perk regulates glioblastoma sensitivity to er stress ... · of a downstream prosurvival mediator of...

PERK Regulates Glioblastoma Sensitivity to ERStress Although Promoting Radiation ResistanceDavid Y.A. Dadey1,2,3, Vaishali Kapoor1, Arpine Khudanyan1,4, Dinesh Thotala1,5,and Dennis E. Hallahan1,5

Abstract

The aggressive nature and inherenttherapeutic resistance of glioblastomamul-tiforme (GBM) has rendered the mediansurvival of afflicted patients to 14 months.Therefore, it is imperative tounderstand themolecular biology of GBM to providenew treatment options to overcomethis disease. It has been demonstrated thatthe protein kinase R–like endoplasmicreticulum kinase (PERK) pathway is animportant regulator of the endoplasmicreticulum (ER) stress response. PERKsignaling has been observed in othermodelsystems after radiation; however, less isknown in the context of GBM, which isfrequently treated with radiation-basedtherapies. To investigate the significance ofPERK, we studied activation of the PERK–eIF2a–ATF4pathway inGBMafter ionizingradiation (IR). By inhibiting PERK, itwas determined that ionizing radiation(IR)-induced PERK activity led to eIF2aphosphorylation. IR enhanced the pro-death component of PERK signaling in cells treated with Sal003, an inhibitor of phospho-eIF2a phosphatase.Mechanistically, ATF4 mediated the prosurvival activity during the radiation response. The data support the notion thatinduction of ER stress signaling by radiation contributes to adaptive survival mechanisms during radiotherapy. The data alsosupport a potential role for the PERK/eIF2a/ATF4 axis in modulating cell viability in irradiated GBM.

Implications: The dual function of PERK as a mediator of survival and death may be exploited to enhance the efficacy ofradiation therapy.

Visual Overview: http://mcr.aacrjournals.org/content/16/10/1447/F1.large.jpg. Mol Cancer Res; 16(10); 1447–53. �2018 AACR.

IntroductionGlioblastoma multiforme (GBM), the most common pri-

mary malignant brain tumor in adults, is characterized by ahigh propensity for invasion and proliferation although beingresistant to therapy (1). Although slight improvements inGBM therapy outcomes were achieved with the advent oftemozolomide and radiotherapy, the 5-year survival rateremains under 10% (2). In efforts to improve therapy forpatients afflicted with GBM, oncologists have sought to iden-tify novel molecular targets involved in promoting the viabil-ity and therapeutic resistance. One approach to therapyfor GBM involves targeting elements of the endoplasmic

1Department of Radiation Oncology, School of Medicine, WashingtonUniversity in St. Louis, St. Louis, Missouri. 2Medical Scientist TrainingProgram, School of Medicine, Washington University in St. Louis, St. Louis,Missouri. 3Department of Neurosurgery, Stanford University School ofMedicine, Stanford, California. 4Oregon Health and Science University,School of Medicine, Portland, Oregon. 5Siteman Cancer Center, School ofMedicine, Washington University in St. Louis, St. Louis, Missouri.

Corresponding Author: Dennis E. Hallahan, Department of RadiationOncology, Washington University in St. Louis, 4511 Forest Park, St. Louis,MO, 63108. Phone: 314-362-9700; Fax: 314-747-5498; E-mail:[email protected].

doi: 10.1158/1541-7786.MCR-18-0224

�2018 American Association for Cancer Research.

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reticulum stress response (ERSR). The ERSR has been increas-ingly shown to be highly deregulated in cancer, where pro-survival aspects of the stress response are preferentially acti-vated and involved in oncogenesis (3). In GBM, upregulationof a downstream prosurvival mediator of the ERSR, GRP78,has been linked to tumor grade, temozolomide resistance, andprognosis (4–6). The ERSR is a broad signaling networkinvolving three major pathways: activating transcription factor6 (ATF6), inositol-requiring enzyme 1 (IRE1), and proteinkinase R–like endoplasmic reticulum kinase (PERK; ref. 7).

