Knockdown Of ERRα Research Articles

Estrogen-related receptor alpha (ERRα) promotes the migration, invasion and angiogenesis of breast cancer stem cell-like cells.

Breast cancer progression, metastasis and recurrence are largely driven by breast cancer stem cells (BCSCs), which constitute a subset of tumor cells exhibiting stem cell characteristics. In this study, we evaluated the role of estrogen-related receptor alpha (ERRα) in the migration, invasion and angiogenesis of BCSCs. The inhibition of ERRα using XCT790 or knockdown of ERRα using shRNA inhibited the mammosphere formation efficiency, as well as the migration and invasion of BCSCs derived from the mammospheres of MCF7 and MDA-MB-231 (MB231) cells. Conversely, the overexpression of ERRα significantly increased the migration and invasion of BCSCs derived from the mammosphere. In addition, the XCT790 treatment or shERRα significantly downregulated the epithelial-mesenchymal transition (EMT), as evidenced by the downregulation in the expression of vimentin, Snail, Slug and N-cadherin in the mammospheres of MCF7 and MB231 cells. The chorioallantoic membrane assay showed that the conditioned media from XCT790-treated and shERRα cells significantly inhibited blood vessel formation and vessel length. Furthermore, XCT790 treatment or shERRα also downregulated the expression of molecular markers of angiogenesis, such as VEGF-A and Ang-2 in the mammospheres. Conversely, the overexpression of ERRα in MCF7 cells significantly increased both EMT and angiogenesis. These findings suggest that ERRα inhibits the migration, invasion and angiogenesis of BCSCs, suggesting as a potential target for breast cancer therapy.

Estrogen-Related Receptor Alpha (ERRα) Promotes Cancer Stem Cell-Like Characteristics in Breast Cancer.

Cancer stem cells drive tumor initiation, invasion, metastasis and recurrence. In the present study, we have evaluated the role of ERRα in the maintenance of breast cancer stem cells (BCSCs) using breast cancer cell lines. The inhibition of ERRα with the inverse agonist, XCT-790, or the knockdown of ERRα in breast cancer cells significantly reduced the mammosphere formation efficiency and mammosphere size along with a significant reduction in the CD44+/CD24- BCSCs. Treatment with XCT-790 significantly downregulated expression of the transcription factors involved in stem cell maintenance such as Oct4, Klf4, Sox2, Nanog and c-Myc in the mammosphere forming stem cells of MCF7 and MDA-MB-231. In addition, XCT-790 induced cell cycle arrest and apoptosis in the mammosphere-forming cells. The knockdown or inhibition of ERRα downregulated the expression of Notch1 and β-catenin, whereas the overexpression of ERRα in MCF7 cells upregulated the expression of these proteins. Moreover, the inhibition of ERRα synergistically enhanced the efficacy of paclitaxel in inhibiting the BCSCs. These results show that ERRα is crucial for the maintenance of BCSCs and suggest that ERRα could be a potential target for breast cancer treatment.

ERRα protects against sepsis-induced acute lung injury in rats

BackgroundSepsis-induced acute lung injury (ALI) is associated with poor survival rates. The identification of potential therapeutic targets for preventing sepsis-induced ALI has clinical importance. This study aims to investigate the role of estrogen-related receptor alpha (ERRα) in sepsis-induced ALI.MethodsLipopolysaccharide (LPS) was used to simulate sepsis-induced ALI model in rat pulmonary microvascular endothelial cells (PMVECs). The effects of ERRα overexpression and knockdown on LPS-induced endothelial permeability, apoptosis and autophagy were determined by horseradish peroxidase permeability assay, TdT-mediated dUTP Nick End Labeling (TUNEL) assay, flow cytometry, immunofluorescence staining, RT-PCR and Western Blotting. The rat model with sepsis-induced ALI was established by cecal ligation and puncture in anesthetized rats to verify the results of in vitro experiments. Animals were randomly assigned to receive intraperitoneal injection of vehicle or ERRα agonist. Lung vascular permeability, pathological injury, apoptosis and autophagy were examined.ResultsOverexpression of ERRα ameliorated LPS-induced endothelial hyperpermeability, degradation of adherens junctional molecules, upregulation of bax, cleaved caspase 3 and cleaved caspase 9 levels, downregulation of anti-apoptotic protein Bcl-2 level, and promoted the formation of autophagic flux, while the knockdown of ERRα exacerbated LPS-induced apoptosis and inhibited the activation of autophagy. Administration of ERRα agonist alleviated the pathological damage of lung tissue, increased the levels of tight junction proteins and adherens junction proteins, and decreased the expression of apoptosis-related proteins. Promoting the expression of ERRα significantly enhanced the process of autophagy and reduced CLP-induced ALI. Mechanistically, ERRα is essential to regulate the balance between autophagy and apoptosis to maintain the adherens junctional integrity.ConclusionERRα protects against sepsis-induced ALI through ERRα-mediated apoptosis and autophagy. Activation of ERRα provides a new therapeutic opportunity to prevent sepsis-induced ALI.

