Transglutaminase 2 Inhibition Reverses ......3.5 mm. The brain of each mouse was harvested andﬁxed in 4% paraformaldehyde. For the subcutaneous mouse model, cells were injected into

Tumor and Stem Cell Biology

Transglutaminase 2 Inhibition ReversesMesenchymal Transdifferentiation of Glioma StemCells by Regulating C/EBPb SignalingJinlong Yin1,2, Young Taek Oh2,3, Jeong-Yub Kim4,5, Sung Soo Kim1, Eunji Choi6,Tae Hoon Kim2, Jun Hee Hong2, Nakho Chang3,7, Hee Jin Cho3,7, Jason K. Sa3,7,Jeong Cheol Kim4, Hyung Joon Kwon6, Saewhan Park1,Weiwei Lin1, Ichiro Nakano8,9,Ho-Shin Gwak1,2, Heon Yoo1,2, Seung-Hoon Lee10, Jeongwu Lee11, Jong Heon Kim1,12,Soo-Youl Kim12, Do-Hyun Nam3,7,13, Myung-Jin Park4, and Jong Bae Park1,2

Abstract

Necrosis is a hallmark of glioblastoma (GBM) and is respon-sible for poor prognosis and resistance to conventional ther-apies. However, the molecular mechanisms underlying necrot-ic microenvironment-induced malignancy of GBM have notbeen elucidated. Here, we report that transglutaminase 2(TGM2) is upregulated in the perinecrotic region of GBM andtriggered mesenchymal (MES) transdifferentiation of gliomastem cells (GSC) by regulating master transcription factors(TF), such as C/EBPb, TAZ, and STAT3. TGM2 expression wasinduced by macrophages/microglia-derived cytokines via NF-kB activation and further degraded DNA damage–inducibletranscript 3 (GADD153) to induce C/EBPb expression, result-

ing in expression of the MES transcriptome. Downregulationof TGM2 decreased sphere-forming ability, tumor size, andradioresistance and survival in a xenograft mouse modelthrough a loss of the MES signature. A TGM2-specific inhibitorGK921 blocked MES transdifferentiation and showed signifi-cant therapeutic efficacy in mouse models of GSC. Moreover,TGM2 expression was significantly increased in recurrent MESpatients and inversely correlated with patient prognosis. Col-lectively, our results indicate that TGM2 is a key molecularswitch of necrosis-induced MES transdifferentiation and animportant therapeutic target for MES GBM. Cancer Res; 77(18);4973–84. �2017 AACR.

IntroductionGlioblastoma multiforme (GBM) is the most malignant glial

tumor and is associated with extremely poor survival (1). Currentstandard treatments, radiotherapy and chemotherapy have notimproved the poor prognosis ofGBM,which has amedian overallsurvival of approximately 14 months and a 2-year survival rate ofless than 10% (2). Analysis of large-scale gene expression andgenomic datasets segregated GBM into four groups—proneural(PN), neural, classical (CL), and mesenchymal (MES; ref. 3).Notably, the MES subtype of GBM is associated with relativelypoor prognosis compared with that of the other subtypes andshows resistance to conventional therapy (4). Moreover, severalreports have suggested that MES transdifferentiation from othersubtypes occurs during GBM progression due to the microenvi-ronment or therapeutic stimuli (4–8). Therefore, it is very impor-tant to elucidate the detailed molecular mechanisms of MEStransdifferentiation during GBM tumor progression to developfuture therapeutics.

Necrosis, a hallmark of GBM, has been suggested to be respon-sible for the poor prognosis and resistance to conventionaltherapies (9, 10). The necrotic microenvironment contains infil-trating macrophages/microglia and glioma stem cells (GSC), andhypoxia-associated genes are expressed (11–14). Interestingly,patients with the MES subtype showed a high level of necrosis(3), and transcriptome analysis of the perinecrotic regions alsoshowed high MES activity (15). Moreover, analysis of The CancerGenome Atlas (TCGA) data set showed that non-MES GBMs

1Department of System Cancer Science, Graduate School of Cancer Science andPolicy, National Cancer Center, Goyang, Korea. 2Specific Organs Cancer Branch,Research Institute and Hospital, National Cancer Center, Goyang, Korea.3Department of Health Sciences and Technology, SAIHST, SungkyunkwanUniversity, Seoul, Korea. 4Division of Radiation Cancer Research, ResearchCenter for Radio-Senescence, Korea Institute of Radiological and MedicalSciences, Seoul, Korea. 5Department of Pathology, College of Medicine, KoreaUniversity, Seoul, Korea. 6Department of Cancer Control and Policy, GraduateSchool of Cancer Science and Policy, National Cancer Center, Goyang, Korea.7Institute for Refractory Cancer Research, Samsung Medical Center, Seoul,Korea. 8Department of Neurosurgery, University of Alabama at Birmingham,Birmingham, Alabama. 9UAB Comprehensive Cancer Center, University ofAlabama at Birmingham, Birmingham, Alabama. 10Department of Neurosurgery,Eulji University School of Medicine, Daejeon, Korea. 11Department of Stem CellBiology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic,Cleveland, Ohio. 12Cancer Cell and Molecular Biology Branch, Research Instituteand Hospital, National Cancer Center, Goyang, Korea. 13Department of Neuro-surgery, Samsung Medical Center, Sungkyunkwan University School of Medi-cine, Seoul, Korea.

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

J. Yin, Y.T. Oh, J.Y. Kim contributed equally to this article.

CorrespondingAuthors: Jong Bae Park, Department of System Cancer Science,Graduate School of Cancer Science and Policy, National Cancer Center, Goyang10408, Korea. Phone: 82-31-920-2450; Fax: 82-31-920-2006; E-mail:[email protected]; and Myung-Jin Park, [email protected]; and Do-Hyun Nam,[email protected]

doi: 10.1158/0008-5472.CAN-17-0388

�2017 American Association for Cancer Research.

