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High-mobility group box 2 is associated withprognosis of glioblastoma by promoting cellviability, invasion, and chemotherapeuticresistance

Zhe Bao Wu†, Lin Cai†, Shao Jian Lin, Zhen Kun Xiong, Jiang Long Lu, Ying Mao,Yu Yao, and Liang Fu Zhou

Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China (Z.B.W., Y.M., Y.Y., L.F.Z.);

Department of Neurosurgery, First Affiliated Hospital of Wenzhou Medical College, Wenzhou, China (Z.B.W.,

L.C., S.J.L., Z.K.X., J.L.L.)

Background. The expression profile of high-mobilitygroupbox2 (HMGB2) inpatientswith glioblastomamul-tiforme (GBM) and its clinical signature with underlyingmechanisms were not fully explored.Methods. HMGB2 protein levels were measured in 51GBM patients by immunohistochemical studies. Toclarify the precise role of HMGB2 on cell invasion and vi-abilityof 3GBMcell lines, wedid invitroand invivoanal-yses with lentivirus vectors and small interfering RNA.Transwell invasion assays and wound-healing assayswere used to analyze the invasion of GBM cells.Expression of p53 and matrix metalloproteinase 2/tissue inhibitors of metalloproteinase 2 (MMP2/TIMP2) protein was analyzed by Western blot.Results. HMGB2 protein expression was significantlyhigher in GBM than in controlled brain tissues(P , .0001). HMGB2 overexpression was significantlycorrelated with shorter overall survival time, which wasthe only independent prognostic factor for overall survivalin a multivariate analysis (P ¼ .017). HMGB2 knockdownby small interfering RNA decreased cell viability and inva-sion in vitro and significantly decreased tumor volume invivo, which might be involved in the change of p53 expres-sion and the balance of MMP2/TIMP2. Moreover,silencing of HMGB2 could significantly increase the sensi-tivity of GBM cells to temozolomide chemotherapy.

Conclusions. Our present data suggest that HMGB2 ex-pression is a significant prognostic factor and might playan important role in cell invasion and temozolomide-induced chemotherapeutic sensitivity of GBM. Thisstudy highlights the importance of HMGB2 as a novelprognostic marker and an attractive therapeutic target ofGBM.

Keywords: glioblastoma, high-mobility group box 2,prognosis, invasion, temozolomide.

Human glioblastoma multiforme (GBM) accountsfor approximately 60% to 70% of malignantgliomas, which are the most common and most

deadly brain tumors.1 The life expectancy of patientswith GBM who undergo the current standard of care(maximal safe surgical resection, radiotherapy with con-comitant temozolomide [TMZ] followed by ≤6 cyclesof adjuvant TMZ) is improved from 12.1 to 14.6months on average after diagnosis.2 Overall survival(OS) in the European Organisation for Research andTreatment of Cancer–National Cancer Institute ofCanada trial was 27.2% in 2 years and 9.8% in 5 yearswith the combined treatment. It was promising thatOS with O6-DNA methylguanine-methyltransferase(MGMT) promoter methylation was 48.9% at 2 yearsand 13.8% at 5 years with the combined treatment.3

However, not all GBM patients benefited from the addi-tion of TMZ to radiation therapy. On the other hand,despite advances in understanding of the molecular path-ogenesis of GBM,4,5 the poor level of OS is not dramati-cally improved, especially for those patients withoutMGMT promoter methylation.

The high-mobility group box (HMGB) family consistsof HMGB1, HMGB2, HMGB3, and HMGB4, whose

†These authors contributed equally to this work.

Corresponding Authors: Liang Fu Zhou, MD, Department of

Neurosurgery, 12 Wulumuqi Middle Road, Shanghai, 200040, China

([email protected], [email protected]); Yu Yao, MD, Department

ofNeurosurgery,12#Wulumuqimiddle Road, Shanghai, 200040, China

([email protected]).

Received November 10, 2012; accepted April 18, 2013.

