Overexpression of MBD2 in Glioblastoma Maintains ......Therapeutics, Targets, and Chemical Biology...

Therapeutics, Targets, and Chemical Biology

Overexpression of MBD2 in Glioblastoma MaintainsEpigenetic Silencing and Inhibits the AntiangiogenicFunction of the Tumor Suppressor Gene BAI1

Dan Zhu1, Stephen B. Hunter2, Paula M. Vertino3,5, and Erwin G. Van Meir1,4,5

AbstractBrain angiogenesis inhibitor 1 (BAI1) is a putative G protein–coupled receptor with potent antiangiogenic and

antitumorigenic properties that is mutated in certain cancers. BAI1 is expressed in normal human brain, but it isfrequently silenced in glioblastoma multiforme. In this study, we show that this silencing event is regulated byoverexpression of methyl-CpG–binding domain protein 2 (MBD2), a key mediator of epigenetic gene regulation,which binds to the hypermethylated BAI1 gene promoter. In glioma cells, treatment with the DNA demethylatingagent 5-aza-20-deoxycytidine (5-Aza-dC) was sufficient to reactivate BAI1 expression. Chromatin immunopre-cipitation showed that MBD2 was enriched at the promoter of silenced BAI1 in glioma cells and that MBD2binding was released by 5-Aza-dC treatment. RNA interference–mediated knockdown ofMBD2 expression led toreactivation of BAI1 gene expression and restoration of BAI1 functional activity, as indicated by increasedantiangiogenic activity in vitro and in vivo. Taken together, our results suggest that MBD2 overexpression duringgliomagenesis may drive tumor growth by suppressing the antiangiogenic activity of a key tumor suppressor.These findings have therapeutic implications because inhibiting MBD2 could offer a strategy to reactivate BAI1and suppress glioma pathobiology. Cancer Res; 71(17); 5859–70. �2011 AACR.

Introduction

DNA methylation is a naturally occurring event that con-sists of the addition of a methyl group to the fifth carbonposition of the cytosine pyrimidine ring by DNA methyltrans-ferases. Alterations in the patterns of DNA methylation arewidespread in human cancers and include genome-widehypomethylation and the hypermethylation of CpG island–associated gene promoters, the latter of which represents onemechanism leading to the epigenetic silencing of genes inhuman cancers (1, 2). DNA methylation alterations have beenwidely reported in human glioblastoma (GBM), a highlyvascularized and aggressive primary intracranial tumor(3–7). A distinct subgroup of primary GBM displays concor-dant hypermethylation at a large number of loci, indicatingthe existence of a glioma CpG island methylator phenotype(gCIMP; ref. 8). Interestingly, the subset of GBM exhibiting

gCIMP is associated with isocitrate dehydrogenase (IDH)mutations, providing a link to an altered metabolic profile (9).

Methyl-CpG–binding domain (MBD) proteins interpret theDNA methylation marks and thus are critical mediators ofmany epigenetic processes (10–12). The MBD family com-prises 5 members; MBD1–4 and MeCP2. MBD1, MBD2, andMeCP2 bind selectively to methylated CpGs and represstranscription from methylated promoters in vitro and in vivo.In contrast, MBD3 binding is not dependent on DNA methyla-tion, and MBD4, while selective for methylated DNA, has beenprimarily characterized as a thymine DNA glycosylase withlittle role in transcriptional repression (10–12). However, theexpression pattern and functional roles of MBDs in glioblas-toma pathogenesis remain yet unidentified.

BAI1 is an orphan G protein–coupled receptor (GPCR)-likereceptor abundantly expressed in normal brain with potentantiangiogenic and antitumorigenic properties that was initi-ally identified in a screen for p53-regulated genes (13–17).Importantly, BAI1 and its related family members BAI2 andBAI3 were recently found to undergo somatic mutation inseveral cancers, including lung, breast, and ovarian cancers(18). BAI1 contains several well-defined protein modules inthe N-terminus such as an integrin-binding RGD motif fol-lowed by 5 thrombospondin type 1 repeats (TSR), a hormone-binding domain, and a GPCR proteolytic cleavage site (GPS;ref. 16). The TSRs within the extracellular region of BAI1mediate direct binding to phosphatidylserine on apoptoticcells, and BAI1 can cooperate with the engulfment and cellmotility 1 (ELMO1)/dedicator of cytokinesis 1 (Dock180)/Racto promote maximal engulfment of apoptotic cells (19).

Authors' Affiliations: Laboratory of Molecular Neuro-Oncology, Depart-ments of 1Neurosurgery, 2Pathology and Laboratory Medicine, 3RadiationOncology, and 4Hematology and Medical Oncology, School of Medicine,and 5Winship Cancer Institute, Emory University, Atlanta, Georgia

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Erwin G. Van Meir, Winship Cancer Institute,Emory University, 1365C Clifton Road NE, Atlanta, GA 30322. Phone: 404-778-5563; Fax: 404-778-5550; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-1157

�2011 American Association for Cancer Research.

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Interestingly, the ELMO1/Dock180 association is also involvedin the invasive phenotype of glioma cells (20). The C-terminusis less well characterized and has a QTEV motif that mediatesbinding to PDZ domain–containing proteins. The N-terminalextracellular domain of BAI1 can be cleaved at the GPS site,and the resulting 120-kDa fragment, known as vasculostatin(Vstat-120), is able to inhibit angiogenesis in vitro and sup-press intracranial tumor growth in vivo (14, 15). A second N-terminal cleavage site was recently identified, generating asmaller vasculostatin (Vstat-40; Cork and colleagues, manu-script submitted).

