Control of the Human Osteopontin Promoter by ERR&alpha

A CM 201 SIP E 3 Pr AJ og P ra m

The American Journal of Pathology, Vol. 183, No. 1, July 2013

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TUMORIGENESIS AND NEOPLASTIC PROGRESSION

Control of the Human Osteopontin Promoter by ERRa in Colorectal Cancer Salah Boudjadi,*y Gérald Bernatchez,* Jean-François Beaulieu,y and Julie C. Carrier*y From the Departments of Medicine* and Cellular Anatomy and Biology,y Faculty of Medicine and the Sciences of Health, University of Sherbrooke, Sherbrooke, Quebec, Canada CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s). Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

Accepted for publication March 7, 2013. Address correspondence to Julie C. Carrier, M.D., Département de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada J1H 5N4. E-mail: Julie. [email protected].

Colorectal cancer is the second leading cause of death from cancer. Osteopontin (OPN) is a component of tumor extracellular matrix identified as a key marker of cancer progression. The estrogen-related receptor a (ERRa) has been implicated in endocrine-related cancer development and progression, possibly through modulation of cellular energy metabolism. Previous reports that ERRa regulates OPN expression in bone prompted us to investigate whether ERRa controls OPN expression in human colorectal cancer. Using a tissue microarray containing 83 tumor-normal tissue pairs of colorectal cancer samples, we found that tumor epithelial cells displayed higher staining for ERRa than normal mucosa, in correlation with elevated OPN expression. In addition, knocking down endogenous ERRa led to reduced OPN expression in HT29 colon cancer cells. Promoter analysis, inhibition of ERRa activity, and expression and mutation of potential ERRa response elements in the proximal promoter of human OPN showed that ERRa and its obligate co-activator, peroxisome proliferator-activated receptor g co-activator-1 a, positively control human OPN promoter activity. Furthermore, chromatin immunoprecipitation experiments confirmed in vivo occupancy of the OPN promoter by ERRa in HT29 cells, suggesting that OPN is a direct target of ERRa in colorectal cancer. These findings suggest an additional mechanism by which ERRa participates in the development and progression of colorectal cancer, further supporting the relevance of targeting ERRa with antagonists as anticancer agents. (Am J Pathol 2013, 183: 266e276; http://dx.doi.org/10.1016/j.ajpath.2013.03.021)

Despite advanced early diagnosis and clinical treatment, colorectal cancer (CRC) remains a worldwide health concern. More than 608,700 deaths were estimated to occur in 2008 from CRC,1 representing the fourth leading cause of death after lung, stomach, and liver cancers. CRC development and progression are complex events that involve multistep accumulations of numerous genetic mutations and epigenetic events that result in unregulated transcription of genes involved in cancer cell growth and in alterations in growth factor-regulated signaling pathways. Osteopontin (OPN) is a secreted matricellular glycophosphoprotein produced by osteoblasts and osteoclasts which plays an important role in bone mineral deposit.2 OPN has also Copyright ª 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2013.03.021

been found to be expressed in various cell types, therefore contributing to numerous physiological processes and to the pathogenesis of a variety of disease states, including chronic inflammation and cancer.3 OPN is involved in almost all steps of tumor progression by regulating cellematrix interactions Supported by the Canadian Institutes for Health Research (CIHR) grants MOP-97836 (J.F.B.) and MOP-89970 (J.C.C.). The CRC biobank was supported by CIHR team grant CTP-82942. All authors are part of the Canadian Institutes for Health Research Team on the Digestive Epithelium. J.F.B. and J.C.C. are members of the Fonds de la Recherche du Québec - Santé (FRQS) funded Centre de Recherche Clinique Étienne-Le Bel. J.F.B. holds the Canada Research Chair in Intestinal Physiopathology. J.C.C. was a scholar from the FRQS.

