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Companion Diagnostic, Pharmacogenomic, and Cancer Biomarkers

The Increased Expression of Estrogen-RelatedReceptor a Correlates with Wnt5a and PoorPrognosis in Patients with GliomaLiudi Zhang1, Yingfeng Zhu2, Haixia Cheng3, Jinsen Zhang4, Yuqian Zhu5,Haifei Chen1, Lu Chen1, Huijie Qi1, Guoqiang Ren2, Jianmin Tang2,Mingkang Zhong1,6,Wei Hua4, Xiaojin Shi1,6, and Qunyi Li1,6

Abstract

Malignant glioma is an often fatal type of cancer. Elevatedexpression of the orphan nuclear receptor estrogen-relatedreceptor alpha (ERRa) is an unfavorable factor for malig-nant progression and poor prognosis in several cancers,although the mechanism by which this receptor affects thepathophysiology of cancers remains obscure. However, fewstudies have been conducted in regard to the role of ERRa inglioma. In the current study, we found that elevated expres-sion of ERRa was observed in 107 glioma cases by means ofIHC. Clinically, high expression of ERRa was associatedwith later stages of disease progression and clinical outcomeof patients with glioma. ERRa had the ability to promote

cell proliferation and migration in glioma cell lines. More-over, in a xenograft model, we also found that silencingERRa had an inhibitory effect on the growth of glioma.Further investigation confirmed that ERRa was involved inthe carcinogenesis of glioma via the regulation of the Wnt5asignal pathway in vitro and in vivo. Our study was first toshow the overexpression of ERRa in glioma tissues and adirect correlation between ERRa expression and clinicalprognosis of glioma. Together, these data reveal that ERRahas prognostic significance in glioma, and targeting ERRamay provide a reliable therapeutic strategy for the treatmentfor human glioma.

IntroductionHuman glioma is the most common primary tumor of the

central nervous system in adults, representing an unmet clinicalchallenge as nearly two thirds of them are high-grade lesions withpoor outcome (1–3). Glioma tumors are histologically classifiedinto grades I to IV in the order of increasingmalignancy accordingto the World Health Organization (WHO) criteria (4). Grade IVglioma, accounting for 54% of all human glioma, is the mostaggressive and lethal type of brain tumors because of its highinvasiveness, robust neovascularization, high relapse after treat-ments, and recurrence rates (5). Despite recent advances intherapeutic modalities comprising maximal surgical resections,adjuvant chemotherapy, and postoperative radiotherapy, themedian survival for patients with glioblastoma (GBM) tumors

remains less than 16 months (6, 7). Thus, it is of significantscientific and clinical value for glioma patients to improve clinicalprognosis by early diagnosis and new rational therapeutic targets.

The orphan nuclear receptor estrogen-related receptor alpha(ERRa) regulates a range of physiologic and biochemical eventstogetherwith its certain transcriptional coactivators in a coordinatedmanner (8–10). The expression pattern of ERRa is characterized byhigh levels in tissueswith increasedmetabolic demands, such as theheart, skeletal muscles, and adipose tissue (11). Interestingly, thereis accumulating evidence supporting that ERRa contributes totumorigenesis of several types of cancers (12). It has been reportedthat thehigh levelofERRa expressioncorrelatedwithcarcinogenesisand poor clinical outcomes in various human tumors, includinghormone-dependent breast or ovary cancers and those hormone-independent tumors. Recently, the results from the early clinicaltrials revealed that a steroidal ERRa inhibitor, SR16388, was effec-tive in patients who have relapsed after the treatment of tamoxifenor an aromatase inhibitor (13). ERRa has been considered to beamong the most promising targets for cancer therapy.

It is demonstrated that activation of ERRa in cancer cells resultsin a significant increase of vascular endothelial growth factor(VEGF) and hypoxia-inducible factor-1 (HIF1) expression (14,15). However, few studies have been conducted in regard to therole of ERRa in glioma. A research suggests that ERRa is differen-tially expressed in severalhumangliomaandastrocytomacell lines(16). Nevertheless, a detailed examination of ERRa expressionlevel and biological function of ERRa in glioma remains unclear.Additionally, its relationship with the pathologic features and theoverall survival in human glioma has not been addressed yet.

Wnt proteins are a large family of cysteine-rich secretedmolecules (17). Wnt5a, a secreted glycoprotein signal transducerof the Wingless/Int1 (Wnt) family that activate the b-catenin

1Clinical Pharmacy Laboratory, Huashan Hospital North, Fudan University,Shanghai, China. 2Department of Pathology, Huashan Hospital North, FudanUniversity, Shanghai, China. 3Department of Pathology, Huashan Hospital,Fudan University, Shanghai, China. 4Department of Neurosurgery, HuashanHospital, Fudan University, Shanghai, China. 5Department of Neurosurgery,Huashan Hospital North, Fudan University, Shanghai, China. 6Clinical PharmacyLaboratory, Huashan Hospital, Fudan University, Shanghai, China.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Liudi Zhang and Yingfeng Zhu are the cofirst authors of this article.

Corresponding Authors: Qunyi Li, Fudan University, Huashan Hospital North,108 Luxiang Road, Shanghai 201907, China. Phone: 21-66895008; Fax: 21-66895009; E-mail: [email protected]; Xiaojin Shi, [email protected];and Wei Hua, [email protected]

doi: 10.1158/1535-7163.MCT-17-0782

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independent pathway (18), has been considered mainly a tumorsuppressor (19). Previous studies had revealed higher Wnt5aprotein levels and mRNA expression in glioma comparedwith nonmalignant control brain tissue. It is also reported thatWnt5a was upregulated by ERRa in prostate cancer cells (20).Furthermore, ERRa regulates osteoblast differentiation via Wnt/b-catenin signaling (21). In glioma cells, Wnt5a stimulated theproliferation,motility, invasiveness, and aggressiveness of humanglioma cell lines, suggesting the oncogenic property of Wnt5a inhuman glioma (22–24).

