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Bone is the most common metastatic site for breast cancer. Although the estrogen-related receptor alpha (ERRα) has been implicated in breast cancer cell dissemination to the bone from the primary tumor, its role after tumor cell anchorage in the bone microenvironment remains elusive. Here, we reveal that ERRα inhibits the progression of bone metastases of breast cancer cells by increasing the immune activity of the bone microenvironment. Overexpression of ERRα in breast cancer bone metastases induced expression of chemokines CCL17 and CCL20 and repressed production of TGFβ3. Subsequently, CD8 + T lymphocytes recruited to bone metastases escaped TGFβ signaling control and were endowed with exacerbated cytotoxic features, resulting in significant reduction in metastases. The clinical relevance of our findings in mice was confirmed in over 240 patients with breast cancer. Thus, this study reveals that ERRα regulates immune properties in the bone microenvironment that contributes to decreasing metastatic growth. SIGNIFICANCE: This study places ERRα at the interplay between the immune response and bone metastases of breast cancer, highlighting a potential target for intervention in advanced disease., (©2020 American Association for Cancer Research.)

Bone is the most common metastatic site for breast cancer. Estrogen-related-receptor alpha (ERRα) has been implicated in cancer cell invasiveness. Here, we established that ERRα promotes spontaneous metastatic dissemination of breast cancer cells from primary mammary tumors to the skeleton. We carried out cohort studies, pharmacological inhibition, gain-of-function analyses in vivo and cellular and molecular studies in vitro to identify new biomarkers in breast cancer metastases. Meta-analysis of human primary breast tumors revealed that high ERRα expression levels were associated with bone but not lung metastases. ERRα expression was also detected in circulating tumor cells from metastatic breast cancer patients. ERRα overexpression in murine 4T1 breast cancer cells promoted spontaneous bone micro-metastases formation when tumor cells were inoculated orthotopically, whereas lung metastases occurred irrespective of ERRα expression level. In vivo, Rank was identified as a target for ERRα. That was confirmed in vitro in Rankl stimulated tumor cell invasion, in mTOR/pS6K phosphorylation, by transactivation assay, ChIP and bioinformatics analyses. Moreover, pharmacological inhibition of ERRα reduced primary tumor growth, bone micro-metastases formation and Rank expression in vitro and in vivo. Transcriptomic studies and meta-analysis confirmed a positive association between metastases and ERRα/RANK in breast cancer patients and also revealed a positive correlation between ERRα and BRCA1 mut carriers. Taken together, our results reveal a novel ERRα/RANK axis by which ERRα in primary breast cancer promotes early dissemination of cancer cells to bone. These findings suggest that ERRα may be a useful therapeutic target to prevent bone metastases.

Bone metastases are one of the main complications of prostate cancer and they are incurable. We investigated whether and how estrogen receptor-related receptor alpha (ERRα) is involved in bone tumor progression associated with advanced prostate cancer. By meta-analysis, we first found that ERRα expression is correlated with castration-resistant prostate cancer (CRPC), the hallmark of progressive disease. We then analyzed tumor cell progression and the associated signaling pathways in gain-of-function/loss-of-function CRPC models in vivo and in vitro. Increased levels of ERRα in tumor cells led to rapid tumor progression, with both bone destruction and formation, and direct impacts on osteoclasts and osteoblasts. VEGF-A, WNT5A and TGFβ1 were upregulated by ERRα in tumor cells and all of these factors also significantly and positively correlated withERRα expression in CRPC patient specimens. Finally, high levels of ERRα in tumor cells stimulated the pro-metastatic factor periostin expression in the stroma, suggesting that ERRα regulates the tumor stromal cell microenvironment to enhance tumor progression. Taken together, our data demonstrate that ERRα is a regulator of CRPC cell progression in bone. Therefore, inhibiting ERRα may constitute a new therapeutic strategy for prostate cancer skeletal-related events.