Our previous results showed that BAI1 expression wasabsent in most human glioma cell lines and primary glio-blastoma samples examined (21), but the underlyingmechanisms remain unknown. In the present study, weprovide evidence that MBD2 is upregulated in glioblastomasand that it plays a central role in the epigenetic silencing ofBAI1 gene expression, thereby suppressing the antiangio-genic activity of BAI1.

Materials and Methods

Primary tumors and cell linesThe primary GBM tumor samples were obtained from

Emory University Hospital and were reviewed by neuropathol-ogists (D.J. Brat and S.B. Hunter) for histologic confirmation ofGBM before being included in this study. Human GBM celllines LN71, LN229, and LN443 were originally established inour laboratory (21). Human GBM cell lines U87MG, SF188, andU251MG were obtained from the American Type CultureCollection (ATCC) and maintained as described (22). All celllines were authenticated by the ATCC for viability, morphol-ogy, and isoenzymology. Human brain microvascularendothelial cells (HBVEC) were purchased from Cell SystemsCorp. For chemical treatment, glioma cells were plated (3 �l05 cells/l00-mm dish) and treated 24 hours later with 5-Aza-dC (5 mmol/L; Sigma) for 1 to 5 days.

DNA methylation analysis of the BAI1 gene exon 1We determined CpG island methylation status by bisulfite

sequencing and methylation-specific PCR (MS-PCR) as pre-viously described (3, 23). Additional details, including primersequences, are provided in Supplementary Materials andMethods.

Reverse transcriptase PCRTo determine the mRNA levels of the BAI1 gene, reverse

transcriptase PCR (RT-PCR) was carried out on the total RNAextracted from the cells or GBM samples as described (13).Additional details are provided in Supplementary Materialsand Methods.

Western blottingWestern blotting was carried out as described (15). The

antibodies used were mouse anti-MBD2 (Abcam; catalog no.ab45027), rabbit anti-MeCP2 (Abcam; catalog no. ab2828),goat anti-actin (Santa Cruz Biotechnology), and rabbit anti-BAI1 (21). The horseradish peroxidase–conjugated secondary

antibodies and enhanced chemiluminescence were fromThermoScientific.

Immunohistochemical analysisImmunohistochemistry (IHC) was carried out on archived

formalin-fixed and paraffin-embedded human GBM resectionspecimens. For the tissue array study, 5 nonneoplastic brainand 54 GBM tumor specimens were sectioned and mountedon 2 slides. Sections were deparaffinized and subjected toantigen retrieval by boiling (20 minutes, 100�C) in 0.01 mol/LTris HCl (pH 10). Slides were then incubated with a 1:200dilution of MBD2 antibody. Immunostaining was detectedwith the avidin-biotin complex method, using diaminobenzi-dine as the chromogen (Abcam). Slides were scanned at �40resolution with a Nanozoomer 2.0 HT slide scanner (Hama-matsu) and staining intensity (5 fields/tumor) was quantifiedby the MetaMorph Premier software; MBD2 status wasassessed on the basis of relative staining intensity unit [absent(0), weak (1; units, 1–75), moderate (2; units, 76–150), strong(3; units, 151–225)] and percentage of positive tumor cells [0%(0), <10% (1), 10%–50% (2), 51%–80% (3), 81%–100% (4)].Immunoreactivity scores (IHC scores) were determined bymultiplying the staining score by the percentage score to givea maximum of 12 (24).

Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) assay was carried

out using a commercial kit (Cell Signaling; catalog no. 9003)with some modifications. After cross-linking, the cells werelysed and sonicated using a Misonix MX2020 sonicator (set-ting 15, 15 seconds for 3 times). Sonicated lysates werecentrifuged at 14,000 rpm at 4�C for 15 minutes to get ridof insoluble fractions. An aliquot of the chromatin preparationwas set aside and designated as input fraction. The clearedchromatin (100 mg) was immunoprecipitated with 2 mg ofeither anti-MBD2 or anti-MeCP2 antibody and incubatedovernight at 4�C with rotation. The second day, salmon spermDNA/Protein A/G agarose slurry was added to these samplesand rocked for 4 hours at 4�C. Protein A/G immune complexeswere collected and washed. Immune complexes were eluted,and DNA was recovered by DNA purification columns andanalyzed by PCR. The primers used were 50-GCT CAC TCTGAC CCT CTG CTC TTTC-30 (forward) and 50-AGT AGC CGAAGA ACT TTC CCT GC-30 (reverse) for BAI1 promoter, theprimers used for MGMT promoter were described previously(25). Acetyl-histone H3 (Lys9) antibody was from Cell Signal-ing (catalogue no. 9649S) and histone H3 (trimethyl-K9) anti-body was from Abcam (catalog no. 8898).

Construction of short hairpin RNA vectors andtransient transfection

Constructs for short hairpin RNA (shRNA) were generatedwith the BLOCK-iTU6 RNAi Entry Vector Kit (Invitrogen) asdescribed (26), and primer sequences are provided in Supple-mentary Materials and Methods. Transient transfection ofglioma cells with plasmid DNA (2 mg/60-mm dish) was carriedout with Lipofectamine 2000 according to the manufacturer'sinstructions (Invitrogen) with minor modification (26).

