In vivo radiation response of proneural gliomacharacterized by protective p53 transcriptionalprogram and proneural-mesenchymal shiftJohn Hallidaya,b,1, Karim Helmya,c,d,e,1, Siobhan S. Pattwellf, Kenneth L. Pittera, Quincey LaPlanta, Tatsuya Ozawaf,g,and Eric C. Hollandf,g,2

aDepartment of Cancer Biology and Genetics, Memorial Sloan–Kettering Cancer Center, New York, NY 10065; bGerstner Sloan–Kettering Graduate Schoolof Biomedical Sciences, New York, NY 10065; cDepartment of Medicine, New Jersey Medical School, University of Medicine and Dentistry of New Jersey,Newark, NJ 07103; dGraduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103; eWeill Medical College ofCornell University, New York, NY 10065; fDivision of Human Biology and Solid Tumor Translational Research, Fred Hutchinson Cancer Research Center,Seattle, WA 98109; and gDepartment of Neurosurgery and Alvord Brain Tumor Center, University of Washington, Seattle, WA 98195

Edited by Mark Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved February 28, 2014 (received for review November 8, 2013)

Glioblastoma is the most common adult primary brain tumor andhas a dismal median survival. Radiation is a mainstay of treatmentand significantly improves survival, yet recurrence is nearly in-evitable. Better understanding the radiation response of glioblas-toma will help improve strategies to treat this devastating disease.Here, we present a comprehensive study of the in vivo radiationresponse of glioma cells in a mouse model of proneural glioblas-toma. These tumors are a heterogeneous mix of cell types withdiffering radiation sensitivities. To explicitly study the gene expres-sion changes comprising the radiation response of the Olig2+ tumorbulk cells, we used translating ribosome affinity purification (TRAP)from Olig2-TRAP transgenic mice. Comparing both ribosome-associ-ated and total pools of mRNA isolated from Olig2+ cells indicatedthat the in vivo gene expression response to radiation occurs pri-marily at the total transcript level. Genes related to apoptosis andcell growth were significantly altered. p53 and E2F were implicatedas major regulators of the radiation response, with p53 activityneeded for the largest gene expression changes after radiation. Ad-ditionally, radiation induced a marked shift away from a proneuralexpression pattern toward a mesenchymal one. This shift occurs inOlig2+ cells within hours and in multiple genetic backgrounds. Tar-gets for Stat3 and CEBPB, which have been suggested to be masterregulators of a mesenchymal shift, were also up-regulated by radia-tion. These data provide a systematic description of the events fol-lowing radiation andmay be of use in identifying biological processesthat promote glioma radioresistance.

Glioblastoma multiforme (GBM) is the most common andaggressive primary brain tumor in adults (1). Current stan-

dard of care for patients with GBM is surgical resection followedby local irradiation and temozolomide chemotherapy. Despiteaggressive therapy, disease progression is universal and medianpatient survival is only 15 months (1). The vast majority of tumorrecurrence occurs within the field of high-dose irradiation, sug-gesting that intrinsic radioresistance of glioma cells is the limitingfactor in treatment effectiveness (2, 3). A more complete knowledgeof the cellular response to radiation in glioma is critically importantto understanding the radioresistant biology of this tumor and de-vising strategies for radiosensitization.Features of the native GBM microenvironment, such as hyp-

oxia and tumor–stroma interactions, are critical modulators ofthe radiation response (4–6). Additionally, interactions betweenGBM cells and the tumor microenvironment have been shown toaffect the stem-like character of GBM cells (7), which has beenlinked to radioresistance (8, 9). Much of the current understandingof the response to radiation of glioma cells comes from work donewith glioma cell lines cultured in vitro, but glioma cells cultured invitro can demonstrate markedly increased radiosensitivity com-pared with the same cells grown as intracerebral xenografts (10).For these reasons it is critical to develop an understanding of theresponse of glioblastoma cells to radiation in vivo.

In vivo studies of radiation response are complicated by sev-eral factors. Glioblastomas are complex mixes of tumor cells andvarious stromal cell types, including astrocytes, immune cells,endothelial cells, and vascular smooth muscle cells (11). Mean-ingful analysis of in vivo gene expression changes after radiationtherefore requires techniques for isolating the expression patternsof individual populations of cells. An important development inrecent years has been the discovery that high-grade human gliomasactually encompass three to four distinct molecular subtypes oftumors (12, 13). These subtypes are associated with differential ac-tivation of oncogenic signaling pathways and correlate with specificgenomic alterations. Because many of these pathways have beenimplicated in the radiation response, in vivo studies of radiationmust take care to use relevant models that resemble the subclassesobserved human patients.Furthermore, in vivo expression profiling has been restricted

to total mRNA levels. Radiation-induced DNA damage has beenreported to regulate translational efficiency both globally and ina transcript-specific manner (14, 15). In glioma cell lines culturedin vitro, irradiation was reported to cause a much larger changein the translating pool of RNA than in total cellular RNA,suggesting a more important role for translational regulationthan transcriptional regulation in the GBM radiation response(16). Traditional polysome isolation methods, however, are notamenable to translational profiling in vivo (11).To study the global radiation response in an in vivo model of

a defined GBM subtype, we combined translating ribosome af-finity purification (TRAP) methodology (17) with an RCAS/tv-a

