O(6)-Methylguanine-DNA Methyltransferase Is a Novel ... · O(6)-Methylguanine-DNA Methyltransferase Is a Novel Negative Effector of Invasion in Glioblastoma Multiforme Manik Chahal1,

Preclinical Development

O(6)-Methylguanine-DNA Methyltransferase Is a NovelNegative Effector of Invasion in Glioblastoma Multiforme

Manik Chahal1, Bassam Abdulkarim1,3, Yaoxian Xu1,3, Marie-Christine Guiot4, Jacob C. Easaw2,Nicolas Stifani3, and Siham Sabri1,3

AbstractThe dismal prognosis of glioblastoma multiforme (GBM) is mostly due to the high propensity of GBM

tumor cells to invade. We reported an inverse relationship between GBM angiogenicity and expression of

the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), which has been extensively

characterized for its role in resistance to alkylating agents used in GBM treatment. In the present study,

given the major role of angiogenesis and invasion in GBM aggressiveness, we aimed to investigate the

relationship between MGMT expression and GBM invasion. Stable overexpression of MGMT in the

U87MG cell line significantly decreased invasion, altered expression of invasion-related genes, decreased

expression of a5b1 integrin and focal adhesion kinase, and reduced their spindle-shaped morphology and

migration compared with the empty vector control. Conversely, short hairpin RNA-mediated stable

knockdown of MGMT or its pharmacologic depletion in the MGMT-positive T98G cell line were required

for increased invasion. The inverse relationship between MGMT and invasion was further validated in

primary GBM patient-derived cell lines. Using paraffin-embedded tumors from patients with newly

diagnosed GBM (n ¼ 59), tumor MGMT promoter hypermethylation (MGMT gene silencing) was

significantly associated with increased immunohistochemical expression of the proinvasive matricellular

protein secreted protein acidic and rich in cysteine (SPARC; P¼ 0.039, x2 test). Taken together, our findings

highlight for the first time the role of MGMT as a negative effector of GBM invasion. Future studies are

warranted to elucidate the role of SPARC in the molecular mechanisms underlying the inverse relationship

between MGMT and GBM invasion and the potential use of MGMT and SPARC as biomarkers of GBM

invasion. Mol Cancer Ther; 11(11); 2440–50. �2012 AACR.

IntroductionThe lethality of glioblastoma multiforme (GBM) stems

from its pronounced infiltrative potential, as cells candiffusely invade beyond the margin of therapeutic resec-tion (1). Consequently, the prognosis of GBM remainspoor, with amedian survival of only 15months followingstandard of care therapy involving surgery, radiotherapy,and chemotherapy with the alkylating agent temozolo-mide (TMZ; 2). Although the putative invasive process(i.e., detachment from the primary tumor site, receptor-

mediated adhesion to the extracellular matrix (ECM),degradation of the ECM, and morphologic alterations) iswell characterized (3), the mechanisms by which cellsinstigate this invasive behavior are still under scrutiny.Therapeutic advances for GBM require a detailed under-standing of the primary mediators of this behavior, asregulators of invasion may play a role in determiningpatient survival (4). Although potential regulators such asthe p75 neurotrophin receptor (5), doublecortex, sema-phorin 3B, secreted protein acidic and rich in cysteine(SPARC; 4), CAAT/enhancer binding protein b (C/EBPb), and STAT3 (6) have been elucidated, their appli-cability as clinical biomarkers or anti-invasive therapeutictargets has not been conclusively determined.

Currently, there are 3 commonly used biomarkers forpatients with brain tumors: isocitrate dehydrogenase 1(IDH1) mutation, 1p/19q codeletion, and most notably,promoter methylation of the gene encoding for the DNArepair protein O6-methylguanine-DNAmethyltransferase(MGMT; ref. 7). Tumoral expression ofMGMT is proposedto mediate resistance to TMZ-induced cytotoxicity viaalkyl transfer at the O6 position of guanine (8). According-ly, correlative studies show that patients with tumorsdisplaying MGMT promoter hypermethylation or lowexpression of MGMT protein [i.e., MGMT(–)] are more

Authors' Affiliations: 1Department of Oncology, Cross Cancer Institute,University of Alberta, Edmonton; 2Department of Oncology, Tom BakerCancer Centre, Calgary, Alberta; 3Department of Oncology, ResearchInstitute of theMcGill University Health Centre; and 4Montreal NeurologicalInstitute, McGill University, Montreal, Quebec, Canada

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

B. Abdulkarim and S. Sabri have contributed equally to this work.

Corresponding Author: Siham Sabri, The Research Institute of the McGillUniversity Health Centre, 1625 Pine AvenueWest, Montreal, Quebec, H3G1A4, Canada. Phone: 514-934-1934; Fax: 514-934-8392; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-11-0977

�2012 American Association for Cancer Research.

MolecularCancer

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likely to benefit from TMZ treatment, compared withpatients with tumors displaying unmethylatedMGMT, orhighMGMT expression [i.e., MGMT(þ); refs. 9, 10]. Thesestudies suggest that MGMT status could be used as apredictive marker for response to alkylating agents.The association betweenMGMT promoter methylation

and a hypermutator phenotype was highlighted in acomprehensive genomic characterization of GBM tumors(11). These alterations could influence functional path-ways dictating tumor phenotype, including invasivebehavior. A potential relationship between MGMT statusand tumor invasion was described in invasive Crooke’scell adenomas, which have decreased expression ofMGMT compared with noninvasive ordinary-type ade-nomas of Cushing’s disease (12), and in gastric carcinoma,in which MGMT promoter methylation was associatedwith lymph node invasion (13). In the context of GBM,Brandes and colleagues (14) recently observed that fol-lowing treatment, GBM tumors with MGMT promotermethylation recurred at more distal sites from the initialradiation field. Although these studies suggest a potentialrelationship between MGMT expression and invasion,thus far, the effect of MGMT expression on GBM inva-siveness has not been explicitly investigated.We have previously characterized a novel inverse rela-

tionship betweenMGMTexpression andGBMangiogeni-city (15). Tumor angiogenesis and invasion, the majorhallmarks of GBM aggressiveness share interdependentmolecular mechanisms of regulation (16–18). We investi-gated the relationship between expression of MGMTprotein andGBM invasiveness using isogenic overexpres-sion and knockdown, pharmacologic depletion ofMGMTin GBM cell lines, and validation in primary patient-derived GBM cell lines. Because of the prominent role ofthe matricellular protein SPARC in GBM invasion in vitroand in vivo (19, 20), we further investigated the relation-ship between tumor MGMT promoter methylation, itsimmunohistochemical expression, and SPARC expres-sion in primary surgical biopsies of patients with GBM.