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Cancer Res; 71(17) September 1, 2011 Cancer Research5860




Scratch-wound endothelial cell migration assayThis assay was conducted as previously described (14). In

brief, conditioned medium (CM) from glioma cells transfectedwith shRNAs was collected and concentrated �100 using anUltraCel filter (Amicon). Confluent HBVECs were incubated in1% serum DMEM medium overnight in 12-well plates andthen wounded with a 10-mL pipette tip, and detached cellswere removed by PBS washes. The cells were then treated withCM collected as previously and diluted to �10 in endothelialcell culture to induce cell migration. Initial wound width wasmeasured; the cells were allowed to migrate for 8 hours, andwound width was measured again. The experiment wasrepeated independently 3 times and the significance wasdetermined by Student t test.

In vivo angiogenesis assayQuantification of the antiangiogenic responses was carried

out using the directed in vivo angiogenesis assay (DIVAA) aspreviously described (27) with the DIVAA Inhibition Assay Kit(Trevigen; catalog no. 3450-096-IK). Collection of CM fromglioma cells transfected with shRNAs was performed as inscratch-wound endothelial cell migration assay. Two micro-liters of�100 concentrated CMwasmixed with 18 mLMatrigelcontaining growth factors and filled into sterile surgicalsilicone tubing (angioreactors). These angioreactors wereincubated at 37�C for 1 hour to allow for gel formation, beforesubcutaneous implantation into the dorsal flank of athymicnude mice [females, 6–8 weeks of age; National CancerInstitute (NCI), Frederick, MD]. Two weeks later, angioreac-tors were harvested and the Matrigel was removed anddigested. Cell pellets and insoluble fractions were collectedby centrifugation at 5,000� g for 2 minutes. The cell pelletswere washed and incubated at 4�C overnight in 200 mL of 25mg/mL of fluorescein isothiocyanate (FITC)-labeled Griffonialectin (FITC-lectin), an endothelial cell–selective reagent. Therelative fluorescence was measured in 96-well plates, using aMolecular Device spectrofluorometer (excitation 485 nm,emission 510 nm). The mean relative fluorescence � SD for8 replicate assays was determined.

Results

Downregulation of BAI1 gene expression inglioblastoma is correlated with aberrant DNAmethylation of exon 1Our previous studies on a limited sample set suggested loss

of expression of the BAI1 tumor suppressor in human GBMspecimens and cell lines (21), although no mechanism wasidentified. To independently confirm and investigate theextent of BAI1 loss in GBM, we analyzed large datasets from2 brain tumor databases, namely, the NCI Repository ofMolecular Brain Neoplasia Data (Rembrandt) and The CancerGenome Atlas (TCGA). The expression of BAI1 was firstdetermined in 28 nontumor brain tissues and 196 GBMsamples (institutional diagnosis) in the Rembrandt dataset.As shown in Fig. 1A, the levels of BAI1 gene expression weresignificantly decreased (P < 0.01) in GBM samples comparedwith the nontumor tissues. In contrast, the expression of

THBS1, which encodes angiogenesis inhibitor thrombospon-din 1 harboring 3 TSRs, showed no change (Fig. 1A). In theTCGA dataset, the expression of BAI1 was available in a totalof 424 GBM samples. Analysis of this dataset also showed aconsistent and dramatic loss of BAI1 expression, with 250samples (59%) showing more than a 2-fold decrease (Fig. 1B).In contrast, the expression of THBS1 increased in 374 samples(88%; Fig. 1B). The variation in relative THBS1 expressionbetween the Rembrandt and TCGA databases may be due tothe different probe sets and array platforms used. Takentogether, these data suggest that a large fraction of primaryGBMs exhibit a significant loss or downregulation of BAI1mRNA expression.

To identify the mechanism underlying BAI1 downregula-tion, we first considered whether BAI1 might be located in aregion of genomic loss in GBM. The BAI1 gene is located onchromosome 8q24, a region that is not reported to exhibit lossof heterozygosity in gliomas, and a fact we confirmed byanalyzing the TCGA genomic dataset (results not shown).We considered next the possibility of epigenetic mechanismsof silencing of the BAI1 gene. Using the MethPrimer software,we identified a CpG island in the first exon of the BAI1 gene(Fig. 1C). Bisulfite sequencing was used to determine themethylation pattern in exon 1 in 2 nontumor brain and 6independent GBM samples; 6 clones per sample weresequenced. Whereas the nontumor samples were mostlymethylation-free, extensive methylation was detected in theGBM samples, with most CpG sites found methylated in 31 ofthe 36 clones sequenced (Fig. 1C). The few clones that showedminimal or no methylation could have been derived fromstromal tissue within the tumor or represent tumor hetero-geneity. To further confirm the bisulfite-sequencing result, theDNA methylation status of the aforementioned 6 tumors, plusan additional 6 tumors and 2 control samples from normalhuman brain white matter, was analyzed by MS-PCR. Eight of12 GBM samples exhibited prominent PCR products with themethylated primer set but no products with the unmethylatedprimer set (Fig. 2A). In contrast, the 2 nontumoral brainsamples exhibited detectable PCR bands only from theunmethylated primer set. Examination of BAI1 expressionin the same tumors showed an inverse correlation betweenmethylation and gene expression in that expression of BAI1was only observed in those samples with some degree ofunmethylated DNA, whereas the GBM samples that werecompletely methylated lacked BAI1 expression altogether(Fig. 2B). These data indicate that an aberrant methylationpattern in exon 1 is associated with the silencing of BAI1expression in a subset of GBM. The glioma-associated silen-cing seemed to be specific for BAI1 as the expression of 2homologs, BAI2 and BAI3, and THBS1 did not vary amonghuman glioma cell lines (Fig. 2C). We next examined the effectof a demethylating agent, 5-Aza-dC, on BAI1 gene expression.Treatment with 5 mmol/L 5-Aza-dC for up to 5 days restoredBAI1 mRNA expression in LN229 cells in a time-dependentmanner (Fig. 2D). Similar reactivation was observed in 3 otherBAI1-silent glioma cell lines (Fig. 2E). Because 5-Aza-dC isknown to be highly effective at inducing the expression ofgenes inappropriately silenced by de novo methylation (28),

Overexpression of MBD2 in Glioblastoma Suppresses BAI1

www.aacrjournals.org Cancer Res; 71(17) September 1, 2011 5861




these results suggest that DNAmethylation of the BAI1 gene islikely involved in the gene silencing.