Significance

Glioblastoma is one of the most radio-resistant tumors, and themechanisms of radioresistance are intensely studied. Here, weuse a mouse model of proneural glioma to evaluate in vivoradiation-induced gene expression regulation at both the trans-lational and transcriptional levels. We found that p53 and E2F aremajor regulators of the in vivo radiation response, and that thereis little contribution from translational regulation, in contrast toprevious in vitro reports. We also found that radiation inducesa rapid shift in subtype from proneural to mesenchymal, whichmay have important implications for attempts to tailor targetedtherapy regimens to tumor subtype.

Author contributions: J.H., K.H., Q.L., T.O., and E.C.H. designed research; J.H., K.H., S.S.P.,Q.L., and T.O. performed research; T.O. contributed new reagents/analytic tools; J.H.,K.H., S.S.P., K.L.P., and Q.L. analyzed data; and J.H., K.H., S.S.P., and E.C.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1J.H. and K.H. contributed equally to this work.2To whom correspondence should be addressed. E-mail: eholland@fhcrc.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1321014111/-/DCSupplemental.

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genetic model of the proneural GBM subtype (18). Irradiationof these tumors induced a rapid and transient cell cycle arrestand asynchronous apoptosis. Interestingly, translational regula-tion was a relatively minor contributor to overall gene expressionchanges. Changes in total transcript levels dominated expressionchanges observed, with genesets for apoptosis and regulation ofcell cycle up-regulated. p53 and E2F targets were among themost frequently altered, with p53 appearing to regulate the mosthighly induced transcripts after radiation. We also observed ashift in glioma subtype from proneural to mesenchymal within 6 hof irradiation, which was highly significant regardless of geneticbackground. The proneural to mesenchymal shift occurred in tumorcells and was not a result of stromal enrichment after radiation.

ResultsResponse to Ionizing Radiation is Heterogeneous by Cell Type in GBM.To study the glioma radiation response in vivo, we introducedplatelet-derived growth factor B (PDGF-B) retrovirus into miceharboring somatic deletion of the Ink4a/Arf locus to generatehighly penetrant high-grade gliomas that closely resemble theproneural subtype of GBM (12, 13, 18–20). We chose to use asingle dose of radiation to enable time course studies after radia-tion, and had previously shown that a single dose of 10-Gy radiationinduces cell cycle arrest in these tumors (21). Tumor-bearing micewere subjected to a single dose of 10-Gy total body ionizing radi-ation (IR), and tumor tissue was collected at time points aftertreatment for immunohistochemical staining. Radiation caused acomplete mitotic arrest, with nearly undetectable levels of phos-phorylated-histone H3 (pH3) at 6 h after treatment (Fig. 1A).Proliferating cells reappeared by 24 h after IR and returned topretreatment levels by 72 h and 6 d after IR (Fig. 1A). By con-trast, the apoptosis marker TUNEL was detected in only a smallpercentage of cells, but apoptosis was observed at all time pointsmeasured after IR, demonstrating an asynchronous mode ofdeath (Fig. 1B). This limited, delayed apoptosis in response to IRcontrasts sharply with the high levels of apoptosis observed in themore radiosensitive medulloblastoma brain tumor (9). Previousexperiments demonstrated that this radiation dose confers onlya modest survival benefit of ∼15 d in this model, consistent withthe radioresistant phenotype of human GBM and the compara-tively minimal apoptosis observed in these experiments (21).Human GBM is a heterogeneous tumor composed of several

cell types and displaying multiple histological features. PDGF-driven mouse gliomas recapitulate this complexity, containingtumor bulk (TB) regions that are nucleus dense and stain posi-tive for Olig2 and PDGF receptor α; and perivascular (PVN)regions that have a splindloid morphology and stain positive for

nestin, smooth muscle actin, and PDGF receptor β (11). Im-munofluorescent costaining for TUNEL and nestin revealed thatIR-induced apoptosis occurs almost exclusively in the TB andminimally in the nestin-positive PVN (Fig. 1E). This accumula-tion of cell death in the TB causes a progressive reduction in thepercentage of tumor cells staining positive for Olig2, droppingfrom 66% before treatment to 43% after 6 d (P = 0.00037) (Fig.1 C and F). The regions of spindloid cells marked by nestinstaining expand concurrently (Fig. 1D).Tumors are composed of both transformed cells and recruited