In this study, we show for the first time that ERRa is morebroadly expressed in grade III and IV tumors than that in grade Iand II brain tissues. Increased ERRa expression levels are corre-lated with a worse overall survival (OS) in glioma patients.Moreover, the inhibition of ERRa expression impairs prolifera-tion and invasion of GBM cell lines, suggesting that ERRamay berequired in the progression of glioma.

Materials and MethodsCase selection

A total of 107 paraffin-embedded tissue samples of gliomawere collected from patients with detailed clinical informationin Huashan Hospital North and Huashan Hospital, FudanUniversity, Shanghai, China. And 11 normal brain (NB) tissueswere obtained from patients who underwent posttrauma sur-gery for severe traumatic brain injury. The Cancer GenomeAtlas (TCGA) mRNA-seq database was downloaded frompublic databases (https://cancergenome.nih.gov/). This studywas approved by the Ethics Committee of Huashan Hospital(2016-229). Written informed consent was obtained from allpatients and the examination of tissue samples used in thisstudy.

The clinical pathologic information of glioma was listed inSupplementary Table S1. All specimens were divided into threegroups: NB, low-grade glioma (LGG, grades I and II), and high-grade glioma (HGG, grades III and IV).

Cell cultureThe human glioma cell lines SNB-19, SF-295, A172, T98G,

LN229, and LN18 were purchased from American Type CultureCollection, without cell line authentication. U87MG and U251were tested on May 22, 2013, and January 7, 2013, respectively(genomic DNA). A172, U87MG, HEK293T, and MDA-MB-231were cultured in Dulbecco's modified Eagle's medium (DMEM,HyClone), while other cell lines were maintained at minimalessential medium (MEM, Invitrogen). Both media were sup-plemented with 10% fetal bovine serum (FBS), 1% l-glutamine,1% sodium pyruvate, and 100 U/mL penicillin/streptomycin(Wisent) at 37�C in a 5% CO2 humidified incubator. PrimaryGBM samples were derived from patients undergoing surgery atHuashan Hospital. Tumors were dissociated and grown asadherent cultures on laminin coated dishes (23). All patient-derived cells (GBM001, 002, 003) were cultured in NeuroCultNS-A basal medium, supplemented with NeuroCult NS-A pro-liferation supplements, 10 ng/mL human bFGF (Sigma) and20 ng/mL human EGF (PeproTech). Human neurons (HN)were maintained in neuronal medium (ScienCell) supplemen-ted with neuronal growth supplement (ScienCell). HMC3 andhuman astrocyte (HA) were cultured in EMEM and DMEMwith10% FBS.

Lentiviral production and infectionThe lentiviral shRNA expression vector targeting hERRa

(NM_004451; pLKD-CMV-G&PR-U6-shRNAERRa, shERRa) andscrambled control (pLKD-CMV-G&PR-U6-shRNAscramble,shCtl) were produced by Obio Technology). Lentiviruses wereobtained by transfecting the shRNA (Supplementary Fig. S1)expression vector targeting hERRa, reducing 85% expressioncompared with shCtl into HEK293T cells. For transduction,U87MG and U251 cells were seeded at 50% to 60% confluenceand infected with viral suspension containing 5 mg/mL polybrene(Sigma-Aldrich) for 6 hours. Two days after infection, cells wereselected for puromycin resistance for 48 hours (2 mg/mL, Sigma-Aldrich) and used for experiments within a week (22). For over-expressing, lentiviral particles were produced with the use of alentivirus packagingmix (SupplementaryMethods). U87MG andU251 cells were treated with lentivirus for 48 hours and harvestedfor examination of gene expression by qRT-PCR and Westernblotting (Supplementary Fig. S1).

RNA extraction and qRT-PCRTotal RNA was extracted from the U87MG and U251 cell lines,

glioma tissues, andNB tissues using TRIzol kit (Takara), accordingto the manufacturer's instructions. Relative quantification of theERRamRNA level was calculated after normalization to GAPDHmRNAusing the 2�DDCtmethod. The primer sequences are shownin Supplementary Table S3.

Western blotting, IHC, and immunofluorescence (IF) assaysCells were treated as presented in the figure legends, and cell

lysates were prepared as previously described (25). The sampleswere blotted on PVDF membranes, and membranes were probedovernight at 4�C with anti-ERRa, anti-b-catenin, anti-Wnt5a,c-myc, p-GSK3b, GSK3b, Cyclin D1, Axin1, and anti-b-actin.

For IHC staining, after routine deparaffinization in xylene,rehydration, and inhibition of endogenous peroxidase activitywith 3%hydrogen peroxide, sectionswere exposed tomicrowave-enhanced antigen retrieval procedure in 0.01 M sodium citratebuffer (pH 6.0). After blocking with 3% BSA, the sections wereincubated with rabbit anti-human antibody directed againstERRa (1: 100; Abcam) for 1 hour, followed by biotin-labeledrabbit anti-goat antibody for 0.5 hours. Sectionswere subsequent-ly incubated with avidin-conjugated horseradish peroxidase(HRP) for 0.5 hours at room temperature.