Estrogen receptor-related receptor alpha (ERRα) is an orphan nuclear receptor with sequence homology to the estrogen receptors, ERα/β, but it does not bind estrogen. ERRα not only plays a functional role in osteoblasts but also in osteoclasts and chondrocytes. In addition, the ERRs, including ERRα, can be activated by coactivators such as peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC1α and β) and are implicated in adipogenesis, fatty acid oxidation, and oxidative stress defense, suggesting that ERRα-through its activity in bone resorption and adipogenesis--may regulate the insulin and leptin pathways and contribute to aging-related changes in bone and cartilage. In this review, we discuss data on ERRα and its cellular and molecular modes of action, which have broad implications for considering the potential role of this orphan receptor in cartilage and bone endocrine function, on whole-organism physiology, and in the bone aging process., (Copyright © 2013 American Society for Bone and Mineral Research.)

Bone metastasis is a complication occurring in up to 70% of advanced breast cancer patients. The estrogen receptor-related receptor alpha (ERRα) has been implicated in breast cancer and bone development, prompting us to examine whether ERRα may function in promoting the osteolytic growth of breast cancer cells in bone. In a mouse xenograft model of metastatic human breast cancer, overexpression of wild-type ERRα reduced metastasis, whereas overexpression of a dominant negative mutant promoted metastasis. Osteoclasts were directly affected and ERRα upregulated the osteoclastogenesis inhibitor, osteoprotegerin (OPG), providing a direct mechanistic basis for understanding how ERRα reduced breast cancer cell growth in bone. In contrast, ERRα overexpression increased breast cancer cell growth in the mammary gland. ERRα-overexpressing primary tumors were highly vascularized, consistent with an observed upregulation of angiogenic growth factor, the VEGF. In support of these findings, we documented that elevated expression of ERRα mRNA in breast carcinomas was associated with high expression of OPG and VEGF and with disease progression. In conclusion, our results show that ERRα plays a dual role in breast cancer progression in promoting the local growth of tumor cells, but decreasing metastatic growth of osteolytic lesions in bone., (©2011 AACR.)

Objective: We reported previously that the orphan nuclear receptor, estrogen receptor-related receptor α (ERRα), is expressed in articular chondrocytes and is dysregulated in a mouse model of inflammatory arthritis. The aim of this study, therefore, was to determine whether ERRα is also dysregulated in patients with osteoarthritis (OA)., Methods: ERRα messenger RNA (mRNA) and protein were quantified in normal and OA cartilage samples and in OA chondrocytes in vitro, with and without short-term treatment with a variety of OA-associated factors and signaling pathway agonists and inhibitors., Results: ERRα expression was lower in OA than in normal articular cartilage. Interleukin-1β (IL-1β) markedly up-regulated ERRα expression in OA chondrocytes in vitro, and agonist or inhibitor treatment indicated that the up-regulation was dependent on cyclooxygenase 2 (COX-2; NS398), prostaglandin E(2), cAMP (8-bromo-cAMP), and protein kinase A (PKA; KT5720). Treatment with the ERRα inverse agonist XCT790 decreased the expression of SOX9 and the up-regulation of ERRα by IL-1β, suggesting autoregulation of ERRα in the IL-1β pathway. Matrix metalloproteinase 13 (MMP-13) expression was also decreased by treatment with XCT790 plus IL-1β versus IL-1β alone, and the down-regulation of MMP-13 mRNA and protein observed with XCT790 alone suggests that the up-regulation of MMP-13 by IL-1β is ERRα-dependent., Conclusion: We report the first evidence that ERRα expression is regulated by IL-1β in COX-2-, cAMP-, and PKA-dependent pathways in OA chondrocytes. We confirmed that SOX9 is an ERRα target gene in human, as in rodent, chondrocytes and identified MMP-13 as a potential new target gene, which suggests that ERRα may both respond to the healing signal and contribute to extracellular degradation in OA cartilage., (Copyright © 2011 by the American College of Rheumatology.)

