Potentiation of ICI182,780 (Fulvestrant)-induced Estrogen Receptor-α Degradation by the Estrogen Receptor-related Receptor-α Inverse Agonist XCT790*

ICI182,780 (Fulvestrant) is a pure anti-estrogen used in adjuvant therapies of breast cancer. This compound not only inhibits the transcriptional activities of the estrogen receptor-α (ERα) but also induces its proteasome-dependent degradation. The latter activity is believed to be required for the antiproliferative effects of ICI182,780. Estrogen receptor-related receptor-α (ERRα) is an orphan member of the nuclear receptor superfamily that is expressed in a wide range of tissues including breast tumors, in which its high expression correlates with poor prognosis. Although not regulated by any natural ligand, ERRα can be deactivated by the synthetic molecule XCT790. Here we demonstrate that this compound also induces a proteasome degradation of ERRα. We also show that although it does not act directly on the steady-state level of ERα, XCT790 potentiates the ICI182,780-induced ERα degradation. We suggest that treatment with XCT790 could thus enhance the efficacy of ICI182,780 in ERα-dependent pathologies such as breast cancer.

Estrogen receptor-␣ (ER␣), 3 a member of the nuclear hormone superfamily, is a ligand-regulated transcription factor that mediates the effects of various estrogenic molecules including 17␤-estradiol (E2) (1). Upon interaction with E2, the ligand binding domain (LBD) of ER␣ adopts an active conformation that allows the recruitment of transcriptional coactivators and the up-modulation of the expression of target genes, on the promoter of which the receptor binds. ER␣ and its cognate ligands are involved in several physiological processes such as metabolism, regulation of female reproduction, and bone homeostasis (2).
Estrogens are also instrumental in various pathological conditions. For example, breast cancers are often dependent upon estrogens that positively regulate cell proliferation (3). For this reason, molecules that block the transcriptional activities of ER␣ in the mammary gland were identified (4,5). These compounds inhibit E2-induced proliferation and some are widely used in adjuvant therapy in breast cancer. This is the case of 4-OH-tamoxifen (OHT), a mixed antagonist (i.e. that displays agonist or antagonist activity depending on the tissue), or ICI182,780 (Fulvestrant), a pure antagonist. Upon interaction with E2, ER␣ not only becomes transcriptionally active but is also targeted for degradation by the proteasome machinery (6 -8). Antagonists affect ER␣ protein stability in a more complex manner. OHT stabilizes the receptor in an inactive conformation, whereas ICI182,780 induces a rapid degradation of the receptor. Various pathways are identified that contribute to ligand-induced ER␣ degradation (9 -15). Some of these pathways are required for the antiproliferative activities of ICI182,780 (11).
Estrogen receptor-related receptor-␣ (ERR␣) is another member of the nuclear receptor superfamily that is identified on the basis of its high level of sequence identity to ER␣ (1,16). ERR␣ is involved in the regulation of metabolism in cooperation with the PGC-1␣ coactivator. Indeed, ERR␣ regulates lipid and glucose metabolism (17)(18)(19)(20)(21)(22). The receptor is also essential to the regulation of mitochondrial biogenesis exerted by PGC-1␣ (23,24). ERR␣ is expressed in a wide variety of tissues, and its high expression correlates with poor prognosis in breast, colon, and ovarian cancers (25)(26)(27)(28)(29), although its roles in tumors have not been determined.
The transcriptional activities of ERR␣ are not regulated by estrogens; however, several levels of interference occur between ERR␣ and estrogen signaling (30,31). For example, estrogens regulate the expression of ERR␣ in mouse uteri (32); in turn ERR␣ positively regulates the expression of aromatase (33), the limiting enzyme in estrogen biosynthesis. Furthermore, ER␣ and ERR␣ display complex positive and negative interactions in the transcriptional regulation of common genes, such as osteopontin, lactoferrin, and TFF1/pS2 (34 -38). In vitro far Western experiments show that ER␣ and ERR␣ physically interact (39). No natural ligand was identified for ERR␣, which is considered an orphan receptor (31). Crystallographic studies show that this receptor spontaneously adopts an active conformation (40). Although ERR␣ regulates transcription in a constitutive manner, some of its activities, such as DNA binding or contact with coactivators, can be regulated by phosphorylation (41). Compounds such as the phytoestrogen genistein can inhibit the transcriptional activities of ERR␣ (42). Based on its capacity to disrupt the interaction between ERR␣ and PGC-1␣, the synthetic molecule XCT790 is identified as an ERR␣ specific ligand and acts as an inverse agonist (43).