For IF staining, sections were deparaffinized and dehydrated ingradient alcohol, and then rinsed in phosphate-buffered saline(PBS). The sections were subjected to antigen retrieval by immer-sion in citric acid antigen retrieval solution and heated in amicrowave oven. Then, the sections were costained with thepatient serum, and a commercial rabbit polyclonal antibody wasdirected against ERRa (red) and Wnt5a (green) and treated withFITC-conjugated rabbit polyclonal antibodies (all purchasedfrom Abcam). All images were captured under a fluorescencemicroscope (Nikon 80i; Nikon).

MTT assay and BrdUrd incorporation assayMTT assay and BrdUrd incorporation assay in U87MG

and U251 cells was measured as previously described (26).Analysis of adenovirus mediated cell proliferation was measuredby MTT (24 hours) and BrdUrd incorporation assay. HN, HA,HMC3, U87MG, and U251 cells were treated with PBS, shCtl,or shERRa. The inhibitory rates were calculated using the formula

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in triplicate: inhibitory rates (%) ¼ [(1 � ODexperimental)/ODcontrol] � 100%.

Colony formation assayFor colony formation assay, U87MGandU251 cells transfected

with shCtl or shERRa were plated in 6-well culture plates at 100cells per well. The cells were washed with PBS and stained withcrystal violet staining. Colonies were counted only if they con-tained >50 cells under a microscope.

Cell-cycle distributionA total of 1 � 106 U87MG and U251 cells were, respectively,

plated in 60-mm culture plates, and the cells were pretreated withshCtl or shERRa. After 48 hours, the cells were trypsinized andthenDNAwas stainedwith propidium iodide (PI; Sigma-Aldrich)in the dark. Then, the samples were run on FACScanII flowcytometry (Biosciences). At least 10,000 cells were counted, andthe percentages of cells within each phase of the cell cycle wereanalyzed using ModFit software.

Transwell migration assayThe cell migration assay was performed using a transwell

chamber (6.5 mm in diameter, 8-mm pore size; Costar), allowingmigrating for 24 hours according to our previous study (26).Analysis of adenovirus-mediated cell was measured by transwellmigration assay. HN, HA, HMC3, U87MG, and U251 cells weretreated with PBS, shCtl o,r shERRa.

Reporter gene assayU87MG cells were transiently cotransfected with TCF/LEF1-

Luc, ERRa, or PGC-1a plasmids and then treated with shERRa forthe indicated times. The pSV-b-galactosidase plasmid (pCH110)was cotransfected and used for data normalization. Subsequently,luciferase activity was measured according to the manufacturer'sprotocol, and expressed as the ratio of luciferase activity tob-galactosidase. For measuring the transcriptional activity ofWnt5a, the pGL3-Basic-Wnt5a-luc, which contains Wnt5a pro-moter with the sequence �2869/þ170, was transfected intoU87MG cells.

Coimmunoprecipitation (co-IP) assayCo-IP assays were performed as previously described (27).

Briefly, whole-cell extracts were prepared using nondenaturinglysis buffer. The proteins were immunoprecipitated using anti-bodies to ERRa, b-catenin, mouse IgG, and protein-A/G PLUS-Agarose beads, then separated by 10%SDS-PAGE and detected byWestern blotting.

Chromatin immunoprecipitation (ChIP)ChIP assays were performed on U87MG as described previ-

ously using primer sequences listed in Supplementary Table S3.Briefly, soluble chromatin fragments were immunoprecipitatedusing antibody to ERRa and then treated with RNase A andproteinase K. Isolated DNA fragments were purified with QIA-quick spin kit, and used as templates to detect the putative ERRresponse elements (ERRE; Supplementary Table S4) by quanti-tative qRT-PCR.

In vivo efficacy and neuropathology of Ad-ERRa-shRNABriefly, male BALB/c nu/nu mice (4–6-week-old, 19 g) were

anesthetized with an IP injection of ketamine (75 mg/kg) and

medetomidine (0.5 mg/kg), placed in a stereotactic apparatus,and a hole was drilled in the skull for cell implantation. Then, 3�105 U87MG cells in 2 mL were injected into the right striatum(þ0.5 mm AP, þ2.1 mm ML, �2.9, �3.2, �3.5, �3.8, �4.1 mmDV from bregma: 1 mL per site) using a 10 mL Hamilton syringewith a 33-gauge needle. The needle was left in place for 3minutesprior to removal to allow tumor cells to settle at the injection site.Five days after tumor implantation,mice received an intratumoralinjection of either 3 mL PBS, inactivated-adenovirus (Ad-Ctl) orAd-ERRa-shRNA solution: 8 mice received PBS, 8 mice receivedAd-Ctl at 1 � 108 pfu/mouse, and 8 mice received Ad-ERRa-shRNA at 1 � 108 pfu/mouse. Treatments were administeredusing the same hole (at �2.9, �3.2, �3.5, �3.8, �4.1 mm DVfrom bregma: 1 mL per site). Mice were euthanized when mori-bund or when survived 71 days after tumor implantation. Mean-while, the mouse OS curves were recorded according to theKaplan–Meier method. Tumor volume was calculated by usinga modified ellipsoidal formula: tumor volume¼ (L�W2)� 0.5,where V is the tumor volume, L is the longitudinal diameter, andW is the transverse diameter (28, 29). The brains of the mice wereextracted andfixed in 10% formalin.Next, theywere embedded inparaffin for IHC of CD68 and myelin basic protein (MBP), asmarkers of macrophages/microglia and an index of oligodendro-cyte integrity, respectively. Nissl staining was used to determinethe histopathologic features of the brains. Brain sections (5 mm)were mounted on gelatinized glass slides and incubated in cresylviolet (0.1%; Sigma) and were passed through destain solution(70% ethanol, 10% acetic acid) and dehydrated (100% ethanoland xylene). Then, tissues were photographed. Human gliomaxenografts were generated according to supplementary methods.

