Regulation of hormone signaling by nuclear receptor interacting proteins.

Nuclear receptors are ligand-activated transcription factors which regulate the expression of genes critical for the growth of hormonedependent cancers. Their expression and activity are controlled by various cofactors which are important players in hormone-dependent carcinogenesis. RIP140 is a negative transcriptional regulator which is recruited by agonist-liganded receptors. Its strong repressive activity involves four silencing domains which interact with histone deacetylases (HDACs), carboxyl-terminal binding proteins (CtBPs) and additional partners. RIP140 positively regulates transactivation when nuclear receptors are recruited to target promoters through interaction with the Sp1 transcription factor. In human breast cancer cells, RIP140 expression is upregulated at the transcriptional level by various ligands of nuclear receptors revealing the existence of regulatory loops. The Mdm2 oncogenic ubiquitin-ligase is another protein which directly interacts with nuclear receptors. It is involved in a ternary complex with ERα and p53 and regulates ERα turn-over. In MCF-7 human breast cancer cells, various p53-inducing agents (such as UV irradiation) abolished E2-dependent turn-over of ERα without affecting its transactivation potential. Altogether, our results show that RIP140 and Mdm2 are two important regulators of ERα expression and activity and could therefore play major roles in hormone-dependent breast carcinogenesis. 1Introduction 1.1 Control of nuclear receptor transactivation Nuclear receptors are ligand-activated transcription factors which subsequently bind to specific responsive elements located in the regulatory region of target gene promoters (1). They stimulate transcription using both a constitutive amino-terminal and a liganddependent carboxyl-terminal activation function (AF1 and AF2, respectively), the latter being associated with the ligand-binding domain. These activation functions act independently or synergistically depending on the cell type and promoter context, by recruiting a number of cofactors (2). 1.2 Role of coregulators in hormone-related cancers Transcription cofactors function usually as part of multimolecular complexes, acting either by stabilisation of the basal transcription machinery or by chromatin remodelling which implicates various enzymatic activities such as histone acetyltransferases and deacetylases (3). Different types of post-translational modifications (phosphorylation, acetylation...) also regulate their activity, localization or interaction with receptors (4). The physiological roles of these cofactors begin to be investigated by invalidation of the corresponding gene in mice. They are also implicated in several pathologies such as hormone insensitivity syndromes or hormone-dependent cancers (5). Clinical studies have investigated the potential interest of these molecules as prognosis or diagnosis markers. They have also described quantitative or qualitative alterations of these genes in tumours. Finally, several approaches are underway to define whether steroid receptor transcription cofactors could be valuable targets in cancer treatment (modulation of their binding to receptors, modification of their expression or inhibition of their enzymatic activity). 2Negative regulation of nuclear receptor activity by RIP140 RIP140 is a widely expressed protein of 1158 residues which was isolated through its recruitment by ERα AF2 in the presence of ligand (6). Subsequently, RIP140 was shown to interact with many nuclear receptors such as ERα, TR, RAR and RXR (7), AR (8), VDR (9), PPARα/LXRα (10), GR (11), SF1 and DAX-1 (12). More recently, gene knockout in mice indicated that it is an essential protein for female fertility and energy homeostasis (13, 14). 2.1 Effects on nuclear receptor activity Our recent work has highlighted both the interaction and transrepression activity of RIP140 on different members of the nuclear receptor superfamily i.e. ERRs (15) and AR (16). Estrogen-receptor-related receptors Estrogen-receptor-related receptors (ERRα, β and γ) exhibit strong sequence similarity to ERα (17, 18) and interfere with estrogen signaling (reviewed in (19)) in part through binding to the same DNA binding elements, namely the ERRE for ERR-response element or the classical estrogen response element (ERE) (20). As expected from results obtained with other nuclear receptors, we have shown that different regions of RIP140 interact in vitro with the three ERRs (15). Indeed, we have observed a significant binding of radiolabelled ERRα, β and γ with chimaeric GST-RIP140 proteins encompassing the aminoterminal (residues 27-439), the central (residues 429-582) or the carboxyl-terminal region of the molecule (residues 683-1158). In transient transfection experiments, RIP140 inhibits ERRα, β and γ activity on EREor ERRE-containing reporter constructs (artificial reporter plasmids and natural promoters harbouring these binding sites such as pS2, ERRα or osteopontin) (15). Androgen receptor The androgen receptor (AR) is a cytoplasmic protein which undergoes nuclear translocation upon hormone binding (21). We have recently evidenced the interaction between AR and RIP140 and delineated the interaction between the LBD of AR and several domains of RIP140 which contain LxxLL motifs (16). In R1881-treated LNCaP prostate cancer cells, RIP140 is recruited to AR-target genes such as the PSA gene and completely relocalized from small nuclear foci to a diffuse pattern. Interestingly, the antagonist-bound AR which also translocates to the nucleus is not able to induce such a redistribution of RIP140. Our results indicate that RIP140 is a bona fide AR repressor since it inhibits AR-mediated transactivation and reverses the TIF2induced overactivation of AR. Moreover, in mouse embryo fibroblasts lacking the RIP140 gene, AR activity is significantly increased as compared to the wild type counterpart cells (22). 