Neuro-Oncology 15(9):1264–1275, 2013.doi:10.1093/neuonc/not078 NEURO-ONCOLOGYAdvance Access publication July 4, 2013

#The Author(s) 2013. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.All rights reserved. For permissions, please e-mail: [email protected].

proteins are ubiquitous in eukaryotic cells andnonspecifically bind to DNA, inducing large-angle DNAbends, enhancing the flexibility of DNA, and likelyfacilitating numerous important biological interac-tions.6–9 Although HMGB2 is a structural analogue ofHMGB1 with an amino acid sequence .85% identicalto that of HMGB1, the HMGB family has different bio-logical functions in the cell.10 HMGB1 overexpressionhasbeen reportedasanovelprognosticmarkerandpoten-tial therapeutic target in a variety of human cancers.11–19

Despite extensive characterization of the diverse roles ofHMGB1 in cancer,20,21 much less is known of HMGB2in carcinogenesis and prognostic value, except for theprognostic significance of HMGB2 expression in hepato-cellular carcinoma,22 squamous cell carcinoma in skin,23

and epithelial ovarian cancer.24

There have been few published reports evaluating thebiological role of HMGB in human glioblastomacells.21,25,26 HMGB1 has been considered as an autocrinefactor capable of stimulating the growth and migrationof human gliomacells.21 Recently, Balani et al26 confirmeda significant elevation of HMGB2 mRNA expression inglioblastoma cells compared with normal brain tissues byreverse transcription (RT)–PCR and then constructed abaculoviral vector expressing the herpes simplex virus thy-midine kinase gene driven by the HMGB2 promoter. TheBalani study26 demonstrated that transduction with theviral vector induced cell death in glioblastoma cell linesin vitro and in vivo. However, the role of HMGB2 inGBM is as yet not entirely clear, especially regarding theprognostic significance of HMGB2 expression in GBM.

The present study investigated the effects of HMGB2knockdown on cell viability and invasion in vitro and invivo and on TMZ-induced chemotherapeutic sensitivity.Furthermore, we explored the underlying mechanisms ofHMGB2 knockdown, which might be involved in theexpression of protein (p)53 and matrix metalloproteinase2 (MMP2)/tissue inhibitors of metalloproteinase 2(TIMP2). Lastly, we analyzed the prognostic significanceof HMGB2 expression in a large number of patients withGBM by immunohistochemistry (IHC). We found thatHMGB2 can be a novel prognostic factor and potentialtreatment target for GBM patients.

Materials and Methods

Cell Culture and Reagents

The human GBM cell lines U87, U251, and Shanghaihuman glioblastoma (SHG)66 were used in this study.SHG66 came from a 47-year-old male with a right parie-tal glioblastoma (World Health Organization gradeIV).27 U87 and U251 cells were purchased from the CellBank of the Shanghai Branch of the Chinese Academy ofSciences. All cell lines were cultured in Dulbecco’s modi-fied Eagle’s medium (Gibco) supplemented with 10%fetal bovine serum (FBS; Hyclone) and 100 U/mL penicil-lin/streptomycin (Gibco) and were maintained in a hu-midified atmosphere with 5% CO2 at 378C. TMZ waspurchased from Sigma.

Western Blot Analysis

Cell lysates were extracted with cell lysis buffer(Beyotime), and the protein concentration in the lysateswas quantified using an enhanced bicinchoninic acidprotein assay kit (Beyotime). Protein samples with 30–50 mg were loaded for immunoblotting using antibodiesagainst p53, MMP2, TIMP2 (Epitomics), and actin(Kangwei).

Cell Viability Assay and Cell Cycle Analysis

Cell viability was assayed using the Cell Counting Kit-8(CCK-8, Dojindo) as described by the manufacturer’sprotocol.

Cells were harvested and fixed in 70% ethanol at2208C overnight, then stained with propidium iodide(36 mg/mL; Sigma) containing 400 mg/mL RNase(Roche) with shaking for 1 h and analyzed by flow cytom-etry (CyAn ADP, Beckman Coulter) for cell cycle profileand apoptosis.

GBM Tissue Collection and Patient Follow-up

A total of 51 patients with GBM, who underwent surgicalremoval of tumor between 2008 and 2010 in theDepartment of Neurosurgery, Huashan Hospital, wereenrolled in this retrospective study. No patient had ahistory of radiotherapy or chemotherapy preoperatively.The study protocol was approved by the local, indepen-dent ethics committee at Huashan Hospital. Clinicaldata were available for all patients and are summarizedin Table 1. There were 30 males and 21 females, rangingin age from 9 to 80 years (mean, 51.7 y). After tumorremoval, all patients had at least 3 cycles of TMZ chemo-therapy with (n ¼ 42) or without radiotherapy (n ¼ 9).The median follow-up time for OS for all patients was18.6 months (range, 5–36 mo). Additionally, 9 con-trolled brain tissues were obtained from adjacent braintissues of contusion and laceration in traumatic braininjury patients.