ERRa and Osteopontin Expression in CRC and cell signaling through binding with integrins and CD44 receptors.4 In particular, OPN has been reported to promote metastasis by increasing tumor growth, cell migration, degradation of the extracellular matrix, cell survival in blood stream, and angiogenesis (reviewed in Wai and Kuo5). OPN is overexpressed in a number of cancers, including CRC in which it has been identified as one of the leading markers in cancer progression.6e9 Of note, OPN can be expressed by both cancer cells and cellular components of the tumor’s microenvironment.10 Nevertheless, in microdissected intestinal adenomas from APCMin/þ mouse, the observed increase in OPN gene expression has been shown to be of epithelial origin,11 suggesting that OPN is a transcriptional target of factors implicated in epithelial carcinogenesis. Of these, it has been reported that activated RAS12 and mutant p5313,14 can induce OPN expression. Furthermore, in silico analysis of the OPN proximal promoter as well as experimental evidences suggest that murine and human OPN gene expressions are regulated by numerous factors, including Sp1, AP-1, c-jun, myc, PEA3, and other ETS family members, SMAD, estrogen receptor a, Nurr1 nuclear receptor, and the Wnt/b-catenin/T-cell factor pathway.15e21 OPN regulation also depends on a number of transcription factors relevant to bone biology, including estrogen-related receptor a (ERRa), for which the contribution to bone formation has been widely studied in the past years.22e24 Despite its structural similarity with estrogen receptors, ERRa is an orphan member of the nuclear receptor superfamily because it is not activated by estrogen-like molecules.25 The activity of ERRa is rather controlled by the interaction with master regulators of energy metabolism such as peroxisome proliferatoractivated receptor g co-activator 1 a/b (PGC-1a/b), which are considered as protein ligands of ERRa.26,27 Although no synthetic ERRa agonist has been reported to date, selective ERRa antagonists, including XCT790, have been useful to delineate ERRa functions in vitro.28,29 ERRa is a regulator of metabolic function that controls multiple biosynthetic pathways involved in energy metabolism.30 In addition, growing evidences support the role of ERRa as a protumorigenic factor. For instance, ERRa is overexpressed and correlates with unfavorable biomarkers and poor clinical outcome in various cancer cell types, including breast,31e34 prostate,35,36 ovarian,37 endometrium38 and colorectal.39 Moreover, in CRC, semiquantitative real-time PCR determination revealed slightly higher ERRa expression in tumors than in matched normal mucosa.40 In support of its oncogenic action, ERRa has also been reported to participate in breast cancer cell proliferation and migration as well as in vascular endothelial growth factorinduced tumor angiogenesis.34,41e44 Although the contribution of ERRa as a transcriptional regulator of energy metabolism is well established, a subset of ERRa targets has also been shown to be highly relevant to tumor biology.42,45 This suggests that ERRa targets key hallmarks of cancer and thus could represent a candidate for the development of new cancer therapeutic agents. In the light of these findings, we investigated whether ERRa protein expression is induced in colon cancer epithelial

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cells and whether ERRa is a transcriptional regulator of human OPN expression.

Materials and Methods Human Tissues Samples of 83 CRC and paired normal tissues (at least 10 cm from the tumor) were obtained from patients undergoing surgical resection without prior neoadjuvant therapy. Tissues were obtained after patients’ written informed consent, according to a protocol approved by the Institutional Human Subject Review Board of the Centre Hospitalier Universitaire de Sherbrooke. Samples were fixed with 4% paraformaldehyde in 0.1 mol/L PBS at 4 C overnight, dehydrated in graded alcohols, and embedded in paraffin. Patient samples were histologically classified and graded according to World Health Organization guidelines and tumor-node-metastasis staging criteria. Furthermore, genomic DNA of tumor cells was extracted from 10-mm thick sections of formalin-fixed paraffin-embedded tissue with the use of a FFPE DNA Isolation Kit for Cells and Tissues (Qiagen, Valencia, CA) and amplified by PCR. Presence of mutations in tumor samples was detected by direct sequencing in 81 patients (Plateforme de Séquençage et de Génotypage des Génomes du Centre de Recherchée du Centre Hospitalier de l’Université Laval, Quebec City, QC, Canada). Molecular characterization included microsatellite instability status (MSI panel marker; NCI, Bethesda, MD), mutation status of p53 (exons 5 to 8 containing mutation hotspots), oncogenic KRAS (alias Ki-ras; codons 12, 13, and 61), and BRAF(V600E) mutations, inactivating mutations of APC (mutation cluster region) and activating mutations of CTNNB1 (exon 3). Clinicopathological and molecular characteristics of the series are described in Table 1.

TMA Construction Tissue microarray (TMA) was performed as previously described.46 Briefly, serial sections were processed for routine hematoxylin and eosin staining to identify tumor and normal mucosal tissue to be punched. Thereafter, tissue cores with a diameter of 2 mm were removed from fixed paraffin-embedded tissue blocks, using a dermatological biopsy punch (Miltex Inc., York, PA), arrayed on a recipient paraffin block, covered with hot paraffin, and cooled down at 4 C for 1 hour to create a new block. From the new TMA block, 4-mm thick sections were mounted on charged glass slides and stored at room temperature.

Immunohistochemistry Paraffin-embedded sections were deparaffinized in xylene. Antigen retrieval was performed in citrate buffer (pH 6). Endogenous peroxidases were quenched with 0.3% H2O2.

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Boudjadi et al Table 1 Clinicopathological Parameters and Molecular Characteristics of Patients in This Series of CRC Samples Parameter

Value

Age, median (range) (years) Sex Male, no. (%) Female, no. (%) Tumor localization Left þ rectum, no. (%) Right þ transverse, no. (%) Tumor grade 1, no. (%) 2, no. (%) 3, no. (%) 4, no. (%) Tumor stage (TNM) 1, no. (%) 2, no. (%) 3, no. (%) 4, no. (%) Microsatellite instability MSS, no. (%) MSI, no. (%) APC/CTNNB1 No mutation, no. (%) Mutation, no. (%) KRAS/BRAF No mutation, no. (%) Oncogenic mutation, no. (%) p53 No mutation, no. (%) Mutation, no. (%)

69 (61-78.8) 48 (57.8) 35 (42.2) 28 (33.7) 55 (66.3) 0 50 25 8

(0) (60.2) (30.1) (9.6)

8 28 33 14

(9.6) (33.7) (39.8) (16.9)

63 (75.9) 18 (21.7) 48 (57.8) 33 (39.8) 46 (55.4) 35 (42.2) 48 (60.2) 33 (37.3)

MSI, microsatellite instability; MSS, microsatellite stable; TNM, tumornode-metastasis.