Materials and MethodsCell culture and drug treatmentGBM cell lines U251, A172, U373, U138, LN18, T98G

(American Type Culture Collection), U87-MG emptyvector (U87/EV), and its MGMT-transfected derivativeU87/MGMT (21) cells were maintained at 37�C, 95%humidifiedair, and5%CO2 inDulbecco’sModifiedEagle’sMedium(DMEM)supplementedwith 10% fetal calf serum(FCS) and 1% penicillin/streptomycin (Invitrogen).Patient-derivedprimaryGBMcell lines originated from

enzymatic dissociation of diagnostic biopsy specimensfrom GBM patients (kindly provided by Dr. KennethPetruk, University of Alberta, Edmonton, Alberta,Canada, and Dr. Joan Turner, Cross Cancer Institute,Edmonton, Alberta, Canada). Primary cell lines weremaintained in DMEM/F12 supplemented with 10% FCSand 1% glutamate (Invitrogen) and were used at thefollowing passages: 10 cell lines less than passage 10, 4

cell lines less than passage 18. All cell lines were tested formycoplasma contamination.

O6-benzylguanine (O6BG, Sigma) was dissolved indimethyl sulfoxide (DMSO). For depletion of MGMTusing O6BG, cells were treated daily for 6 days with 10mmol/L O6BG or DMSO as a control.

Microscopy, immunofluorescence staining, andmorphometric analysis

Brightfield imageswere takenonaZeissAxiovert 200Mmicroscope attached to a Seniscam camera using a ZeissPlan-NEOFLUAR 10�/0.3 or 5�/0.15 lens.

For immunofluorescence analysis, cells were grown oncoverslips coated with 8 mg/cm2 of type I collagen (colla-gen-I) or poly-L lysine (PLL). Cells were then fixed in 3.7%formaldehyde, permeabilized with 0.1% TritonX-100, andstained for filamentous (F)-actin using Alexa Fluor 555phalloidin-TRITC (Invitrogen), or for focal adhesion sitesusing anti-FAK(pY397; BD Biosciences) followed by incu-bation with Alexa Fluor 488 goat anti-mouse IgG (Invitro-gen). The nucleus was visualized with 40, 6-diamidino-2-phenylindole (DAPI; Invitrogen). ImageswerecollectedonaZeissLSM710/ConfoCorObserver.Z1microscopewithaZeiss Plan-APOCHROMAT 40�/1.3 differential interfer-ence contrast oil immersion lens.

Morphologywas analyzed using IntegratedMorphom-etry Analysis in MetaMorph 7.7 imaging software. Cellarea and shape factor (resemblance to a circle) wereassessed, and morphology was determined by dividingcell area by shape factor. High range values indicate amore spindle-shaped morphology.

Western blot analysisWestern blottingwas conducted as described previous-

ly (15).Membraneswere probed forMGMT (cloneMT5.1)CD29/integrin b1, FAK(pY397; all from BD Biosciences),FAK (Upstate), integrin a5 (Cell Signaling), or b-actin(Sigma-Aldrich). Densitometric analysis (Adobe Photo-shop CS3) shows relative band intensities normalized tolevels of b-actin.

Cell motility assayNondirectional cell motility was analyzed by 2-dimen-

sional time-lapse microscopy. Cells were seeded at 75,000cells/well in 6-well plates coated with collagen-I(8 mg/cm2) and allowed to adhere for 45 minutes beforeimaging. Differential interference contrast images wereacquired every minute over a 3-hour period in 2 repre-sentative fields using a Zeiss Axiovert 200M microscopeattached to a CoolSnap camera with a Zeiss Plan-NEO-FLUAR �10/0.3 lens. Composite videos were then con-structed and assessed using the Track Spot Over Timefunction of BitPlane Imaris �64 software.

Invasion assayIn vitro cell invasion was measured using BD BioCoat

Matrigel invasion chambers (8 mmpores; BD Biosciences)followingmanufacturer’s instructions. Cells (25,000)wereserum-starved for 24 hours, seeded in top chambers with

MGMT Negatively Modulates GBM Invasion

www.aacrjournals.org Mol Cancer Ther; 11(11) November 2012 2441




DMEM, and allowed to invade toward a chemoattractant(DMEM þ 10% FCS) for 24 hours. DMSO- or O6BG-treated cells were subjected to invasion assays on day 6in the presence of DMSO or O6BG for 24 hours. Mem-branes were fixed with 3.7% formaldehyde and stainedwith 1% crystal violet. Invasive cells were visualized bybright-field microscopy using a Zeiss Axioskop2 Plusmicroscope attached to an Axiocam color camera with aZeiss FLUAR 5�/0.25 lens and counted using Meta-Morph 7.7 software.

Generation of short hairpin RNA constructs andstable MGMT shRNA transfection

MGMT29mer short hairpinRNA(shRNA; 3 constructs)and control shRNAs were subcloned into the pGFP-V-RSvector (OriGene) at EcoRI and HindIII sites. The shRNA-empty vector plasmid contains a nontargeting sequence.The MGMT subclone containing the 29-mer shRNAsequence (only antisense strands indicated: 50-GGA-CAAGGATTGTGAAATGAAACGCACCA-30) was usedfor further experiments.

T98G cells were seeded into 6-well plates and trans-fected with Lipofectamine 2000 (Invitrogen) followingmanufacturer’s protocol. Stable MGMT shRNA cloneswere generated by puromycin selection (2 mg/mL for2 weeks) and subclones were selected on the basis ofGFP-positivity in single cells. T98shC1.1wasderived fromT98shC1 following further exposure to puromycin.Clonogenic survival assay was used to test TMZ sensi-tivity (15).

Patients’ samples andMGMT promoter methylationTumor tissue paraffin blocks were collected from GBM

patients diagnosed and centrally reviewed in a singleinstitution. Ethical approval was obtained according toinstitutional guidelines (2006, Tom Baker Cancer Center,Calgary, Alberta, Canada). Only patients with newlydiagnosed gliomas (WorldHealthOrganization grade IV)were evaluated (n ¼ 78). MGMT promoter methylationstatus in GBM tumors was characterized by methylation-specific PCR (MS-PCR; 9, 22).

