Genetic Validation of the Protein Arginine ...sor activity. Our ﬁndings provide new information about methods to directly and indirectly affect tumor progression, whereas providing

Therapeutics, Targets, and Chemical Biology

Genetic Validation of the Protein Arginine MethyltransferasePRMT5 as a Candidate Therapeutic Target in Glioblastoma

Fengting Yan1, Lapo Alinari1, Mark E. Lustberg2, Ludmila Katherine Martin1, Hector M. Cordero-Nieves3,Yeshavanth Banasavadi-Siddegowda3, Selene Virk11, Jill Barnholtz-Sloan10, Erica Hlavin Bell8,Jeffrey Wojton3, Naduparambil K. Jacob8, Arnab Chakravarti8, Michal O. Nowicki3, Xin Wu1,Rosa Lapalombella1, Jharna Datta4, Bo Yu5, Kate Gordon1, Amy Haseley3, John T. Patton1,Porsha L. Smith1, John Ryu1, Xiaoli Zhang6, Xiaokui Mo6, Guido Marcucci1, Gerard Nuovo7,Chang-Hyuk Kwon3, John C. Byrd1, E. Antonio Chiocca3, Chenglong Li9, Said Sif4,Samson Jacob4, Sean Lawler3, Balveen Kaur3, and Robert A. Baiocchi1

AbstractGlioblastoma is the most common and aggressive histologic subtype of brain cancer with poor outcomes and

limited treatment options. Here, we report the selective overexpression of the protein arginine methyltransferasePRMT5 as a novel candidate theranostic target in this disease. PRMT5 silences the transcription of regulatorygenes by catalyzing symmetric dimethylation of arginine residues on histone tails. PRMT5 overexpression inpatient-derived primary tumors and cell lines correlated with cell line growth rate and inversely with overallpatient survival. Genetic attenuation of PRMT5 led to cell-cycle arrest, apoptosis, and loss of cell migratoryactivity. Cell death was p53-independent but caspase-dependent and enhanced with temozolomide, a chemo-therapeutic agent used as a present standard of care. Global gene profiling and chromatin immunoprecipitationidentified the tumor suppressor ST7 as a key gene silenced by PRMT5. Diminished ST7 expression was associatedwith reduced patient survival. PRMT5 attenuation limited PRMT5 recruitment to the ST7 promoter, led torestored expression of ST7 and cell growth inhibition. Finally, PRMT5 attenuation enhanced glioblastoma cellsurvival in a mouse xenograft model of aggressive glioblastoma. Together, our findings defined PRMT5 as acandidate prognostic factor and therapeutic target in glioblastoma, offering a preclinical justification fortargeting PRMT5-driven oncogenic pathways in this deadly disease. Cancer Res; 74(6); 1752–65. �2014 AACR.

IntroductionHigh-grade astrocytomas are the most common primary

central nervous system (CNS) malignancy accounting fornearly 14,000 new cases per year in the United States. Althoughsurgery remains the mainstay for the treatment of patientswith these CNS tumors, grade 3 (anaplastic astrocytoma) andgrade 4 (glioblastoma multiforme) astrocytomas exhibit ahighly invasive clinical behavior that, in most cases, precludescomplete surgical resection (1). In contrast withmany cancers,

the outcome of patients diagnosed with glioblastoma hasshown only marginal improvement over the past severaldecades with a median survival of only 15 months despitemultimodal therapy (2). Recent work has contributed to animproved understanding of the pathophysiology of these high-grade malignancies; however, discovery of effective therapieshas been limited by the lack of novel targets that are selectivelyinvolved in the complex pathogenesis of this disease. Numer-ous genome-wide studies have demonstrated that glioblasto-ma possesses a remarkable degree of heterogeneitywith regardto genetic mutation, gene expression profiles, and epigeneticmodifications (3, 4). This degree of biologic heterogeneity hascontributed to the challenge of identifying targets that arecritical to the underlying pathogenesis of this aggressivedisease.

Posttranslational modification of proteins is a commonactivity involved at virtually all levels of cellular regulation.Enzymes in the protein arginine methyltransferase (PRMT)family represent a group of proteins that are evolutionarilyconserved among a wide variety of organisms. PRMT enzymescovalently modify both histone and nonhistone proteins thatare critical to themaintenance of numerous cellular regulatorynetworks (5–7). The PRMT5 enzyme is a type II argininemethyltransferase that uses the cofactor molecule S-adeno-syl-L-methionine to catalyze the transfer of a methyl group to

Authors' Affiliations: 1Division of Hematology, Department of InternalMedicine; 2Division of Infectious Diseases, Department of Internal Medi-cine; Departments of 3Neurosurgery; 4Molecular andCellular Biochemistry;5Chemical Engineering; 6Statistics; 7Pathology; 8Radiation Oncology; and9College of Pharmacy, TheOhio State University (OSU), Columbus; 10CaseComprehensive Cancer Center; and 11Department of Epidemiology andBiostatistics, CWRU School of Medicine, Cleveland, Ohio

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

Corresponding Authors: Robert A. Baiocchi, 400 W. 12th Ave, CCC 453,Columbus, OH 43210. Phone: 614-292-5935; Fax: 614-292-3312; E-mail:[email protected]; and Balveen Kaur, 410 W 12th Ave, CCCRoom 385D, Columbus, OH 43210. Phone: 614-292-3984; Fax: 614-292-3312; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-0884

�2014 American Association for Cancer Research.