In this report, we show that XCT790 not only represses the transcriptionalactivitiesofERR␣butalsoinducesitsproteasomedependent degradation. Although it does not directly affect ER␣ stability, XCT790 also potentiates the ICI182,780-induced ER␣ degradation. This effect is dependent on ERR␣ and on new protein synthesis, indicating an indirect effect. Treatment with XCT790 may enhance the efficacy of ICI182,780 in diseases involving ER␣ such as breast cancer.

EXPERIMENTAL PROCEDURES
Materials-The following antibodies and reagents were used in this study. Anti-ERR␣ raised in a rabbit, using a keyhole limpet hemocyanin-coupled peptide mapping a region conserved between mouse and human ERR␣ but divergent in other ERR subfamily members, was used. Anti-ER␣ (HC-20) and anti-actin antibodies were purchased from Santa Cruz Biotechnology (Tebu, France) and Sigma-Aldrich, respectively. Actinomycin D, MG132, cycloheximide, XCT790, E2, and OHT were purchased from Sigma-Aldrich, and ICI182,780 was purchased from Tocris Cookson Ltd (Bristol, UK).
Cell Lines and Culture Conditions-The human mammary epithelial cell line MCF7 was grown in Dulbecco's modified Eagle's medium supplemented with 2 mM L-glutamine, 50 units/ml penicillin, 50 g/ml streptomycin, and 10% heat-inactivated fetal calf serum. Prior to experiments involving treatment with estrogens, cells were cultured in hormone-free medium (phenol red-free Dulbecco's modified Eagle's medium) with 10% charcoal-stripped fetal bovine serum for 3 days. In all experiments except for dose response a 5 M XCT790 concentration was used.
Transient Transfection and Luciferase Assay-Cells (10 5 ) were seeded in a 24-well plate in hormone-free medium and transfected using 3 l of Exgen500 (Euromedex, Souffelweyersheim, France) with UAS-(Gal4-binding) luciferase reporter constructs, along with Gal4DBD (Gal4) or constructs fusing the Gal4DBD to ERR␣or ER␣-LBD (50 ng). CMV-␤Gal plasmid (50 ng) was added to normalize transfection efficiency, and pSG5 plasmid was added as a carrier up to 500 ng. Six hours later, cells were treated or not with XCT790 or E2 (100 nM) for 48 h and then lysed in 200 l of lysis buffer (125 mM Tris phosphate, pH 7.8, 10 mM EDTA, 5 mM dithiothreitol, 50% glycerol, 5% Triton X-100). Luciferase activity was determined using the luciferase assay system (Promega) and normalized based on ␤-galactosidase levels. Transfections were performed in triplicate.
RNA Interference (siRNA)-The ERR␣ (Dharmacon) and ER␣ (Fisher Invitrogen) siRNAs were tranfected into MCF7 cells with Oligofectamine and Lipofectamine RNAiMax, respectively, according to manufacturer's protocol (Fisher Invitrogen). Cells were transfected twice with ERR␣ or ER␣ siRNA, first in 10-cm plates for 48 h and then in a 6-well plate for 24 h. Cells were then preincubated with XCT790 or ICI182,780 for 48 h and then treated for 16 h with ICI182,780 or XCT790 depending on the first incubation. In other experiments, cells were transfected with siRNA only once for 48 h and treated with drugs as described above.

RESULTS
XCT790 Induces ERR␣ Protein Degradation-To verify that XCT790, an ERR␣ inverse agonist, specifically inactivated this receptor in human mammary cells, an UAS-Luc reporter plasmid was transfected in MCF7 cells with a Gal4-ERR␣LBD fusion plasmid. XCT790 down-regulated the activation driven by the latter construct in a dose-dependent manner, but not in the manner exerted by a Gal4-ER␣LBD plasmid (Fig. 1A). We analyzed the effect of XCT790 on ERR␣ protein expression. As shown by Western blot, XCT790 reduced the amount of ERR␣ protein in a dose- (Fig. 1B) and time-dependent (Fig. 1C) manner. This effect of XCT790 was transient because the expression of ERR␣ was restored to its original level 8 h after withdrawal of the drug (Fig. 1D).