Real-time Reverse Transcription PCR

Total RNA was extracted from GBM and normal braintissues or from the cell lines using Trizol reagent(Invitrogen), according to the manufacturer’s instruc-tions. The first-strand cDNAs were synthesized using ahigh-capacity cDNA archive kit. Each cDNA (2 mL)was amplified in SYBR Green Realtime PCR MasterMix (final volume, 20 mL) and loaded on the AppliedBiosystems 7900 Real-time PCR Detection System.Thermal cycling conditions were as follows: the firststep, 958C for 10 min and the ensuing 40 cycles, 958Cfor 15 s, 608C for 60 s, and 728C for 30 s. PCR primersused were as follows: HMGB2 (forward): 5′-TCGCCCAAAGATCAAAAGTGAAC-3′ and HMGB2(reverse): 5′-GCTGCACTTGAATTCACATTCTTAG-3′.

Wu et al.: Prognostic significance of HMGB2 in GBM

NEURO-ONCOLOGY † S E P T E M B E R 2 0 1 3 1265

Immunohistochemical Analysis

Human GBM formalin-fixed, paraffin-embedded tissuesections were provided and were IHC stained withHMGB2-specific antibody made against COOH-terminal peptide of human HMGB2 (Epitomics), usingthe Dako Cytomation EnVision+ System horseradishperoxidase (diaminobenzidine) (HRP [DAB]) detectionkit. Briefly, the tissue sections in 5 microns were dehydrat-ed and subjected to peroxidase blocking. HMGB2 anti-body was added at a dilution of 1 : 400 and incubated atroom temperature for 30 min on the Dako AutoStainerusing the Dako Cytomation EnVision+ System HRP(DAB) detection kit. The slides were counterstainedwith hematoxylin. The stained slides were observedunder a microscope, and images were acquired.

All the IHC stained sections were evaluated in a semi-quantitative fashion by 2 senior neuropathologistsblinded to the clinical parameters. In cases of occasionalscoring discrepancy, consensus was always achievedafter discussion of findings. Nuclear staining was consid-ered positive. To evaluate the expressions of HMGB2, 10high-power fields (400×) within the tumor showingnuclear staining were selected. The extent of stainingwas calculated according to the percentages of the posi-tive staining tumor cells in relation to the whole tumorcells. The extent of staining was classified as low (+ ¼,25%), medium (++ ¼ ≥25% and ,50%), high(+++ ¼ ≥50% and ,75%), or highest (++++ ¼

≥75%). In this study, in order to analyze the prognosisbetween groups, staining extent ,50% and ≥50%were considered to define the low-score group and thehigh-score group, respectively, and these cutoff valueshad been used in past studies.28–30

IHC staining of MGMT was the same, withMGMT-specific antibody (Abgent) at a dilution of 1 :200. Nuclear staining of tumor cells was considered posi-tive. Nuclear staining of vascular endothelial cells was thepositive control. MGMT protein expression was classifiedinto negative (2) and positive (+) according to the extentof nuclear staining of ,5% and ≥5%, respectively.

Gene Silencing and Overexpression

Three GBM cell lines—U87, U251, and SHG66—weretransfected with small interfering (si)RNA oligonucleo-tides using Lipofectamine 2000. Briefly, siRNA andLipofectamine 2000 were each incubated separatelywith Opti-MEM for 5 min and mixed together for20 min at room temperature, and then the mixture wasapplied to cells plated in 4 mL of medium (final concen-tration of siRNA, 60 nM). The sequences of siRNAswere as follows: for HMGB2, siHMGB2: 5′-CUGAACAUCGCCCAAAGAU-3′;22 for control scrambledsiRNA, siControl: 5′-TTCTCCGAACGTGTCACGTTT-3′. All siRNAs were purchased from GenePharma.

Nucleotide sequence coding for HMGB2 was clonedinto pCDH-CMV-MCS-EF1-Puro vector (SystemBiosciences) at the BamH and EcoRI site. A panel ofhuman cancer cell lines were infected with protocadherin(pCDH)-HMGB2 or pCDH-Control for 72 h, then splitfor the next assays.

Transwell Invasion Assay and Wound-Healing Assay

Matrigel solution (Becton Dickinson) was prepared inserum-free cell culture medium at a dilution of 1 : 8, forcoating in 24-well transwell chambers (8-mm pores;Corning Costar) overnight at 378C before cell seeding.The cells were cultured in the chamber with serum-freemedia containing 1% bovine serum albumin in triplicateat 3 × 105 cells per well. After 24 h of cultivation, themedia from the chamber and the transwell wereremoved and the chamber was gently wiped with acotton swab. Migrated cells were fixed in 4% polyoxy-methylene for 15 min and then washed by phosphatebuffered saline (PBS) twice for a total of 10 min.Staining was done with 0.1% crystal violet, and the cellnumbers on the lower surface of the membrane werequantified.