Genistein mitigates senescence of bone marrow mesenchymal stem cells via ERRα-mediated mitochondrial biogenesis and mitophagy in ovariectomized rats

Senescence of bone marrow mesenchymal stem cells (BMMSCs) induced by chronic oxidative stress is an important factor contributes to the postmenopausal osteoporosis (PMOP). Mitochondrial quality control takes a pivotal role in regulating oxidative stress and cell senescence. Genistein is a major isoflavone in soy products, which is best known for its ability to inhibit bone loss in both postmenopausal women and ovariectomized (OVX) rodents. Here we show that OVX-BMMSCs displayed premature senescence, elevated reactive oxygen species (ROS) level and mitochondria dysfunction, while genistein rescued these phenotypes. Using network pharmacology and molecular docking, we identified estrogen-related receptor α (ERRα) as the potential target of genistein. Knockdown of ERRα greatly abolished the anti-senescence effect of genistein on OVX-BMMSCs. Further, the mitochondrial biogenesis and mitophagy induced by genistein were inhibited by ERRα knockdown in OVX-BMMSCs. In vivo, genistein inhibited trabecular bone loss and p16INK4a expression, upregulated sirtuin 3 (SIRT3) and peroxisome proliferator-activated receptor gamma coactivator one alpha (PGC1α) expression in the trabecular bone area of proximal tibia in OVX rats. Together, this study revealed that genistein ameliorates senescence of OVX-BMMSCs through ERRα-mediated mitochondrial biogenesis and mitophagy, which provided a molecular basis for advancement and development of therapeutic strategies against PMOP.

Efficacy of glucose transporter inhibitors for the treatment of ERRα-overexpressed colorectal cancer.

Colorectal cancer is the most-incidence associated extremely high mortality rate worldwide. The overexpression of estrogen-related receptor α (ERRα) is contributing to a poor prognosis. Obtaining a better understanding of the mechanisms of ERRα in colorectal cancer is important for developing cancer therapies. Western blotting and qRT-PCR were used to determine the protein and mRNA levels of ERRα, OUTB1, and solute carrier family 7 member 11 (SLC7A11) in HCT-116 cells. Short hairpin RNA (shRNA) was used to knockdown ERRα in HCT-116 cells. The level of reactive oxygen species (ROS), the nicotinamide adenine dinucleotide phosphate NADP+/NADPH, and the oxidized glutathione (GSSG)/reduced glutathione (GSH) ratio were measured by HPLC-MS to determine the redox state in HCT-116 cells. Lastly, tumor xenograft experiments were carried out to determine the effect of glucose transporter (GLUT) inhibitor. Knockdown of ERRα decreased the expression of OTUB1 and SLC7A11 in HCT-116 cells. SLC7A11 overexpression induced NADPH-dependent redox system collapse. Aberrant expression of ERRα significantly reduced NADPH level and resulted in collapse of the redox system under glucose deprivation. Furthermore, ERR overexpression of ERRα sensitized cancer cells to inhibition of GLUTs. Treatment with GLUT inhibitor significantly reduced tumor volume after 6 weeks of tumor xenograft experiment. Our study demonstrates that the over-expression of ERRα causes redox system collapses via regulating the expressions of OUTB1 and SLC7A11. Up-regulation of SLC7A11 mediates the disruption of cell metabolism and the balance of redox state in colorectal cancer. Additionally, the GLUT inhibitor significantly reduces colorectal tumor volume, suggesting that the GLUT inhibitor could serve as a potential therapy for colorectal treatment.

Intervention of ERRα Expression on Apoptosis Induction of Multiple Myeloma MM.1S Cells Cultured in Vitro

To investigate the effect of two different approaches ERRα strategy on the apoptosis in multiple myeloma cell line MM.1S. For the one strategy, shRNA was mediated by lentivirus. Stable cell clones were established by transfecting the lentivirus into MM.1S cells and screened by puromycin. For the other strategy, XCT790, a specific reverse agonist of ERRα, was used to treat MM.1S cells. The apoptosis of the cells was analyzed by flow cytometry after ERRα was down-regulated. Western blot assay was used to detect the apoptosis of related proteins. The knocked down ERRα was achieved, lentivirus with shERRα were successfully infected into MM.1S and ERRα was reduced significantly. Knockdown of ERRα could induce MM.1S cell apoptosis dramatically. Meanwhile, the expression of cleaved PARP (a kind of apoptosis related markers) was significantly increased following depletion of ERRα in MM.1S cells. XCT790 could significantly down-regulate the expression of ERRα protein in MM.1S cells, which was consistent with the effect caused by shRNA. Interference the expression of ERRα by shRNA or XCT790 can induce apparent apoptosis in MM.1S cells, which indicating that ERRα is crucial for the survival of myeloma cells.