The methyl-CpG–binding protein MBD2 is selectivelyoverexpressed in GBM

Because the impact of DNAmethylation on gene silencing isoften mediated through the binding of MBDs (29, 30), we thenanalyzed the expression of various MBD proteins in GBM bymining the expression data in the Rembrandt database. Theexpression of MBD1, MBD3, MBD4, and MeCP2 showed nosignificant difference between nontumor control and glioblas-tomas (Fig. 3A). In contrast, we found that MBD2 is signifi-cantly overexpressed in GBM with a more than 2-fold greatermean expression in tumors than in nontumor brain tissues.Comparison of the expression of MBD2 and MeCP2 in 424GBM samples from the TCGA database also showed thatMBD2 expression was markedly increased in a significantfraction of GBM samples (Fig 3B), whereas in a similarcomparison, the mean gene expression of MeCP2 was notsignificantly different. To determine whether MBD2 was alsooverexpressed at the protein level, we applied IHC on 2randomly selected GBM specimens and found dramatically

increased MBD2 immunopositivity in tumor (GBM) versusadjacent nontumor areas (Fig 3C), consistent with the geneexpression data. In contrast, MeCP2 exhibited only low tobackground levels of staining and most GBM tumor cells werenegative for MeCP2 staining. The analysis of MBD2 proteinexpression in GBM was expanded in a tissue array containing5 nontumor brain samples and 54 GBM samples (Fig 3D).Weak to moderate MBD2 expression was detected in less than50% of the tumor cells in 6 of 54 specimens (11%); thesetumors were grouped as low-expressing tumors (IHC score0–4). The remaining 48 of 54 tumors (89%) exhibiting mod-erate or strong MBD2 expression in more than 50% of tumorcells were included in a high-expressing group (IHC score6–12). Taken together, these data suggest that MBD2 issignificantly overexpressed in GBM.

MBD2 is necessary to maintain the silencing of BAI1We next sought to determine whether there was any

relationship between the levels of MBD2 and expression ofBAI1 in glioma cell lines. The protein levels of MBD2 and BAI1were determined, and our data suggested a correlationbetween lack of BAI1 protein expression and elevated

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Figure 1. Aberrant DNA methylation at BAI1 exon 1 in GBM. A, downregulation of BAI1 gene expression in human glioblastomas. Raw data from RembrandtAffymetrix Human Genome HTS U133A 2.0 Array for BAI1 and THBS1 expression in 28 nontumor (NT) brain and 196 GBM samples were analyzedand quantified. *, P < 0.01. B, expression of BAI1 and THBS1 in 424 GBM samples from the TCGA database. The normalized expression values are expressedas log ratios (base 2) for the y-axis, each spot representing 1 sample (x-axis). C, analysis of CpG island methylation in the BAI1 exon 1 in human glioblastomasby bisulfite sequencing. Top, schematic diagram indicates the structure of the BAI1 gene promoter showing the CpG island located in exon 1. TheCpG island was identified by the MethyPrimer software. Bottom, bisulfite-sequencing results on 2 brain samples from nontumor patients and 6 samplesfrom human GBM patients are included. Six sequenced clones per sample are shown. The methylation status of the individual CpG dinucleotides isshown by unmethylated (empty) or methylated (filled) circles.

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MBD2 protein levels (Fig. 4A). We next examined the relation-ship between the expression of MBD2 and BAI1 in primaryGBM. Among the 424 primary GBM samples for which geneexpression data were available from TCGA, there was astatistically significant negative correlation between theexpression of MDB2 and BAI1 (Spearman correlation coeffi-cient of�0.095; P¼ 0.05; n¼ 424; Fig 4B). If one considers onlythose 373 tumors for which MBD2 was overexpressed by 1.4-fold or greater relative to normal tissues (log2 > ¼ 0.5), thisassociation was even more significant (Spearman correlationcoefficient of �0.14329; P ¼ 0.0056; n ¼ 373; Fig. 4C). Takentogether, these data support a negative correlation betweenMBD2 and BAI1. To determine whether MBD2 played a directrole in BAI1 gene silencing, the endogenous levels of MBD2mRNA were knocked down by transient transfection of spe-cific shRNA expression vectors. We tested the effect of 2MBD2-shRNAs first in LN229 cells, a glioma cell line thatexhibits abundant MBD2 expression and is silent for BAI1expression (Fig. 4A). Transfection with either shRNA exhibiteda significant reduction in MBD2 protein expression (Fig. 4D)but had no effect on MeCP2 protein levels, indicating speci-ficity of these shRNAs. Transfection of LN229 cells with the

MBD2-specific shRNA expression vector led to a reactivationof BAI1 mRNA expression as compared with the nonspecificcontrol shRNA (Fig. 4D). Similar results were observed in 2other BAI1-silent glioma cell lines (Fig. 4E). Because MeCP2 isknown to be involved in transcriptional repression of multiplegenes, we also designed MeCP2-specific shRNA vectors anddetermined their effects on BAI1 mRNA expression. Whereasthese vectors potently downregulated MeCP2 protein levels(Fig. 4F), they failed to reactivate BAI1 gene expression(Fig. 4G). Together, these results show that MBD2 contributesto BAI1 silencing.