Three GBM cell migrations (U87, U251, and SHG66)were measured by determining the ability of the cells tomove into acellular space. The cells were treated siRNAagainst HMGB2 or scrambled siRNA as mentioned.After 72 h of culture, the confluent monolayers werewounded using a sterile pipette tip and evaluated underphase contrast microscopy at 0 and 24 h. Photographs

Table 1. Relations between HMGB2 protein levels andclinicopathologic features in GBM

ClinicopathologicParameters

Immunohistochemistry(n 5 51)

P

Low score(n 5 20)

High score(n 5 31)

Age .676

,65y 15 26

≥65y 5 5

Gender .89

Male 12 18

Female 8 13

Preoperative KPS score .078

.80 10 23

≤80 10 8

Tumor resection 1.0

Total 18 27

Subtotal 2 4

TMZ + radiotherapya 1.0

Yes 17 25

No 3 6

MGMT proteinExpression

.311

Positive 5 12

Negative 15 19aAll patients received ≥3 cycles of TMZ chemotherapy.


1266 NEURO-ONCOLOGY † S E P T E M B E R 2 0 1 3

were taken, and the relative distance traveled by the cellsat the acellular front was measured.

Tumor Formation Assay

Five-week-old female athymic nude mice were purchasedfrom the Shanghai Experimental Animal Center. U87cells transfected with siHMGB2 or siControl for 24 hwere trypsinized, resuspended in PBS, and then subcuta-neously injected into the right back for siHMGB2 or leftback for siControl with 106 cells per injection. Tumorsize was measured by a vernier caliper weekly and calcu-lated as (length × width2)/2. All procedures were per-formed in accordance with the National Institutes ofHealth Guide for the Care and Use of LaboratoryAnimals.

In silico REMBRANDT Analysis

The relationship between patients’ prognosis andHMGB2 mRNA expression level was analyzed in silicousing the Repository of Molecular Brain NeoplasiaData (the REMBRANDT database) of the NationalCancer Institute (http://rembrandt.nci.nih.gov).

Statistical Analysis

All statistical analyses were carried out using the SPSS16.0 software package. Continuous variables were ex-pressed as mean+ SE. Correlation between IHC stainingscore and clinicopathologic variables was evaluated usinga x2 test. Kaplan–Meier survival curves were calculatedusing death as the end point. The difference in OS

Fig.1. Thegrowth-suppressiveeffectofHMGB2silencingonGBMcells invitroand invivo. (AandB)TheeffectofHMGB2knockdownvia siRNA

silencing. The cells were transfected with siControl or siHMGB2 for 72 h and subjected to immunoblot analysis and quantitative PCR of HMGB2

expression. (C) HMGB2 knockdown via siRNA silencing inhibited the proliferation of SHG66 (left), U87 (middle), and U251 (right) cells. The cells

were transfected with siControl or siHMGB2 for 24–96 h and subjected to cell proliferation assay by CCK-8. (D and E) HMGB2 knockdown

inhibited the growth of U87 cells in vivo. The representative images for xenograft tumor on the nude mouse are shown in Fig. 1E (top panel),

and the tumor growth curve or tumor weight is shown in Fig. 1D and E (n ¼ 4). *P , .05, **P , .01, ***P , .001.



curves was examined by log-rank test. The significance ofvarious variables for OS was analyzed by the Cox propor-tional hazards model in univariate and multivariate anal-yses to identify which factorswere independent indicatorsfor prognosis. Tumor cell invasion assays and cell viabil-ity assays were tested using independent t-tests. A 2-tailedP-value testwasused with P , .05 considered statisticallysignificant.

Results

HMGB2 Silencing Suppressed the Growth of GBM CellsIn vitro and In vivo

To address the efficacy of HMGB2 on GBM cells, weknocked down HMGB2 in 3 GBM cell lines (U87, U251,and SHG66) and subjected these lines to cell growthanalysis.As shown in Fig.1, each GBMcell line transfectedwith HMGB2 siRNA showed efficient silencing ofHMGB2 expression, as judged by immunoblot analysisand real-time RT-PCR (P , .05; Fig. 1A and B).HMGB2 mRNA expression significantly decreased62.3%, 47.6%, and 87.3% in SHG66, U87, and U251cells, respectively, by siRNA silencing compared withsiControl cells. In cell viability assay, all GBM cell linesshowedsignificantreductionofcellviabilitybyHMGB2si-lencing compared with siControl cells (Fig. 1C). At 96 hafter transfected siRNA, cell viability reduced 48.3%,26.1%, and 20.8% in SHG66, U87, and U251 cells, re-spectively. Furthermore, we found that tumor growthandweight inthesiHMGB2groupweresignificantly inhib-ited compared with those in the siControl group in vivo(Fig. 1D and E). Tumor volumes were 171.4+29.8 mm3