Korean Red Ginseng Improves Astrocytic Mitochondrial Function by Upregulating HO-1-Mediated AMPKα-PGC-1α-ERRα Circuit after Traumatic Brain Injury.

Heme oxygenase-1 (HO-1) exerts beneficial effects, including angiogenesis and energy metabolism via the peroxisome proliferator-activating receptor-γ coactivator-1α (PGC-1α)–estrogen-related receptor α (ERRα) pathway in astrocytes. However, the role of Korean red ginseng extract (KRGE) in HO-1-mediated mitochondrial function in traumatic brain injury (TBI) is not well-elucidated. We found that HO-1 was upregulated in astrocytes located in peri-injured brain regions after a TBI, following exposure to KRGE. Experiments with pharmacological inhibitors and target-specific siRNAs revealed that HO-1 levels highly correlated with increased AMP-activated protein kinase α (AMPKα) activation, which led to the PGC-1α-ERRα axis-induced increases in mitochondrial functions (detected based on expression of cytochrome c oxidase subunit 2 (MTCO2) and cytochrome c as well as O2 consumption and ATP production). Knockdown of ERRα significantly reduced the p-AMPKα/AMPKα ratio and PGC-1α expression, leading to AMPKα–PGC-1α–ERRα circuit formation. Inactivation of HO by injecting the HO inhibitor Sn(IV) protoporphyrin IX dichloride diminished the expression of p-AMPKα, PGC-1α, ERRα, MTCO2, and cytochrome c in the KRGE-administered peri-injured region of a brain subjected to TBI. These data suggest that KRGE enhanced astrocytic mitochondrial function via a HO-1-mediated AMPKα–PGC-1α–ERRα circuit and consequent oxidative phosphorylation, O2 consumption, and ATP production. This circuit may play an important role in repairing neurovascular function after TBI in the peri-injured region by stimulating astrocytic mitochondrial biogenesis.

Knockdown of estrogen-related receptor α inhibits valve interstitial cell calcification in vitro by regulating heme oxygenase 1.

Calcific aortic valve disease (CAVD) is the most common valvular heart disease in adults. The cellular mechanisms of CAVD are still unknown, but accumulating evidence has revealed that osteogenic differentiation of human valve interstitial cells (hVICs) plays an important role in CAVD. Thus, we aimed to investigate the function of estrogen-related receptor α (ERRα) in the osteogenic differentiation of hVICs. We found that the level of ERRα was significantly increased in CAVD samples compared to normal controls. In addition, ERRα was significantly upregulated during hVIC osteogenic differentiation in vitro. Gain- and loss-of-function experiments were performed to identify the function of ERRα in hVIC calcification in vitro. Inhibition of endogenous ERRα attenuated hVIC calcification, whereas overexpression of ERRα in hVICs promoted this process. RNA sequencing results suggested that heme oxygenase-1 (Hmox1) was a downstream target of ERRα, which was further confirmed by western blotting. Additionally, we also found that downregulation of Hmox1 by shHmox1 efficiently reversed the inhibition of calcification induced by ERRα shRNA in hVICs. ChIP-qPCR and luciferase assays indicated that Hmox1 was negatively regulated by ERRα. We found that overexpression of Hmox1 or its substrates significantly inhibited hVIC calcification in vitro. In conclusion, we found that knockdown of ERRα can inhibit hVIC calcification through upregulating Hmox1 and that ERRα and Hmox1 are potential targets for the treatment of CAVD.

Increased ABCC4 Expression Induced by ERRα Leads to Docetaxel Resistance via Efflux of Docetaxel in Prostate Cancer.

Docetaxel is a major treatment for advanced prostate cancer (PCa); however, its resistance compromises clinical effectiveness. Estrogen receptor-related receptor alpha (ERRα) belongs to an orphan nuclear receptor superfamily and was recently found to be closely involved in cancer. In the present study, we found that ERRα was involved in docetaxel resistance in PCa. Overexpression of ERRα conferred docetaxel resistance in PCa cell lines, and cells with ERRα downregulation were more sensitive to docetaxel. Among the drug resistance-related genes, ABCC4 demonstrated synchronous expression after ERRα manipulation in cells. Moreover, both ERRα and ABCC4 were overexpressed in the docetaxel-resistant cell, which could be reversed by ERRα knockdown. The knockdown of ERRα also reversed the reduced drug accumulation in the docetaxel-resistant cell. We also demonstrated for the first time that ABCC4 was a direct target of ERRα as determined by the CHIP and luciferase assays. Bioinformatics analysis revealed high expression of ERRα and ABCC4 in PCa patients, and a number of potential ERRα/ABCC4 targets were predicted. In conclusion, our study demonstrated a critical role for ERRα in docetaxel resistance by directly targeting ABCC4 and stressed the importance of ERRα as a potential therapeutic target for drug-resistant PCa.