MBD2 is recruited to the BAI1 gene promoterin glioma cells

To further examine the role of MBD2 in the BAI1 generegulation, we carried out ChIP using antibodies againstMeCP2 or MBD2. ChIP assays showed that MBD2 wasenriched at the BAI1 promoter in the BAI1-silent cell lines(LN229, U87MG), whereas it was not associated with the locusin BAI1-expressing cells (LN443; Fig. 5A). There was no evi-dence for MeCP2 association with the BAI1 promoter in any ofthe 3 cell lines (Fig. 5A). The inability to detect MeCP2 is not

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Figure 2. Treatment with demethylating agent reactivates BAI1 gene expression. A, analysis of methylation pattern at the BAI1 exon 1 by MS-PCR. Normaltissue was found to be unmethylated (U), whereasmethylation (M) was found in 8 of 12 GBM samples. NT, nontumor brain sample. B, RT-PCR analysis ofBAI1mRNA expression in normal brain and GBM samples, GAPDH mRNA was amplified as a loading control. C, BAI1 mRNA expression in human glioma celllines analyzed by RT-PCR. BAI1 mRNA can be detected only in SF188 and LN443 cells, whereas BAI2, BAI3, and THBS1 mRNAs can be detectedin all 6 glioma cell lines. D, reactivation of BAI1 mRNA expression in a BAI1-silent glioma cell line using demethylation agent 5-Aza-dC (5 mmol/L).LN229 cells were treated for 1, 3, and 5 days, the medium was changed every day with fresh chemicals. Cells mock treated with the same volume of dimethylsulfoxide were used as a negative control. RNAwas then isolated and RT-PCRwas carried out. E, reactivation ofBAI1 gene expression by 5-Aza-dC (5 mmol/L)in glioma cell lines LN71, U87MG, and U251MG after 3 days of treatment.






due to a lack of expression or technical aspects as these cellsexpress high levels of MeCP2. Moreover, MeCP2 was efficientlyrecruited to the promoter of O6-methylguanine-DNA methyl-transferase (MGMT), a gene to which MeCP2 has been shownto bind in a methylation-dependent manner in all 3 cell lines(Fig. 5A; refs. 25, 31). MBDs, and in particular MBD2, arecomponents of NURD/Mi2 corepressor complexes and arethought to direct transcriptional silencing of methylated CpGislands through the recruitment of histone deacetylase(HDAC) activity (10, 11). Consistent with this, aberrantDNA methylation of CpG island promoters is associated withhistone hypoacetylation and the acquisition of H3K9 methyla-tion (1, 32). We therefore examined the status of acetylation oflysine 9 (AcH3K9) and trimethylation of lysine 9 (3MeH3K9)on histone H3 by ChIP in BAI1-expressing (SF188 and LN443)and BAI1-silent (LN229 and U87MG) cell lines. 3MeH3K9, a

marker of condensed chromatin, was enriched at the BAI1promoter in BAI1-silent cells but was absent in BAI1-expres-sing cells (Fig. 5B). In contrast, AcH3K9, a marker for tran-scriptionally active chromatin, was enriched at the BAI1promoter in BAI1-expressing cells but was greatly reducedin BAI1-silent cells (Fig. 5B). These results show a correlationbetween BAI1 expression and changes in histone modification.

Next we investigated the influence of 5-Aza-dC on theassociation of MBD2 with the BAI1 CpG island. Treatment ofthe BAI1-silent cell line U251MG (Figs. 2C and 4A) with 5-Aza-dC caused a significant reduction in MBD2 occupancyat the BAI1 promoter as determined by ChIP (Fig. 5C and D).Concomitant with the loss of MBD2, 5-Aza-dC treatmentalso induced hyperacetylation of histone H3 in the BAI1promoter region and a reduction in H3K9 trimethylation(Fig. 5C and D). Similar results were obtained when a shRNA

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Figure 3. Overexpression ofMBD2mRNA and protein in GBM.A, mRNA expression of MBDfamily members in the Rembrandtbrain tumor database. Expressiondata of 28 nontumor (NT) brain and196 GBM samples were analyzedand quantified from RembrandtAffymetrix Human Genome HTSU133A 2.0 Array. A statisticallysignificant increase in MBD2mRNA expression is shown inGBM as compared withnontumoral brain. *, P < 0.01.B, mRNA expression data ofMBD2 and MeCP2 from TCGAdatabase in 424 GBM samples.The normalized expression valuesare expressed as log ratios (base2) for the y-axis, each spotrepresenting 1 sample (x-axis).C, IHC results of MBD2 andMeCP2 staining in nontumor braintissue (top) and in GBM tissue(middle and bottom). Scale bar,100 mm. D, representative imagesshow the quantification of MBD2staining intensity in GBM tissuearrays. Representative imagesfrom low-expressing (IHC score0–4; 6 cases) and high-expressingGBM samples (IHC score 6–12; 48cases) are shown with therespective scores indicated. Scalebar, 100 mm.