and 1731.4+239.5 mm3 in the siHMGB2 group andthe siControl group, respectively, at end point (P , .01).Furthermore, tumor weights were 0.75+0.14 g and6.38+0.71 g in the siHMGB2 group and the siControlgroup, respectively (P ¼ .0034). These findings showedthat HMGB2 was required for the growth of GBM cellsboth in vitro and in vivo.

HMGB2 Silencing Diminished the Migrationand Invasion of GBM Cells

To address the mechanism underlying the growth sup-pression of GBM cells by HMGB2 knockdown, we firstdetermined the cell cycle profile of the HMGB2-silencedcells by propidium iodide staining. No obvious cell cyclearrest or apoptosis occurred (data not shown). Having es-tablished that HMGB2 knockdown did not induce cellcycle arrest and apoptosis in GBM cells, we next investi-gated whether HMGB2 knockdown inhibited invasion,as a novel cellular response, to regulate cell proliferation.HMGB2 knockdown resulted in significantly decreasedmigration in SHG66 cells in triplicate independentassays by 31.5%, 74.3%, and 66.6% (Fig. 2A) as wellas in U87 and U251 cells (Supplementary Fig. S1).Moreover, in wound-healing assays, we found thatHMGB2 knockdown diminished wound-healing migra-tion in 3 GBM cells (Fig. 2B). The ability of migration

was decreased by 14.4%–43.8% in SHG66 cells,50.5%–82.6% in U87 cells, and 29.6%–44.2% inU251 cells (Fig. 2C).

Effect of HMGB2 on Invasion-Related Biomarkers

To further investigate the downstream molecules ofHMGB2, we performed Western blot to examine the ex-pression of invasion-related biomarkers, including p53,MMP2, and TIMP2. As shown in Fig. 3A, p53 andMMP2 expressions were significantly downregulated;meanwhile, TIMP2 was significantly upregulated inHMGB2-silencing cells. On the other hand, we alsofound thatoverexpressionofHMGB2 upregulated theex-pression of p53 and MMP2, although no change ofTIMP2 levels was observed (Fig. 3B).

HMGB2 Silencing Sensitized GBM Cells to TMZ

Wethen determined the potential chemoresistant effect ofHMGB2 in GBM cells. We first constructed thepCDH-HMGB2 vector in order to exogenously upregu-late the expression of HMGB2 in 3 GBM cells (Fig. 4A).We then confirmed that TMZ treatment indeed causedthe growth inhibition in 3 GBM cell lines (the inhibitoryrate of SHG66 cells was 50.5%, that of U87 cells was39.8%, and that of U251 cells was 10.7% at 48 h;Fig. 4B). HMGB2 silencing was sufficient to increase che-mosensitivityby21.25%,22.34%,and16.74%at48 h inSHG66, U87, and U251, respectively (Fig. 4B). On thecontrary, as shown in Fig. 4C, GBM cells were more resis-tant to TMZ chemotherapy when HMGB2 was overex-pressed. The resistance of SHG66, U87, and U251 cellsto TMZ chemotherapy increased 17.1%, 25.1%, and6.3%, respectively, compared with the negative controlgroupat48 h.Ontheotherhand,HMGB2 down-orover-expression did not induce change of MGMT protein ex-pression (data not shown). These findings demonstratedthat HMGB2 silencing sensitized GBM cells to TMZchemotherapy.

Overexpression of HMGB2 Protein and Its PrognosticSignificance in GBM Patients

To investigate the protein expression profile of HMGB2in GBM, IHC was initially performed in 51 formalin-fixed, paraffin-embedded tissue sections and 9 controlledbrain tissues. Based upon the extent of staining, we classi-fied the samples into 4 groups with increasing stainingextent from the lowest (+) to the highest (++++)(Fig. 5A and B). As shown in Fig. 5A, HMGB2 proteinwas mainly localized to the nuclei of tumor cells (rightpanel). Average IHC scores of HMGB2 expression areshown in Fig. 5B (Supplementary Table S1). Controlledbrain tissue had lower expression of HMGB2 proteincompared with GBM tissues (11.6+9.8 vs 55.9+28.3, respectively, P , .0001). Moreover, HMGB2 ex-pression in the tumor central zone was obviously higherthan that in the invading zone (seen in SupplementaryFig. S2).