CancerResearch

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on March 28, 2020. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst January 22, 2014; DOI: 10.1158/0008-5472.CAN-13-0884

http://cancerres.aacrjournals.org/

two of three guanidino nitrogen atoms within the argininemolecule. PRMT5 drives the formation of both w-NG-mono-methyl andw-NG, NG-symmetric dimethyl arginine residues toaffect protein function (8–12). PRMT5-driven methylation ofarginine residues leads to symmetric dimethylation of histoneproteins H3 (S2Me-H4R3) and H4 (S2Me-H3R8), which in turnalters chromatin structure to promote transcriptional repres-sion (13, 14). A growing number of nonhistone proteinsinvolved in the control of multiple regulatory networks havealso been identified as targets of PRMT5 (6, 15).We have previously reported that PRMT5 overexpression

is involved in the pathogenesis of mantle cell lymphoma andknockdown of PRMT5 expression interfered with growth oftransformed B cells (16). Although this work contributedinformation about the mechanism of PRMT5 overexpres-sion, the functional consequences of PRMT5 inhibition onmaintenance of the malignant phenotype of the transformedcell remain poorly characterized. Here, we show that over-expression of PRMT5 is associated with more aggressivedisease in patients with glioblastoma. Because this enzymeis intimately involved with numerous processes that arefrequently dysregulated in cancer, we sought to determinethe consequences of PRMT5 silencing in glioblastoma. Wedemonstrate that inhibition of PRMT5 overexpression inglioblastoma cells leads to restoration of critical regulatorypathways affecting cell growth, survival, and tumor suppres-sor activity. Our findings provide new information aboutmethods to directly and indirectly affect tumor progression,whereas providing further justification to explore novelapproaches to target PRMT5 overexpression in this incur-able cancer.

Materials and MethodsCell lines and cultureThe human glioblastoma cell lines used in this study are

summarized in Supplementary Table S1. The tet-on–induciblep53 cell line, 2024 (17), was kindly provided by Dr. Erwin VanMeir, Emory University, Atlanta GA.

Evaluation of PRMT5 protein expression in primaryastrocytoma tumorsUnder an Institutional Review Board–approved protocol, 60

patients with astrocytomas treated at The Ohio State Univer-sity (Columbus, OH) from January 2003 to October 2007 wereidentified. Age, gender, race, and previous history of astrocy-tomas were assessed by reviewing themedical records of thesepatients. Reports were reviewed to determine tumor grade,Ki67 proliferation index, as well as clinical characteristics ofdisease. Immunohistochemistry was performed using a Ven-tana Benchmark System (Ventana Medical Systems) and theUltraview Universal Fast Red Kit, following the manufacturer'srecommendations. Optimal conditions for PRMT5 were deter-mined to be 1:125 with antigen retrieval for 30 minutes usingmantle cell lymphoma primary tumor tissues and benign,reactive lymph nodes as the positive and negative controls,respectively. Slides were counterstained with hematoxylin IIfor 4 minutes. See Supplementary Materials for details oncalculating the PRMT5 expression index.

siRNA transfectionsiRNA and scramble (scr) RNA were constructed by silencer

the siRNA Construction Kit by Ambion. si-PRMT5 or scrRNAwere transfected into glioblastoma cells by Lipofectamine 2000according to the manufacturer's instruction. See Supplemen-tary Materials for sequences of inhibitory RNAs used in thisarticle.

Cell cycle, apoptosis, and migration analysisCells were harvested after treatment and fixed in 75% EtOH.

After digestion with RNase, DNA was stained with propidiumiodide and analyzed with a Beckman Coulter flow cytometerandModfit software (Verity). For apoptosis assays, treated cellswere stained with Annexin V and propidium iodide and eval-uated by flow cytometry as described previously (18). Caspaseactivation followed methods previously described (18). Glio-blastomamigration was evaluated as previously described (19).

Real-time quantitative reverse transcription PCR,protein detection, and chromatin immunoprecipitationassay

Total RNA was prepared from untreated and treated glio-blastoma cells using TRizol reagent (Invitrogen) according tothe manufacturer's instructions. The cDNA was prepared withtheMMLVReverse TranscriptionKit (Invitrogen) following themanufacturer's recommendations. Real-time PCR was per-formed using a TaqMan 2 � Universal PCR Master Mix Kitper the manufacture's instructions on an Applied Biosystems7900HT Fast Sequence Detection System. Immunofluores-cence and Western blot analysis used antibody reagents (Sup-plementary Materials) and were performed as described pre-viously (16). Chromatin immunoprecipitation (ChIP) assayswere done using the EZ-MagnaChIPKit (Upstate) according tothe manufacturer's instructions.

ResultsPRMT5 protein is overexpressed in glioblastoma celllines and correlates with proliferation

Aberrant expression of PRMT5 protein has been identifiedin a number of malignancies, including mantle cell lymphoma(16) and germ cell tumors (20). To determine whether PRMT5was overexpressed in astrocytomas, we evaluated a panel ofcell lines (Supplementary Table S1) derived from primaryglioblastoma biopsy specimens for PRMT5 protein expressionby Western blot analysis (Supplementary Fig. S1). Comparedwith normal brain tissue and normal human astrocytes (NHA)that did not express measurable PRMT5 protein, eight astro-cytoma-derived cell lines demonstrated abundant levels ofPRMT5 protein expression (Supplementary Fig. S1). Becausethe various cell lines displayed different growth rates, weexamined whether the degree of PRMT5 protein overexpres-sion correlated with proliferative index (Fig. 1A). Determina-tion of absolute cell numbers was performed in parallel withWestern blot analysis probing for PRMT5 and b-actin proteinsat three different time points (representative blot in Supple-mentary Fig. S1). PRMT5 expression (normalized to b-actincontrol) correlated in a linear fashion with the proliferation ofglioblastoma cell lines (r ¼ 0.81; P < 0.0001).