The orphan nuclear receptor, estrogen receptor-related receptor α (ERRα) is expressed in osteoblasts and osteoclasts (OCs) and has been proposed to be a modulator of estrogen signaling. To determine the role of ERRα in OC biology, we knocked down ERRα activity by transient transfection of an siRNA directed against ERRα in the RAW264.7 monocyte-macrophage cell line that differentiates into OCs in the presence of receptor activator of nuclear factor κB-ligands and macrophage colony-stimulating factor. In parallel, stable RAW cell lines expressing a dominant-negative form of ERRα and green fluorescent protein (RAW-GFP-ERRαΔAF2) were used. Expression of OC markers was assessed by real-time PCR, and adhesion and transmigration tests were performed. Actin cytoskeletal organization was visualized using confocal microscopy. We found that RAW264.7 cells expressing siRNA directed against ERRα and RAW-GFP-ERRαΔAF2 OCs displayed abnormal spreading, and decreased osteopontin and β3 integrin subunit expression compared with the corresponding control cells. Decreased adhesion and the absence of podosome belts concomitant with abnormal localization of c-src were also observed in RAW-GFP-ERRαΔAF2-derived OCs. In addition, RAW-GFP-ERRαΔAF2-derived OCs failed to transmigrate through osteoblast cell layers. Our data show that the impairment of ERRα function does not alter OC precursor proliferation and differentiation but does alter the adhesion/spreading and migration capacities of mature OCs.

Objectives: Subchondral bone loss is a characteristic feature of inflammatory arthritis. Recently, estrogen receptor-related receptor-alpha (ERR-alpha), an orphan nuclear receptor, has been found to be involved in activation of macrophages. We hypothesized that ERR-alpha which is expressed and also functional in articular chondrocytes, osteoblasts and osteoclasts, may be involved in rodent models of inflammatory arthritis., Methods: Erosive arthritis was induced in DBA/1 mice by injection of type II collagen in Freund's complete adjuvant. RNA was isolated from the bone and joints and expression of ERR-alpha and cartilage (GDF5 and Col2a1) and bone [bone sialoprotein (BSP) and osteocalcin (OCN)] markers was analysed by semi-quantitative PCR., Results: We report for the first time that the expression of ERR-alpha is dysregulated in bones and joints in a mouse model of inflammatory arthritis. Specifically, we show that ERR-alpha expression is down-regulated early in bone and later in joints of mice with type II CIA. Concomitantly, temporal changes were observed in GDF-5 and Col2a1 expression in joints following both initial injection and booster injection of type II collagen. Similarly, down-regulation of ERR-alpha mRNA expression in subchondral bone in mice with induced joint inflammation was also paralleled by down-regulation of markers of bone formation (BSP, OCN)., Conclusions: These data suggest that dysregulation of ERR-alpha expression may precede and contribute to the destruction of cartilage and bone accompanying inflammatory arthritis.

We report for the first time the expression of estrogen receptor-related receptor (ERR)-alpha in fetal and adult rat chondrocytes in growth plate and articular cartilage and the rat chondrogenic cell line C5.18 cells in vitro. ERRalpha mRNA and protein were expressed from proliferating chondrocyte to mature chondrocyte stages. We show that overexpressing ERRalpha in C5.18 cell cultures induces an increase in Sry-type high-mobility-group box transcription factor (Sox)-9 expression, a master gene in cartilage formation. In parallel, we report Sox9 promoter regulation by ERRalpha in C5.18 cells. To assess a functional role for ERRalpha in chondrogenesis, its expression was blocked by antisense oligonucleotides in C5.18 cell cultures, and this led to inhibition of cartilage formation associated with down-regulation of Sox9 and Indian hedgehog expression and maturation of proliferating chondrocytes into hypertrophic chondrocytes in vitro. Together these results implicate ERRalpha in the formation and maintenance of cartilage and also suggest that agonists and antagonists of ERRalpha may be useful as therapeutic agents in a wide variety of diseases affecting cartilage and joints.

Estrogen receptor-related receptor-alpha (ERRalpha) is an orphan nuclear receptor with sequence homology to the estrogen receptors, ERalpha/beta, but it does not bind estrogen. However, several recent studies suggest that ERRalpha not only plays a functional role in osteoblasts but also impinges on the estrogen axis in bone, as it does in at least certain other estrogen target tissues. We summarize here data on ERRalpha and its cellular and molecular modes of action that have broad implications for considering the potential role of this orphan receptor as a new therapeutic target in osteopenic disorders such as osteoporosis as well as other estrogen-responsive conditions.