http://neuro-oncology.oxfordjournals.org/lookup/suppl/doi:10.1093/neuonc/not078/-/DC1




We further quantified the expression level of HMGB2in GBM tissues compared with their controlled braintissues (10 GBM vs 8 control) by immunoblot analysis.As shown in Fig. 5C, the level of HMGB2 in tumortissues was significantly higher than that in the controlled

brain tissues (43.8+12.5 vs 11.8+2.4, respectively,P ¼ .039; Fig. 5D, Supplementary Table S2).

Thus, we analyzed the relationship betweenHMGB2 protein overexpression and patients’ prognosis.A Kaplan–Meier survival curve was used to analyze

Fig. 2. HMGB2 silencing suppressed migration and invasion in GBM cells. (A) HMGB2 knockdown suppressed the invasive abilities of GBM cells.

Thecellswere transfectedwith siControlor siHMGB2for72 handsubjected tocell invasionassayby transwell assay.The representative image for

SHG66 cells is shown in Fig. 2A (left), and data from 3 independent experiments are expressed as the mean+SE (right). **P , .01, significantly

different from the siControl group. (B and C) HMGB2 knockdown suppressed the migration abilities of GBM cells. As described, the cells were

subjected to cell migration assay by wound-healing assay. The representative images for GBM cells are shown in Fig. 2B, and data from 3

independent experiments are expressed as the mean+SE (Fig. 2C). *P , .05, **P , .01, significantly different from the siControl group.




the prognostic significance of HMGB2 expression.Expression of HMGB2 protein levels was divided into alow-score group (25.7+16.3,95% confidence interval[CI]: 18.08–33.31) and a high-score group (75.5+12.7,95% CI: 70.83–80.13), which was significantly dif-ferentially expressed between the 2 groups (P , .01;Fig. 5E). The median OS time in patients with low orhigh HMGB2 expression was 20.1+7.6 or 16+5.9months, respectively (P ¼ .033). The log-rank testshowed that patients with high HMGB2 protein expres-sion had a significantly shorter OS time (log-rank teststatistic ¼ 6.724, P ¼ .01; Fig. 5F).

The relationship between clinicopathologic featuresand HMGB2 protein expression in 51 GBM patients issummarized in Table 1. No correlation was observedbetween HMGB2 protein expression and age, gender,preoperative KPS score, tumor resection degree, treat-ment strategy, and MGMT protein expression. To deter-mine whether the expression of HMGB2 was animportant prognostic factor for OS among those 6 clini-copathologic features, a univariate Cox regression analy-sis was used in this 51-patient cohort analyzed by IHC;only a high HMGB2 protein level was identified as an im-portant risk factor forOS (P ¼ .017;Table2).A multivar-iate analysis was also performed with the Coxproportional hazards model, which included those 6 clin-icopathologic features. The results showed that HMGB2protein expression level was the only independent prog-nostic factor of OS (probability for stepwise: entry ¼.05, removal ¼ .05; Table 2).

Furthermore, we used the REMBRANDT database toexamine the relationship between clinical prognosis of

GBM patients and HMGB2 mRNA expression value.According to the results obtained from REMBRANDT,patients with high HMGB2 mRNA-expressing GBM(n ¼ 130) showed statistically poorer prognoses com-pared with patients with intermediate HMGB2 mRNA-expressing GBM (n ¼ 50, P ¼ .015; Fig. 5G).

Taken together, these findings demonstrated thatover-expression of HMGB2 protein predicted a poor prognosisin GBM patients.

Discussion

To the best of our knowledge, this is the first study toanalyze the prognostic significance of HMGB2 expres-sion in GBM patients. Moreover, we confirmedHMGB2 as a signature molecule correlated with the sur-vival and invasion, as well as the TMZ-induced chemo-therapeutic sensitivity, of GBM cells. Silencing ofHMGB2 expression significantly reduced cell viabilityand invasion while increasing the sensitivity of TMZ che-motherapy, which indicates that HMGB2 could be a po-tential therapeutic target of GBM.