stroma. The PDGF oncogene that drives this model containsa hemaglutinin (HA) tag, facilitating discrimination betweenoncogene-expressing tumor cells and nontumor stromal cells.Immunohistochemical staining reveals that most, if not all,PDGF-HA expressing tumor cells reside in the Olig2+ TB bothbefore and after IR (SI Appendix, Fig. S1A). FACS analysis ofgliomas generated with a bicistronic retroviral vector expressingPDGF and RFP in mice expressing GFP driven by the Olig2promoter had confirmed the high degree of coexpression (11).Taken together, these data suggest that PDGF-driven mouse

glioma models the radioresistant phenotype of human GBM.Furthermore, we demonstrate that the cellular response to IR isheterogenous, with oncogene-expressing tumor cells respondingdifferently than nontumor stroma. It is therefore necessary tostudy the radiation biology of the different cell populations in-dependently, because any differences in radiation response bycell type and changes in cellular composition after radiation wouldconfound interpretation of the radiation biology of GBM studiedas homogenous entity.

Regulation of Total Transcript Levels Dominates the Radiation-InducedChanges in Ribosome-Bound RNA. To study global gene expressionchanges, we adapted the TRAP methodology to the RCAS-PDGFglioma model, as described (11). Briefly, PDGF-induced gliomaswere generated in Olig2 bacTRAP mice (22). Tumors were dis-sected from untreated control mice and treated mice 6 h after 10-Gyirradiation. Tumor tissue was divided to collect both total RNAfrom sorted GFP+ cells and translating RNA via immunoprecip-itation of GFP-tagged ribosomes from the same cell population.Paired ribosome-bound and total cellular RNA samples wereanalyzed by expression microarray.Changes in the levels of individual transcripts present on ribo-

somes can be divided into changes in the level of each transcript,and changes in the efficiency with which each transcript is re-cruited to ribosomes for translation, i.e., translation efficiency.Previously, it had been reported that a much larger number oftranscripts showed significant changes in translational efficiencythan in overall transcript level after IR in vitro (16). In our in vivosystem, changes in translation efficiency contributed relativelylittle to overall changes in the mRNAs present on ribosomes afterirradiation (Fig. 2 A and B). Changes in total mRNA and trans-lating mRNAs were highly correlated (R = 0.82) (Fig. 2A). Fifty-eight transcripts showed translational efficiency changes of morethan twofold, compared with 429 transcripts showing twofoldchanges at the total mRNA level. Similarly low levels of trans-lational efficiency changes were seen in irradiated tumors thatadditionally had PTEN deleted by coinjection of an RCAS-Crevirus (SI Appendix, Fig. S2A) (11). For reference, when comparingnormal brain Olig2+ oligodendrocyte precursor cells to theirPDGF-transformed tumor counterparts, 2,239 transcripts exhibi-ted greater than a twofold change in translational efficiency (11).We specifically looked at the translational efficiency of mRNAsshown previously to be translationally regulated in vitro in re-sponse to radiation and found no significant regulation in our invivo system (SI Appendix, Fig. S3). Thus, the gene expression re-sponse to IR of these gliomas does not appear to have a largetranslational regulation component in vivo.

IR-Induced Changes in Gene Expression Primarily Involve GenesetsRelated to Apoptosis and Cell Cycle Arrest. We next sought to char-acterize the gene expression changes induced by radiation in

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Fig. 1. Ten gray of IR induces transient cell cycle arrest, asynchronousapoptosis, and depletion of Olig2+ cells in mouse glioma. Staining forphospho-histone H3 (A), TUNEL (B), Olig2 (C), and Nestin (D). (E) TUNEL+

cells predominantly in tumor bulk rather than Nestin+ perivascular region24 h after IR. (F) Quantification of Olig2+ nuclei in glioma following IR. Errorbars are SD. *P < 0.05, **P < 0.005.