Inhibition of ERRα Aggravates Sepsis-Induced Acute Lung Injury in Rats via Provoking Inflammation and Oxidative Stress.

Inflammation and oxidative stress are critical pathologies that contribute to sepsis-induced acute lung injury (ALI). This study investigated the regulatory role of estrogen-related receptor alpha (ERRα) in an experimental model of sepsis-induced ALI. In vivo, a cecal ligation and puncture- (CLP-) induced ALI model was established in anesthetized rats. Animals were then randomly assigned to receive an intraperitoneal injection of vehicle or ERRα inverse agonist (XCT-790, 2.5 mg/kg). Administration of XCT-790 significantly aggravated a sepsis-induced increase in pathological damage of lung tissues, lung endothelial permeability, myeloperoxidase (MPO) activity in lung tissues, production of serum inflammatory factors, and inflammatory cell accumulation in bronchoalveolar lavage fluid. In addition, XCT-790 treatment exacerbated a CLP-induced decrease in lung superoxide dismutase and an increase in lung malondialdehyde levels. In vitro, the exposure of rat pulmonary microvascular endothelial cells (PMVECs) to lipopolysaccharide (LPS) resulted in increased endothelial permeability and reduced expression of tight junction protein ZO-1, Occludin, JAM-A, and adherens junction protein VE-cadherin, which were further deteriorated by knockdown of ERRα. In addition, LPS-triggered inflammatory factor production and increase in the expression of phosphorylated IκBα and NF-κB p65 were also exacerbated by silencing ERRα gene. Meanwhile, knockdown of ERRα dramatically promoted LPS-activated mitochondrial reactive oxygen species production and LPS-induced downregulation of Sirt3 protein levels in rat PMVECs. Taken together, our present study provides evidences that ERRα functions as a novel negative modulator of sepsis-induced ALI in rats. The underlying mechanisms responsible for ERRα-elicited effects are largely dependent on the regulation of inflammatory response and oxidative stress.

Regulation of PGC-1α expression by a GSK-3β-TFEB signaling axis in skeletal muscle

ObjectiveIn muscle cells, the peroxisome proliferator-activated receptor γ co-activator 1 (PGC-1) signaling network, which has been shown to be disturbed in the skeletal muscle in several chronic diseases, tightly controls mitochondrial biogenesis and oxidative substrate metabolism. Previously, we showed that inactivation of glycogen synthase kinase (GSK)-3β potently increased Pgc-1α abundance and oxidative metabolism in skeletal muscle cells. The current study aims to unravel the molecular mechanism driving the increase in Pgc-1α mediated by GSK-3β inactivation. MethodsGSK-3β was inactivated genetically or pharmacologically in C2C12 myotubes and the requirement of transcription factors known to be involved in Pgc-1α transcription for increases in Pgc-1α abundance mediated by inactivation of GSK-3β was examined. ResultsEnhanced PGC-1α promoter activation after GSK-3β inhibition suggested a transcriptionally-controlled mechanism. While myocyte enhancer factor (MEF)2 transcriptional activity was unaltered, GSK-3β inactivation increased the abundance and activity of the transcription factors estrogen-related receptor (ERR)α and ERRγ. Pharmacological inhibition or knock-down of ERRα and ERRγ however failed to prevent increases in Pgc-1α mRNA mediated by GSK-3β inactivation. Interestingly, GSK-3β inactivation activated transcription factor EB (TFEB), evidenced by decreased phosphorylation and enhanced nuclear localization of the TFEB protein. Moreover, knock-down of TFEB completely prevented increases in Pgc-1α gene expression, PGC-1α promoter activity and PGC-1α protein abundance induced by GSK-3β inactivation. Furthermore, mutation of a specific TFEB binding site on the PGC-1α promoter blocked promoter activation upon inhibition of GSK-3β. ConclusionsIn skeletal muscle, GSK-3β inactivation causes dephosphorylation and nuclear translocation of TFEB resulting in TFEB-dependent induction of Pgc-1α expression.