All experiments were performed according to the institutionalethical guidelines on animal care and approved by the InstituteAnimal Care and Use Committee of Fudan University (approvalnumber: SOP-ICE-033-01.0-AF05).

Statistical analysisStatistical significance of the observed differences was

addressed by statistical tests using GraphPad Prism V7.0. Dataare presented as mean � SEM. Each experiment was repeated aminimum of 3 times. Survival curves were plotted using theKaplan–Meier method, and statistical significance was deter-mined by the log-rank (Mantel–Cox) test. The Cox proportionalhazard model was used for calculating the impact of ERRaexpression in the univariate andmultivariate analysis of variablesand patient's OS duration. P value of < 0.05 was considered to bestatistically significant (�, P ¼ 0.05; ��, P ¼ 0.01; ��, P ¼ 0.001).

ResultsExpression of ERRa in glioma and NB tissues

To assess the role of ERRa in glioma, we first measured theexpressionof ERRamRNA transcripts usingqRT-PCR in34 freshlycollected glioma tissues and11NB tissues. Comparedwith theNBgroup (1.017 � 0.193), the LGG and HGG obtained a muchhigher ERRa mRNA expression level, which were 3.908 � 0.668(P < 0.01) and 6.46 � 0.684 (P < 0.001), respectively (Fig. 1A).In addition, ERRa protein was also found to be upregulated in6 cases of glioma compared with NB tissues (Fig. 1B).

We then examined the protein level of ERRa in 107 achievedparaffin-embedded glioma samples (SupplementaryTables S1andS2) by IHC. A semiquantitative ERRa expression was compiled

Prognostic Significance of ERRa in Glioma

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using intensity of staining aswell as the number of stained cells.Weobserved that protein level of ERRawasmarkedly higher in gliomatissues than that in NB tissues (Fig. 1C). Meanwhile, ERRa expres-sion was significantly high as the glioma pathologic gradeincreased. According to the ERRa staining score, 58 (54.21%)patients were classified as low ERRa group and 49 (45.79%)patients were classified as high ERRa group (Supplementary

Table S2). This result was well validated in TCGA RNA-seq data(Fig. 1D). We analyzed the RNA-sequencing data of glioma fromthe TCGA database and found that ERRa was enriched in HGG.

Survival analysisTo further investigate the potential of ERRa expression to

predict patients' clinical outcomes in glioma, we assessed the

Figure 1.

ERRaoverexpression in humangliomapatients and its correlation with poorprognosis. A, ERRa mRNA expressionin glioma of different grades analyzedusing real-time qRT-PCR; P < 0.01 andP < 0.0001 vs. normal. B, Top,Westernblotting analysis of the ERRa proteinlevel in 3 cases of grade II(L1, L2, and L3), 3 cases of grade IV(H1, H2, and H3) glioma patients and2 NB samples (N1 and N2). Bottom,quantification of ERRa expression.� , P < 0.01 vs. NB. C, Representativeimages of ERRa and CD34 expressionin 107 glioma patients for eachgrades using IHC staining. The arrowindicates ERRa expression. Opticalmagnification is shown; insets, 100 and400; scale bar, 200 mm and 50 mm.D, ERRa expression in the TCGAdatabase. The value represents log2 ofgene-expression value of glioma.P < 0.01 vs. grade II. E, OS and PFS.F, 107 glioma patients according toERRa immunoreactivity (Kaplan–Meier method). G, OS of 551 gliomapatients in TCGA according to ERRaexpression.

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association between ERRa expression and patients' survival usingtheKaplan–Meiermethod. Theprimary endpointwasOS, definedas the time of completion surgery to the date of death or the lastfollow-up contact. We also studied progression-free survival(PFS), defined as the date of completion surgery to tumor recur-rence, death from any cause, or last follow-up contact. TheOS andPFS curves were summarized in Fig. 1E and F.

Highly statistically significant correlations were also observedbetweenOS/PFS and the levels of ERRa immunoreactivity (Fig. 1Eand F; P < 0.01). The increased ERRa immunoreactivity in theHGG group wasmarkedly associated with shortened OS and PFS.We further analyzed the prognostic value of ERRa in a total of 551glioma patients in the TCGA database. As shown in Fig. 1G, highexpression of ERRapredicted significantly poorOS. Taken togeth-er, these results indicated that ERRa expression might affect bothPFS and OS of glioma patients.

ERRa is an independent prognostic factor for gliomaBecause ERRa expression was associated with the pathologic

grade of glioma, we investigated the prognostic significance ofERRa for glioma patients. After univariate analysis by Cox pro-portional hazards model, KPS score (P ¼ 0.031, HR ¼ 1.229),necrosis (P¼ 0.026, HR¼ 0.726), WHO grade (P¼ 0.002, HR¼4.298) andERRa immunoreactivity (P¼0.018,HR¼3.573)wereturned out to be significant prognostic clinical parameters for OSin 107 glioma patients (Table 1).

Additionally, we performed a stepwise multivariate analysisadjusted for the same parameters to determine whether ERRaimmunoreactivity is an independent prognostic factor for gliomapatients. The results reflected that, after the correction for patientKPS score (P ¼ 0.095) and necrosis (P ¼ 0.224), the elevatedexpressionof ERRa (P¼0.021,HR¼1.135) andWHOgrade (P¼

0.031, HR ¼ 3.652) is associated with a shortened OS and PFS,acting as an independent prognostic factor with a relative risk over1.0 for patients.