Targeting PRMT5 in Glioblastoma Multiforme

www.aacrjournals.org Cancer Res; 74(6) March 15, 2014 1753




PRMT5 is overexpressed inprimary glioblastoma tumorsand correlates with more aggressive disease

The direct correlation between glioblastoma cell line growthand PRMT5 expression suggested that this enzyme may con-

tribute toward the malignant phenotype (Fig. 1A). Thus, wenext examined PRMT5 expression in primary astrocytomatumors. High-grade astrocytomas that spontaneously developin a preclinical in vivo murine model allowing for controlled

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Figure 1. PRMT5 overexpression correlates with human glioblastoma (GBM) cell proliferation and inversely correlates with patient survival. A, PRMT5expression correlates with cell growth (r ¼ 0.81 with P < 0.0001). B, hematoxylin and eosin staining of glioblastoma. D–G, PRMT5 is selectivelyexpressed in high-grade (grade 3 and 4) astrocytomas (F and G), whereas normal brain (C) and low- or intermediate-grade astrocytomas (D and E) do not.H, Kaplan–Meier plots by glioma grade. I, the PRMT5 expression index by glioma grade. J, Kaplan–Meier plots of overall survival of patients withglioblastoma as a function of the PRMT5 protein expression index. K, time to death and PRMT5 level in patients who died with glioblastoma. PRMT5 level iscontinuously associated with time to death (Spearman r ¼ �0.57; P ¼ 0.0001).

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Cancer Res; 74(6) March 15, 2014 Cancer Research1754




induction of allelic haploinsufficiency at Nf1, p53, and PTENtumor suppressor gene loci (21) were examined for PRMT5 andKi67 expression (Supplementary Fig. S2). Although controlbrain tissue from wild-type mice failed to demonstrate anydetectable PRMT5 protein expression, all tumor samples takenfrom tumor-bearing Nf1- and p53-deficient mice (Mut3 mice,GFAP-cre; cisNf1�/þ; p53�/�) showed overexpression ofPRMT5 that coincided with the cellular proliferation signalKi67. PRMT5 expression was higher in tumor tissue fromtumor-bearing Mut3 mice compared with uninvolved braintissue proximal to tumor or from brain tissue from controlmice without CNS tumors of the same genetic background(Supplementary Table S3; data not shown).We next examined PRMT5 protein expression profiles in

primary tumor specimens from 60 patients diagnosed withastrocytomas (Supplementary Table S2 and Fig. 1B–G). Toverify that our patient cohort demonstrated clinical behaviorconsistent with the natural history of this disease (22), weevaluated survival in the context of tumor grade (Fig. 1H) andfound median survival to correlate with tumor grade asdescribed previously (23). PRMT5 was overexpressed in grade3 and 4 tumors with the most prominent expression in anuclear distribution (Fig. 1F, G and I). Normal brain tissue ortumors from patients with grade 1 (N ¼ 10) and grade 2astrocytomas (N ¼ 7) did not demonstrate any detectablePRMT5 protein (Fig. 1C–E, and I). The median PRMT5 expres-sion index (definition provided in Supplementary Materials)seen in grade 3 tumors was 0.15 (N ¼ 7; interquartile range ¼0.11–0.25) and for grade 4 tumors was 0.53 (N ¼ 43; inter-quartile range ¼ 0.19–0.78). The difference in PRMT5 expres-sion indices between grade 3 and 4 tumors was statisticallysignificant (P ¼ 0.01, by the standard two-sample Wilcoxonrank-sum test). The differences in the PRMT5 expression indexobserved in grades 1/2 versus grades 3 versus 4 tumors werestatistically significant (P ¼ 0.0001, the Kruskal–Wallis equal-ity-of-populations rank test).Because PRMT5 overexpression was confined to grade 3 and

4 tumors, we excluded patients with grade 1 and 2 tumors fromour evaluation of other clinical variables related to PRMT5expression. In patients with grade 3 and 4 tumors, the PRMT5expression index was not significantly associated with gender,race, or prior diagnosis with astrocytoma. In patients withgrade 4 tumors (glioblastoma), the degree of nuclear PRMT5expression was associated with age, though the result was ofborderline significance (Spearman correlation coefficient ¼0.29; P ¼ 0.057).The heterogeneity in the degree of PRMT5 expression seen

in grade 4 tumors (Fig. 1I) prompted us to examinewhether thePRMT5 expression index correlated with glioblastoma patientsurvival. We stratified grade 4 tumors into three categories ofthe PRMT5 expression index: (i)�0.75; (ii) 0.25 to 0.74; and (iii)<0.25. Patients with grade 4 tumors showing a PRMT5 expres-sion index �0.75 demonstrated shorter overall survival com-pared with patients with lower PRMT5 expression indices.Patients with the PRMT5 expression index <0.25 had a mediansurvival of 726 days (Fig. 1J; 95% confidence interval (CI), 176–1083 days; N ¼ 14) compared with 277 days (95% CI, 156–604days;N¼ 14) for thosewith the PRMT5 expression index¼ 0.25