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vivo. Few genes were highly down-regulated 6 h after 10-Gy IRin the Olig2+ TB, but a number of genes were highly up-regu-lated (Fig. 2C and SI Appendix, Fig. S2B). The most up-regulatedgenes by fold change included several genes known to be in-volved in cell cycle arrest and apoptosis. Fas, Puma, Noxa, andApaf1 are all associated with induction of apoptosis, whereascyclin G and p21 are cell cycle inhibitors, and MDM2 andTRP53INP1 have roles in both functions. Additionally, severalgenes such as Ddit4L, Aldh4A1, and Sestrin2 were up-regulated,which like TRP53INP1, are involved in the response to variousstresses including DNA damage and redox imbalances. Ddit4L isnot well characterized, but its homolog Ddit4 has been shown to beradioprotective (23) and was also up-regulated. Some genes ofunknown relevance were highly up-regulated as well; carbonyl re-ductase 2 (DCXR) and synaptic vesicle 2-related protein were themost up-regulated genes and have no known function in the ra-diation response, whereas PQ loop repeat containing 3 (PQLC3) islargely uncharacterized. To validate the reliability of the arrayresults, we selected some of the most highly up-regulated genes,along with some family members, and performed quantitative PCR(qPCR) on RNA extracted from treated and control neurospheresderived from PDGF-driven Ink4a/Arf−/− gliomas. The fold in-duction in neurospheres at 6 h closely matched the fold changeseen on the array for all transcripts examined (SI Appendix, Fig. S4A and B).To understand what classes of genes are altered by IR, we

performed geneset enrichment analysis (GSEA) (24). In PDGF-driven gliomas with both PTEN intact or deleted, genesets as-sociated with caspase activation and induction of apoptosis werethe highest scoring genesets, whereas the geneset for negative

regulation of growth also was significantly up-regulated (Fig. 2Dand SI Appendix, Fig. S2C). Curiously, the geneset for positiveregulation of cellular proliferation was also the 13th most up-regulated geneset. However, an inspection of the leading edge ofthis geneset revealed that its significance is driven largely by theup-regulation of signaling molecules rather than bona fide cellcycle genes (p21 is also included in this geneset). Olig2+ tumorcells irradiated in vivo increased the expression of a range ofgrowth factors and cytokines, including PDGF-A, CSF1, PGF,LIF, and EGFR.With relatively few individual transcripts significantly down-

regulated, the only geneset highly significantly enriched for down-regulation in PDGF gliomas was “Regulation of Action Potential.”The significance of this geneset was driven by down-regulation ofgenes associated with oligodendrocyte development and myelina-tion, including MBP, MOG, S100b, and SHH, raising the possi-bility of a loss of oligodendrocytic differentiation in these cells inresponse to irradiation (Fig. 2E). PTEN−/− tumors had more sig-nificantly down-regulated transcripts and genesets, and genesetsassociated with mitosis were highly down-regulated (SI Appendix,Fig. S2D). Both PTEN wild-type (WT) and PTEN−/− tumors show-ed down-regulation of RNA processing and DNA replicationgenesets. Additionally, during initial experiments, we also gener-ated expression profiles from the PDGF Ink4a/Arf+/− model (19),which showed similar regulation of both individual transcripts andbroader genesets after radiation (SI Appendix, Fig. S5 A and B).To further determine which gene changes are specific for irra-

diation, and which are general features of cells undergoing arrest,we used an alternative method for generating cell cycle arrest. ThemTOR inhibitor temsirolimus is nongenotoxic and induces cellcycle arrest in PDGF-driven gliomas (25). Olig2+ cells fromPDGF-induced PTEN−/− gliomas treated with either radiation ortemsirolimus shared many of their respective down-regulatedgenes (SI Appendix, Fig. S6 A and B). These shared down-regu-lated genes were enriched for genesets associated with cell cycle,nuclear division, and mitosis. Although many of the down-regu-lated genes were overlapping, the up-regulated genes in each casewere largely unique to the given treatment. The genes up-regu-lated after temsirolimus treatment were not significantly enrichedfor any genesets (SI Appendix, Fig. S6C). By contrast, the genesthat were relatively up-regulated in the irradiated tumors wereenriched for apoptosis, DNA damage, and checkpoint-relatedgenesets (SI Appendix, Fig. S6D).

p53 and E2F Activity Are Major Transcriptional Drivers of the RadiationResponse. Having established that the in vivo radiation response islargely determined by changes in total mRNA levels rather thantranslational modulation, we sought to understand which tran-scription factors might be responsible for the changes in transcriptlevels observed. GSEA was performed to identify transcriptionfactors whose binding motifs were most commonly found in thepromoters of genes regulated by radiation. Genes containing ap53-binding site in their promoter were among the most sig-nificantly up-regulated in both PTEN WT and deleted tumors,consistent with our previous observation that several p53 targetswere among the most significantly up-regulated transcripts (Fig. 3A and B and SI Appendix, Fig. S7 A and B). Immunohistochem-istry of irradiated and control tumors showed that p53 becomesnuclear localized after irradiation and is restricted to the tumorbulk rather than the perivascular regions (SI Appendix, Fig. S7C).To confirm p53 regulation of specific transcripts, we used irradi-ated and unirradiated p53−/− tumorspheres as in ref. 21 and per-formed qPCR for the same panel of genes as in SI Appendix, Fig.S4A and B. Each gene that was up-regulated after IR in p53-intactneurospheres was unaltered in p53−/− neurospheres (SI Appendix,Fig. S4 A–C). Interestingly, the geneset “Negative Regulation ofProgrammed Cell Death” was the 18th and 10th most up-regu-lated geneset in PTEN intact and deleted tumors, respectively.The up-regulation of this set of genes, which includes Fas but alsoBAX, BCL2L1, NFKB1, and IL-7, may underlie some of theradioresistance of these tumors.