Immunohistochemical staining for MGMT andSPARC

Tissue microarray (TMA) sections (3 cores) wereimmunohistochemically stained for MGMT or SPARCon BenchMark XT (Ventana Medical Systems) usingthe technical protocol XT ultraView DAB v3. Antigenretrieval for MGMT or SPARC used an extended CC2protocol or standard CC1 protocol, respectively (Ven-tana Medical Systems). Incubation with anti-MGMTantibody (1:50 for 2 hours, clone MT3.1; Millipore) oranti-SPARC (1:20,000 for 60 minutes; AON-5031, Hema-tologic Technologies) was followed by incubation withUltraView horseradish peroxidase-conjugated anti-body. Antigen detection was carried out using Ultra-View diaminobenzidine chromogen (Ventana MedicalSystems). Primary antibody was omitted in the negative

control. Endothelial cells served as positive internalcontrol for MGMT and SPARC. Immunohistochemistry(IHC) staining was assessed and scored by a neuropa-thologist (M.-C. Guiot), who was blinded for MGMTpromoter methylation status. Sections were digitalizedusing an Aperio scanner scope XT.

Statistical analysisData are reported as mean � SEM and are representa-

tive of at least 3 independent experiments. Student t testwas used to compare between sets of data for cell lines.Spearman rho correlation coefficients (r values) werecalculated with SAS software to determine correlationsbetween MGMT expression, invasion, and integrinexpression. Correlation is significant at the 0.05 level (2-tailed). The relationship betweenMGMT and SPARCwasassessed using x2 test. P values <0.05 denote statisticalsignificance.

ResultsGBM MGMT(þ) cells are less invasive than MGMT(�) cells

Our previous study showed MGMT expressioninversely correlated with angiogenesis (15). Becauseangiogenesis and invasion are critical in glioma aggres-siveness and share common molecular effectors (16, 17),we sought to determine whether MGMT expressionaffects GBM invasive potential. Using the Gene Ontology(GO) Consortium (23) to analyze a previously conductedcDNA microarray, gene expression profiling (GEP) ofU87/EV andU87/MGMT cells revealed several function-al pathways were differentially regulated (15). Interest-ingly, biologic processes involved in invasion were sig-nificantly differentiallymodulated (Table 1). To assess therelationship between MGMT and GBM invasiveness, weinvestigated in vitro invasion of a panel of 6 establishedcell lines, as well as the MGMT(–) cell line U87MG stablytransfected with a vector encoding for MGMT (U87/MGMT) or the control empty vector (U87/EV; 15, 21).Western blot analysis of MGMT showed that MGMTprotein expression was undetectable in U87/EV, A172,U251 cell lines, and low in the cell line U373MG [24; 10%expression; i.e., MGMT(–)]. The MGMT(þ) cell linesinclude U138MG, (intermediate level of MGMT, 40%expression), U87/MGMT, T98G, and LN18, which exhibitcomparable levels of MGMT protein (Fig. 1A).

Using the Matrigel invasion assay, we established thatMGMT(þ) cell lines exhibited significantly less invasioncompared with MGMT(–) cell lines (P < 0.001). Remark-ably, the invasive potential of U87/MGMT was dramat-ically decreased compared with U87/EV cells (P <0.001; Fig. 1B). Furthermore, correlation coefficient anal-ysis showed a significant negative correlation (r ¼ �0.83,P ¼ 0.011) between MGMT expression and invasivenessof GBM cell lines.

b1 integrin, a regulator of glioma invasion, interactswith ECM components of the perivascular basal lamina(25) and mediates intracellular signaling pathways

Chahal et al.

Mol Cancer Ther; 11(11) November 2012 Molecular Cancer Therapeutics2442




controlling cytoskeletal organization and cell move-ment. Western blot analysis of b1 integrin and one ofits binding partners, the a5 subunit revealed a differentpattern of expression in MGMT(þ) and (–) cells. Nota-bly, compared with U87/EV, U87/MGMT cells dis-played doublet bands reminiscent of a differential gly-cosylation process (26). Interestingly, there was a con-sistent trend of low total expression levels for both b1and a5 subunits in MGMT(þ) cells (Fig. 1C) and anegative correlation between expression of MGMTand a5 (r ¼ �0.805, P ¼ 0.016) or b1 integrin subunits(r ¼ �0.903, P ¼ 0.002). Hence, expression of MGMT issignificantly negatively correlated with invasion ofGBM cell lines, which is in accordance with the alter-ation of the pattern of a5b1 integrin expression.

Overexpression of MGMT significantly decreasedmigration, spindle-shaped morphology, andexpression of focal adhesion kinaseNext, we determined whether alterations in genes

involved in migration (Table 1) translated to a functionalmodification of productive cell motility. We used 2-dimensional time-lapse video microscopy to assess non-directional cell migration on type I collagen (collagen-I),an ECM component found in perivascular regions of thebrain (27)whereGBMinvasionoccurs (1). ComparedwithU87/EV cells, we observed a stark contrast in themode ofmigration of U87/MGMT cells. U87/EV cells move byextending elongated lamellipodia with a long and thintrailing edge, characteristic of the typical "stick-slip" pat-tern of GBM migration (28). Conversely, U87/MGMTcells move by extending broad ruffled lamellipodia at theleading edge without a distinguishable trailing process(Supplementary videos 1 and 2, Fig. 2A, left panel).Compared with U87/EV cells, the mean migration speedof U87/MGMT cells was decreased by 29.7% (P < 0.001),and the mean displacement was strikingly decreasedby 61.5% (P < 0.001; Fig. 2A, right panel). Despite activeformation of protrusions, overexpression of MGMTcorrelated with reduced productive movement and

decreased the migratory speed, which may affect inva-siveness of analyzed cells.

Because motility and invasion necessitate an alter-ation of morphology (29), we next assessed cytoskeletalF-actin organization of cells seeded on coverslips coatedwith PLL or collagen-I. As shown by rhodamine-labeledphalloidin staining, adhesion of U87/EV cells for 1 hourinduced a spindle-shaped morphology (30) character-ized by irregular lamellipodia and filopodia projectionson PLL and increased spreading with evidence of clearactin stress fibers on collagen-I. In sharp contrast, U87/MGMT cells retained their round appearance regardlessof substrate (Fig. 2B, left panel). In addition, quantita-tive evaluation of morphology revealed that U87/EVcells had a more spindle-shaped appearance comparedwith U87/MGMT cells when plated on PLL (P < 0.001)or collagen-I (P < 0.001; Fig. 2B, right panel), which wasmaintained at 24 hours and 48 hours (SupplementaryFig. S1).