to 0.74, and 108 days (95% CI, 17–173 days; N ¼ 15) for thosewith the PRMT5 expression index �0.75 (Fig. 1J; log-rank P <0.0001). Cox proportional hazards were used to model mor-tality in patients with glioblastoma. In a multivariable model(with age, gender, Ki67, and PRMT5 as effects), the PRMT5expression index (represented as a single continuous variable)was significantly associated with decreased overall survival. Inthe model a 10% increase in the PRMT5 expression index onaverage was associated with an unadjusted HR, 1.37 (95% CI,1.18–1.57; P < 0.001) and an adjusted HR, 1.40 (95% CI, 1.18–1.66; P< 0.001) formortality. In themodel, age, gender, andKi67were not significantly associated with mortality. We evaluatedthe association of PRMT5 expression with time to deathgraphically in patients with glioblastoma who died over fol-low-up (only 4 of 43 individuals, 9.3%, were alive at the end offollow-up). We found that PRMT5 expression was strongly andcontinuously associated with time to death in patients withglioblastoma who died during follow-up (Fig. 1K; Spearmanrank correlation coefficient ¼ �0.57; P < 0.0001).

Previous glioblastoma tumor history was not significantlyassociated with the PRMT5 expression index or survival inindividuals with glioblastoma. Within the 43 patients diag-nosed with glioblastoma, 11 had a previous glioblastomahistory. We did not observe any difference in survival overfollow-up for primary glioblastoma or recurrent glioblastoma(median survival ¼ 259 days in primary glioblastoma and 254days in recurrent glioblastoma, the log-rank test ¼ 0.6 on thesurvival curves). The PRMT5 expression index was not signif-icantly associated with prior glioblastoma history, the medianPRMT5 expression index ¼ 0.56 in primary glioblastoma and0.31 in recurrent glioblastoma (Wilcoxon rank-sum P ¼ 0.5).

PRMT5 mRNA in glioblastoma cell lines did not differ fromNHAs (not shown); however, the amount of protein differedmarkedly between glioblastoma primary tumors, cell lines,NHA, and normal brain (Fig. 1 and Supplementary Fig. S1),suggesting that PRMT5 dysregulation occurred at the level ofmicroRNA (miRNA), similar to that observed with mantle celllymphoma (16). We, therefore, evaluated PRMT5 expression asa function of survival and found that in the Rembrandt datasetfor glioblastoma cases (total n ¼ 181 cases), no significantcorrelation between PRMT5 transcript levels and survival(Supplementary Fig. S1B). We, therefore, evaluated twomiRNAs, miR-96 and miR-92b, previously shown to be under-expressed in mantle cell lymphoma allowing for efficientPRMT5 translation and upregulation of PRMT5 protein levels(16). We used nanostring methodology to quantify miR-96(average ¼ 44 counts) and miR-92b (average ¼ 108 counts)and found bothmiRs to be underexpressed compared with thetop 50 detectable miRs (average counts ranging from 601–123,068) for 11 primary glioblastoma RNA samples (Supple-mentary Fig. S1C). These observations indicate that miRdysreulation may contribute toward more efficient PRMT5translation and elevated protein levels.

Consequences of PRMT5knockdown in glioblastoma celllines in vitro

Because expression of PRMT5 correlated with proliferationrate of glioblastoma cells and survival of glioblastoma patients,






we next determined the consequences of PRMT5 silencing invitro. We developed a panel of nine small inhibitor RNAmolecules (siRNA, and control scrambled: scrRNA) designedto knockdown PRMT5 transcript and protein expression. Sig-nificant decreases in mRNA levels (90% and 80% in U1242 andU251, respectively) and in PRMT5 protein expression wereachieved with three lead siRNAs (Fig. 2A and B and Supple-mentary Fig. S3A). Because PRMT5 and PRMT7 have each beenshown to possess type II methyltransferase activity, we exam-ined the specificity of our si-PRMT5 reagent. As shown inSupplementary Fig. S3B, si-PRMT5 did not affect the expres-sion of the other type II PRMT enzyme, PRMT7. More impor-tantly, PRMT5 knockdown by si-PRMT5 led to demethylationof target histone protein arginine residues (H4R3) as deter-mined by immunofluorescence using an antibody specific forsymmetric dimethyl arginine of histone 4 (S2Me-H4R3, Fig. 2B).Twenty-four hours following siRNA treatment, cytoplasmicand nuclear PRMT5 protein levels were undetectable and thiscorrelated with the loss of the PRMT5-associated epigeneticmark, S2Me-H4R3 (14).