We established whether XCT790 exerted its effect on ERR␣ expression at the RNA or protein level. As determined by quantitative PCR, no reduction in the steady-state level of ERR␣ mRNA was evidenced upon XCT790 treatment even after 48 h ( Fig. 2A, left panel). It has been reported that expression of the ERR␣ gene can be enhanced by its own product (44). A reduction of ERR␣ protein level is expected to result in a drop of ERR␣ mRNA expression, which could be masked by a high stability of ERR␣ mRNA. However, when cells were treated with actinomycin D, a global transcriptional inhibitor, the steady-state level of ERR␣ mRNA was rapidly reduced ( Fig. 2A,  right panel). The half-life of this mRNA was clearly shorter than 2 h, comparable with that of c-Myc (used as an unstable mRNA control) and well beyond that of hypoxanthine-guanine phosphoribosyltransferase (used as a stable mRNA control). ERR␣ protein might not participate in the basal expression level of its corresponding mRNA in the conditions used here. Furthermore, XCT790 does not act on ERR␣ expression at the mRNA level. Treatment with MG132, a proteasome inhibitor, blocked the effect of XCT790 (Fig. 2B) indicating that the drug induced the ERR␣ protein degradation in a proteasome-dependent manner. Treatment by cycloheximide did not impair the effect of XCT790 (Fig. 2C), implying an accelerated ERR␣ protein turnover induced by the drug in a direct manner (i.e. new protein synthesis-independent). These data show that the effect of XCT790 on ERR␣ receptor is comparable with that of ICI182,780 on ER␣ (i.e. down-regulation of the activity as well as acceleration of receptor degradation).
XCT790 Effects on Antagonist-induced ER␣ Degradation-We next addressed the specificity of action of XCT790. When used alone the drug reduced the expression of ERR␣ protein but had no effect on ER␣ (Fig. 3A, left panel). As expected, a 16 h treatment with ICI182,780, an ER␣ antagonist, reduced the protein expression of ER␣, but not ERR␣. Strikingly, exposure to both molecules led to a complete disappearance of ER␣ signal, indicating that XCT790, inactive per se on ER␣, greatly potentiated the degradation-inducing effect of ICI182,780. XCT790 acted at the protein level because the steady-state level of ER␣ mRNA was not modulated (Fig. 3A, right panel). In contrast ICI182,780 treatment was accompanied by an up-modulation of ER␣ mRNA level, an expected result because ER␣ is autoregulated in MCF7 cells (45). We next tested the possibility of the converse effect, i.e. an effect of ICI182,780 on XCT790-induced ERR␣ degradation (Fig. 3B). To this end, we modified the order and length of exposure to the drugs. After 48 h, ICI182,780 reduced the expression of ER␣ protein but not that of ERR␣ (Fig. 3B, left panel). XCT790 induced a moderate reduction of ERR␣ expression, an effect that was dramatically enhanced upon ICI182,780 treatment. Again this potentiation effect was at the protein, but not the mRNA, steady-state level  does not act on ERR␣ mRNA level. RNA was extracted after the indicated time of XCT790 treatment. ERR␣ mRNA level were measured by quantitative PCR and normalized to the level of 36B4 mRNA (right panel). ERR␣ mRNA is unstable. RNA was extracted after the indicated time of actinomycin D treatment. mRNA level were measured using specific primers in quantitative PCR. The experiments were performed twice in triplicate. Error bars indicate standard deviation. B, XCT790 acts on ERR␣ protein in a proteasome-dependent manner. Cells were treated for 16 h with XCT790 and/or MG132 (a proteasome inhibitor) and processed for Western blot analysis. C, XCT790 accelerates ERR␣ protein turnover. MCF7 cells were treated for 16 h with XCT790 and/or cycloheximide (CHX; a protein synthesis inhibitor) and processed for Western blot. (Fig. 3B, right panel). We concluded that XCT790 and ICI182,780 potentiate the degradation-inducing effect of one another in addition to their direct action on their cognate receptor. In contrast to ICI182,780, OHT, another ER␣-antagonist in MCF7 cells, stabilizes ER␣, thus leading to an elevation of the steady-state level of the receptor (7). We examined the relationships between OHT and XCT790 (Fig. 3C). Pretreatment with the latter drug did not enhance or reverse the stabilizing effect of OHT on ER␣ (Fig. 3C, left panel). Similarly, OHT did not enhance the XCT790-induced degradation of ERR␣ (Fig 3C, right panel).