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became transcriptionally similar to theMES subtype with increas-ing levels of necrosis (15). Collectively, these results indicate thatthe necrotic microenvironment may be involved in MES trans-differentiation during GBM progression. A recent analysis oflongitudinal genomic and transcriptomic data showed thatexpression-based subtype change is a major event of GBM recur-rence (16). Because subtype transdifferentiation is affected bytherapy-induced necrosis, we hypothesized that therapy-inducedMES subtype change due to the necrotic microenvironment is themajor cause of acquired resistance to conventional therapy andrecurrence.

To test our hypothesis, we identified the molecular mechan-isms underlying MES transdifferentiation in the necrotic regionsof GBM. We analyzed common population of genes betweennecrosis-associated genes and MES GSC-enriched genes. Here,we showed that perinecrotic areas highly expressed transgluta-minase 2 (TGM2), which contributed to the MES transdiffer-entiation of GSC via regulation of master transcription factors(TF) of MES GBM.

Materials and MethodsCell culture

Astrocyte and 293T cells were maintained in DMEM supple-mented with 10% FBS (HyClone). Human NSCs were pur-chased from Millipore and cultured according to the manufac-turer's instructions. Patient-derived GBM stem cells (83NS,131, 528NS, 84NS, 047T, and 352T2) were maintained inDMEM/F-12 supplemented with B27 (Invitrogen), EGF (10ng/mL; R&D Systems), and bFGF (5 ng/mL; R&D Systems).Astrocyte was obtained from ScienCell Research Laboratories.293T was obtained from ATCC. 83NS, 528NS, and 84NS wereobtained from Dr. Ichiro Nakano (University of Alabama atBirmingham, Birmingham, AL). 131, 047T, and 352T2 wereobtained from Dr. Do-Hyun Nam (Samsung Medical Center,Korea). All experiments using GSC were performed on cells inpassages 5-30 (2015-2016). The cell cultures have not beenauthenticated but all cells were repeatedly screened for myco-plasma (e-Myco Mycoplasma PCR Detection Kit, iNtRON) andmaintained in culture for less than 6 months after receipt.

Plasmids, transfection, and lentivirus production and infectionLentivirus production was performed as previously reported

(17). Briefly, 3 to 4 � 106 293T cells were plated on 100 mmculture dish 24 hours before transfection. Then, 4.5 mg oflentiviral construct (pHRST-IRES-TGM2, pLL3.7-shTGM2-B,and pLL3.7-shTGM2-C), 3 mg of psPAX2 (Addgene), and 1.5mg of pMD2.G (Addgene) were cotransfected into 293T cellsusing 27 mL of Lipofectamine 2000 (Invitrogen). The mediumwas changed 6 hours after transfection. The medium contain-ing lentivirus was harvested 48 hours after transfection. Viralparticles were concentrated and purified using a Lenti-X con-centrator (Clontech). Cells were infected with lentivirus in thepresence of 6 mg/mL polybrene. siRNAs against humanC/EBPb, GADD153 and negative control siRNA (Genolution)were transfected in GSC using Lipofectamine 2000 (Invitro-gen). The nucleotide sequences used for target-specific siRNAor shRNA are as follows: anti-GADD153 siRNA, 50-GAAAGAA-CAGGAGAAUGAAUU-30; shTGM2-B, 50-GCCTCGTGGTTAT-TAGCAAGG-30; and shTGM2-C, 50-GCCATTGACCACCCAC-CATAT-30.

Quantitative RT-PCRSemiquantitative RT-PCR was performed as previously

described (17, 18). Total RNAwas isolated from cells using Trizolreagent (Invitrogen) according to themanufacturer's instructions.Total RNA (1 mg)was used as a template to synthesize cDNAusingM-MLV reverse transcriptase (Invitrogen). The PCR primers areshown as follows: TGM2, sense 50-AACATGGGCAGTGACTTTGA-30 and antisense 50-AGAGAAAGGCTCCAGGTTGA-30; C/EBPb,sense 50-GACAAGCACAGCGACGAGTA-30 and antisense 50-CAGCTGCTCCACCTTCTTCT-30; TNFa, sense 50-ACGGCATG-GATCTCAAAGAC-30 and antisense 50-GTGGGTGAGGAGCACG-TAGT-30; GADD153, sense 50-AGATGGCAGCTGAGTCATTG-30

and antisense 50-GTTCTGGCTCCTCCTCAGTC-30; and GAPDH,sense 50-GGAGTCCACTGGCGTCTTCAC-30 and antisense 50-GAGGCATTGCTGATGATCTTGAGG-30. The PCR products wereanalyzed on a 1% agarose gel.

Limiting dilution assayFor in vitro limiting dilution assays (LDA), GSCwith decreasing

numbers of cells (200, 100, 50, and 10 or 100, 50, 25, and 5) perwell were plated in 96-well plates containing DMEM/F-12 withB27, EGF (10 ng/mL), and bFGF (5 ng/mL). Extreme limitingdilution analysiswas performedusing software available at http://bioinf.wehi.edu.au/software/elda/.