The orphan nuclear estrogen receptor-related receptor alpha (ERRalpha) is expressed by osteoblastic cells and plays a functional role in osteoprogenitor proliferation and differentiation. To dissect further the role of ERRalpha in bone, we investigated the effects of estrogen (E2) on ERRalpha both in vitro and in vivo. Chronic treatment of fetal rat calvaria cells with E2-stimulated bone nodule formation and up-regulated ERRalpha mRNA expression at early (10 h and d 8) but not later times in culture, suggesting a link between ERRalpha and E2 during osteoprogenitor proliferation. ERRalpha mRNA levels were significantly lower in ovariectomized adult rat bones vs. those of sham-operated rats early (1 d and 1 wk) post surgery, but levels returned to control levels thereafter. ERRalpha is also expressed in osteoclasts (tartrate-resistant acid phosphatase + multinucleated cells) in vivo and in vitro (RAW 264.7 cells) and ovariectomization lowered the OPG/receptor activator of nuclear factor kappaB ligand expression ratio. Down-regulation of ERRalpha expression via antisense treatment of rat calvaria cells not only inhibited osteogenesis but also increased adipocyte colony formation and changed the OPG/receptor activator of nuclear factor kappaB ligand ratio. These data suggest that ERRalpha is regulated by estrogen in bone in which it may play a functional role at several levels (osteoblasts, adipocytes, and osteoclasts) in E2 deficiency diseases such as osteoporosis.

The orphan nuclear estrogen receptor-related receptor (ERR) alpha is expressed by osteoblastic cells, is known to transactivate at least one osteoblast-associated gene osteopontin (OPN) and plays a functional role in osteoprogenitor cell proliferation and differentiation. To dissect further the role of ERR-alpha in bone formation, we compared its expression to that of the estrogen receptor (ER) alpha and ER-beta in rat calvaria (RC) and fetal tibia in vivo and in RC and rat bone marrow (RBM) cells in vitro. We found that ERR-alpha is highly and widely expressed in most, if not all, cells in RC cell cultures from early proliferation stages through mineralized nodule formation; ER-alpha was localized similarly but at lower levels and ER-beta, although present, was barely detectable. These patterns of expression in vitro correlated with what we observed in vivo in sections of 21-day fetal RC, in which ERR-alpha appeared to be more highly expressed than either of the ERs. Interestingly, ERR-a also is highly expressed in RBM cells, while ER-alpha and ER-beta mRNA is expressed, but at lower levels. Moreover, we found that ERR-alpha, ER-alpha, and ER-beta were all expressed in osteoblasts in fetal and adult tibia whereas they were expressed differentially in calvaria in vivo in subsets of osteoblasts, supporting the hypothesis that ERR-alpha may interact with one or both of the ERs in those osteoblasts in which they are coexpressed and that all three receptors may be required for bone formation but at different times and for different functions.

The orphan nuclear estrogen receptor-related receptor alpha (ERRalpha), is expressed by many cell types, but is very highly expressed by osteoblastic cells in which it transactivates at least one osteoblast-associated gene, osteopontin. To study the putative involvement of ERRalpha in bone, we first assessed its expression in rat calvaria (RC) in vivo and in RC cells in vitro. ERRalpha mRNA and protein were expressed at all developmental stages from early osteoprogenitors to bone-forming osteoblasts, but protein was most abundant in mature cuboidal osteoblasts. To assess a functional role for ERRalpha in osteoblast differentiation and bone formation, we blocked its expression by antisense oligonucleotides in either proliferating or differentiating RC cell cultures and found inhibition of cell growth and a proliferation-independent inhibition of differentiation. On the other hand, ERRalpha overexpression in RC cells increased differentiation and maturation of progenitors to mature bone-forming cells. Our findings show that ERRalpha is highly expressed throughout the osteoblast developmental sequence and plays a physiological role in differentiation and bone formation at both proliferation and differentiation stages. In addition, we found that manipulation of receptor levels in the absence of known ligand is a fruitful approach for functional analysis of this orphan receptor and identification of potential target genes.