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against MBD2 was employed (Fig. 5C and D). Interestingly,the depletion of MBD2 did not allow for the binding ofanother methyl-CpG–binding protein MeCP2 (Fig. 5C).Taken together, these findings support the conclusion thatMBD2 selectively binds to the BAI1 promoter and that itspresence is necessary to maintain characteristics of closedchromatin and transcriptional silencing at the BAI1 locus.These data also suggest that the targeting of MBD2 may beas effective as DNA demethylating agents in restoring chro-matin conformation and prompting the reactivation of BAI1gene expression.

Reactivated BAI1 expression inhibits endothelial cellmigration and in vivo angiogenesisThe aforementioned data, combined with our previous

demonstration that the cleaved 120-kDa N-terminal fragmentof BAI1 (vasculostatin or Vstat-120) can suppress angiogenesisin vitro and inhibit tumor growth in vivo (14, 15), suggest thatreactivation of BAI1 expression byMBD2 knockdownmay be anovel therapeutic approach for GBM. However, whether the

levels or functionality of reactivated BAI1 are sufficient torestore the antiangiogenic activity is not known. Therefore, wefirst determined whether reactivation of BAI1 gene expressionby MBD2 silencing could restore BAI1 protein synthesis andantiangiogenic activity as measured in an in vitro endothelialcell migration assay. BAI1-silent U251MG cells were transi-ently transfected with control or MBD2-specific shRNA vec-tors as previously, and CM was collected 3 days later.Confluent HBVECs were wounded and incubated with CMfrom transfected U251MG cells. Eight hours later, phase-contrast images were captured to monitor the distance tra-veled by HBVECs from the wound edge to the center of thewound. As a positive control, we used CM from U251MG cellsstably transfected with a BAI1-expressing vector. U251MG-BAI1 cell CM dramatically inhibited wound closure as com-pared with untreated control (Fig. 6A; compare photographs 2and 3), consistent with our previous results (14). Similarly,HBVECs treated with CM from MBD2-shRNA–transfectedU251MG cells exhibited clearly reduced migration ascompared with control shRNA-transfected cells (Fig. 6A);

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Figure 4. Reactivation of BAI1 by MBD2 knockdown. A, determination of protein expression of MBD family members in human glioma cell lines by Westernblotting. Low MBD2 levels are shown in SF188 and LN443 cells, the only cell lines in which BAI1 protein is detected. B and C, correlation between theexpression ofMBD2 and BAI1 in TCGAGBM samples. The relative expression level ofMBD2mRNA is plotted against that ofBAI1mRNA in 424 total availableTCGA GBM samples and in 373 GBM samples in which MBD2 is overexpressed (C). Expression values are expressed as log2 ratio of tumor/normal. D,knockdown of MBD2 reactivates BAI1 gene expression. LN229 cells were transiently transfected with 2 different shRNA expression vectors against humanMBD2 or control-scrambled shRNA. Two days later, Western blot (WB) analysis was carried out to determine the reduction in MBD2 levels and reactivation ofBAI1 gene expression by RT-PCR. E, reactivation of BAI1 by knockdown of MBD2 using one shRNA expression vector in glioma cell lines LN71 andU251MG. Procedures as in B. F, the MeCP2 shRNA expression vector suppresses expression of MeCP2 but not of MBD2. LN229 and U251MG cells weretransfected with MeCP2-shRNA or control shRNA. Lysates of transfected cells were subjected to immunoblotting using indicated antibodies after 48 hours.G, knockdown of MeCP2 expression is not able to reactivate BAI1 gene expression. BAI1 gene expression was examined by RT-PCR on total mRNAsextracted from the cells transfected in D. Expression of BAI1 mRNA in SF188 was used as a positive control.






quantification of the migration speed showed more than 50%reduction in wound closure (Fig. 6B).

Because MBD2 knockdown has the potential to reactivatethe expression of other genes that are silenced in a DNAmethylation–dependent manner in addition to BAI1, it ispossible that the inhibitory effect on wound closure deter-mined previously could result from the reexpression of othergenes. To address this issue, we designed BAI1-shRNAs todetermine whether the effect is BAI1 specific. BAI1-specificshRNAs suppressed BAI1 protein expression substantially,resulting in strongly reduced levels of Vstat-120 in the CM(Fig. 6C). We then repeated the scratch-wound assay with CMfrom cells transfected with both MBD2-shRNA and BAI1-shRNA. The migration of HBVECs treated with CM fromU251MG cells transfected with both MBD2-shRNA andBAI1-shRNA was more than 50% faster than that of HBVECstreated with CM from cells transfected with both MBD2 andcontrol shRNAs (Fig. 6A and B). These results show that aconcomitant reduction in BAI1 expression partially abolishedthe antiangiogenic effect of MBD2 knockdown. We furtherdetermined the antiangiogenic activity of reactivated BAI1 in

an in vivo angiogenesis assay. CMs from U251MG cells trans-fected with either MBD2-shRNAs alone or in combinationwith BAI1-shRNAs were mixed with Matrigel plus FGF-2 andVEGF (angioreactor) and s.c. implanted into athymic nudemice. Two weeks later, angioreactors were dissected andvisually inspected for evidence of angiogenesis. Vasculariza-tion was readily observed, and the angiogenic responsehad penetrated deep into the angioreactor with CM fromcontrol shRNA transfected U251MG cells (Fig. 6D, top). Incontrast, whereas an occasional angiogenic response wasobserved in angioreactors exposed to CM from MBD2-shRNA–transfected cells, the extent of this response wasusually minimal and had only superficially penetrated theangioreactor. To directly measure the angiogenic response, wedissociated the cells in the angioreactor and used a fluores-cein-labeled lectin (FITC-lectin) that specifically binds toendothelial cells to quantify the number of murine endothelialcells infiltrated into the angioreactor. A statistically significantdifference in endothelial cell content was found betweenangioreactors exposed to CM from cells transfected withMBD2-shRNA versus MBD2-shRNA in combination with