Focal adhesion kinase (FAK) is a primary mediator ofthemolecular link between ECM-bound integrins and thecell cytoskeleton, and thus is an important regulator of cellspreading, migration, and invasion (31). GEP analysisrevealed that the expression of the protein tyrosine kinase2 gene (PTK2) encoding FAK mRNA was 2.25-fold lowerin U87/MGMT cells (P ¼ 0.007). Accordingly, immuno-blotting showed that regardless of substrate, the expres-sion of total FAKprotein andFAK(pY397)were decreasedin U87/MGMT compared with U87/EV cells at both 1hour and 24 hours after plating (Fig. 2C). Furthermore,immunofluorescence staining of FAK(pY397) showedprominent focal adhesion sites along the elongated lamel-lipodia of U87/EV cells, but not in U87/MGMT cellsplated on collagen-I (Fig. 2D).

Thus, our results show that overexpression of MGMTcorrelated with profoundmorphologic alterations, differ-ential cytoskeletal F-actin reorganization, and focal adhe-sion turnover, which may account for the reduced migra-tory and invasive phenotype in U87/MGMT comparedwith U87/EV cells.

Table 1. Differential regulation of Gene Ontology biologic processes and KEGG pathways involved ininvasion

Term Count % of gene list P value

Biologic processes involved in invasionGO:0006928 � cell motility 78 3.48% 1.15E-07GO:0016477 � cell migration 56 2.50% 2.52E-07GO:0032989 � cellular structure morphogenesis 70 3.12% 3.48E-03GO:0009611 � response to wounding 61 2.72% 5.33E-03GO:0007155 � cell adhesion 114 5.08% 2.27E-05

KEGG pathways involved in invasionhsa04512 � ECM-receptor interaction 19 0.85% 1.79E-02hsa04510 � focal adhesion 36 1.60% 1.49E-02

NOTE: Gene Expression Omnibus accession no. GSE40776.Abbreviation: KEGG, Kyoto Encyclopedia of Genes and Genomes.






Depletion of MGMT is associated with increasedGBM invasiveness

To establish proof-of-concept that MGMT expressioninfluences invasion in GBM cells, we used stable shRNA-mediated knockdown of endogenous MGMT in T98Gcells. Western blot analysis showed similar levels ofMGMT protein in T98G cells stably transfected withempty vector (T98/EV) compared with parental cells,although MGMT was decreased in 2 randomly selectedclones T98shC1 and T98shC8 (by 70% and 60%, respec-tively). Further knockdown of MGMT was induced inT98shC1.1, a subclonederived fromT98shC1 (by 90%; Fig.3A),which functionally increased their sensitivity to TMZtreatment in clonogenic survival assays (data not shown),although U87/MGMT and T98/EV cell lines were almostcompletely resistant to TMZ treatment (15).

Remarkably, T98shC1, T98shC8, and T98shC1.1 clonesdisplayed a more spindle-shaped morphology comparedwith the cobblestone-like morphology of T98G and T98/EV cell lines (Supplementary Fig. S2A). These phenotypic

alterations translated into increased invasiveness only inthe T98shC1.1 cell line (comparedwith T98/EV,P¼ 0.046;compared with T98G, P ¼ 0.008; Fig. 3B).

The stark difference between invasion of either U87/MGMT and U87/EV (Fig. 1B) or T98/EV and T98shC1.1cell lines (Fig. 3B), which had no detectable levels ofMGMT protein, suggests that MGMT shRNA-inducedknockdown in T98shC1 and T98shC8 cell lines was notsufficient to induce a shift in their invasion. Therefore, wefurther depleted MGMT using O6BG, a pseudosubstrateof MGMT that induces its degradation (32). Comparedwith their respective DMSO control conditions, treatmentwith O6BG (10 mmol/L for 6 days) decreased MGMTexpression by 80% in both T98G and T98/EV cells (Fig.3C). This depletion was associated with acquisition ofspindle-shaped morphology in both cell lines (Supple-mentary Fig. S2B). When compared with DMSO control,treatment with O6BG further depleted MGMT protein by20% and 30% in T98shC1 and T98shC8 cell lines, respec-tively, thereby depleting the total amount of MGMTprotein by 80% (Fig. 3C). Remarkably, although O6BGtreatment did not affect invasion ofU87/EVcell line in theabsence of endogenous MGMT, invasion was significant-ly increased by a fold change of 2.72 for T98G cells (P ¼0.027), 2.07 for T98/EV cells (P ¼ 0.026), 3.63 for T98shC1cells (P ¼ 0.017), and 2.06 for T98shC8 cells (P ¼ 0.019)treated with O6BG compared with respective DMSOcontrols (Fig. 3D). Thus, as shown by shRNA-mediatedknockdown of MGMT (T98shC1.1) and depletion byO6BG treatment, decreased MGMT expression by 80%seems to be a prerequisite for increased in vitro invasive-ness of GBM cell lines.

MGMT(þ) primary patient-derived GBM cellsdisplay an invasive phenotype compared withMGMT(�) cells

To validate our findings in isogenic overexpression andknockdownmodels and determine their potential clinicalvalidity, we examined invasion of 14 primary patient-derived GBM cell lines. Western blotting revealed that 9out of 14 cell lineswereMGMT(þ) whereas the remaining5 cell lineswereMGMT(–; Fig. 4A). Overall, theMGMT(–)cell lines were significantly more invasive compared withtheMGMT(þ) cell lines (P¼ 0.048), and notably, 3 of the 5MGMT(–) cell lines displayed levels of invasion compa-rable to U87/EV. Interestingly, 6 of the 9 MGMT(þ) celllines exhibited a similar invasive profile as U87/MGMT,although the remaining 3 were intermediately invasive(Fig. 4B).