Immunoblot analysisProteins were extracted with RIPA buffer with complete pro-

tease inhibitors (Roche), separated by electrophoresis, transferredto PVDFmembranes (Millipore), and blockedwith 5% skimmilk(BDBiosciences). Primary antibodies against TGM2 (Chemicon),CD44 (R&D Systems), Sox2 (R&D Systems), TAZ (Cell SignalingTechnology), C/EBPb (Santa Cruz Biotechnology), p-STAT3 (CellSignaling Technology), STAT3 (Santa Cruz Biotechnology), IkBa(Santa Cruz Biotechnology), GADD153 (Santa Cruz Biotechnol-ogy), and a-tubulin (Santa Cruz Biotechnology) were incubatedovernight at 4�C. Immunoreactive bands were visualized usingperoxidase-labeled affinity purified secondary antibodies (KPL)and the Amersham ECL primeWestern blotting detection reagent(GE Healthcare).

Polymerization of GADD153 by TGM2Purified guinea pig liver transglutaminase (Zedira) was diluted

in buffer containing 10 mmol/L Tris-HCl (pH 8.0), 1 mmol/LEDTA (pH 8.0), and 0.1 mol/L NaCl. The TGM2 2 was incubatedin 40 mL of reaction buffer [50 mmol/L Tris-HCl (pH 8.0), 50mmol/L NaCl, 0.5 mmol/L EDTA, 1 mmol/L MgCl2, 5 mmol/LDTT, 10 mmol/L CaCl2] with human GADD153 (Biosource) at37�C for 10 minutes. The reaction mixture was separated using4% to 12% Tris-glycine gels (Invitrogen).

In vivo studyAll animal experiments were conducted in accordance with

protocols approved by the Institutional Animal Care and UseCommittee at the National Cancer Center, Republic of Korea.For the orthotopic mouse model (19, 20), cells were trans-planted following resuspension in DMEM/F-12 with B27, EGF(10 ng/mL), and bFGF (5 ng/mL). Cells were injected stereo-tactically into the left striatum of 5-week-old female BALB/cnude mice. The injection coordinates were 2.2 mm to the left ofthe midline and 0.2 mm posterior to the bregma at a depth of

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3.5 mm. The brain of each mouse was harvested and fixed in4% paraformaldehyde. For the subcutaneous mouse model,cells were injected into the hip area on both sides of eachmouse. Tumor growth was measured two times a week usingelectronic caliper to measure two diameters using the followingformula: length�width2 � 0.5. The mean tumor volume at thestart of the experiment was approximately 200 mm3. Mice weresacrificed 4 weeks after the cell injections. The tumors wereextracted, pooled for each experimental group, and mechan-ically disaggregated using steel operating scissors. GK921(2 mg/kg; MedChem Express) was orally administered dailyand radiation treatment was administered at a dose of 2.5 Gyfor 4 days. Survival and tumor size were analyzed usingGraphPad PRISM software (version 7).

Histology and IHC stainingFor observation of the histologic features, the brains were

removed, fixed with 4% paraformaldehyde for 24 hours at 4�C,and stained with hematoxylin (DaKo) and 0.25% eosin(Merck). For IHC staining of cancer stem cell markers andTGM2-associated genes [CD44 (Cell Signaling Technology),Sox2 (R&D Systems), TGM2 (Cell Signaling Technology),C/EBPb (Santa Cruz Biotechnology), GADD153 (Santa CruzBiotechnology)], after the antigen retrieval process with citratebuffer (pH 6.0) and endogenous peroxidase blocking with 3%hydrogen peroxide, tissue sections were incubated in primaryantibody overnight at 4�C in a humidified chamber with IHCworld antibody diluent buffer. We developed samples using3,30-diaminobenzidine (DAB, Vector Laboratories) as the chro-mogen. To quantify the IHC staining, immunostained cellswere analyzed using a Tissue FAXS system (Tissue Gnostics).The scanned images were quantified using the HistoQuestcytometry software in the Tissue FAXS system. The cut-offthreshold was determined by the stained density of the negativecontrol. For patient tissue staining, GBM specimens wereobtained from patients undergoing surgery with the approvalof the Institutional Review Board of the Samsung MedicalCenter (No. 2010-04-004) and with informed consent. ThisInstitutional Review Board follows the guidelines of Declara-tion of Helsinki and Belmont Report.

Bioinformatics analysisGenomic data and clinicopathologic data for GBM samples

were downloaded from TCGA data portal (http://cancergenome.nih.gov/). For TCGA RNA sequencing data analysis,BAM files were available from the Cancer Genomics Hub(http:/cghub.ucsc.edu). The expression measurements, RPKMvalues were estimated using R package DEGseq. The nearesttemplate prediction algorithm was used to predict the MESactivity of a given TCGA samples with statistical significanceusing a predefined set of markers that are specific to MESsubtype (21, 22). We also analyzed clinical information for thepatients with untreated GBM who had survival informationavailable.

As shown in Fig. 1A, to reanalyze the molecular characteristicsof cancer stem cells in our study, we acquired microarray data inraw form from the Gene Expression Omnibus (GEO) database(GEO number, GSE67089), published at Ichiro Nagano Labora-tory (7). Affymetrix Human U219 CEL files (GSE67089) werenormalized using the robust multiarray average method from theAffy packages (23). For unbiased analysis, unspecific filtering was

performed with the SD filter (24), and genes that were notchanged or were expressed at a very low level at all microarrayswere removed by the cut-off value (SD less than 2). The Hopkinsstatistic (25) was used to assess the clustering tendency of thefiltered dataset. The filtered dataset was highly clusterable (Hvalue, 0.1900499). The Elbow method (26) was used to deter-mine the optimal number of the clusters and four clusters weresuggested. All samples from the dataset were grouped into foursubgroups (astrocytes, glioma cell lines, cancer stem cells of twosubtypes, PN and MES) using the filtered genes (total 901 genes)through principle component analysis (PCA), K-means (27),PAM (28), and hierarchical clustering.

