}-estradiol (E2) and the indicated dosages of RA, alone, or in combination, prior to assessing luciferase activity. Each transfectant also contained an equivalent quantity of a cytomegalovirus {beta}-galactosidase expression plasmid and results were corrected for {beta}-galactosidase activity. Each point is the average of three independent determinations. Error bars are S.D. This is representative of three independent experiments. B, RIP140 siRNA reverses RA-mediated inhibition of E2-dependent ERE-TK-Luc reporter activity. Cells were transfected with an ERE-TK-Luc reporter in the absence or presence of RIP140 siRNA and treated with 10 µM RA and 100 nM estrogen as described above. Each point is the average of three independent determinations. Error bars are S.D.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Breast cancer is one of the most common malignancies among women with a cumulative risk of 1 in 8 for women in the United States by the age of 85. Worldwide, 10–15% of all women will be diagnosed with breast cancer during their lifetimes. Although adjuvant chemotherapy improves survival of radically resected breast cancer, ~50% of all patients will eventually relapse (47). Resistance to anticancer agents is thought to be responsible for chemotherapy failures in breast cancer. The identification of novel approaches for the prevention and treatment of breast cancer is urgently needed. Despite the limited effectiveness of retinoids as a single agent, there is currently great interest in using retinoids in combination therapy for breast cancer, as well as other cancers. This is highlighted in studies showing synergistic effects of retinoids combined with selective estrogen receptor modulators for prevention of breast cancer (48, 49).

Current adjuvant therapies for breast cancer include chemotherapy and the anti-hormone therapy, tamoxifen. Unfortunately, a large proportion of women eventually fail tamoxifen therapy (50). Of interest is the clinical finding that many breast cancers developing tamoxifen resistance remain ER+, hormone dependent, and sensitive to other forms of endocrine therapy, such as aromatase inhibition (50). This implies a plasticity of ER function that is independent of ER expression levels or mutations and may involve changes in the ER coregulatory machinery. Furthermore, it has been proposed that the selectivity and tissue-specific actions of selective estrogen receptor modulators may be due to variations in coregulator composition (51, 52). Evidence for the importance of coregulator levels in ER function include the finding that the coactivator SRC-3 is amplified in breast cancer (53) and that disparate expression of SRC-1 in uterine and breast cancer cells may in part explain the tissue-specific effects of selective estrogen receptor modulators (52).

One strategy for designing more effective chemopreventive agents is to uncover new pharmacologic targets based on a mechanistic understanding of the beneficial and harmful downstream targets of existing chemopreventive agents such as the retinoids. Retinoids inhibit both the incidence and number of carcinogeninduced mammary tumors in animal models (54–56). ER-dependent target gene activation is critical to support continued proliferation of ER+ breast cancers and several reports suggest that breast cancer growth suppression mediated by retinoids is anti-estrogenic in nature (33, 34, 36, 39, 40) with RAR-{alpha} playing a major role in initiating RA signaling (35, 57, 58). Data presented in this study further confirm these findings. We report here that within an estrogen-dependent context RA can induce cell cycle arrest and abrogate estrogen-mediated cell cycle reentry. In addition, RA treatment of estrogen-dependent breast cancer cells is able to oppose estrogen-dependent reporter activity, further indicating that a proportion of RA effects are anti-estrogenic.

RIP140 has previously been shown to inhibit a number of ligand-activated nuclear receptors through direct receptor binding and recruitment of histone deacetylases and the transcriptional repressor, C-terminal-binding protein, to receptors in a ligand-dependent fashion (13, 14, 59). Fernandes and colleagues (60) showed that ligand-dependent corepressor (LCoR) has similar properties to RIP140 in repressing the activity of ligand-bound nuclear receptors (60). LCoR does not share extensive sequence homology with RIP140, but like RIP140, functions in a histone deacetylase- and C-terminalbinding protein-dependent manner (60). Data previously published by our laboratory identified the nuclear co-repressor, RIP140, as highly and rapidly induced following RA treatment of human breast cancer cells (24, 25). Whereas it has been suggested that RIP140 is induced by estrogen and vitamin D (61, 62), we have only seen very modest increases in RIP140 levels with estrogen as compared with RA in MCF-7 cells (data not shown). In contrast to RIP140, LCoR does not appear to be regulated by ligand-activated nuclear receptors (23).