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Figure 5.ChIP assay shows the specific binding ofMBD2 to theBAI1 promoter. A, cross-linked chromatin was prepared fromBAI1-silent (LN229, U87MG) andBAI1-expressing (LN443) glioma cell lines, sonicated to shear DNA fragments, and immunoprecipitated with anti-MBD2 or MeCP2 antibodies or controlimmunoglobulins (IgG). The immunoprecipitates were then subjected to PCR analysis using primer pairs spanning the BAI1 or MGMT promoters. Bindingof MBD2 to the BAI1 promoter was found only in BAI1-silent cells (LN229, U87MG), whereas MeCP2 did not bind altogether. Binding to theMGMT promoterwas used as a positive control for both proteins. B, the presence of acetylation (AcH3K9) and trimethylation (3MeH3K9) on lysine 9 of histone H3 associateswith the activation status of the BAI1 promoter. ChIPs were carried out in BAI1-expressing (SF188, LN443) and in BAI1-silent (LN229, U87MG) cells. The markof active chromatin (AcH3) is associated with BAI1-expressing cells, whereas that of inactive chromatin (MeH3) with silent cells. C, release of MBD2from the BAI1 promoter in the presence of demethylation agent 5-Aza-dC and MBD2 knockdown. U251MG cells were treated with either 5-Aza-dC(5 mmol/L) for 5 days or transfected with MBD2-shRNA or nonspecific shRNA control for 72 hours before ChIP assays. Note that both 5-Aza-dC andshMBD2 treatments led to decrease of MBD2 binding and concomitant switch from inactive to active chromatin markers on the BAI1 promoter.D, quantification of the MBD2, AcH3K9, and 3MeH3K9 ChIP fractions on the BAI1 promoter from C. ChIP quantification was plotted as a ratio of boundMBD2, AcH3K9, or 3MeH3K9 to input signal and expressed on the y-axis.

Zhu et al.





BAI1-shRNA (Fig. 6D, bottom). Taken together, these resultsshow that restoration of BAI1 expression by MBD2 knock-down reactivates BAI1 synthesis and antiangiogenic activity.

Discussion

Here we provide evidence that the BAI1 gene is epigeneti-cally silenced in GBM and that manipulation of the silencingevent can reactivate BAI1 expression and antiangiogenic

tumor suppressor activity, which can be exploited for ther-apeutic means.

In glioma cell lines, BAI1 silencing could be reversed bytreatment with the demethylating agent 5-Aza-dC. Therefore,our data support the notion that DNAmethylation contributesto inactivation of the BAI1 gene. Transcriptional silencingthrough promoter DNA methylation has been proposed tooccur through several different molecular mechanisms, suchas by direct interference with transcription factor binding, by

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Figure 6. Reactivation of BAI1 expression by MBD2 knockdown restores antiangiogenic activity of BAI1 in the cell CM. A, CM of U251MG glioma cells withreactivated BAI1 expression inhibits HBVEC migration in a scratch-wound assay. Representative photographs of wounded HBVECs before (0 hour) and 8hours after treatment with CM are shown. CM from cells stably transfected with a BAI1 expression vector (U251-BAI1; clone B12) inhibited cell migrationsubstantially (compare second and third photographs). CM from cells transiently transfected for 72 hours with shRNAs against MBD2 or BAI1, singly or incombination, inhibited cell migration differentially (panels 4–6). ct-shRNA, control shRNA. B, quantification ofmigration speed in the scratch-wound assay of A.Final wound width was measured after 8 hours and the distance migrated was calculated. The experiment was repeated 3 times with similar results. Columns,mean; bars, SD (n ¼ 6 for each condition); ***, P < 0.001. C, Western blot showing the levels of BAI1 and secreted vasculostatin (Vstat-120) 72 hours aftertransfection with shRNA expression vectors in U251MG cells used in A and B. WCE, whole-cell extract. D, quantitative in vivo angiogenesis assay.Angioreactors containing CM from U251MG cells transfected with indicated shRNAs were implanted s.c. in athymic nu/numice. Representative images showvascularization of angioreactors from each group 2 weeks later (top). The relative mouse endothelial cell invasion in the angioreactor was quantified bydissociating the cells in the angioreactor, incubating themwith a fluorescently labeled plant lectin specific for endothelial cells, andmeasuring the fluorescenceof cell pellets derived from each group (n ¼ 8, bottom). ***, P < 0.001.






altering the structure of chromatin, and/or by recruiting MBDproteins (10, 11, 29, 30, 33). MBD proteins are critical med-iators of many epigenetic processes in that they interpret themethylation marks on DNA and facilitate the establishment ofa repressive chromatin environment. ChIP assays showedbinding of MBD2 to the CpG island region in the BAI1promoter specifically in cell lines where the gene was methy-lated and silenced, whereas there was no association ofMeCP2 with the methylated BAI1 promoter. Furthermore,in BAI1-silent glioma cell lines, shRNA-directed knockdownof MBD2 resulted in the local depletion of MBD2 and restoredthe chromatin state to one similar to that of expressing celllines (e.g., hyperacetylated at H3K9) and reactivated BAI1expression. Taken together, these results support a mechan-ism wherein the specific binding of MBD2 to the methylatedBAI1 promoter is necessary to maintain transcriptionalrepression of BAI1.