HMGB2 has been shown to have high-level expressionin GBM.26,31 Balani et al26 reported that HMGB2 mRNAlevels were found to be 11- to 79-fold higher in primaryglioblastoma tissue samples than in the normal humanbrain. In the recent study by Lin et al,31 massively parallelsignature sequencing was used to find that HMGB2 wasoverexpressed in GBM compared with normal braintissue in protein level (ratio of GBM/normal ¼ 6.45)31

despite the lack of confirmation by Western blot or

Fig. 3. The effect of HMGB2 on invasion-related biomarkers. (A) Cells were transfected with siControl or siHMGB2 for 72 h and subjected to

immunoblot analysis for expression of indicated proteins. (B) Cells were infected with pCDH-vector (control) or pCDH-HMGB2 (HMGB2) for

72 h and then subjected to immunoblot analysis for expression of indicated proteins.



IHC. In our study, we further confirmed that HMGB2 ex-pression was significantly 5 times higher in GBM than incontrolled brain tissues in protein level. To explore the bi-ological function of HMGB2 overexpression in GBM, wedownregulated the expression of HMGB2 by siRNA,which could significantly reduce the proliferation and vi-ability in U87, U251, and SHG66 cells in vitro.Furthermore, downregulation of HMGB2 expressioncould also significantly reduce the invasion of thosecells. In nude mice, U87 cell lines transfected withHMGB2-siRNA could significantly inhibit tumorgrowth in vivo. These observations support our conclu-sions that HMGB2 overexpression is correlated withcell survival and poor prognosis of GBM.

Since MMPs are involved in tumor invasion andmetastasis,32–37 we explored whether levels of MMP2and their inhibitors (TIMP2) were changed whenHMGB2 down- or overexpression was regulated inGBM cells. Silencing of HMGB2 expression resulted ina decrease in the expression of MMP2 in GBM celllines, while an increase in TIMP2 in the same cells indicat-ed that invasion of GBM cells was significantly reduced.Conversely, exogenous overexpression of HMGB2induced the upregulation of MMP2 and thus increasedthe invasion of GBM cells. In this condition, TIMP2was not downregulated, which indicated that it was notalways the case that an increase in the active form ofMMP2 found in glioma cells was accompanied by a

Fig. 4. HMGB2 silencing sensitized GBM cells to TMZ chemotherapy. (A) The transfection efficiency of pCDH-HMGB2. The cells were infected

withpCDH-vector (control)orpCDH-HMGB2(HMGB2) for72 handsubjectedto immunoblotanalysis. (BandC)Thecellswere transfectedwith

siHMGB2 (B) or infected with pCDH-HMGB2 (C) for 24 h, treated with TMZ (12.8 mM) for 48–96 h, and then subjected to cell proliferation

assay by CCK-8. *P , .05, **P , .01, ***P , .001.



decrease in TIMP2 in the same cells.33 On the other hand,it is now clear that p53 regulates the key stages of meta-static progression, such as cell migration and inva-sion.38,39 HMGB2 participates in DNA damageresponseby modulatingp53phosphorylationand thus in-teracts with p53.40–42 HMGB2 suppressed the transcrip-tional activity of p53 in osteosarcoma,41 whereasHMGB2 enhanced the transcriptional activity of p53 in

HeLa cells,40 which indicated that HMGB2 possiblyacted in a tissue-specific manner. In this study, ectopicoverexpression of HMGB2 led to accumulation of thep53 protein, whereas HMGB2 knockdown with siRNAresulted in a substantial decrease in the p53 protein levelin GBM cell lines. These results explain the underlyingmechanism that HMGB2 overexpression is correlatedwith cell invasion and poor prognosis of GBM.

Fig. 5. HMGB2 was overexpressed in GBM and negatively correlated with patient survival. (A and B) IHC staining of human GBM tissues using

HMGB2-specific antibody, as described in Material and Methods. HMGB2 protein is expressed in nucleus (A, right panel). Classification of

samples according to the extent of staining of HMGB2 expression (n ¼ 51). (C) Immunoblot analysis to determine expression of HMGB2 in

GBM and controlled brain tissues. T: GBM tumor tissues (n ¼ 10); C: controlled brain tissues (n ¼ 8). (D) Quantification of HMGB2

expression in GBM and controlled brain tissues. (E) The extent of staining in the low- and high-score group of HMGB2. Low: low-score group

of HMGB2 with staining extent , 50%; High: high-score group of HMGB2 with staining extent ≥50%. (F and G) Correlation analysis of

HMGB2 protein expression (F, n ¼ 51) and mRNA levels (G, n ¼ 180) and patient OS. **P , .01.