During ER stress, ATF6 translocates to the Golgi where itis cleaved by proteases (7). The cleaved ATF6 cytosolic fragmentcan then act as a transcription factor and drive the synthesis of ERchaperones, including GRP78 (8). IRE1 splices Xbox bindingprotein1 (XBP1) mRNA to yield XBP1-S, which then upregulatestranscription of genes involved in protein degradation (7, 8).PERK phosphorylates initiation factor 2a (eIF2a) and transientlyhalts the global protein translation although upregulating ATF4translation (7). ATF4 then triggers expression of genes involved inamino acids metabolism and suppression of oxidative stress (8).However, during prolonged ER stress, ATF4 promotes CHOPexpression, which is involved in promoting ER stress–inducedcell death (7). Characterization of the physiologic roles of ATF6,IRE1, and PERK in the radiation response may lead to theidentification of novel molecular targets to improve therapeuticefficacy.

Previously, we have shown that radiation can induce ERstress and downstream signaling associated with the ERSR(8). Studies have shown that PERK signaling is also inducedby radiation in endothelial cells (9). In this study, we foundthat radiation activates the PERK pathway in GBM. We showthat radiation induction of VEGF-A, a recently identified targetof ATF4 (10), occurs in GBM and can be attenuated byinhibition of PERK. We also found that radiation can sensitizeGBM to chemical inducers of ER stress in a PERK-dependentmanner. Furthermore, we found that knockdown of ATF4 incombination with radiation led to reduced proliferation andcolony formation. This study supports a potential role forthe PERK/eIF2a/ATF4 axis in modulating cell viability in irra-diated GBM.

Materials and MethodsCell cultures and chemicals

The human glioblastoma cell line D54 was a gift fromDr. Yancey Gillespie (University of Alabama at Birmingham,Birmingham, AL). Human glioblastoma cell line LN827 was agift fromDr. JoshuaRubin (WashingtonUniversity in St. Louis, St.Louis, MO). Mouse glioblastoma cell line GL261 was obtainedfrom NCI. Human embryonic kidney 293T cells were obtainedfrom ATCC. D54 and GL261 cells were cultured in DMEM/F12media containing 10% FBS and 1% penicillin–streptomycin.LN827 cells were cultured in DMEM containing 10% FBS and1% penicillin–streptomycin. 293T cells were cultured in DMEMlow-glucose media containing 10% FBS and 1% penicillin–strep-tomycin. All cell cultures were grown in a humidified incubator at37�C with 5% CO2. 2-Deoxy-glucose (2DG) was purchased fromSigma. GSK2606414 and Sal003 were purchased from EMDMillipore. Radiation of cells was performed at a dose rate of2.5 Gy/minute with RS2000 160 kV X-ray Irradiator using a 0.3-mm copper filter (Rad Source Technologies).

Lentiviral transductionThe pLenti-CHOP-mCHerry plasmid was a gift from

Dr. Fumihiko Urano (Washington University in St. Louis,St. Louis, MO). Lentiviruses were generated by cotransfectingpLenti-CHOP-mCherry, the envelope plasmid (pCMV-VSV-G),and the packaging plasmid (pCMV-dR8.2) into 293T cells usingFugene 6. The target cells were incubated with virus supernatantfor 18 hours, afterwhich theywere allowed to recover for 24 hoursbefore selection with 2 mg/mL puromycin for 72 hours.Puromycin-resistant cells were then used in downstream assays.

Transfection of siRNASilencer-select predesigned siRNAs against ATF4 and nontar-

geting control siRNA were purchased from Life Technologies/Ambion. Lipofectamine RNAiMax transfection reagent (LifeTechnologies/Ambion) was used to deliver siRNAs, according tomanufacturer's protocol. Gene silencing was confirmed 48 hoursafter transfection by qRT-PCR.

Quantitative real-time PCR analysisOne microgram of RNA was used to produce cDNA with High

Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems).Primer sequences were as follows: PERK primers 50-ACGATGA-GACAGAGTTGCGAC-30 (forward), 50-ATCCAAGGCAGCAATTC-TCCC-30 (reverse); VEGF-A primers 50-AGGGCAGAATCATCACG-AAGT-30 (forward), 50-AGGGTCTCGATTGGATGGCA-30 (reverse);actin primers 50-CATGTACGTTGCTATCCAGGC-30 (forward), 50-CTCCTTAATGTCACGCACGAT-30 (reverse); GAPDH primers 50-GGAGCGAGATCCCTCCAAAAT-30 (forward), 50-GGCTGTTGTCA-TACTTCTCATGG-30 (reverse). The DDCt method for quantitationof relative gene expression was used to determine the meanexpression of each target gene normalized to the geometric meanof actin and GAPDH.