We further investigated whether depletion of MGMTprotein was associated with increased invasiveness inprimary cells. In the absence of endogenous MGMT pro-tein, O6BG treatment of P-GM1 cells (10 mmol/L for 6days) did not significantly alter invasion. Depletion ofMGMT by O6BG by 80% in P-GM7 and in P-GM13 cellswas associated with a significant increase in invasioncompared with DMSO control (1.85-fold, P ¼ 0.018 and2.46-fold, P < 0.001), respectively). Conversely, depletion

Figure 1. Inverse relationship between MGMT protein expression andinvasion in human GBM cell lines. A, Western blotting showing MGMTprotein levels normalized to actin in 8 human GBM cell lines includingU87MG cells stably transfected with empty vector (U87/EV) or MGMT(U87/MGMT). B, histogram shows the number of invading MGMT(–)compared with MGMT(þ) cells assessed by Matrigel invasion assay(means� SEM; n¼ 3); ��, P < 0.001. Representative photomicrographs(inset) illustrate decreased invasion of U87/MGMT compared with U87/EV cells. Scale bar, 300 mm. C, immunoblots of the same panel of GBMcell lines shows expression of b1 and a5 integrin subunits. Note thatoverexpression ofMGMTdecreased invasion and reduced expression ofa5b1 integrin in U87/MGMT compared with U87/EV cells.

Chahal et al.





of MGMT by only 60% in O6BG-treated P-GM5 cells (Fig.4C) did not induce a significant increase in invasion (Fig.4D). These findings corroborate that drastic decrease ofMGMT expression is associated with increased in vitroinvasion of established andprimary patient-derivedGBMcell lines.

Relationship between MGMT status and expressionof SPARC in GBM patientsTo substantiate the clinical relevance of our findings,

we sought to investigate the relationship betweenMGMT and the expression of SPARC, a well-knownproinvasive molecule (33) in a series of tumor biopsiesfrom newly diagnosed GBM patients with no prior

history of radiotherapy or chemotherapy (n ¼ 78).Because the clinical value of immunohistochemicaldetection of MGMT protein is still controversial, wefirst investigated the correlation between MGMTexpression and SPARC using analysis of MGMT pro-moter methylation by MS-PCR, which has been pro-spectively carried out in formalin-fixed paraffin-embed-ded (FFPE) tumors form this cohort of patients. MS-PCRresults were not available in a total of 14 cases (insuf-ficient tissue for analysis or technically unable to obtainresults). Patients were dichotomized as methylated orunmethylated. MGMT promoter was found methylatedin 58% (37/64) of cases, in accordance with previousreports [34%–68%, with a mean of 46% (34)].

Figure 2. Overexpression of MGMT inU87MG cells decreased the migratoryphenotype, expression, and activationof FAK. A, left, high-magnificationimages of U87/EV and U87/MGMTcells migrating on collagen-I andrecorded by time-lapse video. Right,histograms show average migrationspeed and displacement in U87/EVand U87MGMT cells (>50 cells). B, left,U87/EV and U87/MGMT cells platedon collagen-I or PLL for 1 hour werestained for F-actin (red) and nuclearDNA (blue). Right, histogram showsquantitative evaluation of morphologyas determined by cell area and shapefactor (>100 cells; means � SEM; n ¼3); ��, P < 0.001. C, Western blottingshowing FAK(Y397) and total-FAKlevels normalized to actin in U87/EVand U87/MGMT cells plated oncollagen-I or PLL for 1 hour and 24hours. D, immunofluorescence of FAK(pY397; green) and nuclear DNA (blue)in U87/EV and U87/MGMT cells platedon collagen-I or PLL for 1 hour. Notethat overexpression of MGMT (U87/MGMT) decreased migration, thespindle-shaped appearance,activation, and expression of FAK andthe number of focal adhesion sitescompared with U87/EV cells. Scalebars, 100 mm.






For the purpose of this study, we assessed immunohis-tochemical expression of MGMT using TMA sections ofthe same series ofGBMpatients (n¼ 78). Twenty-five of 78cases (32%) were scored negative (0) or showed a hetero-geneous tumor staining (1þ and 2þ), whereas 53 tumorsamples (68%) showed homogeneous nuclear MGMTimmunostaining (�90% MGMT(þ) tumor cells: 3þ; Fig.5A–F).

As previously reported, we observed a concordancebetween MGMT expression and MGMT promoter meth-

ylation status in 56% of analyzed samples (35), althoughthe subgroup of patients with unmethylatedMGMT pro-moter displayed a strong concordance with MGMT-immunopositivity (74%).

IHC staining of SPARC showed cytoplasmic localiza-tion in GBM tumor cells. On the basis of the percentage ofcytoplasmic positive tumor cells, of the 70GBMcaseswithavailable data (insufficient tissue on TMA sections, n¼ 8),9were scored as negative toweakly positive (13%, score¼0 or 1), 17 were moderately positive (24%, score ¼ 2), and44 were strongly positive (63%, score ¼ 3; Fig. 5G–N).

Strikingly, MGMT promoter methylation was signifi-cantly associated with increased immunohistochemicalexpression of SPARC (score¼ 3 vs. score¼ 0, 1, and 2; n¼59 cases with available data for both MGMT status andSPARC by IHC). Up to 76% of cases (25 out of 33) withMGMT promoter methylation were strongly positive forSPARC (P ¼ 0.039, x2 test). In contrast, immunopositivityof MGMT failed to correlate with SPARC expression byIHC staining (P ¼ 0.405).

DiscussionIn the present study, we identified a novel role for

MGMT protein as a potential negative molecular andphenotypic regulator of GBM invasion, the main causeof treatment failure for patients with GBM. Using estab-lished and isogenic GBM cell lines differing in MGMTprotein expression, primary GBM cell lines, and archivedFFPE tumors from GBM patients, we provide the firstdirect evidence of an inverse relationship betweenMGMTexpression and GBM invasiveness.