Tissues were treated with streptavidin/biotin blocking reagent (Vector Laboratories Inc., Burlingame, CA) and blocking serum [PBS 1X, 0.1% BSA, 0.2% Triton X-100, 0.1% donkey serum, 0,1% goat serum (all from SigmaAldrich, Oakville, ON, Canada)]. Sections were incubated with anti-OPN (1:200; R&D Systems, Minneapolis, MN) and anti-ERRa (1:100; NLS5402; Novus Biologicals, Littleton, CO) overnight at 4 C and with the corresponding biotinylated goat (Millipore, Bedford, MA) and rabbit (GE Healthcare, Baie d’Urfé, QC, Canada) secondary antibodies for 1 hour at room temperature. Slides were incubated with horseradish peroxidase-streptavidin and stained with 3,30 -diaminobenzidine (Vector Laboratories Inc.), then counterstained with light hematoxylin, dehydrated, and protected with a coverslip. Representative images were acquired with a Leitz Orthoplan microscope (Leica, Wetzlar, Germany).

Expression Analysis The scoring system for protein expression in tumor and normal samples was based on staining intensity (no staining Z 1; weak staining Z 2; moderate staining Z 3;

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strong staining Z 4) and percentage of expression in epithelial cells (0% to 25% Z 1; 25% to 50% Z 2; 50% to 75% Z 3; 75% to 100% Z 4). Final immunostaining score (intensity percentage of expression) ranged from 1 to 16. The expression index for each patient was defined as the difference between the expression score for tumor and normal paired samples: no change (0), increased expression in cancer (positive index Z 1), and decreased expression in cancer (negative index Z 1).

Cell Culture and Treatment Conditions The human transformed embryonic kidney cells (HEK293T) and the human colon carcinoma cell line HT29 were originally obtained from the American Type Culture Collection (Manassas, VA). The HT29 cell line was selected because of its high ERRa and OPN expression. Cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Burlington, ON, Canada) supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 10 mmol/L HEPES, 100 U/mL penicillin, and 100 mg/mL streptomycin (Wisent, St-Bruno, QC, Canada) and maintained in a 5% CO2-humidified atmosphere at 37 C. Cells were also incubated with the selective ERRa inverse agonist XCT790 for 48 hours at 5 mmol/L (Sigma-Aldrich), known to reduce ERRa activity and to increase its degradation.28

Lentivirus-Mediated RNA Interference The PLK0.1 lentiviral shRNA expression vector targeting hERRa (NM_004451) and control scrambled (shCTL) were obtained from Sigma-Aldrich. Lentiviruses were produced by cotransfecting the shRNA expression vector and the Vira Power Lentiviral Packaging Mix (Invitrogen) in HEK293T cells. HT29 cells were infected with viral suspension for 2 days and maintained in medium supplemented with 5 mg/ mL puromycin (Sigma-Aldrich).

RNA Extraction and Quantitative Real-Time RT-PCR Analysis Total RNA was isolated with the Totally RNA kit (Invitrogen) according to the manufacturer’s instructions. RNA was reverse-transcribed with AMV RT (Roche Diagnostics, Laval, QC, Canada), and cDNA samples were amplified with the following primer pairs: OPN forward, 50 -TTGCAGTGATTTGCTTTTGC-30 , and reverse, 50 -GTCATGGCTTTCGTTGGACT-30 ; ERRa forward, 50 -CGAGAGGAGTATGTTCTA-30 , and reverse, 50 -CCCGCCCGCCGCCGCTCAGCA-30 ; human acidic ribosomal phosphoprotein P0 (RPLPO) forward, 50 -GCAATGTTGCCAGTGTCTG-30 , and reverse, 50 -GCCTTGACCTTTTCAGCACAA-30 . Quantitative real-time PCRs were performed in triplicate with the use of Brilliant II SYBR Green QPCR master mix in a MX3000P Real-Time System (Stratagene, Cedar Creek, TX). Thereafter, mRNA expression levels

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ERRa and Osteopontin Expression in CRC were calculated with the DDCT method,47 normalized to RPLPO expression and expressed as fold difference relative to that of negative control cells (shCTL).