The enhanced expression of estrogen‐related receptor α in human bladder cancer tissues and the effects of estrogen‐related receptor α knockdown on bladder cancer cells

Estrogen-related receptor α (ERRα) belongs to the superfamily of nuclear orphan receptors. However, the role of ERRα in bladder cancer remains unknown. This study examined the expression of ERRα in bladder cancer tissues and explored the molecular mechanisms of ERRα in bladder cancer progression. The expression of ERRα in bladder cancer tissues from 61 patients was determined by immunohistochemistry. We performed quantitative real-time polymerase chain reaction assay to detect the gene expression levels and carried out Western blot assay to measure protein levels. In vitro functional assays, including colony formation, Cell Counting Kit-8, Transwell invasion, and migration assays, were performed to detect bladder cancer cell growth, proliferation, invasion, and migration, respectively. Flow cytometry was used to determine the cell apoptotic rate of bladder cancer cells. Among the 61 detected bladder cancer tissues, 39 bladder cancer tissues showed positive ERRα immunoreactivity. Higher ERRα immunoreactivity score was significantly associated with TNM stage, tumor grade, distant metastasis, and poor survival in patients with bladder cancer. Univariate and multivariate analyses showed that ERRα immunoreactivity was an independent prognostic factor for overall survival in patients with bladder cancer. ERRα was found to be upregulated in bladder cancer cell lines, and knockdown of ERRα suppressed bladder cancer cell growth, proliferation, invasion, and migration; promoted bladder cancer cell apoptosis; and inhibited the epithelial-mesenchymal transition of bladder cancer cells. On the other hand, bladder cancer cell proliferation, invasion, and migration were significantly enhanced after cells were transfected with an ERRα-overexpressing vector. In vivo tumor growth and metastasis assays showed that ERRα knockdown resulted in remarkable inhibition of tumor growth and tumor metastasis in nude mice. Collectively, our results suggest that the enhanced expression of ERRα may play a key role in the development and progression of bladder cancer and ERRα may serve as an important prognostic factor for bladder cancer.

Long non-coding RNA SPRY4-IT1 promotes development of hepatic cellular carcinoma by interacting with ERR\u03b1 and predicts poor prognosis

Hepatocellular carcinoma (HCC) has become one of the most common leading causes of cancer-related deaths worldwide. This study investigates the role of lncRNA, SPRY4-IT1 in the development of HCC. Quantitative real-time PCR (qRT-PCR) was performed and the results showed that SPRY4-IT1 expression was up-regulated in HCC tissues and high expression of SPRY4-IT1 was associated with poor 5-year overall survival in the HCC patient cohort. Clinicopathological analysis showed that the expression of SPRY4-IT1 was significantly correlated with TNM stage in HCC patients. In vitro CCK-8 assay, colony formation assay, cell invasion and migration assays demonstrated that knock-down of SPRY4-IT1 suppressed cell proliferation, colony formation, cell invasion and migration in HCC cells. Flow cytometric analysis showed that knock-down of SPRY4-IT1 induced cell cycle arrest at G0/G1 phase and induced apoptosis. In addition, knock-down of SPRY4-IT1 also suppressed the mRNA and protein expression of estrogen-related receptor α (ERRα). Similarly, knock-down of ERRα inhibited cell proliferation, colony formation, cell invasion and migration in HCC cells. More importantly, ERRα overexpression antagonized the effects of SPRY4-IT1 knock-down on cell proliferation, colony formation, cell invasion and migration in HCC cells. Taken together, our data highlights the pivotal role of SPRY4-IT1 in the tumorigenesis of HCC.

Estrogen/ERR-α signaling axis is associated with fiber-type conversion of upper airway muscles in patients with obstructive sleep apnea hypopnea syndrome.

Estrogen is related with the low morbidity associated with obstructive sleep apnea hypopnea syndrome (OSAS) in women, but the underlying mechanisms remain largely unknown. In this study, we examined the relationship between OSAS and estrogen related receptor-α (ERR-α). We found that the expression levels of ERR-α and Myh7 were both downregulated in palatopharyngeal tissues from OSAS patients. In addition, we report that ERR-α is dynamically expressed during differentiation of C2C12 myoblasts. Knockdown of ERR-α via instant siRNA resulted in reduced expression of Myh7, but not Myh4. Furthermore, differentiation of C2C12 cells under 3% chronic intermittent hypoxia, a model resembling human OSAS, was impaired and accompanied by a obvious reduction in Myh7 expression levels. Moreover, activation of ERR-α with 17β-estradiol (E2) increased the expression of Myh7, whereas pretreatment with the ERR-α antagonist XCT790 reversed the E2-induced slow fiber-type switch. A rat ovariectomy model also demonstrated the switch to fast fiber type. Collectively, our findings suggest that ERR-α is involved in estrogen-mediated OSAS by regulating Myhc-slow expression. The present study illustrates an important role of the estrogen/ERR-α axis in the pathogenesis of OSAS, and may represent an attractive therapeutic target, especially in postmenopausal women.