Flow cytometryCells (D54) were transfected with ATF4 specific and non-

targeting control siRNA and radiated with 3 Gy. Ninety-sixhours after IR, cells (1 � 105) were collected and stained withCleaved-PARP PE antibody (BD Biosciences) according to themanufacturer's protocol. Stained cells were analyzed by flowcytometry. Similarly, cells transduced with CHOP-mCherrywere treated with specified doses of radiation prior to collectionafter 96 hours for analysis by flow cytometry.

Western immunoblot analysisProtein extracts (40 mg) were analyzed using antibodies for the

detection of phospho-eIF2a, total-eIF2a (Cell Signaling Technol-ogy), and ATF4 (ProteinTech). Antibody against tubulin (Sigma)or GAPDH (Cell Signaling Technology) was used to normalizeprotein loading in each lane. Blots were imaged with ChemiDoc-MP Imaging System (Bio-Rad), and analyzed with Image LabSoftware (Bio-Rad).

Colony formation assaysCells were seeded at defined cell densities depending on the

radiation dose and allowed to attach overnight. Cells were thenirradiated with 0 or 3 Gy. After incubating for 7–10 days, plateswere stained with 0.5% crystal violet. Colonies comprised of50 cells or more were counted as a colony. The colony countswere normalized to plating efficiency and represented as asurviving fraction relative to control (sham/nontargetingsiRNA).

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Mol Cancer Res; 16(10) October 2018 Molecular Cancer Research1448




Cell proliferation assaysGlioma cells (3,000 per/well) were seeded in 96-well plates

48 hours after siRNA transfection. Cells were irradiated with 3 Gyand allowed to grow for 96 hours. Proliferation was determinedby reading the optical density by adding 10 mL of PrestoBlue cellviability reagent (Life Technologies) to each well. The cells withpresto blue were incubated at 37�C for 15 minutes; the absor-bance was measured at 560ex/590em with a microplate reader.

Statistical analysisWhere indicated, statistical analyses were performed using the

Student t test and one-way or two-way ANOVA. Bonferroni's mul-tiple comparisons test was applied where necessary. These analyseswere performed in Prism 6 (GraphPad Software), and statisticalsignificance was indicated in each graph where appropriate.

ResultsIrradiation of glioma cells induces phosphorylation of eIF2a

PERK-mediated phosphorylation of eIF2a occurs duringthe ER stress response (11). Thus, to begin our study of PERK

signaling in the radiation response, we investigated theeffect of radiation on the phosphorylation status of eIF2a.We observed that the levels of phospho-eIF2a were elevat-ed in a dose-dependent fashion in D54 and GL261 celllines 24 hours post 3 Gy and 6 Gy IR (Fig. 1A). These observa-tions support the IR induction of the eIF2a component ofthe ERSR.

Radiation induction of p-eIF2a and VEGF-A is downstreamof PERK

Phosphorylation of eIF2a is downstream of several otherkinases, including controlled nonrepressed kinase (CGN2),heme-regulated eIF2a kinase (HRI), and protein kinase R (PKR;ref. 12). To determine whether PERK is involved in radiation-induced eIF2a phosphorylation, we used an inhibitor of PERK,GSK2606414 (PERKi). PERKi specifically inhibits PERK overother eIF2a kinases with an IC50 of 0.4 nmol/L (13). PERKi hasbeen used in animal models to study Alzheimer's disease (14)and Parkinson disease (15). PERKi was found to beneuroprototective with minimal systemic neurotoxicity (15).

Figure 1.