Protein Expression and Immunoblotting Cells were lyzed in chilled Triton buffer (50 mmol/L TrisHCl, pH 7.5, 1% Triton X-100, 100 mmol/L NaCl, 5 mmol/L EDTA) supplemented with protease inhibitors. Fiftymicrogram proteins were subjected to electrophoresis on 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride membrane (GE Healthcare). Membranes were incubated overnight at 4 C with primary antibodies, followed by incubation with horseradish peroxidase-coupled secondary antibodies. Detection was performed with the Pierce ECL Western Blotting Substrate (Pierce, Rockford, IL). Antibodies used were anti-hERRa NLS5402 (1/2000; Novus Biologicals); anti-OPN AF1433 (1/2000; R&D Systems), and anti-actin MAB1501R (1/5000; Chemicon International, Billerica, MA). Secondary antibodies were anti-rabbit IgG horseradish peroxidase and anti-mouse IgG horseradish peroxidase (1/5000; GE Healthcare).

luciferase vector. In some experiments, 200 ng of PLK0.1 shCTR versus 200 ng of PLK0.1 shERRa were transfected together with expressing vectors, and 5 mmol/L XCT790 versus dimethyl sulfoxide was added to the medium concomitantly with cell transfections. Transfections were allowed to proceed for 3 hours at 37 C, followed by replacement with Dulbecco’s modified Eagle’s medium 10% fetal bovine serum media. Cells were incubated an additional 48 hours before assaying for luciferase activities, using a dual luciferase assay kit (Promega, Madison, WI). All results represent experiments conducted at least three times.

ChIP Assay Chromatin immunoprecipitation (ChIP) assay was performed on HT29 cells as previously described.48 Briefly, after sonication, soluble chromatin fragments of 500 to 1000 bp in length were incubated with 5 mg of anti-hERRa antibody, rabbit anti-IgG antibody, or no antibody for 16 hours at 4 C, followed by incubation with 40 mg of salmon sperm DNA/ protein A-agarose for 1 hour at 4 C. Immunoprecipitates

OPN Promoter Analysis and Site-Directed Mutagenesis The human OPN promoter sequence was analyzed for ERRa response elements (ERREs) with the use of the MatInspector version 2.7 web-based search algorithm from Genomatix Software (Munich, Germany). A 1-kb OPN human promoter inserted in the pSGG plasmid vector was purchased (Switch Gear Genomics, Menlo Park, CA) and named OPN-luc. Mutations of ERRE-S1, ERRE-S2, ERRES3, and ERRE-S1 þ ERRE-S2 potential ERRa response elements were introduced with the use of the Geneart SiteDirected Mutagenesis System according to the manufacturer’s instructions (Invitrogen). Pairs of oligonucleotide primers used were ERRE-S1 forward, 50 -CCCTCAAAAACCCCACCATTTGGGTAATAGTATTGCATT-30 , and reverse, 50 -AATGCAATACTATTACCCAAATGGTGGGGTTTTTGAGGG-30 ; ERRE-S2 forward, 50 -30 , and reverse, 50 TCCGCTGTGTCACTGCAAATATGTGCAACCTTGGGC-30 ; ERRE-S3 forward, 50 -AAAGCTAAGCTTGAGTAGTAGACCAGTGAGGCAAGTTTTCTG-30 , and reverse, 50 -CAGAAAACTTGCCTCCATGGTCTACTACTCAAGCTTAGCTTT-30 . The integrity of each construct was confirmed by sequencing.

Plasmids and Cell Transfections HEK293T cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Cells were plated in 12-well plates and transfected in triplicates 24 hours later with the following amounts of plasmid DNAs: 100 ng of pSGG OPN-luciferase or mutated pSGG OPN-luciferase reporters, 200 ng of plenti hERRa, 200 ng of plenti HA-hPGC1a, and 2 ng of pRL-SV40 renilla


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Figure 1 ERRa and OPN expression scores are higher in CRC than in paired normal tissues. Distribution of immunostaining scores for ERRa (A) and OPN (B) in normal margin tissues and CRC tissues. Boxes represent lower quartile, median, and upper quartile, whereas whiskers represent the lowest and highest immunostaining scores. ERRa and OPN expressions are significantly higher in CRCs than in normal paired tissues. ***P < 0.001, Wilcoxon test.

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Boudjadi et al were washed and eluted as previously described.48 Samples were then treated with RNase A (Roche Diagnostics) for 30 minutes at 37 C and with proteinase K (Roche Diagnostics) for 2 hours at 42 C. Isolated DNA fragments were purified with QIAquick spin kit (Qiagen), and quantitative PCRs were performed. A region located between 784 and 593 bp upstream of OPN initiation start site was amplified with the following primer pair: forward 50 -CGCAGACGATTTGCATCTAA-30 and reverse 50 -GTGGTTCTGAATTCCGCTGT-30 . Enrichment for this region was normalized against a negative control segment, and enrichment of the ERRa promoter region was used as positive control, as previously described.48

compare scores between tumor and normal samples for each marker. The weighted k was calculated for correlation analysis between each marker. Fisher’s exact test was used to study the statistical association between clinicopathological parameters and the expression index for ERRa and OPN. Data were expressed as the means SE of at least three different experiments. Statistically significant differences were defined as P < 0.05.