A web tool for Venn diagrams (bioinformatics.psb.ugent.be/webtools/Venn/) was used to select common genes between theabove 919 genes (unique gene, 561 genes) and 1,150 positivenecrosis-related genes (unique gene, 864 genes). The toolidentified 90 common genes and the variable factors andseparated all samples into four groups using PCA. Throughthis analysis, the 90 genes were revealed to be importantvariables associated with each group (astrocytes, glioma celllines, and PN and MES subtypes). Next, due to the associationbetween the MES subtype and necrotic characteristics of GBM,significant analysis of microarray (SAM) was performed usingthe freely available MeV software (version 4.9.0, http://mev.tm4.org) to determine whether there were differentiallyexpressed genes in MES subtype cancer stem cells comparedto PN subtype. Twenty-seven genes were found to be statisti-cally significant in the analysis, and the order of genes wasranked by fold change. This analysis was performed by R andMeV software in all bioinformatics analyses.

Statistical analysisAll the data are expressed as the mean � SEM from at least

three independent experiments. For survival curve analysis, theKaplan–Meier method was used. In the case of patients whowere alive at the time of last follow-up, the survival recordswere censored in our analysis. Statistical Package for the SocialSciences software, version 16 (SPSS) was used for statisticalanalysis. Spearman's correlation coefficients and significance(two-tailed) were calculated using SPSS software for each pairof genes. In the case of the mouse experiments, results of themulti-dataset were compared by analysis of variance using thelog-rank (Mantel–Cox) test. The results of the two-datasetexperiments were compared using a two-tailed Student t test.The level of statistical significance stated in the text is basedon the P values. �, P < 0.05 or ��, P < 0.01 was consideredstatistically significant.

ResultsTGM2 is upregulated in MES GBM

Given that patients with the MES subtype exhibit a high degreeof tumor necrosis, we compared the gene signature between thetumor necrotic regions andMESGSC (3, 7). TGM2was selected asa strong candidate because of its important role in tumorigenesis(Fig. 1A). By using tissue microarray (TMA) analysis, we con-firmed that TGM2 was upregulated in MES GBM samples andenriched in the perinecrotic regions of human GBM tissues(Supplementary Fig. S1A–C and Fig. 1B). Furthermore, recurrentMES patient samples showed significant increases in TGM2expression compared to initial MES samples (Fig. 1C). Our results

TGM2 Regulates Mesenchymal Transdifferentiation of GBM

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raised possibility that TGM2might play an important role for theMES transdifferentiation in recurrent MES GBM.

Among the members of the transglutaminase family, TGM2 isexpressed in various cancer cell types and associated with poorpatient survival (29–34). However, the functions of TGM2 inGBM, especially in the MES transdifferentiation of GSC, areunclear. To analyze clinical importance of TGM2 expression inGBM, we carried out an expression analysis from TCGA data set.TGM2 was highly expressed in the MES subtype (Fig. 1D) andshowed strong MES activity in the MES samples (Fig. 1E). Fur-thermore, the prognosis for GBM patients with high TGM2expression was poor (Fig. 1F).

Taken together, the results showed that TGM2 can be used as aprognostic marker for GBM, because of the markedly higherexpression of TGM2 in GBM patients with the malignant MESsubtype and a poor prognosis.

TGM2 regulates the transdifferentiation of MES GSCTo address functional significances of TGM2,wefirst confirmed

TGM2 expression in subtypes of GSC. In immunoblot analysis,TGM2 was detected in the MES GSC but not in astrocytes, neuralstem cells (NSC), or PN and CL GSC (Fig. 2A). The expression ofCD44, a MES subtype marker, was similar to that of TGM2,whereas Sox2, a marker of the PN subtype, was inversely relatedto the expression of TGM2 (Fig. 2A; refs. 35–37). Similar patterns

were also observed in an IHC analysis of a GSC-injected ortho-topic mouse model (Fig. 2B).

To determine the role of TGM2 in transdifferentiation of MES,we silenced TGM2 in MES GSC (83NS and 131) using shRNA,examined the levels of master TF, including C/EBPb, TAZ, andSTAT3, and performed LDAs. When TGM2 was silenced in MESGSC, both the MES marker CD44 and master TF decreased (Fig.2C and Supplementary Fig. S2A). The sphere-forming ability wasdecreased when TGM2 was suppressed in MES subtype cells, asshown in LDAs measuring the sphere-forming ability of cancerstem cells (Fig. 2D and Supplementary Fig. S2B; ref. 38). Next, weoverexpressed TGM2 in PN 528NS and 84NSGSC (7). The resultsshowed that overexpression of TGM2 increased the MES markerCD44 and master TF (Fig. 2E and Supplementary Fig. S2C).Similarly, the GSC sphere-forming ability was enhanced as TGM2expression increased (Fig. 2F and Supplementary Fig. S2D). Weobtained similar results using 047T and 352T2 cells, CL subtypecells (Supplementary Fig. S2E–H). These results indicate thatTGM2 regulate self-renewal activity of MES GSC by modulationof master TF, which is critical for maintaining the transcriptomeprofiling of the MES subtype.

TGM2 regulates tumor progression in vivoTo further analyze the functional significance of TGM2 in vivo,

we generated an orthotopic mouse model using TGM2-silenced83NS cells. Depletion of TGM2 in MES GSC significantly

Figure 1.