In particular, stable overexpression of MGMT decreas-ed invasiveness of U87/MGMT cells comparedwith theircounterpart and induced profound alterations of genetic,molecular, and phenotypic features. First, GEP analysisrevealed modulation of a plethora of key genes encodingstructural and signaling proteins involved in cytoskeletonremodeling, cell adhesion, and movement. Second,molecular determinantswhichmay account for decreasedinvasion could be related to (i) a differential integrinprofile, i.e., decreased total expression levels and presum-ably alterations of thematuration process ofa5b1 integrin,which may reduce cell spreading and migration (26) and(ii) decreased expression and activation of FAK. Owing tothe role of FAK in cell motility, we selected FAK for in-depth quantitative studies and validated decreased totalexpression of FAK shown by GEP analysis. In accordancewith decreased FAK-pTyr397 byWestern blotting, immu-nofluorescence showed a reduced number of focal adhe-sions sites in U87/MGMT. Third, morphologic changeswere evidenced by (i) a differential cytoskeletal F-actinreorganization and cell spreading on PLL and collagen-Isubstrates and (ii) motility reminiscent of a mesenchymalmode of migration for U87 cells (36), while quantitativemorphometric analysis and monitoring cell migrationby time-lapse showed an amoeboid motility for U87/MGMT cells. Failure to extend long polarized pseudopo-dia and decreased tyrosine phosphorylation of FAK in

Figure 3. MGMT-shRNA knockdown and its depletion in T98G cells areassociated with increased invasion. A, Western blotting of MGMTnormalized to actin in T98G, T98/EV, T98shC1, T98shC1.1, and T98shC8cells. B, histogram shows that MGMT-shRNA knockdown significantlyincreased invasion only in T98shC1.1 cells as determined by Matrigelinvasion assay (means � SEM; n ¼ 3). C, Western blotting showsdepletion of MGMT protein by O6BG (10 mmol/L, 6 days) compared withDMSO control. D, histogram shows O6BG does not alter MGMT(–) U87/EV cell invasion, but increased invasion of T98G and T98/EV cells, asillustrated in representative photomicrographs (right; scale bar, 300 mm).Additional depletion of MGMT by O6BG is required for increasedinvasiveness of T98shC1 and T98shC8 cells (means � SEM foldincrease; n ¼ 3; �, P < 0.05).

Chahal et al.





U87/MGMT agrees with studies showing the require-ment for regulated focal adhesion turnover for mesen-chymal motility (37). Understanding how MGMT affectsthe dynamics of actin cytoskeleton turnover and elicits anamoeboid mode of migration deserves further validationon 3-dimensional substrates and may ultimately identifynew targets to efficiently reduce invasiveness in vivo.Overall, forced expression of MGMT provided some

mechanistic insights into potential concerted effectorsleading to decreased invasiveness. Conversely, shRNA-mediated knockdown of endogenousMGMT by 90%wasrequired for increased invasion of T98shC1.1 cell line. Inaddition, drastic depletion of MGMT protein by O6BG inT98G, as well as T98shC1 and T98shC8 cell lines wasrequired for their increase invasiveness compared withparental untreated cells.

Although we establish for the first time the relevanceof MGMT to invasion in vitro, validation of our findingsin primary GBM cells and patients archived tumorshighlights their potential clinical significance.We showedfor the first time an inverse relationship between MGMTand the proinvasive protein SPARC in a series of primaryGBM patients. Interestingly, SPARC has been shownto promote migration and invasion through direct phys-ical interactions with b1 integrin (38, 39) and activationof important signaling molecules for glioma cell motility,such as integrin-linked kinase and FAK (40, 41). Inparticular, MGMT promoter hypermethylation (which isexpected to reflect low levels of MGMT protein) wassignificantly associated with high levels of tumoralSPARC expression. As previously reported in otherstudies, we found a limited concordance between

Figure 4. MGMT(þ) primary patient-derived GBM cells are less invasivethan MGMT(–) cells. A, Westernblotting shows MGMT expressionnormalized to actin in 14 primaryGBM cell lines. B, histogram showsthe number of invading MGMT(–)compared with MGMT(þ) cellsassessed by Matrigel invasion assay(means � SEM; n ¼ 3;�, P < 0.05). C, Western blottingshows depletion ofMGMTprotein byO6BG (10 mmol/L, 6 days) wasdrastic in P-GM7 and P-GM13, butnot in P-GM5 cells. D, histogramshows O6BG significantly increasedinvasion of P-GM7 and P-GM13, butnotP-GM5cells, andhasnoeffect onMGMT(–) P-GM1 cell invasion(means � SEM fold increase; n ¼ 3;��, P < 0.001).






immunopositivity of MGMT and promoter methylation(42) despite our caution in analyzing immunohistochem-ical expression of MGMT in patients with similar highglioma grade and without prior treatment with chemo-therapy or radiotherapy (43). Potential limiting factors forthe validation of immunohistochemical analysis ofMGMT as amarker of GBM invasiveness could be relatedto the relatively small sample size in our cohort andMGMT intratumoral heterogeneity. In particular, MGMTprotein expression has been shown to decrease progres-sively from the inner to the peripheral layer in GBMsamples (44). Interestingly, tumor cells located in the brainparenchyma (45) or beyond themargin of tumor resection(residual tumor cells; 46) are more invasive than cellswithin the tumor core.

Our study reveals that SPARCmight be involved in themolecular mechanisms underlying the inverse relation-ship between MGMT and GBM invasion. In a recentreport, high expression of the membrane–cytoskeleton

linker protein, Ezrin was correlated with loss of MGMTexpression and increased invasion in esophageal cancer(47). Physical interactions of MGMT with binding part-ners may also account for downstream alterations ofbiologic processes, such as the transcription integratorCREB-binding protein CBP/p300 (48) and the Histoneacetyltransferase p300 (EP300) known to regulate tran-scription via chromatin remodeling (Protein InteractionNetwork Analysis; ref. 49).

In sum, our data establish MGMT as a potential newnegative effector of GBM invasion beyond its well-knownrole in response to alkylating agents. Future preclinicalstudies are warranted to investigate the inverse relation-ship between MGMT and SPARC, and validate the prog-nostic value of MGMT and SPARC as new invasionbiomarkers in prospective studies testing anti-invasivetherapies targeting invasive glioma cells with high levelsof SPARC.

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

Authors' ContributionsConception and design: M. Chahal, B. Abdulkarim, J.C. Easaw, S. SabriDevelopment of methodology: M. Chahal, Y. Xu, S. SabriAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Chahal, B. Abdulkarim, M.-C. GuiotAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis):M.Chahal, B.Abdulkarim,M.-C. Guiot, J.C.Easaw, N. Stifani, S. SabriWriting, review, and/or revision of themanuscript:M.Chahal, N. Stifani,S. SabriAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): B. Abdulkarim, M.-C. Guiot, J.C.Easaw, N. StifaniStudy supervision: B. Abdulkarim, J.C. Easaw, S. Sabri

AcknowledgmentsThe authors thankDr. Roseline Godbout andDr.ManishAghi for GBM

cell lines, Bonnie Andrais for technical assistance, andDr. Xeujun Sun andGeraldine Barron for cell imaging assistance.