Statistical Analysis

To evaluate the expression of ERRa and OPN at the protein level in our tissue collection, we constructed a TMA that contained one spot of normal mucosa and one spot of CRC for 83 patients, in which the mutation in key genes involved in colorectal carcinogenesis were characterized (Table 1). ERRa was found to be expressed in the epithelium of most CRC samples, the expression score being significantly higher in cancerous tissues than in margin tissues (median Z 9

Statistical analysis was performed with SPSS software version 18.0 (SPSS Inc., Chicago, IL) and StatXact software version 6 (Cytel Software Corporation, Cambridge, MA). Comparisons between groups were performed with one-way analysis of variance, with Bonferroni correction for multiple comparisons. A nonparametric Wilcoxon test was used to

Results ERRa and OPN Expression Are Co-Overexpressed in Human CRC Samples

Figure 2 Representative co-increased expressions of (ERRa and OPN immunohistochemical staining in tumoral compared with adjacent nonmalignant (normal) tissue from four patient samples (patient numbers: 4, 66, 57, and 64). Column 1: Cancer tissues stained with anti-ERRa antibody. Column 2: Normal tissues stained with anti-ERRa antibody. Column 3: Cancer tissues stained with anti-OPN antibody. Column 4: Normal tissues stained with anti-OPN antibody. The identification number of representative patients is indicated on the left. Scale bars: 50 mm (columns 1e4). TMA sections were used for subsequent ERRa and OPN immunostaining.

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ERRa and Osteopontin Expression in CRC Table 2 ERRa and OPN Expressions Are Correlated in Colorectal Cancer and Paired Normal Tissues OPN index ERRa index

Total

1 0 1

1

0

1

Total

3* 3 4 10

1 10* 13 24

4 8 37* 49

8 21 54 83

Correlation table for ERRa and OPN immunohistochemical expression indices in cancer relative to nontumor tissue (index: 0 Z no change; 1 Z increased in cancer; 1 Z decreased in cancer). ERRa and OPN expressions were higher in 65% (54 cases) and 59% (49 cases) of tumors compared with paired normal tissues, respectively. The relative expressions of both proteins were significantly concordant in 60.24% of cases (k Z 0.27, P Z 0.01). ERRa and OPN both increased in 44.57% of tumors studied (37 cases). *The number of cases in which ERRa and OPN indices are correlated with each other.

and 6, respectively; P < 0.001, Wilcoxon test) (Figure 1A). In a paired comparison, immunostaining scores for ERRa expression were observed to be higher in cancerous than in noncancerous tissues in 54 patients (65.1%), equal in 21 patients, and lower in only 8 patients. Interestingly, ERRa expression was also observed in the cytoplasm from samples with strong epithelial nuclear ERRa expression (Figure 2). Although not as strong as epithelial cells, fibroblasts, endothelial cells, and lymphocytes within the tumor microenvironment displayed noticeable ERRa immunostaining (Figure 2). As a second protein of interest, only few CRC displayed no OPN immunostaining in epithelial cells (four samples), and expression scores were significantly higher in cancerous tissues than in normal colon (median Z 6 and 4, respectively; P < 0.001, Wilcoxon test) (Figure 1B). In the 83 matched samples assessed, staining scores for epithelial OPN were higher in cancerous than in noncancerous tissues in 49 patients (59.02%), equal in 24 patients, and lower in only 10 patients. Although not scored, variable OPN immunostaining in endothelial and mesenchymal cells were observed from normal and cancerous tissues, being in some samples either stronger or weaker than in epithelial cells (Figure 2). Expression indices for ERRa and OPN immunostaining did not correlate with clinicopathological or molecular parameters, including tumor grade and stage as well as microsatellite instability and mutations in APC/CTNNB1, RAS/RAF, and p53. Of particular interest, scores for ERRa and OPN immunostaining were significantly correlated in 60.24% of tissues in this series (weighted k Z 0.27, P Z 0.014), and both proteins were coeup-regulated in 44.57% of tumors relative to adjacent normal tissues (Table 2).

ERRa Regulates OPN Expression in HT29 Colon Cancer Cells To investigate whether OPN expression is regulated by ERRa in colon cancer cells, its endogenous expression was


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silenced in HT29 cells, using a lentivirus-based shRNA strategy. Figure 3 shows that specific shRNAs (shERRa) efficiently knocked down ERRa mRNA and protein expression, compared with HT29 cells infected with shCTR. Silencing ERRa during 4 days resulted in a marked reduction in OPN gene and protein expression (Figure 3, A and B) which persisted up to 12 days after infection of HT29 cells (Figure 3B). These findings support a positive contribution of ERRa to the expression of OPN in colon cancer cells.

OPN Is a Direct Target of ERRa In view of reports that ERRa controls the murine OPN promoter in osteoblastic cells through either a derivative of an ERRa response element23 or a noncanonical response element,24 we next analyzed the human OPN promoter sequence in silico to detect potential ERRE composed of the extended ERE half-site AGGTCA (or its reverse complement). Three potential ERRa response elements were found located within the proximal promoter of hOPN: ERRE-S1, GACCTAGG located at 705 to 696 bp; ERRE-S2,

Figure 3

Silencing ERRa down-regulates OPN expression in HT29 colon cancer cells. HT29 cells were infected with lentivirus encoding for a nontargeting shRNA (shCTL) or shRNA targeting ERRa (shERRa) A: After 4 days, ERRa and OPN gene expression was measured by quantitative real-time PCR and normalized to RPLPO expression. Data are presented as the means SD expression, relative to shCTL-treated cells, from three experiments performed in triplicate. *P < 0.05, ***P < 0.001. Numbers are exact expression ratio. B: HT29 cells were lyzed 4 and 12 days after infection, and ERRa and OPN expression were assayed by Western blot analysis and normalized to actin expression.