TGM2 is highly expressed in the MES subtype of GBM. A, The overlapping genes correlated with necrosis (TCGA database) and the MES subtype (I. Nakanodatabase). Details of the analysis are described in Materials and Methods. B, IHC analysis of TGM2 staining in GBM patient tissues. N, necrotic region; PN,perinecrotic region. Scale bar, 100 mm. C, TGM2 expression fold changes in non-MES and MES tissues were determined by RNA sequencing in the initial andrecurrent samples. D, TGM2 expression in each GBM subtype from TCGA database. E, MES activity ratio of TGM2 high and low expression in TCGA database.F, GBM patient survival graphs comparing between TGM2 high expression group (high in 20% of the samples in TCGA database) and others.

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increased mouse survival and reduced tumor size compared tothat of the controlGSC (Fig. 3A andB). In addition, the expressionlevels of TGM2 and the MES marker CD44 were specificallydecreased in 83NS cells with TGM2 knockdown as determinedby IHC (Fig. 3B). Moreover, overexpression of TGM2 in PN GSCsignificantly decreased mouse survival and increased tumor sizeand the level of TGM2andCD44 compared to those of the controlGSC (Fig. 3C and D). Taken together, these results suggest TGM2to be an important therapeutic target in MES GBM.

TGM2 regulates C/EBPb expression directly by polymerizationof GADD153

To determine whether TGM2 is the key molecular switch ofMES transdifferentiation, we first analyzed the correlationbetween TGM2 and the master TF using TCGA dataset. BecauseC/EBPb was most strongly correlated with TGM2 expressionamong MES master TF (Fig. 4A and Supplementary Fig. S3A),we assessed the effect of TGM2 on the regulation of C/EBPb.Previous reports demonstrated that TGM2 degrades target pro-teins by crosslinking; therefore, we speculated that theremust be a

negative upstream regulator of C/EBPb as a TGM2 substrate, andthis protein may be degraded by TGM2-mediated crosslinking(31, 32). GADD153, a nuclear protein that avidly dimerizes withC/EBP isoforms a and b, is a transcriptional inhibitor of C/EBPbsignaling (39, 40). Moreover, induction of GADD153 was alsoassociated with decreased expression of C/EBPb (40). AsGADD153 expression showed anticorrelation with C/EBPb inTCGA dataset (Fig. 4B), we hypothesized that TGM2-mediatedGADD153 crosslinking may induce C/EBPb expression. WhenTGM2 was overexpressed in 528NS cells, the expression of C/EBPb increased, andGADD153 expression decreased (Fig. 4C). Incontrast, suppressing TGM2 expression in 83NS cells led to anincrease in GADD153 (Fig. 4D). More importantly, silencing ofGADD153 in 528NS cells strongly induced C/EBPb expression,suggesting that GADD153 is a negative regulator of C/EBPb (Fig.4E). To confirm the degradation of GADD153, we added protea-some inhibitors to MES subtype cells and found that the expres-sion of GADD153 was augmented (Fig. 4F and SupplementaryFig. S3B and C). To further determine whether GADD153 is adirect substrate of TGM2, we incubated recombinant human

Figure 2.

TGM2 regulates stemness of MES and PN GSC. A, Immunoblot (IB) and PCR analysis showing the concentration of TGM2, CD44, and Sox2 in GBM patient-derivedcells. a-Tubulin and GAPDH were used as loading controls. B, IHC analysis of TGM2, CD44, and Sox2 in a xenograft mouse model established using GSC.C, Immunoblot analysis of TGM2, CD44, TAZ, C/EBPb, pSTAT3, and STAT3 in GSC (83NS) infected with shTGM2-expressing lentiviral or control construct. a-Tubulinwasused as a loading control.D, LDAwasperformed in83NS-Con, 83NS-shTGM2-B, and83NS-shTGM2-C cells.E, Immunoblot analysis of TGM2,CD44, TAZ, C/EBPb,pSTAT3, and STAT3 in GSC (528NS) infected with TGM2-expressing lentiviral or control construct. a-Tubulin was used as a loading control. F, LDA wasperformed in 528NS-Con and 528NS-TGM2 cells.






GADD153 with purified TGM2 in vitro. TGM2 induced the poly-merization of GADD153 in a Ca2þ -dependent manner (Fig. 4Gand H).

These collective data indicate that increased expression ofTGM2 in MES subtype cells leads to the direct degradation ofGADD153 and thus induces C/EBPb expression.

Radiation-induced TGM2 regulates MES transdifferentiationAmong the current therapeutic interventions for GBM, radio-

therapy often leads to recurrence, largely due to the survival ofradioresistant GSC (41, 42). Following radiotherapy, GSC shiftfrom the PN to the MES subtype, mainly due to the secretion ofTNFa by infiltrating macrophages/microglia (4). However, themolecular link between macrophages/microglia-secreted cyto-kines and MES transdifferentiation has not been clearly demon-strated. To determine the possible role of TGM2 in radiation-induced MES transdifferentiation by macrophages/microgliainfiltration, we analyzed macrophages/microglia infiltration andTGM2 expression in 83NS-derived xenograft tumors with orwithout radiation treatment. As expected, radiation treatment

induced TGM2 expression and macrophages/microglia infiltra-tion (IBA1 staining) in the perinecrotic regions (Fig. 5A). Fur-thermore, in response to irradiation treatment on 528NS cells, theexpression of TGM2 was increased and, consequently, the expres-sion of master TF (Fig. 5B) and sphere forming ability was alsoenhanced (Fig. 5C). More importantly, we confirmed that radi-ation induced TGM2 and TNFa expression in 528NS-derivedxenograft tumor (Fig. 5D). When the recombinant TNFa wasapplied, the expression of TGM2, CD44, and master TF increasedin a dose-dependent manner (Fig. 5E). In contrast, expression ofIk-Ba, a negative regulator of NF-kB, decreased (Fig. 5E). Todemonstrate the molecular link between NF-kB and this event,we treated Bay 11-7085, an irreversible inhibitor of Ik-Ba phos-phorylation, to PN GSC and found that the decreased expressionof Ik-Ba by TNFa treatment was rescued, and TGM2 expressionwas downregulated (Fig. 5F). Moreover, downstream targets ofTGM2, CD44, and master TF were also downregulated (Fig. 5F).Furthermore,we investigatedwhethermaster TFwere regulatedbyTGM2 following NF-kB activation. As shown in Fig. 5G, TNFainduced master TF expression was blocked by TGM2 silencing.