ERRα Is a Marker of Tamoxifen Response and Survival in Triple-Negative Breast Cancer.

Estrogen-related receptor alpha (ERRα) signaling has recently been implicated in breast cancer. We investigated the clinical value of ERRα in randomized cohorts of tamoxifen-treated and adjuvant-untreated patients. Cox proportional hazards regression was used to evaluate the significance of associations between ERRα gene expression levels and patient DMFS in a previously published microarray dataset representing 2,000 breast tumor cases derived from multiple medical centers worldwide. The 912 tumors used for immunostaining were from a tamoxifen-randomized primary breast cancer trial conducted in Stockholm, Sweden, during 1976-1990. Mouse model was used to study the effect of tamoxifen treatment on lung colonization of MDA-MB-231 control cells and MDA-MB-231 cells with stable knockdown of ERRα. The phenotypic effects associated with ERRα modulation were studied using immunoblotting analyses and wound-healing assay. We found that in ER-negative and triple-negative breast cancer (TNBC) adjuvant-untreated patients, ERRα expression indicated worse prognosis and correlated with poor outcome predictors. However, in tamoxifen-treated patients, an improved outcome was observed with high ERRα gene and protein expression. Reduced ERRα expression was oncogenic in the presence of tamoxifen, measured by in vitro proliferation and migration assays and in vivo metastasis studies. Taken together, these data show that ERRα expression predicts response to tamoxifen treatment, and ERRα could be a biomarker of tamoxifen sensitivity and a prognostic factor in TNBC.

Abstract A07: Novel effectors of K-Ras-mediated and KSR1 dependent colon tumorigenesis

Abstract Purpose of the study: The purpose of the study is to identify new effectors of K-Ras mediated and KSR1 dependent colon tumorigenesis. Background: Gene targeting and RNA interference identified Kinase suppressor of Ras1 (KSR1) as an effector of oncogenic Ras-induced tumor formation in mouse embryonic fibroblast (MEF) and human colon tumor cells but dispensable for normal development. Therefore, KSR1 and its downstream signaling molecules are potential targets for therapy in colon tumorigenesis. Previous analyses identified nuclear co-activator Peroxisome proliferator-activated receptor gamma co-activator 1(PGC1) and transcription factor Estrogen Related Receptor α (ERRα) as effectors of Ras-induced and KSR1-dependent cell transformation in MEFs. To identify molecules that are essential for Ras-driven and KSR1-dependent colon tumorigenesis, we performed high-throughput screens with the human colon tumor cell line HCT116, using genome-wide siRNA and natural product libraries. The siRNA screen identified AMP-activated protein kinase subunit γ1 (AMPKγ1) as an effector of KSR1-dependent signaling. Results: Compared to non-targeting siRNA, RNAi of AMPKγ1 in HCT116 cells decreased anchorage-independent growth in polyHEMA-coated plates by 6-fold in 24 h (p<10-3) and by 11-fold in 48 hours (p<10-3). Moreover, RNAi of AMPKγ1 differentially induced apoptosis of HCT116 cells (21%) compared to immortalized normal human colon epithelial cells (HCEC) (0.7%; p<10-4). Similarly, RNAi of AMPK catalytic α subunits (AMPKα1/AMPKα2) induced the death of HCT116 cells (21.5%) compared to non-targeting control siRNA (1.3%). RNAi of K-Ras, KSR1 and AMPKγ1 decreased protein expression of PGC1β and ERRα in HCT116 cells. Protein and mRNA expression of PGC1β and ERRα were significantly up-regulated in colon cancer cell lines compared to HCEC, indicating these are important effectors of K-Ras mediated KSR1 and AMPK-driven cell transformation. Furthermore, stable knockdown of PGC1β and ERRα decreased colony formation by 7-fold (p<10-3) and 3.5-fold (p<10-3) in soft agar and delayed tumor formation in nude mice (p<10-4). In-situ hybridization in human tumor samples revealed PGC1β mRNA to be significantly up-regulated in primary tumor stage I, stage II, and stage IV metastatic tumor samples (p<0.05) compared to normal colonic epithelium. ERRα mRNA was up-regulated in primary tumor stage I, II, III and stage IV metastatic tumors (p<0.05). Assay of natural product library for molecules that phenocopy RNAi of KSR1 and AMPK inhibitor Compound C identified 4-OH-Staurosporine. 4-OH-Staurosporine is a competitive inhibitor ATP binding to recombinant AMPK (α1β1γ1) (Ki=23.5 nM). 4-OH-Staurosporine treatment decreased phosphorylation of AMPK substrates acetyl-CoA carboxylase (ACC) at Ser89 and Raptor at Ser792 and expression of PGC1β and ERRα in a panel of human colon tumor cell lines (HCT116, HCT15, SW480, CACO2, GEO). Treatment with 3.75μM 4-OH-Staurosporine also decreased viability of colon tumor cells (HCT116, 52%; HCT15, 15%; SW480, 12%, CACO2, 58%; DLD1, 16%; GEO, 15%) compared with HCEC (p<10-3). Conclusion: These results demonstrate the importance of AMPK and its downstream signaling effectors PGC1β and ERRα in maintaining colon tumor cell survival driven by oncogenic Ras and demonstrate the value of using KSR1 as a reference standard to detect genes essential to colon tumor survival. Citation Format: Binita Das, Kurt Fisher, Hyunseok Kim, Deanna J. Volle, Deandra R. Smith, Robert S. Livergood, John MacMillan, Michael White, Robert Lewis. Novel effectors of K-Ras-mediated and KSR1 dependent colon tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A07. doi: 10.1158/1557-3125.RASONC14-A07