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Figure 4 ERRa, co-activated by PGC1a, induces hOPN promoter activity. A: HEK293T cells were transiently transfected with 100 ng of hOPN-Luc reporter and with 200 ng of ERRa and/or PGC1a expression vector. Two ng per well of pRL-SV40 renilla luciferase reporter was used as an internal control in 12-well plates. Promoter-dependent luciferase activities were determined as fold activation over the individual luciferase activity with PV alone. Results are represented as the means SEM from three independent experiments, each performed in triplicate. ***P < 0.0001 (one-way analysis of variance). B: OPN luciferase assay in HEK293T cells cotransfected with 200 ng of ERRa and PGC1a expression vector, together with plasmid vector containing shERRa DNA sequence targeting ERRa or shCTR. C: OPN luciferase assay in HEK293T cells cotransfected with 200 ng of ERRa and PGC1a expression vector and treated with 5 mmol/L XCT790 or DMSO as control for 48 hours. **P < 0.001, t-test (B and C). DMSO, dimethyl sulfoxide; PV, Plenti vector.

CACAGGTCA, located at 630 to 621; and ERRE-S3, TAAAGGACA, located at 400 to 391 bp from the transcription initiation site. As a first step in evaluating the significance of these imperfect sites, the human proximal 1-kb OPN region cloned in front of a luciferase reporter gene was studied. The binding capacity of ERRa to this region was characterized by transient cotransfection in HEK293 cells, using PGC1a as putative ERRa co-activator. As previously reported, ERRa was a weak activator, and its overexpression did not significantly affect hOPN promoterdependent luciferase activity (Figure 4A). In contrast, PGC1a transfection led to a moderate induction of luciferase activity (Figure 4A), possibly via co-activation of endogenous ERRa in HEK293T cells. Furthermore, co-expression of PGC1a and ERRa strongly induced hOPN promoter activity over luciferase activity with vector alone. Of note, cotransfection of increasing doses of PGC1a and ERRa resulted in synergistic activation of the hOPN promoter (data not shown). To test whether ERRa is required for PGC1a-mediated hOPN promoter induction,

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transiently transfected cells were incubated with shERRa and with the ERRa inverse agonist XCT790. As shown in Figure 4B, silencing or inhibiting ERRa activity and expression resulted in substantial drops in PGC1a/ERRainduced hOPN promoter activity (by 55% and 62%, respectively). Because three imperfect ERRa response elements could be found in the proximal hOPN promoter region, we next sought to map in further detail the region required for ERRa control of hOPN expression. We therefore performed mutation analysis of the hOPN-Luc reporter in HEK293T cells. As shown in Figure 5, A and B, disruption of ERRE-S1 led to 68.3% reduction of the hOPN promoter activity induced by ERRa þ PGC1a, relative to the wild-type hOPN promoter. Interestingly, hOPN reporter constructs harboring mutation in either ERRE-S2 or ERRE-S1 þ ERRE-2 sequences displayed markedly reduced basal and ERRa þ PGC1a-induced luciferase activity (Figure 5, C and D). These findings suggest that ERRa, co-activated by PGC1a, is able to control hOPN through the ERRE-S1 and/or the ERRE-S2 sites. Furthermore, the ERRE-S2 site may be more functional than the ERRE-S1 site because its disruption impaired basal hOPN activity, whereas mutation in the ERRE-S1 sequence did not further affect the drop in luciferase activity observed in ERRE-S2emutated reporter. In contrast, mutating the ERRE-S3 sequence did not affect basal or induced luciferase activity, suggesting that this potential binding site is not a functional ERRE (Figure 5E). Interestingly, ERRa could also modulate hOPN promoter activity through a noncanonical element because disruption of the ERRE-S1 and ERRE-S2 site did not completely abolish ERRa þ PGC1a-induced luciferase activity. Taken together, these results suggest that ERRa, co-activated by PGC1a, positively regulates the activity of the human OPN proximal promoter.

In Vivo Occupancy of the Human OPN Promoter with ERRa in Colon Cancer Cells After showing that the ERRE-S1 and ERRE-S2 sites are critical for ERRa responsiveness in hOPN and that ERRa modulates OPN expression in HT29 colon cancer cells, we next performed a ChIP assay to test whether ERRa binds to the hOPN promoter in the native chromatin environment of HT29 colon cancer cells. As shown in Figure 6, ChIP with a human ERRa-specific antibody resulted in a significant enrichment in the genomic fragment that contained the ERRE-S1 and ERRE-S2 sites in the hOPN promoter (2.88fold). As a strong positive control, we found endogenous ERRa to be tethered to autoregulation sites within the promoter of the human ERRa gene (11.34-fold enrichment). As negative control, weak precipitation was detected with the use of no antibody or anti-rabbit IgG. Together, these data suggest that ERRa binds to the cis-regulatory domain

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ERRa and Osteopontin Expression in CRC