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Fig. 2. In vivo gene expression changes induced by IR in RCAS-PDGF Ink4a/Arf−/− gliomas collected 6 h after 10-Gy IR. (A) Log2 change in total mRNA vs.log2 change in ribosome-bound translating mRNA 6 h after 10-Gy IR. R,Pearson correlation. (B) All probes altered more than twofold in translatingmRNA, and corresponding changes in total mRNA and translational effi-ciency. (C) Volcano plot of expression changes 6 h after 10-Gy IR. Genesetsmost significantly enriched (D) and depleted (E) among translating mRNAsafter IR, with selected enrichment plots. FDR q-val, calculated false discoveryrate; NES, Normalized Enrichment Score.

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Several other transcription factor binding motif genesets weresignificantly up-regulated after 6 h, including genesets for CEBP,myocyte enhancer factor 2, IFN regulatory factor (IRF), andSTAT3. IRF is a tumor suppressor that has been shown toenhance the transcriptional activity of p53 (26). Genesets fordown-reglated genesets were dominated by those associated withE2F-binding motifs. E2F family transcript levels also fell afterirradiation, driven by E2F1, which fell more than twofold by 6 h(SI Appendix, Fig. S7B).In our preliminary experiments with Ink4a/Arf+/− mice, we

collected RNA at both 2 h and 6 h after 10-Gy IR. The Ink4a/Arf+/− dataset was consistent with the Ink4a/Arf−/− dataset; fortranscription factors with targets up-regulated in Ink4a/Arf−/−

tumors, their targets were already up-regulated by 2 h in Ink4a/Arf+/− tumors and further up-regulated at 6 h as the radiationresponse progressed (SI Appendix, Fig. S7B). Likewise, E2Ftargets were down by 2 h and further decreased at 6 h, and E2Ftranscript levels decreased along a similar time course.

p53 Drives Most of the Highly Induced Genes in the in Vivo RadiationResponse. The gene encoding p53 is altered in 35–50% of pro-neural tumors, including tumors that exhibit down-regulation ofp53 expression without genomic changes (13, 27). These alterationsin p53 expression may co-occur with Ink4a/Arf loss and are in-dependent of Ink4a/Arf status. Because p53 is such a large com-ponent of the radiation response in our PDGF-Ink4a/Arf−/− gliomamodel, we compared the radiation response at 6 h of tumors withcompromised p53 to p53 WT tumors. We generated gliomas byusing a p53shRNA-RFP RCAS retroviral vector in combination

with RCAS-PDGF in Ink4a/Arf−/− Olig2-TRAP reporter mice.Consistent with previous work, p53shRNA-containing tumors ex-hibited shorter latency compared with PDGF-only tumors, anda single dose of 10-Gy radiotherapy conveyed a survivalbenefit of only 8 d, compared with 15 d for PDGF-only tumors(Fig. 3D; ref. 21). Immunohistochemistry of p53shRNA-PDGF tumors revealed lower levels of nuclear p53 after IR(SI Appendix, Fig. S8B). To perform expression profiling, wecollected RFP/GFP double-positive cells via FACS to isolatetumor cells definitively infected with the p53shRNA virus.p53 transcript levels were significantly lower in p53shRNA-PDGF tumors compared with PDGF-only tumors (SI Appendix,Fig. S8A). Interestingly, unsupervised clustering placed p53shRNAtumors 6 h after irradiation with unirradiated tumors rather thanirradiated p53 intact tumors, indicating the importance of p53 inshaping the overall radiation response (Fig. 5C).The most up-regulated transcripts after IR in the p53shRNA-

PDGF gliomas were similar to those of the PDGF-only tumors,but the p53 short hairpin markedly inhibited the up-regulation ofvirtually all of the most highly up-regulated transcripts in p53intact tumors (Fig. 3E), suggesting that p53 is required for thevast majority of the largest gene expression changes in p53 intacttumors. The induction of each previously ex vivo validated p53target was also lower in the p53shRNA/PDGF tumors (SI Ap-pendix, Fig. S4A). p53 likely drives much of the caspase pathwayup-regulation seen in these tumors, because caspase activationwas the most highly enriched geneset when comparing relativeexpression changes after radiation in PDGF-only and p53shRNAgliomas (SI Appendix, Fig. S8E).