Abstract 1385: Orphan nuclear receptor estrogen-related receptor alpha (ERRα) characterized as a novel transcriptional regulator of the oncogenic fusion gene TMPRSS2:ERG in prostate cancer

Abstract The expression of androgen-responsive TMPRSS2:ERG fusion gene, which results in aberrant expression of the oncogenic transcription factor ERG under the regulation of androgen receptor (AR), is characterized as the predominant factor responsible for the initiation and also advanced progression of prostate cancer (PCa). However, its upstream transcriptional regulation in androgen-independent PCa is still not fully understood. Recent studies show that the orphan nuclear receptor estrogen-related receptor alpha (ERRα/NR3B1) plays certain regulatory roles in the advanced progression of prostate cancer, such as its cross-talk with AR signaling. Based on this, we hypothesize that besides AR, ERRα could also regulate or target to the promoter of fusion gene TMPRSS2:ERG in prostate cancer cells. Here we showed that treatment with an ERRα-specific inverse agonist (XCT790) or knockdown of ERRα could decrease the TMPRSS2:ERG expression in AR-positive VCaP prostate cancer cells. Besides, the expression levels of TMPRSS2:ERG could be elevated by ectopic ERRα expression, with further elevation by co-expression with a genetically modified ERRα-specific coactivator PGC1α(2X9), in AR-negative NCI-H660 prostate cancer cells. By ChIP and luciferase reporter analysis, three putative ERRα transactivation DNA binding sites were identified in the promoter region of TMPRSS2:ERG fusion gene. Besides, in vitro phenotype characterization studies showed that ectopic expression of TMPRSS2:ERG, using an ERRα-responsive and TMPRSS2:ERG fusion gene promoter-driven expression plasmid in AR-negative prostatic cell lines, including PC-3 prostate cancer cells and BPH-1 immortalized prostatic epithelial cells, could promote their invasion capacity. Moreover, both ERRα and TMPRSS2:ERG showed elevated expressions in a xenograft model of castration-resistant prostate cancer cells (VCaP-CRPC). Besides, luciferase reporter assays showed that both TMPRSS2:ERG promoter and ERRα-binding element (ERRE) transcriptional activities were elevated in VCaP-CRPC cells as compared to the VCaP parental cells; and their transcriptional activities could be suppressed by XCT790 or sh-ERRα. Based on these results, we conclude that ERRα could directly transactivate the TMPRSS2:ERG fusion gene in prostate cancer and may contribute to its transactivation in AR-negative prostate cancer cells and the advanced castration-resistant prostate cancer. This study is supported by a General Research Fund from the Research Grants Council of Hong Kong (Project code: 2140629). Citation Format: Zhenyu Xu, Shan Yu, Franky Leung Chan. Orphan nuclear receptor estrogen-related receptor alpha (ERRα) characterized as a novel transcriptional regulator of the oncogenic fusion gene TMPRSS2:ERG in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1385. doi:10.1158/1538-7445.AM2014-1385