Grant SupportThis study has been funded by the Alberta Cancer Foundation and a

generous donation from St John Bosco Elementary School (Edmonton,AB). M. Chahal is the recipient of a Graduate Studentship Award fromAlberta Cancer Foundation.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedDecember 5, 2011; revisedAugust 6, 2012; acceptedAugust 22,2012; published OnlineFirst September 17, 2012.

References1. Giese A, Bjerkvig R, Berens ME, Westphal M. Cost of migration:

invasion of malignant gliomas and implications for treatment. J ClinOncol 2003;21:1624–36.

2. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B,Taphoorn MJ, et al. Radiotherapy plus concomitant and adjuvanttemozolomide for glioblastoma. N Engl J Med 2005;352:987–96.

3. Nakada M, Okada Y, Yamashita J. The role of matrix metalloprotei-nases in glioma invasion. Front Biosci 2003;8:e261–9.

4. Rich JN, Hans C, Jones B, Iversen ES, McLendon RE, Rasheed BK,et al. Gene expression profiling and genetic markers in glioblastomasurvival. Cancer Res 2005;65:4051–8.

5. Johnston AL, Lun X, Rahn JJ, Liacini A, Wang L, Hamilton MG, et al.The p75 neurotrophin receptor is a central regulator of glioma invasion.PLoS Biol 2007;5:e212.

6. CarroMS, LimWK, AlvarezMJ, Bollo RJ, Zhao X, Snyder EY, et al. Thetranscriptional network for mesenchymal transformation of braintumours. Nature 2010;463:318–25.

MG

MT

A B C

D E F

G H I J

K L M NSPA

RC

Figure 5. Representative IHC staining of MGMT and SPARC.Photomicrographs display expression of MGMT (A–F; score: 0, 2þ, and3þ, respectively) and SPARC (G–N; score: 0, 1, 2, and 3, respectively) inTMA of GBM patients (A–C and G–J). The insets show highermagnification (D–F and K–N). Endothelial cells were used as internalpositive control (arrows). Scale bars, 200 mm (A–C and G–J), 50 mm(D–F and K–N).

Chahal et al.





7. Tabatabai G, Stupp R, van den Bent MJ, Hegi ME, Tonn JC, Wick W,et al. Molecular diagnostics of gliomas: the clinical perspective. ActaNeuropathol 2010;120:585–92.

8. Esteller M, Garcia-Foncillas J, Andion E, Goodman SN, Hidalgo OF,Vanaclocha V, et al. Inactivation of theDNA-repair geneMGMTand theclinical response of gliomas to alkylating agents. N Engl J Med2000;343:1350–4.

9. Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M,et al. MGMT gene silencing and benefit from temozolomide in glio-blastoma. N Engl J Med 2005;352:997–1003.

10. Kreth S, Thon N, Eigenbrod S, Lutz J, Ledderose C, Egensperger R,et al. O-Methylguanine-DNA methyltransferase (MGMT) mRNAexpression predicts outcome in malignant glioma independent ofMGMT promoter methylation. PLoS One 2011;6:e17156.

11. Network TCGAR. Comprehensive genomic characterization defineshuman glioblastoma genes and core pathways. Nature 2008;455:1061–8.

12. Takeshita A, Inoshita N, Taguchi M, Okuda C, Fukuhara N, Oyama K,et al. High incidence of low O(6)-methylguanine DNA methyltransfer-ase expression in invasive macroadenomas of Cushing's disease. EurJ Endocrinol 2009;161:553–9.

13. Park TJ, Han SU, Cho YK, Paik WK, Kim YB, Lim IK. Methylation of O(6)-methylguanine-DNA methyltransferase gene is associated signif-icantly with K-ras mutation, lymph node invasion, tumor staging, anddisease free survival in patients with gastric carcinoma. Cancer 2001;92:2760–8.

14. Brandes AA, Tosoni A, Franceschi E, Sotti G, Frezza G, Amista P, et al.Recurrence pattern after temozolomide concomitant with and adju-vant to radiotherapy in newly diagnosed patients with glioblastoma:correlation With MGMT promoter methylation status. J Clin Oncol2009;27:1275–9.

15. Chahal M, Xu Y, Lesniak D, Graham K, Famulski K, Christensen JG,et al. MGMT modulates glioblastoma angiogenesis and responseto the tyrosine kinase inhibitor sunitinib. Neuro Oncol 2010;12:822–33.

16. Vajkoczy P, Goldbrunner R, Farhadi M, Vince G, Schilling L, Tonn JC,et al. Glioma cell migration is associated with glioma-induced angio-genesis in vivo. Int J Dev Neurosci 1999;17:557–63.

17. Eccles SA. Parallels in invasion and angiogenesis provide pivotalpoints for therapeutic intervention. Int J Dev Biol 2004;48:583–98.

18. OnishiM, Ichikawa T, Kurozumi K, Date I. Angiogenesis and invasion inglioma. Brain Tumor Pathol 2011;28:13–24.

19. Schultz C, Lemke N, Ge S, Golembieski WA, Rempel SA.Secreted protein acidic and rich in cysteine promotes gliomainvasion and delays tumor growth in vivo. Cancer Res 2002;62:6270–7.

20. Thomas SL, Alam R, Lemke N, Schultz LR, Gutierrez JA, Rempel SA.PTEN augments SPARC suppression of proliferation and inhibitsSPARC-induced migration by suppressing SHC-RAF-ERK and AKTsignaling. Neuro Oncol 2010;12:941–55.

21. Aghi M, Rabkin S, Martuza RL. Effect of chemotherapy-induced DNArepair on oncolytic herpes simplex viral replication. J Natl Cancer Inst2006;98:38–50.

22. Hamilton MG, Roldan G, Magliocco A, McIntyre JB, Parney I, EasawJC. Determination of the methylation status of MGMT in differentregions within glioblastoma multiforme. J Neurooncol 2010;102:255–60.

23. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al.Gene ontology: tool for the unification of biology. The Gene OntologyConsortium. Nat Genet 2000;25:25–9.

24. Kanzawa T, Germano IM, Kondo Y, Ito H, Kyo S, Kondo S.Inhibition of telomerase activity in malignant glioma cells correlateswith their sensitivity to temozolomide. Br J Cancer 2003;89:922–9.