Although DNA methylation has been extensively studied inGBM, the expression and regulation of MBDs have not. For thefirst time, we provide evidence that MBD2 is specificallyoverexpressed in GBM. TheMBD2 gene is located on chromo-some 18q21 (34); no amplification of this region has beenreported in GBM, nor have genomic copy number changes orsomatic mutations for MBD2 been observed in the data fromthe Rembrandt and TCGA glioma databases (data not shown).Therefore, the overexpression of MBD2 observed in a subset ofGBM may result from a different mechanism, such asenhanced transcription. MBD2 has the greatest binding affi-nity for methylated DNA among MBD family proteins in vitro(34), suggesting that MBD2 may be the MBD family memberwith the greatest effect on gene silencing (35). Accumulatingevidence shows that MBD2 is involved in the suppression ofaberrantly methylated tumor suppressor genes by binding tomethylated promoters (36, 37), including p14/ARF and p16/Ink4A (38, 39), 14-3-3s (24), and GSTP1 (glutathione S-trans-ferase p1; ref. 40). In all cases, siRNA-mediated knockdown ofMBD2 resulted in the reactivation of the corresponding genetarget, similar to what we observed for BAI1. Consistent with arole in tumor promotion, previous work has shown thatknockout of MBD2 strongly suppresses intestinal tumorigen-esis in ApcMin mice (41). The underlying mechanism may bethat deficiency of MBD2 elevates levels of the Wnt targetLect2, a Wnt pathway repressor (42).

The recent discovery of 5-hydroxymethylcytosine (5-hmC)in mammalian DNA has added a new dimension to theregulation of DNA methylation and may be particularly rele-vant in the pathogenesis of glioblastomas as the TET enzymesthat catalyze the hydroxylation of methyl cytosine residues areamong those affected by the accumulation of 2-hydroxyglu-tarate that accompanies IDH gene mutations (43). Althoughthe precise function of 5-hmC in epigenetic regulation is notyet completely understood, recent work suggests that it mayfacilitate DNA demethylation through a base excision repairmechanism (44). Furthermore, there is emerging evidence thatthe binding of some MBD proteins, including MBD2b, to DNAis inhibited by 5-hmC (45, 46), and thus the conversion of 5-mCto 5-hmC may play a functional role in the dynamic regulation

of gene expression. Future studies are warranted to determinethe distribution of 5-hmC in the BAI1 gene regulatory regionsand its role in BAI1 gene transcription.

At present, epigenetic approaches in cancer therapy havefocused primarily on inhibitors of the DNA methyltrans-ferases and histone modifiers (e.g., HDACs). Our data sug-gest that MBD2 may also represent a promising cancertherapeutic target. MBD2-null mice display a surprisinglyweak phenotype, and global DNA methylation levels andgenomic imprinting are relatively unaffected by the absenceof MBD2 (47). The fact that MBD2 knockout mice are viableand resistant to tumorigenesis, coupled with the finding thatdownregulation of MBD2 could restore a functional BAI1with potent antiangiogenic activity, makes MBD2 a parti-cularly attractive target for therapeutic intervention forGBM and/or in the prevention of glioma progression.Sequence-specific antisense inhibitors of MBD2 have beenshown to inhibit both anchorage-independent growth ofhuman cancer cell lines in vitro and the growth of humantumor xenografts in vivo (48, 49). Validation of MBD2 as aviable target in GBM will require careful examination of theglobal impact on gene expression, as the potential thera-peutic benefit will depend upon the reprogrammed tran-scriptome tipping the tumor toward an anti- orprotumorigenic biological response. Our data show thatantagonizing MBD2 elicits a global antiangiogenic responsethat is largely dependent upon BAI1 expression and wouldbe expected to suppress tumor growth. In summary, ourresults show a functional role of MBD2 in the repression ofBAI1 in GBM. This study could lead to new therapeuticprospects for the treatment of patients with brain tumors.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors’ Contributions

D. Zhu and E.G. Van Meir conceived experiments, D. Zhu carried out allexperiments, S.B. Hunter provided the GBM tissue array and histologic con-firmation, and P.M. Vertino provided experimental advice. D. Zhu, P.M. Vertino,and E.G. Van Meir wrote the manuscript.

Acknowledgments

We thank all the Van Meir laboratory members for helpful comments, theEmory Biomarker CORE facility for DNA sequencing services, Dr. Daniel J. Bratfor neuropathology diagnosis of GBM samples, Dr. Yuan Liu for help withstatistical analysis, Dr. Y. Nakamura for the BAI1 expression vector, and Narra S.Devi and Zhaobin Zhang for technical support.

Grant Support

NIH R01-CA86335 (E.G. Van Meir), NIH RO1-CA077337 (P.M. Vertino), theSoutheastern Brain Tumor Foundation (D. Zhu and E.G. Van Meir), and theUniversity Research Council of Emory University (E.G. Van Meir).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 1, 2011; revised June 29, 2011; accepted June 30, 2011;published OnlineFirst July 1, 2011.

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2011;71:5859-5870. Published OnlineFirst July 1, 2011.Cancer Res Dan Zhu, Stephen B. Hunter, Paula M. Vertino, et al.

BAI1Suppressor Gene Silencing and Inhibits the Antiangiogenic Function of the Tumor Overexpression of MBD2 in Glioblastoma Maintains Epigenetic

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