Several studies have demonstrated that HMGB wasassociated with the sensitivity of cancer cells to chemo-therapy agents, including cytarabine, cisplatin, and oxali-platin.22,42–44 Overexpression of HMGB2 resulted inmore resistance of SK-Hep-1 and SH-J1 cells to cisplatinchemotherapy.22 However, other studies demonstratedthat the introduction of the HMGB2 gene into humanlung cancer cells increased cisplatin sensitivity,45 whichindicates that the role of HMGB2 might be dependenton cancer type. TMZ, an oral alkylating agent, wasmost commonly used in GBM’s standard of care.2,3 Weinvestigated the effects of HMGB2 down- or overexpres-sion, in particular whether it would change the sensitivityof GBM cells to TMZ. This study confirmed that GBMcells with HMGB2 overexpression were more resistantto TMZ chemotherapy. On the contrary, downregula-tion of HMGB2 would obviously increase sensitivity toTMZ treatment. However, the magnitude of chemo-therapeutic responses with modulation of HMGB2 ex-pression was not great—the change in U87 and SHG66was about 20% and in U251 was lower. One of themajor reasons might be that HMGB2 down- or overex-pression did not induce a change of MGMT proteinexpression. On the other hand, modulation of HMGB2expression mainly changed the ability of GBM cells toinvade, which resulted in a change in chemotherapeuticresponses and cell proliferation to a certain degree. Theseresults further support our conclusions that HMGB2overexpression is correlated with the resistance of chemo-therapy and poor prognosis in GBM.

In GBM, MGMT promoter methylation is correlatedwith improved progression-free survival and OS in pa-tients treated with alkylating agents. Thus, it is widely ac-cepted that MGMT promoter methylation is the strongestpredictor for outcome and benefit from TMZ chemother-apy.3,46–48 However, it is controversial whether MGMTprotein expression is also a prognostic factor, as isMGMT promoter methylation. Several studies confirmedthatMGMT protein expression levelwasnot aprognosticfactor,49–52 including an interesting study involvingChinese GBM patients,53 because there was a poor corre-lation between the IHC staining results and the methyla-tion status of the MGMT promoter in GBM samples.

In our study, univariate Cox regression analysis for OSfound that HMGB2 protein expression was an indepen-dent prognostic factor of outcomes, despite MGMTprotein expression having the tendency to be near statisti-cal significance (P ¼ .092). InamultivariateCoxanalysis,high HMGB2 protein expression was found to be the onlyindependent poor prognostic factor for OS (P ¼ .017),which further confirmed that HMGB2 protein expressionhad a significant correlation with the prognosis of GBMpatients. Of course, MGMT promoter methylationshould be further analyzed as a possible prognosticfactor in patients with GBM in our series.

Taken together, our findings demonstrated thatHMGB2 protein expression was significantly increasedin GBM and that HMGB2 might play an important roleby promoting invasion and proliferation of GBM cells.Moreover, our present data suggest that HMGB2 expres-sion is involved in the sensitivity of TMZ chemotherapyand is a significant independent prognostic factor ofGBM. This study highlights the importance of HMGB2as a novelprognostic markerand an attractive therapeutictarget of GBM.

Supplementary Material

Supplementary material is available online at Neuro-Oncology (http://neuro-oncology.oxfordjournals.org/).

Funding

This project was based upon work funded by grants fromthe Shanghai Science and Technology Research Program(10JC1402201 to L.F.Z.), from the National NaturalScience Foundation of China (81271523 to Z.B.W.),from the Zhejiang Provincial Natural ScienceFoundation of China (R2091137 to Z.B.W.), and fromthe Zhejiang Provincial Program for the Cultivation ofHigh-level Innovative Health Talents (Z.B.W.).

Conflict of interest statement. None declared.

Table 2. Summary of OS Analyses by Univariate and Multivariate Cox Regression Analysis (HR ¼ Hazard Ratio)

Variable Univariate Analysis Multivariate Analysisb

HR (95% CI) P HR (95% CI) P

Age (,65 y vs ≥65 y) 0.53 (0.17–1.58) .251

Gender (male vs female) 0.84 (0.37–1.92) .68

Preoperative KPS score (.80 vs ≤80) 0.68 (0.30–1.53) .349

Tumor resection (total vs subtotal) 1.93 (0.72–5.16) .192

TMZ vs TMZ + radiotherapya 0.52 (0.22–1.24) .14

MGMT protein expression (positive vs negative) 1.98 (0.90–4.37) .092

HMGB2 (low vs high) 3.35 (1.25–9.02) .017 3.35 (1.25–9.02) .017aAll patients received ≥3 cycles of TMZ chemotherapy.bProbability for stepwise: entry ¼ .05, removal ¼ .05.







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