Radiation Triggers a Shift from Proneural Toward MesenchymalExpression Patterns. When Phillips et al. first described the ma-jor high-grade glioma subtypes, they noted that some proneuraltumors tended, upon recurrence, to shift toward a mesenchymalgene signature (12). Because that report and subsequent workshave relied on gene expression profiling of whole tumor lysates,and because the mesenchymal subgroup tends to express highlevels of markers typical of glioma stromal cell types (11, 12), it isunclear whether any shifts in subtype, and indeed whether sig-nificant differences between the groups in general, are duelargely to varying amounts of stroma. It was reported that Stat3and CEBPB acted as master regulators of mesenchymal trans-formation, whose expression could drive neural stem cells alonga mesenchymal lineage, and whose inhibition in tumor cellscaused both a loss of mesenchymal signature and tumor ag-gressiveness (28). As such, we noted with interest the presence ofboth CEBPB and Stat3 targets high on our list of most up-reg-ulated transcription factor motifs in the Olig2-expressing cellsafter radiation (Figs. 3A and 4 A and B). Further, pStat3 levelswere elevated at 6 h after IR, as detected by IHC (SI Appendix,Fig. S9).To determine whether radiation did, in fact, induce a shift

from proneural to mesenchymal lineages in these tumor cells invivo, we performed GSEA on proneural and mesenchymal genesignatures from Verhaak et al. (13). Although the Olig2 tran-script was reduced by 1.7-fold, 6 h after radiation, the produrancefor the GFP-L10a protein is longer than 6 h (SI Appendix, Fig. S10A and B), allowing expression profiling of the cells that were Olig2expressing at the time of radiation. Olig2+ tumor cells 6 h after invivo irradiation did, in fact, exhibit highly significant enrichment ofgenes in the mesenchymal gene signature, and equally significantloss of proneural signature genes [false discovery rate (FDR)q value of 0.000 for both genesets] (Fig. 4C). The shift is detectedin RNA isolated from Olig2+ tumor bulk cells via either cellsorting or cell type-specific TRAP, and also occurred in tumorswith PTEN deleted (Fig. 4C). Thus, the proneural to mesenchymalshift induced by IR occurs in the tumor cells themselves and is notan artifact of changing stromal content. In fact, the shift had oc-curred within 2 h in Ink4a/Arf +/− tumors, before any selection hashad time to occur (SI Appendix, Fig. S11 A and B).

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Fig. 3. p53 and E2F are major drivers of in vivo radiation response. (A)Transcription factors whose targets are most significantly enriched and de-pleted among translating mRNAs after IR. (B) Enrichment plots for targets ofp53 and E2F. (C) Unsupervised hierarchical clustering of genes significantlyaltered by IR in PDGF and PDGF/p53shRNA gliomas. (D) Kaplan–Meier sur-vival curves of unirradiated and irradiated PDGF/p53shRNA gliomas in Ink4a/Arf−/− mice after postnatal day 18 randomization and single-dose 10-Gy IR.(E) Fold changes of the 50 most induced transcripts 6 h after 10-Gy IR in totalRNA from PDGF-driven gliomas, and the corresponding fold change in totalRNA from PDGF/p53shRNA tumors.

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Unlike many of the changes observed after IR, the shift fromthe proneural to mesenchymal character does not appear to behighly sensitive to p53 activity. p53shRNA tumors also showedhighly significant loss of proneural markers and gain of mesen-chymal markers (Fig. 4C). GSEA on the relative expressionchanges after irradiation of PDGF-only vs. PDGF+p53shRNAtumors showed nonsignificant enrichment in the PDGF-onlytumors for both proneural and mesenchymal genesets after IR(Fig. 4D). Thus, it does not appear that the decrease in p53activity in the p53shRNA tumors coherently inhibits or reversesthe proneural to mesenchymal shift in those tumors.Previously, we had shown that treatment of a Ras/Akt glioma

model with temsirolimus shifted surviving tumor cells towarda more oligodendroglial phenotype, including induction of theproneural marker Olig2 (29). Because mTOR activity as mea-sured by pS6 activity is higher in PTEN−/− PDGF-driven tumors(11), we wondered whether our PTEN-deleted tumors weremore mesenchymal. Indeed, PTEN−/− tumor cells are shiftedfrom proneural toward mesenchymal in our expression dataset(SI Appendix, Fig. S11C). However, temsirolimus treatment ofPTEN−/− tumors did not trigger a reversal of the proneural tomesenchymal shift (SI Appendix, Fig. S11D), and IHC of irra-diated tumors does not suggest an increase in mTOR activity(SI Appendix, Fig. S11E). Taken together, these findings in-dicate that the IR-induced proneural to mesenchymal shift isnot driven by mTOR signaling.Overall, these results show that radiation rapidly induces a

shift in expression profile from proneural to mesenchymal overthe course of a few hours. This shift occurs specifically in pro-neural tumor cells and is not a function of stromal enrichment.Furthermore, unlike much of the response to radiation, theproneural to mesenchymal transformation does not appear to becoherently affected by p53 activity.