Figure 3.

TGM2 regulates tumorigenicity of MES and PN GSC in an orthotopic xenograft mouse model. A, Kaplan–Meier survival graph of mice implanted with 83NS-Con,83NS-shTGM2-B, and 83NS-shTGM2-C cells (n ¼ 7, 1 � 104 cells injected in each mouse). B, Hematoxylin and eosin (H&E) staining of the whole brain andIHC analysis of TGM2 and CD44 in the orthotopic xenograft mouse model. Scale bar, 100 mm. C, Kaplan–Meier survival graph of mice implanted with 528NS-Conand 528NS-TGM2 cells (n ¼ 6, 5 � 104 cells injected in each mouse). D, Hematoxylin and eosin (H&E) staining of the whole brain and IHC analysis of TGM2and CD44 in the orthotopic xenograft mouse model. Scale bar, 100 mm.

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Taken together, the results indicate that after irradiation of PNGSC, TGM2 is upregulated via NFkB signaling to enhance trans-differentiation to MES subtype and acquire radioresistance.

Knockdown of TGM2 suppresses radiation-induced MEStransdifferentiation in vivo

To interrogate the functional relations of TGM2 in radio-resistance, we confirmed the combined effect of TGM2 knock-down and radiation treatment in the orthotopic mouse model. Acombination of TGM2 silencing and radiation treatment furtherprolonged mouse survival, and decreased in tumor size of an83NS-derived model (Fig. 6A and B). Notably, IHC analysisshowed that TGM2, CD44, and IBA1 expression further increasedafter radiation treatment and blocked by TGM2 silencing (Fig.6B). These results suggest that TGM2 induction by radiation mayplay a critical role in radioresistance of MES GSC.

In previous results, we demonstrated that PN toMES transitionwas mediated by TGM2 expression; thus, we postulated thatTGM2 expression may increase during irradiation of PN GSC toinduce recurrence and radioresistance viaMES subtype change. Toverify this hypothesis, we measured the expression of TGM2 in a528NS animalmodel after irradiation. As expected, TGM2 expres-sion was increased in the irradiated tissues of the 528NS-injectedmouse model (Fig. 6C and D). These results suggest that PN

subtype patients may express TGM2 during irradiation to pro-mote theMES subtype change. Thus, inhibition of TGM2 to blockPN to MES transition could be a good therapeutic strategy. Toassess this hypothesis, we analyzed the combination treatment ofirradiation and TGM2 silencing using the 528NS animal model;survival of the 528NS-injected mice was prolonged in the radi-ation treatment group, but this group eventually showed radio-resistance due to TGM2 induction, as expected (Fig. 6C and D).TGM2 silencing in the radiation combined group substantiallyincreased survival of the 528NS-injected mice (Fig. 6C). Takentogether, our results strongly suggest that a combinationof TGM2-specific suppression and conventional radiotherapy may be effec-tive not only in MES GBM but also non-MES GBM patients.

The TGM2 inhibitor GK921 specifically induces cell death andreduces the MES signature and tumorigenesis of MES GSC

To explore possible clinical application, we evaluated theeffects of blocking TGM2 using GK921, a TGM2-specific inhibitor(43, 44). When GK921 was applied, cell growth of the astrocytesand PN subtype cells was not significantly affected, whereas inMES subtype cells, cell viability was reduced even at a lowconcentration of 0.5 mmol/L (Fig. 7A). In addition, CD44 andmaster TF were reduced following treatment with GK921 (Fig. 7Band C). To further confirm in vivo efficacy of its treatment, we

Figure 4.

TGM2 regulates C/EBPb expression directly by polymerization of GADD153. A, A correlation dot plot of C/EBPb and TGM2 in TCGA database. B, A correlationdot plot of GADD153 and TGM2 in TCGA database. C, Immunoblot analysis of TGM2, GADD153, and C/EBPb in 83NS-Con, 83NS-TGM2 cells. a-Tubulin wasused as a loading control. D, Immunoblot analysis of TGM2, GADD153, and C/EBPb in 83NS-Con, 83NS-shTGM2-B, and 83NS-shTGM2-C cells.a-Tubulin was used asa loading control. E, Immunoblot (IB) and PCR analysis of GADD153 and C/EBPb in 528NS transfected with siC/EBPb or siControl. a-Tubulin was used as aloading control. F, Immunoblot analysis of TGM2 and GADD153 in 83NS cells treated with the proteasome inhibitor MG132 (10 mmol/L, 12 hours) and epoxomicin(2 mmol/L, 12 hours). a-Tubulin was used as a loading control. G and H, Immunoblot analysis of TGM2 (G) and GADD153 (H) in vitro cultured with CaCl2 (10 mmol/L),TGM2 recombinant protein (0, 50, 250, and 500 ng), or GADD153 recombinant protein (500 ng).






generated an orthotopic xenograft mouse model using MES GSC(83NS) and orally administered GK921. The results showed thatthe median survival increased, and the tumor size was alsoreduced by GK921 treatment (Fig. 7D and E). Notably, IHCanalysis demonstrated that CD44 and C/EBPb expression wasreduced, and GADD153 expression increased in the GK921treatment group (Fig. 7E). Because we suggested significant ther-apeutic benefit of radiation therapy and TGM2 inhibition in non-MESGSCby blocking non-MES toMES subtype change, we testedGK921 drug effect combined with irradiation in 528NS-derivedsubcutaneous mouse model. Although GK921 or irradiationtreatment partially reduced tumor volume compared with vehiclegroup, but these groups showed relapse of tumor growth (Fig. 7F).However, combined treatment of GK921 and irradiation signif-icantly decreased tumor size and prolonged duration of treatmentresponse (Fig. 7F).