25. D'Abaco GM, Kaye AH. Integrins: molecular determinants of gliomainvasion. J Clin Neurosci 2007;14:1041–8.

26. Gu J, Isaji T, Sato Y, Kariya Y, Fukuda T. Importance ofN-glycosylationon alpha5beta1 integrin for its biological functions. Biol Pharm Bull2009;32:780–5.

27. Gladson CL. The extracellular matrix of gliomas: modulation of cellfunction. J Neuropathol Exp Neurol 1999;58:1029–40.

28. Ulrich TA, de Juan Pardo EM, Kumar S. The mechanical rigidity of theextracellularmatrix regulates the structure,motility, andproliferation ofglioma cells. Cancer Res 2009;69:4167–74.

29. Demuth T, BerensME.Molecular mechanisms of glioma cell migrationand invasion. J Neurooncol 2004;70:217–28.

30. Zhong J, Paul A, Kellie SJ, O'Neill GM. Mesenchymal migrationas a therapeutic target in glioblastoma. J Oncol 2010;2010:430142.

31. Natarajan M, Hecker TP, Gladson CL. FAK signaling in anaplasticastrocytoma and glioblastoma tumors. Cancer J 2003;9:126–33.

32. Dolan ME, Moschel RC, Pegg AE. Depletion of mammalian O6-alkyl-guanine-DNA alkyltransferase activity by O6-benzylguanine providesa means to evaluate the role of this protein in protection againstcarcinogenic and therapeutic alkylating agents. Proc Natl Acad SciU S A 1990;87:5368–72.

33. Arnold SA, Brekken RA. SPARC: a matricellular regulator of tumori-genesis. J Cell Commun Signal 2009;3:255–73.

34. Weller M, Stupp R, Reifenberger G, Brandes AA, van den Bent MJ,Wick W, et al. MGMT promoter methylation in malignantgliomas: ready for personalized medicine? Nat Rev Neurol 2010;6:39–51.

35. Cao VT, Jung TY, Jung S, Jin SG, Moon KS, Kim IY, et al. Thecorrelation and prognostic significance of MGMT promoter methyla-tion and MGMT protein in glioblastomas. Neurosurgery 2009;65:866–75; discussion 75.

36. Yamazaki D, Kurisu S, Takenawa T. Involvement of Rac and Rhosignaling in cancer cell motility in 3D substrates. Oncogene 2009;28:1570–83.

37. Carragher NO, Walker SM, Scott Carragher LA, Harris F, Sawyer TK,Brunton VG, et al. Calpain 2 and Src dependence distinguishesmesenchymal and amoeboid modes of tumour cell invasion: a link tointegrin function. Oncogene 2006;25:5726–40.

38. Nie J, Chang B, Traktuev DO, Sun J, March K, Chan L, et al. IFATScollection: combinatorial peptides identify alpha5beta1 integrin as areceptor for the matricellular protein SPARC on adipose stromal cells.Stem Cells 2008;26:2735–45.

39. Weaver MS, Workman G, Sage EH. The copper binding domain ofSPARC mediates cell survival in vitro via interaction with integrinbeta1 and activation of integrin-linked kinase. J Biol Chem 2008;283:22826–37.

40. Barker TH, Baneyx G, Cardo-Vila M, Workman GA, Weaver M, MenonPM, et al. SPARC regulates extracellular matrix organization throughits modulation of integrin-linked kinase activity. J Biol Chem2005;280:36483–93.

41. Shi Q, BaoS, Song L,WuQ, Bigner DD,HjelmelandAB, et al. TargetingSPARC expression decreases glioma cellular survival and invasionassociated with reduced activities of FAK and ILK kinases. Oncogene2007;26:4084–94.

42. Preusser M, Charles Janzer R, Felsberg J, Reifenberger G,Hamou MF, Diserens AC, et al. Anti-O6-methylguanine-methyl-transferase (MGMT) immunohistochemistry in glioblastoma multi-forme: observer variability and lack of association with patientsurvival impede its use as clinical biomarker. Brain Pathol 2008;18:520–32.

43. Capper D, Mittelbronn M, Meyermann R, Schittenhelm J. Pitfalls in theassessment of MGMT expression and in its correlation with survival indiffuse astrocytomas: proposal of a feasible immunohistochemicalapproach. Acta Neuropathol 2008;115:249–59.

44. Della PuppaA, Persano L,Masi G, Rampazzo E, Sinigaglia A, PistollatoF, et al. MGMT expression and promoter methylation status maydepend on the site of surgical sample collection within glioblastoma:a possible pitfall in stratification of patients? J Neurooncol 2011;106:33–41.

45. Hoelzinger DB, Mariani L, Weis J, Woyke T, Berens TJ, McDonoughWS, et al. Gene expression profile of glioblastoma multiformeinvasive phenotype points to new therapeutic targets. Neoplasia2005;7:7–16.






46. GlasM, Rath BH, SimonM, Reinartz R, Schramme A, Trageser D, et al.Residual tumor cells are unique cellular targets in glioblastoma. AnnNeurol 2010;68:264–9.

47. SuY, LiuR, Sheng J, LiuH,WangY, PanE, et al.Malignant progressionin O(6)-methylguanine-DNA methyltransferase-deficient esophagealcancer cells is associated with Ezrin protein. DNA Cell Biol 2011;31:856–66.

48. Teo AK, Oh HK, Ali RB, Li BF. Themodified human DNA repair enzymeO(6)-methylguanine-DNA methyltransferase is a negative regulator ofestrogen receptor-mediated transcription upon alkylation DNA dam-age. Mol Cell Biol 2001;21:7105–14.

49. WuJ, Vallenius T,OvaskaK,Westermarck J,Makela TP,Hautaniemi S.Integrated network analysis platform for protein-protein interactions.Nat Methods 2009;6:75–7.

Chahal et al.





2012;11:2440-2450. Published OnlineFirst September 17, 2012.Mol Cancer Ther Manik Chahal, Bassam Abdulkarim, Yaoxian Xu, et al. Effector of Invasion in Glioblastoma MultiformeO(6)-Methylguanine-DNA Methyltransferase Is a Novel Negative

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O(6)-Methylguanine-DNA Methyltransferase Is a Novel ... · O(6)-Methylguanine-DNA Methyltransferase Is a Novel Negative Effector of Invasion in Glioblastoma Multiforme Manik Chahal1,