XCT790 Acts Indirectly on ER␣ Stability in an ERR␣-dependent Manner-XCT790 acts on both the transcriptional activity and expression of ERR␣ protein. It is possible that the modulation of antagonist-induced ER␣ degradation by XCT790 is because of the absence of ERR␣ receptor. If so, knocking down ERR␣ should also result in a potentiation of the ER␣-degrading effect of ICI182,780. Transfection of an siRNA directed against ERR␣ resulted in a nearly complete elimination of the receptor (Fig. 4A, left  panel). However, under these conditions ICI182,780 did not display any enhanced ER␣-degrading effect as compared with cells transfected with a control siRNA. This indicates that the indirect effect of XCT790 on ER␣ stability is not simply a consequence of ERR␣ disappearance. This result also questions the requirement of ERR␣ in XCT790 effect on ICI182,780-induced ER␣ degradation. Cells were thus transfected with ERR␣-directed siRNA and subsequently treated with XCT790 (Fig. 4A, right panel). Under these conditions, the drug was unable to enhance ICI182,780induced ER␣ degradation, indicating that ERR␣ is absolutely required for the effect of XCT790. We also tested whether the absence of ER␣ resulted in an enhanced ERR␣ degradation effect of XCT790. XCT790 was not more potent in inducing ERR␣ degradation in cells treated with an ER␣-targeting siRNA than in siRNA-treated controls (Fig. 4B,  left panel). However, ICI182,780 absolutely required ER␣ to modulate XCT790-induced ERR␣ degradation (Fig. 4B, right panel).
Cross-modulation of ERR␣ and ER␣ Stability Requires New Protein Synthesis-The above results show that receptor absence and treatment by antagonist drugs are not equivalent. Furthermore, a pretreatment by a given drug is required to enhance the capacity of the other drug to degrade its cognate receptor (i.e. a cotreatment is inefficient for this promotion, data not shown). This 48-h pretreatment (Fig. 3) could be reduced to 12 h (Fig. 5). This suggests an active role of the drugs in modulating the expression of an intermediate factor involved in receptor stability. To evaluate this possibility, cells were treated with cycloheximide, subsequently with XCT790, and then with ICI182,780 (Fig. 5A). Under these conditions, XCT790 did not up-modulate ER␣ sensitivity to ICI182,780, indicating that new protein synthesis was required for the indirect effect of XCT790 on ER␣. We also performed the converse experiment in which cells were treated with cycloheximide, subsequently with ICI182,780, and then with XCT790 (Fig. 5B). Our results show that ICI182,780 required the new protein synthesis of an intermediate protein to up-modulate ERR␣ sensitivity to XCT790.

DISCUSSION
XCT790 as an ERR␣ Degradation-inducing Agent-XCT790, a synthetic compound identified on the basis of its capacity to disrupt the interactions between ERR␣ and the PGC-1␣ coactivator, down-regulates the constitutive transcriptional activity of ERR␣ (43). The effect has been observed in CV-1 and also in COS1 cells (46). This observation can be extended to other cell types: XCT790 acts as a powerful ERR␣ inverse agonist in MCF7 and MDA-MB231 cells (this study and data not shown). XCT790 promotes the degradation of ERR␣ protein. Expression of the corresponding mRNA is unaffected. Induction of cognate receptor protein degradation, independent of any effect on the corresponding mRNA, was demonstrated for other NR/ligand pair such as ER␣/ICI182,780 or PPAR␥/pioglitazone (47)(48)(49). The ERR␣ mRNA is very unstable (halflife Ͻ 2 h), which is consistent with the circadian regulation of its expression in vivo (50). In contrast the ERR␣ protein is very stable, as its half-life is estimated to be over 24 h based on cycloheximide treatment (data not shown). The ERR␣ gene is positively auto-regulated (41,44). Lowering the level of ERR␣ protein could result in reduction of the level of the corresponding mRNA, a phenomenon we could not observe. However, if ERR␣ clearly binds to its own promoter in the absence of any given stimulus (41), activation of the promoter is only seen in the presence of PGC-1␣, a factor that we could not detect in the MCF7 strain we used (data not shown). Another nonexclusive hypothesis is that ERR␣ activity on its own promoter requires additional factors or stimuli. XCT790 requires the proteasome, but no new protein synthesis, to induce the degradation of ERR␣, but the precise mechanism of this degradation remains to be determined. XCT790-induced degradation also requires the N-terminal domain of ERR␣, because a deletion mutant lacking this part of the receptor is resistant to this effect (data not shown). It could be hypothesized that the phosphorylation events that occur in this region of the receptor (41) may be involved in regulating the stability of ERR␣. However, blocking various phosphorylation pathways such as mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, or PKC␦ did not alter the potency of XCT790 (data not shown). The effect of XCT790 on ERR␣ is comparable with that of ICI182,780 on ER␣ in that each compound blocks the transcriptional effect of its cognate receptor and induces its proteasome-dependent degradation (7). It is distinct from the effect of OHT, a mixed ER␣ antagonist that stabilizes ER␣.