We next determined the effects of PRMT5 depletion ongrowth properties of glioblastoma cells. As shown in Fig. 3A, si-PRMT5 elicited a significant antiproliferative effect on cellgrowth (P < 0.0001). Invasiveness is a characteristic feature ofglioblastoma and a major contributing factor that preventssuccessful surgical resection leading to the low progression-free survival observed in patients with glioblastoma. siRNAinduced loss of PRMT5 expression led to cell morphologicchanges in glioblastoma cell lines demonstrating a transitionfrom a cellular morphology with elongated cell shape anddendritic-like projections to a more rounded cellular shape(Fig. 3B). To determine whether this change in cellular mor-phology correlated with decreased invasiveness of glioblasto-ma cells, we evaluated cellular migration by both wound-healing and Transwell migration assays. Treatment of glio-blastoma cell lines with lipofectamine or scrRNA controls didnot alter cellularmigration compared with untreated cells (notshown). Inhibition of PRMT5 overexpression with siRNA,however, resulted in marked reduction in migratory activityof both cell lines in both Transwell (Fig. 3C) andwound-healingassays (not shown). Altered cellular proliferation, morphology,and migratory activity led us to next examine whether cell-cycle progression was affected with PRMT5 knockdown (Fig.3D). Using parallel immunofluorescence assays to confirmPRMT5 knockdown and loss of the S2Me-H4R3 epigeneticmark, we were able to confirm a correlation between loss ofPRMT5 expression and cell-cycle arrest (Fig. 3D). Furthermore,reduced expression of PRMT5 led to an overall increase in thenumber of hypodiploid cells, indicating endonucleolytic DNAcleavage associated with cell death (2.05% in scrRNA and10.80% in si-PRMT5; P < 0.001), a decrease in the percentageof cells in G1–G0 (60.2% in scrRNA and 35.5% in si-PRMT5; P <0.0001), and an increased accumulation of cells in S (9.2% inscrRNA and 14.9% in si-PRMT5; P < 0.0001) andG2–M(24.3% inscrRNA and 32.7% in si-PRMT5; P ¼ 0.0001) phases of the cellcycle (Fig. 3D). These results demonstrate that PRMT5 silenc-ing promotes cell-cycle arrest and leads to hypodiploid nuclearcontent consistent with programmed cell death.

Knockdown of PRMT5 leads glioblastoma cells toundergo caspase-dependent apoptosis

PRMT5 silencing can induce apoptosis in different types ofcancer (6, 24). To investigate the effect of PRMT5 knockdownon glioblastoma cell viability, three different cell lines (A172,U1242, and U251) were transfected with either scrRNA or si-PRMT5, and cell viability was determined by Annexin V–fluorescein isothiocyanate (FITC)/propidium iodide stainingand flow cytometry. As shown in Fig. 4A and B, PRMT5knockdown resulted in the induction of apoptosis in all celllines comparedwith scrRNA control (P¼ 0.002 across cell linesand time points).

The BCL2 family of proteins is known to play a central role inthe regulation of apoptosis (25). As shown in Fig. 4C, si-PRMT5significantly led to an increase in the BAX/BCL2 ratio associ-ated with activation of downstream caspase enzymes andapoptosis (26). ChIP experiments were performed and failedto show PRMT5 recruitment to the BAX gene promoter (datanot shown), suggesting other pathways are involved in thechange of BAX/BCL2 following PRMT5 depletion.

As caspase-3 is a key protease associated with DNA frag-mentation, we investigated whether apoptosis induced byPRMT5 knockdown was caspase-3 dependent. Flow cytometryanalysis of glioblastoma cells stained with an antiactivatedcaspase-3 antibody showed increased caspase-3 activationfollowing PRMT5 silencing (Supplementary Fig. S4A). Pretreat-ment of glioblastoma cell lines with the pan-caspase inhibitorsZ-VAD-FMK (100 mmol/L) and Q-VD-OPh (20 mmol/L) led toblockade of caspase-3 activation (Supplementary Fig. S4A) andprevented apoptosis (Fig. 4D andE;P< 0.0001), thus, consistentwith a caspase-dependent mechanism of cell death.

The addition of temozolomide to radiotherapy hasimproved the survival of patients with glioblastoma and isnow considered standard in the management of these patients(27). Glioblastoma cells were preincubated with 100 mmol/L oftemozolomide for 72 hours and transfected with scrRNA or si-PRMT5 (Fig. 4F and Supplementary Fig. S4B). PRMT5 knock-down led to greater than additive induction of programmedcell death when combined with temozolomide.

Apoptosis induced by PRMT5 depletion occursindependent of p53 mutational status

The p53 pathway is commonly deleted or mutated in glio-blastoma and associatedwithmore aggressive disease and poorprognosis (28). Our initial observations suggested that inductionof glioblastoma cell line cell death following PRMT5 depletionoccurred independent of p53 mutational status (Fig. 5, A172p53wt vs. U1242 and U251 p53 mut and Supplementary TableS1). Recentworkhasdescribed themechanismbywhichPRMT5modulates the activity of p53 (6, 7). The p53 wild-type cell line,A172 (29), was cotransfected with siRNA targeting p53 (si-p53)and si-PRMT5.As shown inFig. 5AandB, changes in cell viabilitydue to PRMT5 depletion remained unchanged regardless of p53expression level. We confirmed these studies using pifithrin-a,an inhibitor of p53 transcriptional activity (Supplementary Fig.S5). In addition, in p53-null human glioblastoma cells with adoxycycline-inducible p53 expression system (2024 cell line), theloss of cell viability due to PRMT5 depletion was similar despite

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absence or presence of p53 expression, even after inducing DNAdamage with ionizing radiation (Fig. 5C and D). Furthermore,the cells seemed to be sensitized to radiation-induced cell death

upon depletion of PRMT5, regardless of p53 expression (Fig. 5Cand D). We found all cell lines, regardless of p53 mutationalstatus, underwent G2–M cell-cycle arrest (Fig. 3D and data not

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Figure 2. siRNA-mediated silencingof PRMT5 expression. A, humanglioblastoma cell lineswere treatedas indicated and expression ofPRMT5 detected by real-time PCRand Western blot analysis.B, si-PRMT5 reduced expressionof PRMT5 and its epigenetic markS2Me-H4R3 as determined byimmunofluorescence microscopy.






shown) and apoptosis followingPRMT5depletion, an importantfindingwhen taken in the context thatp53defects areassociatedwith chemo- and radioresistance and portend a poor prognosisin patients with glioblastoma.