DiscussionGBM is a devastating disease for which there is no cure. Radi-ation is standard therapy for patients with glioma, however,therapeutic response is limited and disease progression occursuniversally. A better understanding of the radiation biology of

glioma is critical to making progress in treating this destructivedisease. Much of our current understanding of the radiationresponse of glioma is based on data from cultured glioma cells.The in situ tumor microenvironment differs greatly from thatprovided by cell culture, and work has shown that cells grown invivo may have a markedly different radiation response than thosegrown in culture (10). However, interpretation of in vivo gliomaradiation studies is complicated by the highly heterogeneouscellular composition of glioma.In this study, we used a PDGF-driven mouse model of glioma

to study the in vivo radiation response in a defined population ofproneural tumor cells. It should be noted that although we be-lieve our model is fairly representative of proneural gliomas asa whole, ∼50% of proneural gliomas have Ink4a/Arf intact. Al-though 35% exhibit PDGFRA amplification, the extent to whichPDGF ligand is a driver of this subtype is not known (13).Radiation of PDGF-driven gliomas with a single 10-Gy dose of

radiation resulted in transient cell cycle arrest and asynchronousapoptosis. The effects of radiation vary by cell type, becauseapoptosis is largely restricted to the Olig2+ tumor cells and isaccompanied by a decrease in the proportion Olig2+ cells overtime. We used the TRAP system targeted to Olig2+ cells to studyboth translating and total RNA populations specifically in theOlig2+ proneural tumor bulk before and after radiation. Some-what surprisingly, and in contrast with previous work done in cellculture, the translational contribution to overall gene expressionchanges is minimal in this in vivo system.Consistent with our histological data, we find that apoptotic

gene expression programs are highly induced within 6 h by IRand are accompanied by a loss of expression of genes associatedwith cell cycle progression. Several transcripts are also highlyinduced that are involved in stress responses, including redoximbalances that may be important in modulating the genotoxiceffect of radiation (5). A range of transcripts for secreted growthfactors and cytokines are also up-regulated after radiation.The transcription factors contributing most to these effects

appear to be p53 and E2F. p53 activity drives the largest ex-pression differences observed and modulates tumor resistance toIR. The targets of both were among the most significantly alteredsets of transcription factor targets in each of the tumor typeswe studied. The targets of IRF, a transcription factor known tocooperate with p53, were also among the most heavily inducedby radiation. The largest expression differences observed are p53dependent, and suppression of p53 activity by shRNA modulatestumor resistance to IR. p53 controls the overall expressionchanges after radiation to such a large extent that irradiated p53knockdown tumors cluster more closely with unirradiated tumorsthan with irradiated p53 intact tumors.A shift toward mesenchymal subtype at recurrence in pre-

viously proneural gliomas has been noted (12). In this work, wehave identified a clear shift in expression pattern from proneuralto mesenchymal within 6 h in response to radiation. This shiftwas observed after radiation in each of the tumor types we ex-amined. The proneural to mesenchymal shift we see is tumor cellspecific and is not the result of stromal enrichment via higherrates of cell death in the tumor bulk. Although changes in thestromal compartment may also affect tumor subtype, as mea-sured by profiling of whole tumor lysate, we show here thatthe tumor bulk cells themselves undergo a subtype shift afterradiation treatment. CEBPB and Stat3 have previously beensuggested to act as master regulators of a proneural to mes-enchymal shift in glioma cells, but the signaling network inthat case was inferred from expression data from wholetumor lysates. In RNA isolated from Olig2+ tumor cells, bothCEBPB and Stat3 were among the transcription factors whosetargets were highly induced by radiation. Our data are evi-dence that a proneural-to-mesenchymal shift occurs in thesame subset of cells in which CEBPB and Stat3 activity is el-evated, supporting the reported link between CEBPB andStat3 and the proneural-to-mesenchymal shift.