2.2 Mechanisms of transrepression The strong transcriptional repressive activity of RIP140 which was initially attributed to competition with coactivator binding to nuclear receptors (23) also involves an intrinsic inhibitory effect due to several domains which recruit various repressive effectors. We have identified two evolutionary conserved CtBP (Carboxyl-terminal Binding Proteins) binding motifs (PIDLS and PINLS motifs located respectively between residues 440 to 444 and 565 to 569) which explain partly the repressive action of RIP140 (24). RIP140 also interacts with class I and II HDACs. We show that RIP140 directly binds HDACs through a sequence comprised between residues 115 and 199 and using fluorescent chimaeric proteins, we have evidenced its colocalization with HDAC5 in intact cells (24). However, our results indicate i) that the repressive activity of RIP140 could be partially affected by inhibition of HDAC enzymatic activity and ii), that two additional domains in the Cterminal region of the protein support strong repressive activity but do not require HDAC activity or CtBPs (24), thus suggesting that the global repressive activity of RIP140 may depend on the interplay among several negative regulatory modules. 2.3 Effect on HRE-independent transactivation RIP140 also regulates HRE-independent transactivation by nuclear receptors which involves their indirect recruitment on target genes through protein-protein interaction in particular with AP1 or Sp1 factors (25). We previously reported the inhibition by RIP140 of estradiolinduced AP-1-dependent transcription of ERα (26). RIP140 antagonizes the stimulatory effect of GRIP1 and competes for binding to c-Jun and ERα both in vitro and in intact cells. More recently, we have demonstrated that overexpression of RIP140 strongly increases ERRαand ERRγ-mediated transactivation via Sp1-response sites, on both isolated sites and natural promoters (15). This positive regulation exerted by RIP140 involves HDAC either directly or indirectly as suggested by overexpression of HDAC1 or treatment with TSA. 2.4 Regulatory loops involving RIP140 Cloning of the RIP140 gene and analysis of transcriptional regulatory mechanisms revealed that it is involved in several feed-back loops. RIP140 mRNA levels are rapidly and directly increased by 17βestradiol (E2) in MCF-7 human breast cancer cells (27). This estrogenic regulation which is preferentially mediated by ERα and not restricted to mammary cancer cells, involves a consensus ERE which allows efficient binding of ERα, both in vitro and in intact cells (28). Very interestingly, the regulatory feed-back loop that we have demonstrated for ERα also exists for several other nuclear receptors (such as retinoid (29) and androgen (16) receptors) or for the dioxin receptor (AhR) (28, 30). 3Effect of Mdm2 on ER signaling Several studies have shown that binding of E2 to ERα significantly decreases its stability. This shorter half-life in the presence of hormone appears to implicate the ubiquitin/proteasome pathway since ERα is ubiquitinated (31) and its ligand-dependent down-regulation is blocked by proteasome inhibitors (32-34). Previous studies have suggested that ubiquitin-conjugating enzymes or ATPase subunits of the proteasome complex bind nuclear receptors and modulate their functions. In a recent study, we have shown that the Mdm2 oncoprotein is also involved in the ligand-dependent decrease of ERα stability (35). 3.1 Regulation of ERα expression and activity The Mdm2 oncogene is overexpressed in a wide variety of human cancers (36) and its role in tumorigenesis is linked to its ability to act as an E3 ubiquitin-ligase which is required for the ubiquitination and proteasome-dependent degradation of several growth regulatory proteins including p53 (37-39). Our data indicate that Mdm2 regulates ERα expression as a ternary complex with p53 (35). Using a modified mammalian two-hybrid system and an in vitro protein-protein interaction assay, we have shown that Mdm2 coexists with ERα and p53 within the same protein complex in intact cells. Using transient transfection into p53/Mdm2-/cells, we have demonstrated that p53 and Mdm2 are required for liganddependent ERα turn-over. By chase experiments using cycloheximide, we have found that Mdm2 overexpression decreases the apparent stability of the ERα protein thus confirming its role in the posttranslational regulation of ERα expression. Finally, a mutant of Mdm2 (Mdm2 RING) deleted in the carboxyl-terminal part of the protein which contains the RING domain required for its ubiquitin-ligase activity (40) still interacts with ERα in GST-pull down experiment but does not decrease ERα accumulation as compared with the effect of its wildtype counterpart suggesting that the E3 ubiquitin-ligase activity of Mdm2 is directly involved in ERα degradation. 3.2 Effect of stress inducing agents Since cellular stress results in an increased accumulation of p53 due mainly to the inability of Mdm2 to degrade the protein, we have tested the effect of various stress-inducing agents (which stabilize p53) on ERα turn-over (35). We show that treatments that increase p53 levels in MCF-7 human breast cancer (such as UV irradiation or treatment with RITA which inhibits the interaction of p53 with Mdm2) concomitantly suppress the hormone-dependent down-regulation of ERα. In the case of UV irradiation, our data demonstrate that the effect results from an increase in ERα stability. 3.3 Ligand-dependent turn-over and transactivation Previous studies proposed that the E2-dependent decrease of ERα accumulation was required for transcriptional activity of the receptor (41). However, our data provide several lines of evidence showing that the E2-dependent turnover of the receptor is not necessary for EREmediated transactivation (35). Indeed, we have found (using p53/Mdm2-/cells or UV irradiation of MCF-7 cells) that ERα strongly activates transcription in cells where E2 up-regulates receptor levels thus dissociating the effect of E2 on ERα degradation and activity.