A New Level of Cross-talk between ER␣ and ERR␣-Previous research shows that XCT790 specifically targets ERR␣ (43). Consistently, XCT790 did not exert a direct effect on ER␣ either by modulating transcriptional activity or by modifying its stability. On the contrary, XCT790 potentiated the degradation-inducing effect of ICI182,780 on its cognate receptor in a strictly ERR␣-dependent manner. Far Western  experiments show that ERR␣ and ER␣ could physically interact with each other in vitro (39). It could be that these receptors heterodimerize in vivo and that these contacts could "protect" each other from antagonist-induced degradation. Eliminating one receptor by a degradation-inducing antagonist would result in oversensitivity of the other receptor to its own antagonist. In this respect, we noted that ERR␣ is more sensitive to XCT790 in MDA-MB231 (ER␣-deficient cells) than in MCF7 (ER␣-positive cells) (data not shown). However, restoration of ER␣ expression in the former cells did not yield a reduced sensitivity of ERR␣ to XCT790. Furthermore, treatment of MCF7 cells with a siRNA directed against one receptor does not result in enhanced sensitivity of the other receptor to its antagonist (Fig. 4).
The above hypothesis assumes that ERR␣ and ER␣ heterodimerize, a phenomenon that we could not observe by glutathione S-transferase pull-down or by coimmunoprecipitation in vivo (data not shown). Altogether, this suggests that XCT790 plays an indirect but active role (i.e. more than through inducing ERR␣ degradation) in enhancing the effect of ICI182,780 on ER␣ degradation. Consistent with this, the effect of XCT790 could only be observed after a minimum 12-h pretreatment time and not upon cotreatment, suggesting additional factors must be produced. This hypothesis was confirmed when cycloheximide treatment blocked the enhancing effect of XCT790. We tested the effect of XCT790 on the expression of factors involved in proteasome-dependent ICI182,780-induced ER␣ degradation (6, 8 -11, 13, 51). No response to XCT790 was observed for Uba3 (ubiquitin-activating enzyme 3, the catalytic subunit on the NEDD8 pathway), E6AP (an E3 ubiquitin ligase), or UbcH7 (an ubiquitin-conjugating enzyme) (data not shown). The potentiation of XCT790-induced degradation by ICI182,780 also requires both ER␣ and new protein synthesis of an unknown factor. ICI182,780 and XCT790 behaved in a similar manner, although it is not known whether the intermediate factor required is identical in both cases. XCT790 potentiates the degradation induced by ICI182,780, but not the degradation induced by E2, the natural agonist of ER␣ (data not shown). This phenomenon is, however, not intrinsically linked to the antagonist nature of ICI182,780. OHT, which behaves as an ER␣ antagonist in MCF7 cells, induces ER␣ stabilization, and this effect could not be reverted by XCT790. ICI182,780 treatment leads to the inhibition of ER␣-dependent transcriptional activity. Pretreatment with XCT790 did not enhance this blocking effect (data not shown), indicating that the effect of ICI182,780 on transcription can be separated from that on ER␣ degradation with only the latter being targeted by XCT790 through ERR␣. Our results indicate a new level of interference between ER␣ and ERR␣, in that a destabilizing antagonist to one receptor modulates the degradation of the other induced by a cognate antagonist, although the precise mechanism by which they do so remains to be determined.
Possible Consequences in Anti-estrogenic Therapies-ICI182,780 is used in neoadjuvant therapy of breast cancer because it blocks estrogen-dependent cell proliferation. In MCF7 cells, blocking the NEDD8 pathway, an essential component of ICI182,780-induced ER␣ degradation, reduces the antiproliferative efficacy of the drug, suggesting that receptor deg-radation is an integral part of the antiproliferative effect (11). It can be expected that treatment with XCT790, as potentiating the degradation of ER␣ induced by ICI182,780, could also enhance the antiproliferative effect of the anti-estrogen. However, in MCF7 cells, pretreatment with XCT790 did not potentiate the negative effect of ICI182,780 on proliferation. Each ER␣ moiety contacted by ICI182,780 is transcriptionally blocked immediately and rapidly degraded by the proteasome. This "all-or-nothing" approach may not be effective in vivo, particularly with patients with intact tumors. The kinetics of ER␣ occupancy by ICI182,780 might not be identical in cells and in vivo. A high ERR␣ expression in breast tumors correlates with poor prognosis. An inverse correlation between ERR␣ and ER␣ has been suggested (25) but not confirmed in a larger screen (26). Tumors should thus exist that express both ER␣ and ERR␣. For these tumors, our data suggest that pretreatment with XCT790 could induce a component involved in ICI182,780-induced ER␣ degradation and thereby enhance the efficacy of the latter drug and reduce its efficient dose.