ERRa selectively promotes behavior of glioma cellsTo understand the significance of ERRa expression in glioma,

we measured ERRa expression in a panel of human glioma celllines usingWestern blotting (Fig. 2A). Among the glioma cell linestested, protein levels of ERRa revealed higher in the grade IV–derived glioma cell lines U251, U87MG, and A172, which was inaccordance with a previous study (16).

We therefore assessed whether the downregulation of ERRa byits specific inverse agonist XCT790 contributed to the reduction ofproliferation in 5 glioma cell lines. Downregulation of ERRawithXCT790 selectively decreased the proliferation of glioma cell linesthat have high level of ERRa expression (Fig. 2B). The estrogenreceptor–negative MDA-MB-231 human breast cancer cell actedas a positive control. The LN229 cells did not respond to XCT790,consistent with their lack of ERRa expression. The regressionanalysis indicated that there was a statistically strong correla-tion between inhibition rates of XCT790 and ERRa expression(Supplementary Fig. S2).

We then performed knockdown assays by using shERRa inHNs, HA, human microglia cell (HMC3), and GBM patient-derived primary cells (GBM001, GBM002, and GBM003),respectively. Silencing of ERRa slightly reduced the HA andHMC3 cell growth compared with glioma cells (Fig. 2C) in theMTT assay. In contrast, as shown in Fig. 2D, ERRa depletiondecreased proliferation of GBM patient-derived cells. We alsofound that knockdown of ERRa decreased proliferation ofU87MG and U251 after transfection of shERRa at 48 and 72hours (Fig. 2E). On the contrary, overexpression of ERRa

Table 1. Cox proportional hazard regression analyses of ERRa expression and clinicopathologic factors affecting OS duration in pGBM patients

Variable Univariable regression Multivariable regressionHR (95% CI) P value HR (95% CI) P value

GenderMale 1.921 (0.806–4.578) 0.141 1.178 (0.77–1.804) 0.45Female

Age�50 1.786 (0.465–3.024) 0.721 2.05 (1.263–3.327) 0.067<50

KPS score<85 1.229 (0.489–3.09) 0.031 1.508 (0.932–2.441) 0.095�85

Tumor originPrimary 0.572 (0.21–1.559) 0.275 1.123 (0.665–1.896) 0.664Secondary

NecrosisNo 0.726 (0.427–3.392) 0.026 0.717 (0.419–1.225) 0.224Yes

EdgeNot clear 1.363 (0.469–3.961) 0.569 0.663 (0.361–1.218) 0.185Clear

Resection�98% 0.724 (0.209–2.513) 0.611 0.648 (0.368–1.141) 0.133<98%

WHO gradeIIIþIV 4.298 (5.634–10.584) 0.002 3.652 (1.254–3.658) 0.031IþII

ERRa expressionHigh 3.573 (1.247–10.237) 0.018 1.135 (0.746–1.727) 0.021Low

Abbreviations: HR, hazard ratio; CI, confidence interval; KPS, Karnofsky performance status. P value of < 0.05 (bold in Table 1) was considered to be statisticallysignificant.






remarkably promoted cell proliferation in U87MG and U251cells at 24, 48, and 72 hours after transfection of lenti-ERRa(Fig. 2F; Supplementary Fig. S3). The colony formation assayshowed that the colony formation rates of U87MG andU251cells transfected with shERRa were lower than in thesame cells transfected with the scramble (Fig. 2G). In addition,we measured the effect of ERRa knockdown on the glioma cellcycle. The data presented in Fig. 2H showed that reduced ERRa

expression delayed the progression of the cell cycle and inhib-ited cell proliferation by arresting cells at G0–G1 phase.

As shown in Fig. 2I, overexpression of ERRa after the transfec-tion of lenti-ERRa increased the cell migration of the correspond-ing cells. Cell migration was decreased in the shERRa group(Fig. 2J, P < 0.05). These results indicated ERRa promoted tumorcell growth andmetastasis, functioning as anunfavorable factor inglioma.

Figure 2.

The role of ERRa in regulatingbehaviors of glioma cells. A, ERRaexpression in various human gliomacell lines usingWestern blotting. MDA-MB-231 was used as a positive control.B, Effects of XCT790 on glioma cellproliferation. � , P < 0.01 vs. 5 mmol/Ltreatment. C and D, BrdUrdincorporation assay of human cells HN,HA, HMC3, GSC83 U87MG, U251,GBM001, GBM002, and GBM003transfected with shERRa. � , P < 0.05and �� , P < 0.01 vs. shCtl treatment. Eand F, Effects of knockdown (E) andoverexpression (F) of ERRa on cellproliferation at the indicated time(Supplementary Fig. S4) aftertransfection. � , P <0.05 and ��, P <0.01vs. shCtl or lenti-ERRa treatment. G,Colonies were stained and countedafter indicated time in the U87MG andU251 cells only if they contained >50cells under amicroscope. � ,P<0.05 vs.shCtl. H, Left, cell-cycle distributionwas examined by flow cytometry.Right, quantification of cell-cycledistribution. I and J, Effects of ERRaoverexpression (I) or knockdown (J)on migration of U87MG and U251 cellsfor 24 hours by transwell migrationassay. �, P < 0.05; �� , P < 0.01 vs. shCtlor lenti-ERRa treatment. All data(mean � SEM) are representative ofthree independent experiments.