On the basis of these results, we concluded that GK921increased response to radiation therapy of GSC by blocking MEStransdifferentiation. Our results strongly support the therapeuticpotential of TGM2 inhibition in GBM patients.

DiscussionSubtype changes of GBM are strongly associated with acquisi-

tion of resistance to conventional therapy and poor prognosis.Because MES transdifferentiation leads to recurrence and resis-

tance to conventional therapies, the underlying mechanisms ofthis shift require elucidation for therapeutic improvement (4, 7).Our study demonstrated TGM2 as a key molecular switch for thenecrosis-induced MES transdifferentiation. TGM2modulates thisprocess by inducing proteasomal degradation through the poly-merization of GADD153 and by modulating C/EBPb. Silencingand pharmacologic inhibition of TGM2 significantly increasedsurvival rate and therapeutic efficacy of irradiation in mousemodels. In addition to extensive in vivo data, clinical relevanceof our findings was supported by in silico analysis of TGM2expression in a large glioma genomic dataset and IHC analysisof patient-derived specimens. Especially, TGM2 expression washighly upregulated in recurrentMES patient samples compared tothat of non-MES and initial GBM samples, and correlated withpoor prognosis. These results suggested that TGM2might play animportant role for the recurrence and radioresistance of GBM byinducing MES transdifferentiation and could be an importanttherapeutic target for the recurrent GBM.

Infiltrating macrophages/microglia in necrotic regions havebeen linked to poor prognosis via secretion of cytokines (45–48). TNFa, one of the major cytokines secreted by infiltratingmacrophages/microglia in the perinecrotic region, induced mas-ter TF of the MES transition and promoted conversion from a PNsubtype to a MES subtype in an NF-kB signaling-dependentmanner (4). However, the molecular link between NF-kB andmaster TFwas unclear.Here, we demonstrated that TNFa-induced

Figure 5.

Radiation-induced TGM2 regulates MES transdifferentiation. A, Immunocytochemistry analysis of TGM2 and IBA1 expression in GBM xenografts derivedfromMES 83NSwith or without radiation treatment. TGM2was labeled in green and IBA1 was labeled in red. Nuclei were counterstainedwith DAPI (blue). N, necroticregion. Scale bar, 100 mm. B, Immunoblot analysis of TGM2, CD44, TAZ, C/EBPb, pSTAT3, and STAT3 in 528NS cells, which were treated with radiation (3 Gy).a-Tubulin was used as a loading control. C, LDA was performed in 528NS-Con and 528NS-IR cells. D, PCR analysis showing the RNA concentration of TNFaand TGM2 in 528NS-derived xenograft tumor. GAPDHwere used as loading controls. E, Immunoblot analysis of IkBa, TGM2, CD44, TZA, C/EBPb, pSTAT3, and STAT3in 528NS cells treated with TNFa recombinant protein (0, 10, 20, and 50 ng/mL). a-Tubulin was used as a loading control. F, Immunoblot analysis of IkBa,TGM2, CD44, C/EBPb, pSTAT3, and STAT3 in 528NS cells treated with TNFa recombinant protein (100 mmol/L) and an IkBa inhibitor (Bay 11-7085, 10 mmol/L).a-Tubulin was used as a loading control. G, Immunoblot analysis of TGM2, CD44, C/EBPb, pSTAT3, and STAT3 in 528NS cells infected with shTGM2-expressinglentiviral or control construct treated with TNFa recombinant protein (100 mmol/L). a-Tubulin was used as a loading control.

Yin et al.





NF-kB activation was responsible for the TGM2 expression, andthis event ultimately led to MES differentiation via regulation ofmaster TF. Therefore, our results indicate that TGM2 is amolecularmediator between NF-kB and master TF for the necrosis-inducedMES transdifferentiation.

Recent studies have introduced an algorithm to identify tran-scriptional interactions based on GBM expression classes (16).From the transcriptomic findings, several TF, including C/EBPb,C/EBPd, TAZ, STAT3, FOSL2, bHLHE40, andRUNX1,were shownto be regulators of the transition to the MES subtype (15, 49). Ofthese TF, C/EBPb, TAZ, and STAT3 were identified as mastertranscriptional regulators. These TF regulate expression of keyfactors and downstream signaling, including the MES gene sig-nature (15). A high degree of necrosis is observed in theMES classof GBM, andmaster TF were strongly correlated with the degree ofnecrosis (3, 15). All three TF are closely involved in self-renewal ofMES GSC and maintenance of MES characteristics (49, 50). By

blocking TGM2, we effectively downregulated the master TF.Interestingly, TGM2 directly targeted GADD153, a negative reg-ulator of C/EBPb, to induce protein degradation (40). Our resultssuggest that TGM2 is a key molecular switch for MES subtypechange by regulating master TF.