Figure 5

Mutation analysis of potential ERREs in the hOPN promoter. The functionality of three potential ERREs was determined by mutation analysis. A: Luciferase assay in HEK293T cells with the use of wild-type hOPN-luc reporter. BeE: ERRE-S1, ERRE-S2, ERRE-S1 þ ERRE-S2, and ERRE-S3 mutated reporters, respectively. Underlined letters are components of the putative ERRa response element (TNAAGGTCA, in which N can be any base) and mutated nucleotides are labeled as white letters in gray frames. The five reporters (100 ng) were cotransfected with 200 ng of ERRa and/or PGC1a expression vector. Two ngs per well of pRL-SV40 renilla luciferase reporter was used as an internal control in 12-well plates. Promoter-dependent luciferase activities were determined as fold activation over the individual luciferase activity with vector alone in wild-type hOPN-luc reporter (top column in A). The means SD of three experiments performed in triplicate wells are shown. PV, Plenti vector.

of the endogenous OPN promoter in HT29 and transactivates this gene.

Discussion In the present study, we demonstrated that the immunohistochemical expression of the nuclear receptor ERRa is elevated in neoplastic epithelial cells of a large subset of CRC samples, in correlation with higher OPN expression in tumors versus normal mucosa. Furthermore, we showed with the use of loss-of-function approaches that ERRa contributes to OPN expression in HT29 colon cancer cells. Results from the functional and mutation analysis of the OPN promoter and ChIP assay also suggest that OPN is a direct target gene of ERRa in colon cancer cells. ERRa shares considerable structural similarity with ERa, and reports of functional cross talk between these nuclear receptors prompted the first investigations of ERRa expression in hormone-responsive cancers. Indeed, ERRa is overexpressed in several cancer types, and elevated expression has been correlated with poor clinical outcome in breast and ovarian cancers.31,32,37 Furthermore, ERRa gene expression has been determined by semiquantitative real-time PCR in CRCs and found to be modestly but significantly higher in tumors than in matched normal mucosa and to correlate with higher histological grade and tumor-node-metastasis stage of CRC.39 In the present study,


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we constructed a tissue microarray with 83 tumor-normal tissue pairs from patients with CRC and analyzed the samples by ERRa immunostaining. This powerful approach allows the simultaneous analysis of tens of samples, and its use to validate transcriptomic analysis is rapidly growing. In agreement with previously reported ERRa gene expression analysis, we first observed that scores for ERRa expression were significantly higher in tumors than in normal tissues. Although our analysis focused on epithelial cells, the examination of the tumor microenvironment revealed ERRa immunostaining in fibroblasts, endothelial cells, and lymphocytes. This is consistent with known functions of ERRa in immunity via reactive oxygen species production in macrophages and T-cell activation and differentiation.49,50 Although ERRa is mostly located in the nucleus, noticeable cytoplasmic staining was also observed in tumor cells with high nuclear ERRa expression. As recently proposed, this could involve a feedback loop in which activated ERK8 inhibits ERRa and promotes its nuclear exclusion in cancer cells.51 Interestingly, we did not observe any correlation between ERRa expression index and clinicopathological parameters, including tumor stages and grades. Further investigation in a larger set of CRC samples would be needed to test any association between ERRa overexpression and oncogenic pathways. OPN has been considered an important determinant in bone formation and tumor progression. In keeping with

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Figure 6 ERRa occupies the hOPN promoter in HT29 colon cancer cells. ChIP assay with a human ERRa-specific antibody (black columns) was performed in HT29 cells. Mock immunoprecipitations with the use of rabbit anti-IgG (white columns) and without the use of antibody (gray columns) were performed as negative control. Data were calculated with PCR quantification of DNA fragments located between 784 and 593 bp upstream of the OPN initiation start site relative to a negative control DNA fragment. Enrichment for the autoregulation sites within the promoter of the human ERRa gene was used as a positive control. Results represent the mean of three independent experiments, with real-time quantitative PCR performed in triplicate. *P < 0.05, **P < 0.01 compared with negative control ChIP (t-test). Ab, antibody.

previously reported analyses in whole colorectal tumor mass,7e9,14 a statistically significant increase in epithelial OPN expression was detected in tumors herein compared with normal mucosa. Our data did not find any correlation between OPN immunostaining index and either clinicopathological parameters or oncogenic molecular alterations. Of note, OPN expression significantly correlated with ERRa expression, being co-overexpressed in 44.57% of paired tumors. Taken together, these results suggest that ERRa and OPN could participate in CRC biology and that either the expression of the two proteins is positively regulated by common oncogenic factors or OPN is a target of ERRa in CRC. Increasing evidences also support a role for ERRa as a protumorigenic factor, possibly by contributing to the coordination of energy-generating and biosynthetic pathways to effectively promote cancer cell growth. In addition, functional and physiological genomic approaches have found that the ERRa/PGC-1 complex can positively regulate the expression of a subset of genes which are key to tumor biology. Interestingly, silencing ERRa in colon cancer cells significantly affects many biological gene cluster processes, including biological adhesion gene ontology, together with reduced cell proliferation, anchorage-independent cell growth, and tumorigenicity in nude mice.52 Taking into consideration the known role of ERRa in bone physiology and the coinduction of ERRa and OPN in CRC samples, we postulated that ERRa contributes to OPN expression in HT29 colon