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Cross-talk of ERRa and Wnt signaling pathwayTo explore the effects of ERRa on the transcriptional activity

of b-catenin, U87MG cells were cotransfected with TCF/LEF1-Lucand GFP, PGC-1a, or ERRa vectors. It is noted that both PGC-1a

and ERRa significantly enhanced transcriptional activity ofb-cate-nin upon ERRa overexpression compared with GFP in U87MGcells (Fig. 3A and B, P < 0.05). As expected, when U87MG cellswere treated with PGC-1a and shERRa, PGC-1a/ERRa-evoked

Figure 3.

The expression of ERRawas positively correlated withWnt5a. A and B, TCF/LEF1-Luc reporter plasmid was cotransfected into U87MG cells plus with PGC-1a (A) orERRa (B) vectors and the luciferase was monitored after 4-day treatment. � , P < 0.05 vs. GFP. C, When U87MG cells were treated with PGC-1a and shERRa,a little changewas examined comparedwith GFP. � ,P <0.05 vs. GFP.D, co-IP of endogenous ERRa and b-catenin fromU87MG cell extractswas followed byWesternblotting for the indicated proteins. E, The transcription level of Wnt pathway genes was detected after the transfection of shERRa or shCtl in U87MG cellsusing qRT-PCR. The dotted line indicated changes upon the shERRa treatment. F, Immunofluorescent staining of low, medium, and high levels of ERRa (red),together withWnt5a (green) in human glioma tissues (grades I–IV). Nucleuswas stained by DAPI (blue). Scale bar, 200 mm.G,Quantitative summary and P values ofERRa and Wnt5a immunoreactivity. � , P < 0.05 vs. ERRa low expression.






transactivation ofWnt/b-catenin signaling pathwaywasmarkedlysuppressed (Fig. 3C). These results were further ascertained by co-IP of endogenous ERRa and b-catenin from U87MG cell extractsfollowed by Western blotting (Fig. 3D). Following the observa-tion ofmodulation, we detected the transcription of a panel of theWnt pathway to gain a detailed understanding of whichWnt genewas changed by ERRa inactivation. After the transfection ofshERRa or shCtl in U87MG cells, qRT-PCR was performed toquantify mRNA of Wnt. Interestingly, we found a significantdownregulation of Wnt5amRNA in U87MG cells compared withshCtl treatment (Fig. 3E). Thus, we have confirmed that Wnt/b-catenin and ERRa signaling pathway converge, a finding of highsignificance in carcinogenesis.

The expression of ERRa is positively correlated with Wnt5ain vivo

Todetect the associationof ERRawithWnt5a expression in vivo,we checked the expression of ERRa and Wnt5a in 77 humanglioma tissues by immunofluorescence assays (Fig. 3F). Of the 75glioma cases, 47 (61.03%) and 49 (63.64%) had elevated ERRaandWnt5a, respectively (Fig. 3G). The analysis showed that scoresfor ERRa and Wnt5a immunostaining were significantly corre-lated in tissues tested (P < 0.05). This result suggested ERRaexpression is positively correlated with Wnt5a in clinical glioma.

Wnt5a, a direct target of ERRa, partly rescues the phenotypescaused by knockdown of ERRa

To further decipher the mechanism involved in the role ofERRa, we examined the relative protein expression level ofWnt5ain U87MG cells treated by XCT790 or shERRa. After treatment ofXCT790 or transfection with shERRa, the expression of ERRa andWnt5a in U87MG was significantly decreased, respectively(Fig. 4A). To ascertain whether Wnt5a was directly regulated byERRa, we analyzed the humanWnt5a promoter sequence in silicoto detect potential ERRE composed of the extended ERE half-siteAAGGT. Four potential ERREs were found located within theproximal promoter of Wnt5a (Fig. 4B). Then, we transfected theWnt5a promoter reporter gene plasmid (pGL3-Basic-Wnt5a-luc)into U87MG to evaluate the effect of ERRa on Wnt5a promoteractivity. The data showed that the activity of pGL3-Basic-Wnt5a-luc was significantly decreased by treatment with shERRa(Fig. 4C) or XCT790 (Fig. 4D).

AChIP assaywas performed to examinewhether ERRabinds toWnt5a promoter in the native chromatin environment of gliomacells. ChIP with anti-ERRa in U87MG cells showed that knock-down of ERRa resulted in a significant decrease in the genomicfragment that contained the ERRE-1 and ERRE-3 sites in theWnt5a promoter (Fig. 4E). Together, these data suggested thatERRa can bind to the cis-regulatory domain of the endogenousWnt5a promoter and thus regulates the transcription of Wnt5a.

To elucidate whether ERRa influences glioma growth andmigration through regulating the expression of Wnt5a, we usedWnt5a protein (500 ng/mL) to perform the rescue experiments.The data showed that inhibitory effects on cell migration (Fig. 4F)and growth (Fig. 4G) caused by shERRa were both partly abol-ished by Wnt5a.

Inactivation of the Wnt/b-catenin pathway reversed the effectsof ERRa overexpression on cell proliferation

To investigate the possible involvement of ERRa/Wnt signalingin glioma, we used U87MG cells. shRNA were used to target

Wnt5a and b-catenin, an essential component of the canonicalWnt signaling pathway. Depletion of Wnt5a and b-cateninreversed the effects of ERRa overexpression on cell proliferationof U87MG (P < 0.01; Fig. 4H and I). The results suggest a role forthe Wnt pathway in regulating glioma cell proliferation.