Radiation-induced necrosis has long been considered a majorcause of radioresistance (9, 51). However, there are no clearmolecular explanations for this event. In this paper, we demon-strated that irradiation induced TGM2 expression in perinecroticregions is responsible for the radioresistance by triggering MESsubtype change. TGM2 can be a therapeutic biomarker for notonly the MES subtype but also non-MES subtypes because ther-apy-induced necrosis can easily convert non-MES toMESpatients.When we compared therapeutic efficacy between MES GSC andnon-MES GSC using a combination of radiation and TGM2silencing, non-MES GSC-injected mice showed better therapeuticbenefit following the combination treatment despite their initial

Figure 6.

Suppression of TGM2 inhibits radiation-induced MES transdifferentiation in an orthotopic xenograft mouse model. A, Kaplan–Meier survival graph of miceimplanted with 83NS cells infected with shTGM2-expressing lentiviral or control construct with combinational treatment of radiation (n ¼ 4, 1 � 104 cellsinjected in each mouse). B, Hematoxylin and eosin (H&E) staining of the whole brain and IHC analysis of TGM2, CD44, and IBA1 in the orthotopic xenograftmouse model of A. Scale bar, 100 mm. C, Kaplan–Meier survival graph of mice implanted with 528NS cells infected with shTGM2-expressing lentiviralor control construct and treated with radiation (n ¼ 6, 1 � 104 cells injected in each mouse). D, Hematoxylin and eosin staining of the whole brain and IHCanalysis of TGM2 in the orthotopic xenograft mouse model of C. Scale bar, 100 mm.






low level of TGM2 compared to that of MES GSC. Our resultsstrongly indicate that TGM2 is a therapeutic target that canbe usedto improve the response to radiotherapy not only MES GBMpatients but also for non-MES GBM patients by preventing non-MES to MES transition.

In conclusion, we demonstrated that TGM2as a key therapeutictarget in GBM by regulating the master TF of the MES subtype toincrease radioresistance and recurrence. Therefore, our results willprovide important therapeutic implication for recurrent GBM.

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

Authors' ContributionsConception and design: J. Yin, Y.T. Oh, H.-S. Gwak, H. Yoo, S.-H. Lee, J.H. Kim,S.-Y. Kim, D.-H. Nam, J.B. Park

Development ofmethodology: Y.T. Oh, J.-Y. Kim, S.S. Kim, E. Choi, J.H. Hong,N. Chang, H.J. Kwon, W. Lin, J.H. Kim, S.-Y. Kim, J.B. ParkAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N. Chang, S. Park, H.-S. Gwak, D.-H. Nam,M.-J. Park, J.B. ParkAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T.H. Kim, N. Chang, H.J. Cho, J.K. Sa, H.-S. Gwak,S.-H. Lee, J.H. Kim, S.-Y. Kim, D.-H. Nam, J.B. ParkWriting, review, and/or revision of the manuscript: J. Yin, Y.T. Oh, J.-Y. Kim,E. Choi, T.H. Kim, H.-S. Gwak, J.H. Kim, M.-J. Park, J.B. ParkAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Yin, I. Nakano, J. Lee, J.H. Kim, S.-Y. Kim,D.-H. Nam, J.B. ParkStudy supervision: J.H. Kim, S.-Y. Kim, D.-H. Nam, J.B. Park

Grant SupportThis research was supported by grants from the National Cancer Center,

Republic of Korea (NCC-1410290, NCC-1510061), Basic Science Research

Figure 7.

GK921 inhibits MES GSC growth and stemness. A, Cell viability assays of astrocytes and GBM patient-derived cells treated with different concentrations ofGK921 (48 hours).B andC, Immunoblot analysis of TGM2, CD44, TAZ, C/EBPb, pSTAT3, and STAT3 inMESGSC [83NS (B) and 131 (C)] treatedwith GK921 (24 hours).D, Kaplan–Meier survival graph of mice implanted with 83NS cells treated with GK921 (n ¼ 12, 5 � 102 cells injected in each mouse). E, Hematoxylin andeosin (H&E) staining of the whole brain and IHC analysis of CD44, C/EBPb, and GADD153 in a GK921-treated orthotopic xenograft mouse model. Scale bar, 100 mm.F, A graph comparing tumor volume in a vehicle, GK921 treatment, radiation treatment, and the combinational treatment at subcutaneous xenograft mousemodel [vehicle (n ¼ 4), GK921 (n ¼ 4), IR (n ¼ 3), GK921þIR (n ¼ 4), 1 � 106 cells injected in each mouse].

Yin et al.





Program through theNational Research Foundation of Korea (NRF) funded by theMinistry of Science, ICT & Future Planning (NRF-2015H1D3A1036090, NRF-2015R1A2A1A15054865,NRF-2015M3A9D9067485,NRF-2017R1A2B4011741,and NRF-2015R1C1A1A01054963), the Korea Institute of Radiological andMed-ical Science (KIRAMS), funded by theMinistry of Science, ICT and Future Planning,Republic of Korea (1711045557, 1711045538, 1711045554/50531-2017), andthe Korea Health Technology R&D Project through the Korea Health IndustryDevelopment Institute (KHIDI), funded by the Ministry of Health & Welfare,Republic of Korea, HI14C3418.

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.

Received February 13, 2017; revised May 30, 2017; accepted July 11, 2017;published OnlineFirst July 28, 2017.

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2017;77:4973-4984. Published OnlineFirst July 28, 2017.Cancer Res Jinlong Yin, Young Taek Oh, Jeong-Yub Kim, et al. Signaling

βTransdifferentiation of Glioma Stem Cells by Regulating C/EBPTransglutaminase 2 Inhibition Reverses Mesenchymal

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Transglutaminase 2 Inhibition Reverses ......3.5 mm. The brain of each mouse was harvested andﬁxed in 4% paraformaldehyde. For the subcutaneous mouse model, cells were injected into