PRMT5 inhibition promotes restoration of regulatorygene expression

Several reports have shown that PRMT5 contributes totranscriptional repression of tumor suppressor genes ST7

(16), NM23 (14), RBL2 (p130; ref. 30), and DOC-1 (31). Toinvestigate potential genes targeted by PRMT5 in glioblastoma,we treated cell lines with PRMT5 siRNA or scrRNA control, andcarried out transcriptome analysis using Affymetrix cDNAexpression arrays (Supplementary Table S4, genes showing>1.5-fold expression with PRMT5 knockdown). Consistentwith the findings of Pal and colleagues (16) who reported asimilar experiment using a murine cell line, we found 84 genesto become upregulated >1.5-fold compared with only four

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Figure 3. Consequences of PRMT5silencing in glioblastoma in vitro. A,proliferationwas evaluated byMTSassay. B, change of cellmorphology with PRMT5knockdown. C, PRMT5 silencingleads to decreased migratoryactivity of glioblastoma cellsmeasured by Transwell assay.D, cell-cycle analysis by flowcytometry.

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genes downregulated after PRMT5 knockdown, verifyingPRMT5 as a global transcriptional repressor in human glio-blastoma cells. We observed a wide variety of genes with

functions ranging from tumor suppressor (ST7) to immunemodulatory activity (CXCL10, CXCL11, and IL6). Similar to datawith mantle cell lymphoma cell lines and tumors (16) in which

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Figure 4. PRMT5 Knockdown promotes cell death of human glioblastoma cells. A, U251 cells were treated as indicated and cell death assessed flowcytometry. B, A172, U1242, and U251 were treated as indicated, resulting in decreased cell viability of siPRMT5 compared with scrRNA (P ¼ 0.002).C, Western blot analysis for BCL2 and BAX. D, caspase inhibitors blocked siPRMT5-induced cell death. E, cell death of three human glioblastoma cell linespretreated with caspase inhibitors. F, cells treated with temozolomide, replated, treated as indicated, and cell death determined by flow cytometry.






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we characterized a PRMT5-dependent repressive complex(PRMT5, MBD2, and HDAC2), we noted a 1.4-fold increase inST7mRNA expression in glioblastoma cell lines treatedwith si-PRMT5.On the basis of this cell line in vitro data suggesting that ST7

is a functional downstream target of PRMT5, we sought tofurther examine ST7 expression levels as they related to clinicaloutcomes in glioblastoma. We used the Rembrandt dataset toanalyzemicroarray gene expression data to associated survivaldata. Kaplan–Meier analysis was performed on all patientswith glioma (grade 1–4; Supplementary Fig. S6) and glioblas-toma only (Fig. 6A). Both analyses demonstrated that ST7expression correlated with statistically significant differencesin overall survival in patients with glioma. Specifically, 2-foldupregulation of ST7 was associated with a statistically signif-icant increase in overall survival. This supports our hypothesisthat lower ST7 expression levels attributed to increased levelsof PRMT5 and transcriptional silencing of ST7 correlate with amore aggressive and worse clinical outcome. It also supportsthe notion that ST7 contributes to the pathogenesis in sup-porting the malignant behavior of glioblastoma.Given that ST7 repression in glioblastoma tumors associated

with significant reduction in survival (Fig. 6A) and the roleplayed by ST7 in maintenance of extracellular matrix andmodulation of genes that preventmetastasis (32), we examinedthe functional consequences of PRMT5 inhibition and mod-ulation of this anticancer target gene. To verify that PRMT5was physically recruited to the promoters of these genes andassociated with a corepressor complex, we isolated chromatinpreparations and performed ChIP studies. Glioblastoma celllines were grown in the presence of si-PRMT5 (validated withthree separate siRNA constructs as shown in SupplementaryFig. S3), scrRNA or left untreated, and 24 hours later cross-linked chromatin was prepared for ChIP assays. Using anti-bodies specific for PRMT5 and nonreactive antibody controland primers specific for regions within target gene promoters,we confirmed PRMT5 recruitment to the ST7 promoter (Fig.6B). When PRMT5 levels were knocked down, enrichmentof PRMT5, MBD2, DNMT3a, and HDAC2 on the ST7 promoterwas markedly diminished consistent with our prior findings(14, 16, 32; Fig. 6B, and data not shown). Loss of PRMT5recruitment on target promoters led to enhanced transcrip-tional (Fig. 6C) and translational activity (Fig. 6D). Further-more, we noted that overexpression of ST7 in three glioblas-toma cell lines (A172, U1242, and U251) was able to induce celldeath and reduce cell proliferation (Supplementary Fig. S7),indicating that ST7 played a role as a downstream effector ofPRMT5 dysregulation.