Recent microarray studies suggest extensive and early ligand-dependent repression of gene expression by nuclear receptors (63, 64). However, the mechanism for such repression is unclear. Ligand-dependent corepressors like RIP140 and LCoR are candidate mediators of such repression. This is particularly relevant for the steroid nuclear receptors such as ER, progesterone receptor, and glucocorticoid. In contrast to non-steroidal receptors like RARs, which bind to DNA in the presence or absence of ligand, ER is sequestered from DNA in the absence of bound ligand (3). Thus, unlike the non-steroidal nuclear receptors that are predicted to actively repress gene expression in the absence of ligand by corepressor recruitment, there is no mechanism to account for repression by ER under native physiological conditions (6, 7, 65). RIP140 and LCoR provide a mechanism for active repression of agonist-bound ER. Whether there is a subset of ER-target genes preferentially targeted for RIP140 repression is currently unknown.

There is overwhelming evidence supporting the importance of ER as a therapeutic target for hormone-dependent breast cancer. Thus, our finding that retinoid receptors can directly activate the unique ER corepressor RIP140 has obvious implications for the efficacy of retinoid therapies in breast cancer. The mechanism underlying proposed anti-estrogenic effects of RA is unclear. Suggested past mechanisms have included sequestration of limiting cofactors, complex interplay of growth, and stress signaling pathways including AP1, or retinoid opposition of ER downstream components (66, 67). More recent models incorporating coregulator dynamics have been proposed to explain both transrepression between nuclear receptors and self-limitation of transcriptional activation by a given nuclear receptor. In one model nuclear receptors compete for limiting pools of coactivators (6, 7). Thus, for example, retinoid activation of RARs would deprive ER of necessary coactivators for maximal transcriptional activation (65). Evans and colleagues (68) proposed a model to explain the phasic nature of nuclear receptor activation in the constant presence of ligand. In this model ligand-induced post-translational modification of coactivator complexes attenuate receptor signaling. The model that we are proposing, RA-dependent activation of a ligand-dependent corepressor RIP140, may contribute to attenuation of retinoid receptor signaling in the continued presence of ligand (24, 25).

Here we propose a straightforward mechanism of cross-talk mediated by RA induction of a ligand-dependent corepressor of the estrogen receptor. We demonstrate that similar to RAR signaling (25), RIP140 is rate-limiting for ER signaling. Interestingly, we show that the net effect of RIP140 knockdown is growth promotion in ER+ breast cell lines. These data support a role for RA-induced RIP140 in transrepressing ER. Induction of RIP140 may be a general regulatory mechanism for RAmediated signaling in diverse cell contexts through a mechanism of self-limitation and repressional cross-talk among nuclear receptor family members. Further studies are needed to fully understand the role of RIP140 in cross-talk between RAR and ER signaling to optimize the design of retinoid-based therapies for the prevention and treatment of cancer.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant R01-CA104312 (to M. J. S.) and American Cancer Society Grant RSG-01-144-01 (to M. J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Recipient of Pre-graduate Fellowship BC010159 [GenBank] from the Department of Defense. Back

§ To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, Dartmouth Medical School, 7650 Remsen, Hanover, NH 03755. Tel.: 603-650-1920; Fax: 603-650-1129; E-mail: michael.spinella{at}dartmouth.edu.

1 The abbreviations used are: RAR, retinoic acid receptor; RA, all-trans-retinoic acid; RIP140, receptor interacting protein 140 kDa; ER, estrogen receptor; RARE, RA responsive element; ERE, estrogen responsive element; siRNA, small interfering RNA; E2, 17 {beta}-estradiol; HA, hemagglutinin; LCoR, ligand-dependent corepressor. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Jan-Ake Gustafsson (Karolinska Institute, Sweden) for providing the HA-RIP140 expression plasmid as well as Dr. Sarah Freemantle (Dartmouth Medical School) and Dr. James DiRenzo for helpful discussions throughout the course of this work and during the preparation of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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