To explore the mechanism of ERRa/Wnt in U87MG, b-cateninand target genes (c-myc and cyclin D1) expression in the Wnt/b-catenin pathwaywere examined byWestern blotting inU87MGtransfected with shERRa. The results showed that knockdown ofERRa and Wnt5a significantly decreased levels of these genes(Fig. 4J and K). We also found that Wnt5a suppression decreasedthe expression of Axin1, b-catenin, andDvl2 as well as the ratio ofp-GSK3b/GSK3b in U87MG (Fig. 4L). Moreover, overexpressionof Wnt5a could rescue the decreased expression of above genes,which was induced by ERRa depletion (Fig. 4M). Our dataindicate that ERRa suppression inhibits cell proliferation bysuppressing the WNT/b-catenin pathway.

ERRa promotes tumorigenesis in vivoTo determine whether Ad-ERRa-shRNA affects neuronal, glial,

and immune cells as well as glioma cells in the brain, weperformed theMTTassay.We found that Ad-ERRa-shRNA slightlyreduced HA growth compared with U87MG and U251 (Fig. 5A),in which cell proliferation was obviously inhibited. The result inthe MTT assay is validated by the BrdUrd Assay (Fig. 5B). More-over, Ad-ERRa-shRNA led to tumor regression and long-termsurvival in over 71% of the animals and increased the mediansurvival from25 days and 28 days in the PBS and Ad-Ctl groups to45 days (Fig. 5C). Ad-ERRa-shRNA can also inhibit the growth ofglioma (Fig. 5D). Neuropathological analysis of the brains fromlong-term survivors showed complete tumor regression and didnot reveal signs of neurotoxicity (Fig. 5E). We then verified thatreduced expression of ERRa, Wnt5a, Axin1, and Dvl2 in the Ad-ERRa-shRNA group compared with Ad-Ctl (Fig. 5F). Here, weshow that ERRamay function as amolecule that is associatedwiththe Wnt5a signaling pathway, leading to the degradation of thecanonical b-catenin signaling pathway and the consequent sup-pression of its downstream genes, such as Axin and GSK3b, bothin vitro and in vivo, which promotes glioma cell proliferation, cellcycle, andmigration (Fig. 5G). In summary, a single intratumoralinjection of Ad-ERRa-shRNA resulted in significant improvementin survival and no sign of neurotoxicity in intratumoral gliomaxenografts. These data suggest that ERRa may be a promisingtherapeutic agent for glioma. It is consistent with the results in thesubcutaneous xenograft model (Supplementary Fig. S4).

DiscussionTo our knowledge, this is the first study that applied clinical

glioma tissues to elucidate the clinical signature and biologicalfunction of ERRa and Wnt5a in glioma. We showed that ERRapromoted cell proliferation andmigration in human glioma cells.More importantly, ERRa was identified for the first time as acritical marker not only for tumorWHO classification but also forclinical outcomes in glioma patients.

An abnormal Wnt pathway is associated with many humandiseases, including cancer and degenerative diseases (30). Wntsignaling via b-catenin has a recognized role in controlling theproliferation of cells in human cancers (31, 32). The role ofWnt5ain cancer is dual and controversial in various cancer types. Pre-vious reports indicate that Wnt5a inhibits cell proliferation,

Zhang et al.





Figure 4.

ERRa suppression inhibits cell proliferation by suppressing the WNT/b-catenin pathway. A, Western blotting analysis of Wnt5a and ERRa expression in U87MGcells treated with XCT790 (5 mmol/L) or shERRa. � , P < 0.05 and �� , P < 0.01 vs. Ctl or shCtl. B, Diagram of the potential ERREs (boxed sequences) in thepromoter of the human geneWnt5a.C andD, Effect of ERRa onWnt5a promoter activity after treatment with shERRa (C) or XCT790 (D). � , P < 0.05 and �� , P < 0.01vs. Ctl or shCtl.E,Recruitment of ERRa to ERREs of humanWnt5apromoterwas evaluatedbyChIP assayafter treatment of shERRa for 48h. The immunoprecipitatedproducts were detected by qRT-PCR. � , P < 0.05 vs. Ctl. F and G, Effects of Wnt5a (500 ng/mL) and shERRa on FBS (10%, 48 hours) induced U87MG cellmigration (F, transwell assay) and proliferation (G). � , P < 0.05 and �� , P < 0.01. H and I, Depletion of Wnt5a and b-catenin inhibited the proliferation of U87MGinduced by ERRa overexpression. �, P < 0.05; �� , P < 0.01; �� , P < 0.001 vs. Ctl. J and K, Effects of ERRa (J) and Wnt5a (K) depletion on b-catenin and targetgenes (c-myc and cyclin D1) expression. � , P < 0.05 and �� , P < 0.01 vs. shNC or shCtl. L, Effects of ERRa depletion on Axin 1, b-catenin, and Dvl2 as well asthe ratio of p-GSK3b/GSK3b expression. � , P < 0.05 and �� , P < 0.01 vs. shCtl.M, Overexpression of Wnt5a rescued the decreased expression of above proteins (M)induced by ERRa depletion. � , P < 0.05 and �� , P < 0.01 vs. shCtl þ Lenti-Ctl. Data (mean � SEM) are representative of three independent experiments.






Figure 5.