Abstract 4570: Role of ERRalpha in ovarian cancer

Abstract Objective. The estrogen receptor-related receptor-alpha (ERRα, encoded by ESRRA) has emerged as a key transcriptional regulator of mitochondrial function and has been implicated as a prognostic marker in many cancers, including epithelial ovarian cancer. ERRα has shown promise as a therapeutic target in breast cancer where ERRα transcriptional activity regulates growth of breast cancer cells. This study aims to evaluate whether ERRα expression in ovarian cancer correlates with patient outcomes, determine the effect of inhibiting ERRα transcriptional activity on ovarian cancer cell growth, and identify molecular signaling pathways regulated by ERRα in ovarian cancer cells. Methods. Affymetrix U133A gene expression microarray analysis from frozen tumor specimens of serous epithelial ovarian cancer was used to compare ESRRA transcripts levels in patients with short-term (<3 years) verses long-term survival (>7 years). ESRRA transcript levels were also measured in normal ovarian surface and fallopian tube epithelial tissues. Protein expression levels were assessed in ovarian cancer cell lines and patient tumor samples. Overall survival was estimated using the Kaplan-Meier Method. The effect of ERRα inhibition on proliferation and anchorage-independent growth was determined after treatment with the ERRα antagonist, XCT790. The activity of 49 different phospho-receptor tyrosine kinases (RTKs) was analyzed after down-regulation of ERRα with shRNA. Results. ESRRA expression was significantly higher in cancer tissues verses normal ovarian surface and fallopian tube epithelial tissues. Expression was also higher in patients with short-term versus long-term survival. Inhibition of ERRα with XCT790 led to a >50% reduction in proliferation and anchorage-independent growth. The induced knockdown of ERRα decreased the phosphorylation of RTKs, including HER2, suggesting that important growth promoting pathways are downstream of ERRα. Conclusions. In this study we determined that higher expression of ESRRA correlates with worse survival in ovarian cancer patients. However, inhibition of ERRα results in decreased growth potential. This effect may be manifest through the regulation of receptor tyrosine kinase activity. Thus, ERRα may be an important therapeutic target for the treatment of ovarian cancer and warrants future studies to determine the role of ERRα regulated signaling networks in ovarian oncogenesis. Citation Format: Ellen V. Stevens, Regina Whitaker, Audrey Guinet, Ching-Yi Chang, Carole Grenier, Jeffrey Marks, Donald P. McDonnell, Susan K. Murphy, Andrew Berchuck, Stephanie Gaillard. Role of ERRalpha in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4570. doi:10.1158/1538-7445.AM2014-4570

Mechanism of cardiomyocyte PGC-1α gene regulation by ERRα

Peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α (PGC-1α) regulates critical genes involved in cardiac mitochondrial biogenesis and fatty acid oxidation, and its loss is associated with impaired metabolism and various cardiac pathologies. Estrogen-related receptor α (ERRα) targets many of the same genes as PGC-1α, and extensive cross talk exists between these 2 regulators. Here we report the identification of an evolutionarily conserved ERRα binding site within the PGC-1α promoter. Using luciferase reporter assays and overexpression, inhibition, or knockdown of ERRα, we show that PGC-1α expression is critically dependent upon ERRα in primary cardiomyocytes. We demonstrate that short-term hypoxia results in reduced ERRα mRNA expression, which precedes a similar loss of PGC-1α mRNA. However, chromatin immunoprecipitation reveals that despite a key role for ERRα in regulating PGC-1α in normoxic cardiomyocytes, ERRα loss is not responsible for PGC-1α loss in hypoxia. Histone deacetylase 5 (HDAC5) has previously been demonstrated to strongly inhibit expression of PGC-1α, and we show that overexpression of ERRα is sufficient to overcome this repressive effect. Our data elucidates the mechanism by which ERRα regulates cardiac PGC-1α gene expression, and suggests that ERRα may provide a means to normalize PGC-1α expression that could be useful in the development of strategies aimed at improving cardiac metabolism in disease.

The Estrogen-Related Receptor Alpha Upregulates Secretin Expressions in Response to Hypertonicity and Angiotensin II Stimulation

Osmoregulation via maintenance of water and salt homeostasis is a vital process. In the brain, a functional secretin (SCT) and secretin receptor (SCTR) axis has recently been shown to mediate central actions of angiotensin II (ANGII), including initiation of water intake and stimulation of vasopressin (VP) expression and release. In this report, we provide evidence that estrogen-related receptor α (ERRα, NR3B1), a transcription factor mainly involved in metabolism, acts as an upstream activator of the SCT gene. In vitro studies using mouse hypothalamic cell line N-42 show that ERRα upregulates SCT promoter and gene expression. More importantly, knockdown of endogenous ERRα abolishes SCT promoter activation in response to hypertonic and ANGII stimulations. In mouse brain, ERRα coexpresses with SCT in various osmoregulatory brain regions, including the lamina terminalis and the paraventricular nucleus of the hypothalamus, and its expression is induced by hyperosmotic and ANGII treatments. Based on our data, we propose that both the upregulation of ERRα and/or the increased binding of ERRα to the mouse SCT promoter are two possible mechanisms for the elevated SCT expression upon hyperosmolality and central ANGII stimulation.