PRMT5 inhibition leads to enhanced survival in apreclinical glioblastoma xenograft modelTo further examine whether PRMT5 protein overexpression

is relevant to glioblastoma pathogenesis, we studied the con-sequences of PRMT5 inhibition in an aggressive in vivo glio-blastoma xenograft model. Nude mice underwent intracranialimplantation of Gli36 delta cells that were treated either withscrRNA or si-PRMT5 (equivalent cell number and viability wasvalidated before engraftment, n ¼ 8/group). Mice engrafted

with Gli36 cells treated with a single exposure to the PRMT5short hairpin RNA (shRNA) group were shown to have pro-longed survival (P¼ 0.0004; Fig. 7B) compared withmice in thecontrol shRNA group. Tumor burden was tracked by clinicalassessment and by MRI at day 28 (Fig. 7A). These in vivo dataprovide further support of the relevance PRMT5 plays insupporting the malignant phenotype of glioblastoma.

DiscussionHigh-grade astrocytomas are the most common primary

brain tumors and are associated with a poor prognosis despiteaggressive surgical and medical management (23). Over thelast several decades, we have observed improved outcome andsurvival for patients with a wide variety of cancers; however,the minimal improvement in survival of patients with glio-blastoma illustrates the need to discover novel targets todevelop new therapeutic approaches. In addition to classicgene mutations, inactivation of regulatory and tumor suppres-sor genes by epigenetic repression is frequently observed inthese tumors. Histone deacetylase inhibitors have been shownto possess antitumor activity in malignant astrocytomas (33),clinical trials investigating these agents have been disappoint-ing, highlighting the need to identify other promising epige-netic targets.

The PRMT family of enzymes represents a group of proteinsthat are highly conserved in nature and involved with a widevariety of developmental and biologic processes (34). PRMT5, atype II argininemethyltransferase, silences the transcription ofgenes by symmetric dimethylation of arginine residues onhistone proteins and works more efficiently when associatedwith other corepressor enzymes such as HDAC and MBDproteins (13, 35). Recent studies have shown PRMT5 to be amajor prosurvival factor regulating eIF4E expression and p53function (7). Furthermore, PRMT5 depletion sensitizes humancolon cancer, lung cancer, and fibrosarcoma cells to TRAILwithout affecting TRAIL resistance in nontransformed cells(24). Here, we show that PRMT5 is selectively overexpressed inhigh-grade astrocytomas and that the degree of PRMT5 expres-sion correlated with cell growth and patient survival. Our workexamining potential mechanisms of PRMT5 overexpressionpoint toward miRNA dysregulation, and we are currentlyinvestigating how PRMT5 translation is efficiently supportedin glioblastoma.

Our findings showing a correlation between PRMT5 over-expression and poor clinical outcome of patients with glio-blastoma are intriguing with regard to potential utility as aprognostic factor to identify patients with more aggressivedisease. Although several abnormalities affecting epigeneticprocesses have been described in glioblastoma tumors, includ-ing expression of DNMT enzymes (36), and methyl-CpG–binding domain proteins (37), the prognostic relevance ofthese markers remains unclear. Identifying biomarkers likePRMT5 that are of prognostic and biologic significance willassist in developing novel approaches to this disease. Futureprospective analysis will be essential to determine the trueprognostic value of PRMT5 overexpression in patients withglioblastoma.






Apart from epigenetic modifications, PRMT5 has beenshown to mediate arginine methylation of p53 to regulate itsfunction (7). Because p53 has been shown to be highlyrelevant as a critical tumor suppressor for both primaryand secondary subtypes of glioblastoma (38), we exploredthe role of p53 in cell death triggered by PRMT5 knockdown.We observed equivalent degrees of cell death induced byPRMT5 silencing in both p53 wild-type, mutant, and deletedglioblastoma cells. Work by several groups has demonstrat-ed that PRMT5 directly targets p53 function by methylatingR333, R335, and R337, modifications that negatively affectsp53 recruitment to target gene promoters during genotoxic

stress and cell death. Scoumanne and colleagues (7) showedthat PRMT5 knockdown led to decreased p53 stability,reduced expression of the p53 target gene CDKN1A, anddecrease of breast cancer cell proliferation. Other work byJansson and colleagues (6) demonstrated that the effect ofPRMT5 depletion with siRNA on DNA damage–inducedapoptosis was more significant in p53 wild-type comparedwith p53-null colorectal carcinoma cell lines. Our resultssuggest that cell death induced by PRMT5 silencing occursindependent of p53 mutational status, indicating that therelevance of arginine methylation of p53 may differ betweentumor types and, perhaps, PRMT5 might serve as an equally

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Figure 6. ST7 is a PRMT5 target gene. A, Kaplan-Meier plot for glioblastoma caseswith differential ST7 gene expression (lowST7 n¼ 169; high ST7 n¼ 12). B,ChIP assays using IgG control or anti-PRMT5 antibody performed on chromatin from U251 or U1242 treated as indicated. Fold enrichment with eachantibody was calculated relative to the IgG control (in triplicate). The data were normalized by the input DNA ½ðDCtÞ¼CtðIPÞ � CtðinputÞ� and one-wayANOVA was used to analyze the normalized data for each cell line. C and D, expression of ST7 is upregulated by si-PRMT5 determined by real-time PCR (C)and Western blot analysis (D).