Ad-ERRa-shRNA promotes glioma therapy in vitro and in vivo. A and B, Analysis of adenovirus-mediated cell proliferation using MTT (24 hours). HN, HA, HMC3,U87MG, and U251 cells were treated with PBS, shCtl, or shERRa, respectively. The result of the MTT assay was also validated by the BrdUrd assay (B). � , P < 0.05and �� , P <0.01 vs. shCtl or PBS. C, Kaplan–Meier survival curves for OS in Ad-ERRa-shRNA (108 pfu), Ad-Ctl and PBS-treated nude mice bearing U87MGintracranial xenografts. Intracranial implantation of U87MGwas followed by one intratumoral injection (day 5) of Ad-ERRa-shRNA (108 pfu; n¼ 8), Ad-Ctl (n¼ 8), orPBS (n ¼ 8). The P values were determined and represent a comparison of survival of Ad-ERRa-shRNA–treated mice with that of mice treated with PBSand Ad-Ctl (both < 0.01). D, The tumor volume of glioma xenografts was measured after death of mice. Tumor volumes were calculated according to theformula V ¼ tumor length (L) � tumor width (W)2 � 0.5, where L is the longitudinal diameter and W is the transverse diameter. E, Neuropathologicalanalysis was assessed in moribund mice and long-term survivors by Nissl staining and immunocytochemistry of CD68 and MBP (scale bar, 2,000 and 200 mm).Note complete tumor regression in long-term survivors and the absence of neuropathological or inflammatory side effects. F, ERRa, Wnt5a, Axin1, and Dvl2expression was determined by Western blotting analysis in the removed tumors in the brain. � , P < 0.05 and ��, P < 0.01 vs. Ad-Ctl. G, Graphical summary ofthe ERRa/Wnt5a pathway analysis.


Zhang et al.




invasiveness, and aggressiveness in some cancer cells, acting as atumor suppressor (33, 34). In contrast, it has been suggested thatWnt5a has oncogenic properties. For instance, Wnt5a promotesinvasion and migration in breast, gastric cancer cells (35) andmelanoma (36). A report indicated thatWnt5a is highly expressedin high grades of glioma, and its expression is correlated withinvasive activities (23). Taken together, these findings demon-strate that the role of Wnt5a is dependent on the cell type.

Wnt5a worked as an essential player in maintaining the pro-liferative andmigratory capacity of glioma cells. Our data are alsoconsistent with previous findings that the protein expression ofWnt5a was higher in the HGG group compared with the LGGgroup (22, 23, 37). Before this study, direct evidence for theassociation between ERRa and Wnt5a was scant. We hypothe-sized that knockdown of ERRa decreased malignant behavior ofglioma cells through the Wnt/b-catenin pathway. We detectedexpressions of b-catenin and downstream target genes (c-myc andcyclin D1) of the Wnt/b-catenin pathway (38), and our datashowed that significant reductions in b-catenin, c-myc, and cyclinD1were observed in ERRa-knockdown glioma cells. The data alsodemonstrated that inactivation of the Wnt/b-catenin pathwayreversed the effects of ERRa overexpression on cell proliferation,showing that canonical and noncanoncialWnt signaling pathwayis required to maintain the proliferative capacity of glioma cells.These data suggested that knockdown of ERRa inhibited theproliferation and migration of glioma cells through suppressingthe Wnt/b-catenin pathway.

We provided compelling data for the involvement of ERRa inthe carcinogenesis via the regulation of Wnt5a signaling pathwayin vitro and in vivo. Further, this study confirmed thatWnt5a acts asa key molecule in the biological functions of ERRa in humanglioma. The versatile role of Wnt/b-catenin signaling in control-ling tumor cell properties such as motility, invasion, and clono-genicity has attracted significant attention of researchers for ther-apeutic strategies targeting this pathway (39, 40).

In our work, ERRa, as an upstream molecule of Wnt5a, mayprovide powerful target candidate for the treatment of glioma.Therefore, the identification of Wnt5a as a direct transcriptionaltarget of ERRa was of great significance as the expression of this

protein has previously been correlated with increased cell prolif-eration and migration (37).

In conclusion, our study demonstrates on the first attempt thatERRa is a potent independent prognostic factor to predictpatients' cancer-specific outcomes and guide personalized treat-ment decisions. Increased ERRa expression indicated worse prog-nosis and correlated with short survival time. As a consequence,understanding the regulation of ERRa/Wnt5a activity in gliomacould lead to the identification of a reliable therapeutic strategyfor the treatment of glioma andwarrants investigation to improvepatient survival.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L. Zhang, Y. Zhu, W. Hua, Q. LiDevelopment of methodology: L. Zhang, Y. Zhu, H. Chen, L. Chen, H. Qi,G. Ren, J. Tang, W. HuaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Zhu, Y. Zhu, H. Chen, G. Ren, J. Tang, W. HuaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Zhu, Y. Zhu, H. Chen, L. Chen, H. Qi, G. Ren,J. Tang, X. ShiWriting, review, and/or revision of the manuscript: L. Zhang, Y. Zhu,H. Cheng, W. Hua, Q. LiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. Cheng, J. ZhangStudy supervision: M. Zhong, W. Hua, Q. Li

AcknowledgmentsThe authors thank Drs Yu Zhang, Jinsen Zhang, and Li Chen for valuable

discussions.This study was supported by a grant from the National Natural Science

Foundation of China (No.81302853, Q. Li).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 14, 2017; revised January 10, 2018; accepted October 10,2018; published first October 15, 2018.

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2019;18:173-184. Published OnlineFirst October 15, 2018.Mol Cancer Ther Liudi Zhang, Yingfeng Zhu, Haixia Cheng, et al. Correlates with Wnt5a and Poor Prognosis in Patients with Glioma

αThe Increased Expression of Estrogen-Related Receptor

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