A

C D

B

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Chg

CEBP Targetsqval = 0.084

STAT3 Targetsqval = 0.163

CEBP Targets STAT3 Targets

PDGF + Cre hs35p+FGDPFGDPWT vs p53sh

Relative IR Chgsqval = 0.000 qval = 0.000qval = 0.000

Mes

ench

ymal

qval = 0.113

Mes

ench

ymal

qval = 0.008 qval = 0.000qval = 0.000

Pron

eura

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Fig. 4. IR induces proneural-to-mesenchymal shift in PDGF gliomas. (A)Enrichment plots of CEBP and STAT3 targets in translating mRNA 6 h after10-Gy IR in PDGF Ink4a/Arf−/− gliomas. (B) Time course of CEBP and STAT3target up-regulation in PDGF Ink4a/Arf+ /− gliomas. Average of total andtranslating mRNA pools is shown; dotted lines are SE. (C) Enrichment plots ofmesenchymal and proneural signature genes in translating mRNA after 10-GyIR in PDGF-driven Ink4a/Arf−/−, Ink4a/Arf−/−/PTEN−/−, and Ink4a/Arf−/−/p53shRNAgliomas. (D) Enrichment plots of mesenchymal and proneural signature genesin the relative changes of translating mRNA after IR of PDGF Ink4a/Arf−/−

tumors vs. PDGF Ink4a/Arf−/−/p53shRNA tumors.

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The subtype shift we observe is not clearly affected by p53 ac-tivity and is also not likely to be driven my mTOR signaling. Stat3can be phosphorylated through a variety of pathways, includingSrc, Abl, mTOR, and JAKs. IL-15, LIF, and IL-7 are all amongthe most up-regulated transcripts after IR in Olig2+ cells them-selves, and all are known activators of Stat3 via gp130/JAK (30,31), suggesting that in irradiated proneural gliomas, cytokine ac-tivation of JAKs may drive the proneural-to-mesenchymal shift.Further studies are necessary to fully elucidate the mechanism bywhich radiation induces a proneural-to-mesenchymal shift.Recent work indicates that mesenchymally shifted cells are

more radioresistant (32). Given that therapeutic radiationis given in fractions over a period of days, a radiation-inducedmesenchymal shift may represent an important mechanism ofresistance to fractionated therapy, with important consequencesfor optimal design of radiation schedules.The recent discernment of distinct subtypes of high-grade

glioma and the identification of distinct growth factor pathwaysactive in each subtype suggested that targeted pharmacologicalinhibition of these pathways on a subtype-specific basis mightlead to better clinical outcomes. Subtyping of patient gliomas hasalready begun in an effort to develop more targeted individualizedcourses of therapy. However, the shift in subtype from proneuralto mesenchymal that we observe here within hours of IR has im-portant implications for this paradigm. The speed of the shift isevidence that the subtypes may not be as hardwired as previouslybelieved. The apparent plasticity of gliomas with regard to subtypemay indicate a need to target not only the oncogenic pathwaystypically associated with a given subtype, but also to identify andtarget other progrowth pathways that may become activated bytreatment. Radiation-induced shifts are of particular importance,because radiation is a nearly universal component of glioma ther-apy. As various targeted therapies are being tested in combinationwith radiation, understanding the shifts in signaling patterns in

response to radiation will be crucial to interpreting the resultsof these trials.

MethodsGeneration and Treatment of Murine Gliomas. PDGF-driven gliomas weregenerated in neonatal mice by RCAS-mediated retroviral transduction asdescribed (18, 19). Symptomatic (lethargy, weight loss, macrocephaly)tumor-bearing mice were subjected to total body irradiation by using aCs-137 source (Gammacell 40 Exactor; MDS Nordion). For survival studies, 10-Gysingle-dose cranial IR was delivered on postnatal day 18 by an X-RAD 320(Precision X-Ray) as in ref. 21. Temsirolimus was administered for 3 d at40 mg/kg as in ref. 25.

Collection and Labeling of Ribosome-Bound and Total Cellular RNA. Tumorsgenerated in Olig2 bacTRAP mice (22) were grossly dissected. Translating RNAwas collected by immunoprecipitation, and total RNA was collected via FACS asin ref. 11. RNA was amplified and biotin-labeled according to manufacturerprotocol (Ambion AMIL1791) and hybridized to Illumina MouseRef-8 ExpressionBeadChips. RNA quality and labeling efficiency was assessed by Agilent 2100Bioanalyzer.

Microarray Analysis and Geneset Enrichment. Partek Genomics Suite softwarewas used to quantile normalize and log2 transform all data. For translationalefficiency analysis, as in ref. 11, a detection P value filter of 0.05 was applied tobackground subtracted expression values before quantile normalization andlog2 transformation. GSEA was performed with the javaGSEA Desktop Applica-tion by using 1,000 geneset permutations, the provided gene ontology biologicalprocess and transcription factor motif genesets, and all other default settings(24). Genesets for glioma subtype signatures were obtained from ref. 13.

ACKNOWLEDGMENTS. This study was supported by the National Institutesof Health Grants R01 CA100688, U54 CA126518, U01 CA141502-01, and U54CA143798. K.H. was a Howard Hughes Medical Institute Medical ResearchTraining Fellow.

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