Yan et al.





attractive target in glioblastoma tumors in which p53 isoften mutated or deleted and associated with a poorprognosis.Changes observed in the glioblastoma transcriptome fol-

lowing PRMT5 depletion helped us identify PRMT5 as amastertranscriptional repressor in this disease. We identified thetumor suppressor gene ST7 as a potential target as it becamederepressed with PRMT5 inhibition. Consistent with theseresults, ChIP experiments identified the ST7 promoter to bedirect targets of PRMT5. The human ST7 gene was firstrecognized as a candidate tumor suppressor based on itschromosomal location (7q31.1), a site of frequent loss ofheterozygosity in transformed cells (39) and its reducedexpression in cancer (40). Here, we show that reduced ST7expression correlates with poor outcome in patients withglioblastoma. ST7 protein activity is also involved with extra-cellular matrix remodeling in several solid tumor models andmay mediate tumor suppression by altering the tumor micro-environment and affecting tumor invasion (32). The invasivenature of glioblastoma tumors precludes complete surgical

resection in the majority of patients with this disease. Thus,development of experimental therapeutic strategies to targetPRMT5 overexpression promoting restoration of tumor sup-pressors that negatively regulate malignant cell invasion andmicroenvironment may affect metastatic potential of thisdisease in vivo. We are currently examining the direct roleST7 plays in glioblastoma invasiveness.

Previous work has shown that PRMT5 associates with SWI/SNF chromatin remodeling complexes along with other core-pressor molecules like HDAC2 (35), DNMT3a (13), and MBDproteins (31). Biochemical assays have demonstrated thatPRMT5 activity on target H4R3 and H3R8 histone arginineresidues is markedly enhanced when lysine residues becomedeacetylated by HDAC2 (35). Our results showed that inhibi-tion of PRMT5 leads to loss of recruitment at target genepromoters (ST7,CXCL10, andCXCL11, data not shown). Impor-tantly, our prior work and current data suggest that by inhibit-ing PRMT5 overexpression alone, additional corepressor pro-teins contributing to anticancer gene silencing may also beaffected, providing rationale to further investigate the

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antitumor activity of combination strategies incorporatingagents that target the epigenome (HDAC inhibitors, hypo-methylating agents) with PRMT5 inhibitors.

Finally, PRMT inhibitionwas shown toprolong survival in anaggressive preclinical mouse glioblastoma model. Collectively,our data suggest that PRMT5 overexpression represents anunfavorable prognostic marker and an attractive therapeutictarget for glioblastoma. Experimental therapeutic strategiesaimed at inhibiting the effects of PRMT5 overexpression incancer might lead to a better understanding of how to directlyand indirectly affect glioblastoma tumor progression. Thesefindings further justify our current efforts to explore novelapproaches to target PRMT5 activity in glioblastoma and othercancers in which this enzyme is dysregulated.

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

Authors' ContributionsConception and design: F. Yan, L. Alinari, A. Chakravarti, H. Kwon, J.C. Byrd,C. Li, S. Lawler, R.A. BaiocchiDevelopment of methodology: F. Yan, L. Alinari, Y. Banasavadi-Siddegowda,A. Chakravarti, X. Wu, G. Nuovo, C.-H. Kwon, J.C. Byrd, C. Li, R.A. BaiocchiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Yan, L. Alinari, M.E. Lustberg, L.K. Martin, H.M.Cordero-Nieves, Y. Banasavadi-Siddegowda, S. Virk, J. Barnholtz-Sloan, J.Wojton,

N.K. Jacob, A. Chakravarti, M.O. Nowicki, B. YuK. Gordon, A. Haseley, J.T. Patton,P. L. Smith, J. Ryu, G.Marcucci, G. Nuovo, C.-H. Kwon, J.C. Byrd, S. Jacob, S. Lawler,B. Kaur, R.A. BaiocchiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Yan, L. Alinari, M.E. Lustberg, H.M. Cordero-Nieves, Y. Banasavadi-Siddegowda, S. Virk, J. Barnholtz-Sloan, E.H. Bell, J.Wojton,N.K. Jacob, B. YuK. Gordon, A. Haseley, J.T. Patton, X. Zhang, X. Mo, G. Marcucci,C.-H. Kwon, J.C. Byrd, E.A. Chiocca, C. Li, S. Lawler, R.A. BaiocchiWriting, review, and/or revision of the manuscript: F. Yan, L. Alinari, M.E.Lustberg, L.K. Martin, J. Barnholtz-Sloan, E.H. Bell, J. Wojton, N.K. Jacob,A. Chakravarti, R. Lapalombella, J. Datta, B. Yu, K. Gordon, X. Zhang, X. Mo,G. Marcucci, C.-H. Kwon, E.A. Chiocca, C. Li, S. Sif, S. Lawler, R.A. BaiocchiAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): B. Yu, K. Gordon, P. L. Smith, G.Marcucci, J.C. Byrd, E.A. Chiocca, C. Li, S. SifStudy supervision: G. Nuovo, C.-H. Kwon, R.A. Baiocchi

Grant SupportThis work was supported by grants from 1R21NS071346-01 (R.A. Baiocchi and

C. Li), The Ohio Cancer Research Associates (R.A. Baiocchi), V Foundation ( R.A.Baiocchi), 2009 AACR-National Brain Tumor Society Fellowship, inMemory ofBonnie Brooks (F. Yan), and the European Society of Hematology/AmericanSociety of Hematology (L. Alinari). F. Yan is a 2013 recipient of the AmericanAssociation for Cancer Research—Millennium Scholar in Training Award.

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

Received March 27, 2013; revised November 19, 2013; accepted December 14,2013; published OnlineFirst January 22, 2014.

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2014;74:1752-1765. Published OnlineFirst January 22, 2014.Cancer Res Fengting Yan, Lapo Alinari, Mark E. Lustberg, et al. PRMT5 as a Candidate Therapeutic Target in GlioblastomaGenetic Validation of the Protein Arginine Methyltransferase

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