Estrogen receptor-related receptor alpha (ERR alpha) is an orphan nuclear receptor closely related to the estrogen receptor (ER), whose expression covers various stages of embryonic development and persists in certain adult tissues. We show that ERR alpha binds as a homodimer on a specific target sequence, the SFRE (SF-1 response element), already known to respond to the orphan nuclear receptor SF-1. Target sequences that are related to the SFRE and that discriminate between ERR alpha and SF-1 were identified. We have also analyzed the transcriptional properties of the ERR alpha originating from various species. All ERR alpha orthologs act as potent transactivators through the consensus SFRE. ERR alpha activity depends on the putative AF2AD domain, as well as on a serum compound that is withdrawn by charcoal treatment, suggesting the existence of a critical regulating factor brought by serum.

The superfamily of nuclear receptors comprises transcription factors that depend on a ligand for their activity. In addition, the superfamily includes a number of orphan receptors, for which no ligand is known. We report here that the orphan receptor estrogen receptor related alpha receptor (ERR alpha) stimulates the expression of the thyroid hormone receptor alpha (TR alpha) gene promoter. We characterized a responsive site that is both necessary and sufficient for ERR alpha-induced transactivation. In addition, we show that both TR alpha and ERR alpha are coexpressed in embryonic intestine, brown fat and heart as well as in the adult gonads. In the testis, expression of both receptors can be found in the seminiferous tubes where it is totally restricted to spermatocytes I. Altogether this suggests that TR alpha is an in vivo target of ERR alpha.

We studied the expression of the estrogen-related receptor 1 (ERR-1) during mouse embryonic development. ERR-1 is expressed at very early stages in ES cells and at E8.5 in the mesodermal cells of the visceral yolk sac. ERR-1 continues to be expressed later in mesodermal tissues and particularly in heart and in skeletal muscles. This expression persists during all the embryonic development and in adult stage. ERR-1 transcripts level increases during muscle differentiation. Accordingly, we show that ERR-1 expression increases during the myoblast to myotube transition in differentiating C2 myoblastic cells. ERR-1 has also been detected in the nervous system during embryonic development. At E10.5, a high level of ERR-1 transcripts can be observed in differentiated cells of the intermediate zone of the spinal cord which also suggests a role of ERR-1 in the differentiation of the nervous system. The same is observed in the telencephalon vesicules at E13.5. Later, at E15.5 and E17.5, expression persists in the spinal cord but decreases dramatically in the central nervous system. Moreover, ERR-1 expression increases during skin formation and is detected in the stratum spinosum which contains differentiated Malpighian cells. Finally, we also observed ERR-1 in endodermal derivatives such as the epithelium of intestine and urogenital system. The DNA target of ERR-1 has been identified to be the SF-1/FTZ-F1 responsive element (SFRE) and we show in this paper that SF-1/FTZ-F1 and ERR-1 bind to and activate transcription independently through the SFRE element. Our study suggests that ERR-1 may be implicated in numerous physiological or developmental functions, particularly in the muscle, the central and peripheral nervous system and the epidermis. Interestingly, in these various systems ERR-1 expression is correlated with post-mitotic cells stage, suggesting that ERR-1 may play a role in the differentiation process.

We studied the expression of estrogen-related receptor ERR-1 during mouse embryonic development. ERR-1 mRNA is present in bones formed by both the endochondral and intramembranous routes, and the onset of its expression coincides with bone formation. By RT-PCR experiments, we found that ERR-1, but not the related receptor ERR-2, is expressed in osteoblastic osteosarcoma cell lines as well as in primary osteoblastic cell populations derived from normal human bone. By gel shift analysis we found that ERR-1 binds as a monomer specifically to the SFRE sequence (SF-1-responsive-element; TCAAGGTCA). Mutation analysis revealed that both the core AGGTCA motif and the TCA 5'-extension are required for efficient ERR-1 binding. In transient transfection assays, ERR-1 acts as a potent transactivator through the SFRE sequence. This effect is cell-specific since ERR-1 activates transcription in the rat osteosarcoma cell line ROS 17.2/8 as well as in HeLa, NB-E, and FREJ4 cells but not in COS1 and HepG2 cells. Notably, the osteopontin (a protein expressed by osteoblasts and released in the bone matrix) gene promoter is a target for ERR-1 transcriptional regulation. Our findings suggest a role for ERR-1 in bone development and metabolism.