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cancer cells, in which both proteins are well expressed. In support of our hypothesis, silencing ERRa expression significantly reduced OPN gene and protein expression in this cell line. Other ERRa target genes have been proposed to contribute to the malignant phenotype of cancer cell lines, including vascular endothelial growth factor,34,53 osteoprotegerin, and the matrix metalloproteinases 1 and 1334 and WNT11.43 Of interest, the regulation of OPN expression by ERRa has mostly been investigated in the context of bone biology. In particular, Bonnelye et al54 reported that either siRNA-mediated silencing of ERRa or the expression of a dominant-negative form of ERRa resulted in lower mouse OPN levels in the RAW264.7 monocyte-macrophage cell line that differentiates in osteoclasts. Furthermore, the characterization of the regulation of OPN expression by ERRa has been mainly performed in cell lines of rodent origin. In the present study, luciferase assays showed that the ERRa/PGC-1a complex positively regulated the human OPN promoter. In our hands, transient overexpression of ERRa alone did not affect human OPN promoter activity, likely because its transcriptional activity is strongly linked to the availability of its co-activators, including PGC1a.26 Indeed, in addition to potentiating ERRa transcription of its targets, including OPN, PGC1a can also increase ERRa expression through a positive loop because its ownpromoter harbors three ERRa functional response elements.27 PGC1a co-activates several other nuclear receptors, including peroxisome proliferator-activated receptor g, ERa, glucocorticoid receptor, thyroid hormone receptor, liver X receptor, retinoid X receptor a, and farnesoid X receptor.55 The fact that the induction of OPN by the ERRa/PGC-1a complex is strongly attenuated by pharmacologic or shRNA-mediated knockdown of ERRa supports a specific role for ERRa in OPN transactivation. The role of ERRa in colorectal tumor growth may therefore be mediated in part by increased expression of OPN. There is some controversy as to whether ERRa regulates OPN expression in a cell- and species-dependent manner, through either canonical or noncanonical response elements.24 The putative ERREs contain the core AGGTCA sequence common to nuclear receptors, preceded by a TNA sequence, in which N can be any nucleotide base.30 The present analysis of the proximal human OPN promoter found three imperfect ERREs, named ERRE-S1 (TGACCTAGG), ERRE-S2 (CACAGGTCA), and ERRE-S3 (TAAAGGACA), in which only the first two harbor the core AGGTCA sequence (or its reverse complement). This nuclear receptor half-site appears to be essential for ERRa/PGC-1a complex-mediated transactivation because our mutation analysis showed that disruption of this ERRE-S3 site did not affect hOPN promoter-dependent luciferase activity. In turn, disruption of either ERRE-S1 and/or ERRE-S2 sites resulted in attenuated induction of hOPN activity, whereas mutation of the ERRE-2 sequence reduced basal hOPN activity, possibly related to the presence of endogenous ERRa and PGC1a in HEK293T cells. These two response elements are located 80 pairs apart,

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ERRa and Osteopontin Expression in CRC suggesting a possible coordination between these sites in the activation of the OPN promoter by ERRa. Overall, our findings suggest that ERRa binds to the ERRE-S1 and ERRE-S2 sequence within the proximal promoter of human OPN and that the ERRa/PGC-1a complex positively regulates its activity. Although relatively straightforward, conditions associated with luciferase assay remain artificial and may not necessarily reflect the in vivo cell-specific environment and native chromatin structure. In fact, a recent study reported that the activity of the OPN promoter can be repressed by Trichostatin, a potent histone deacetylation, in HeLa cells.56 We therefore investigated whether ERRa occupies the human OPN promoter in HT29 cells, using a validated ChIP assay. Consistent with results from our luciferase assay, we found that ERRa binds to the human OPN promoter in a region containing the ERRE-S1 and ERRE-S2 sites. These sites are however too close to accurately discriminate ERRa binding of the ERRE-S1 versus the ERRE-S2 site. ERRa could collaborate with transcription factors pertinent to CRC biology to induce OPN expression. In particular, the hOPN promoter has been reported to be activated by Wnt/bcatenin/T-cell factor 418 involved in early stages of CRC. Furthermore, cooperation between ERRa and b-catenin has been shown to contribute to the expression of WNT11 factor and to MDA-MB-231 breast cancer cell migration.43 Further studies are required to fully understand the cross talk between ERRa and CRC-enriched oncogenic factors as well as the contribution of each of the latter in the regulation of gene expression involved in CRC development and progression. In conclusion, with the use of TMA samples of CRC, our results show that ERRa and OPN are co-expressed and higher in tumor epithelial cells as opposed to normal mucosa. Furthermore, our data provide compelling evidence that OPN is a direct target of ERRa, the induction of which could clearly contribute to CRC pathogenesis. These findings also support the growing interest in developing ERRa antagonists as therapeutic options in cancer treatment.

Acknowledgments We thank Nathalie Carrier for assistance in statistical analysis and Pierre Pothier for reviewing the manuscript.

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