Radiation induction of p-eIF2a andVEGF-A is downstream of PERK. A,D54 and GL261 cells were irradiatedwith 3 Gy or 6 Gy and harvested24 hours after IR. Shown are theimmunoblot, showing eIF2aphosphorylation in D54 and GL261cells lines.B,Cellswere incubatedwith1 mmol/L PERKi for 1.5 hours before6 Gy IR and harvested 24 hours afterIR. Shown are the immunoblots ofeIF2a phosphorylation in D54 andLN827 cells. C, D54 cells wereincubated with 1 mmol/L PERKi for1.5 hours before 6 Gy IR, and RNA wasanalyzed 24 hours after IR. Shown arethe qRT-PCR analysis of PERK andVEGF-A gene expression in D54 cells.In all graphs, data shown are themeans � SD (n ¼ 4). ��, P < 0.0001,

GBM Sensitivity to ER Stress Requires PERK

www.aacrjournals.org Mol Cancer Res; 16(10) October 2018 1449




We treated D54 cells with 1 mmol/L PERKi prior to 6 Gy IR andmeasured phospho-eIF2a levels 24 hours after IR. Pretreatmentof D54 and LN827 cells with PERKi resulted in attenuation ofbaseline phosphorylation of eIF2a, and abrogation of radia-tion-induced phosphorylation of eIF2a (Fig. 1B). VEGF-A is arecently identified downstream target of the PERK/ATF4 axis(16). We examined the impact of PERK inhibition on VEGF-ARNA expression by qRT-PCR. We found that radiation-inducedPERK 4.1-fold and VEGF-A 3.9-fold compared with untreatedcontrol (DMSO; P < 0.0001; Fig. 1C). Inhibition of PERK-attenuated induction of both PERK and VEGF-A by IR(Fig. 1C). These results suggest that PERK plays a role inradiation-induced eIF2a phosphorylation and associateddownstream signaling.

IR potentiates ER stress which reduced proliferation in aPERK-dependent manner

PERK is a well-characterized switch between survival anddeath during persistent ER stress and is known to mediate celldeath through induction of CCAAT/-enhancer–binding proteinhomologous protein (CHOP; ref. 11). Because we observedradiation-induced activation of the PERK and phospho eIF2a,we hypothesized that IR could affect the sensitivity of cells tochemical ER stressors. To test this hypothesis, we irradiated D54and LN827 cells with PERK inhibitor (PERKi), ER stress inducer(2DG) and GADD34 phosphatase inhibitor (Sal003). We foundthat treatment with 5 mmol/L 2DG or 10 mmol/L Sal003 reducedcell viability by 23% and 33%, respectively, in D54, and 36% and40%, respectively, in LN827 (Fig. 2A). Combination of 3 Gy IR

Figure 2.

IR potentiates ER stress which reducedproliferation in a PERK-dependentmanner. A, D54 and LN824 cells weretreated with 1 mmol/L PERKi for 1 hourprior to 3 Gy IR. Twenty-four hours afterIR, cellswere treatedwith 5mmol/L 2DGor 10 mmol/L Sal003. Shown is the cellviability of D54 and LN824, 72 hourspostdrug treatment with PrestoBlue cellviability reagent.B,D54 and LN824 cellswere treated with Sal003 for 1 hour priorto 3 Gy IR. The fluorescence (mCherry)was measured 96 hours postIR usingflow cytometry. Shown are the CHOPpromoter reporter assays of D54 andLN824 treated with Sal003.

Dadey et al.





and 5 mmol/L 2DG resulted in 49% and 51% attenuation in cellviability in D54 and LN827, respectively (Fig. 2A). Similarly, thecombination of 3Gy IR and 10 mmol/L Sal003 leads to a 48% and51%decrease in cell viability inD54 and LN827, respectively (Fig.2A). In bothD54 and LN827, treatmentwith PERKi abrogated theadditive effect of IR and 2DG or Sal003 on cell viability. Todetermine whether IR was enhancing prodeath signaling whencombined with chemical induction of ER stress, we transducedD54 and LN827 with a CHOP-mCherry promoter reporter. IR(3 Gy) led to 1.13- and 1.2-fold increase in CHOP levels in D54and LN827, respectively (Fig. 2B). Combining IR (3 Gy) withSal003 lead to an increase of 6.8- and 1.8-fold expression ofCHOP in D54 and LN827, respectively (Fig. 2B). These resultsindicate that IR can potentiate the effect of ER stressors on prodeath signaling downstream of PERK.

Knockdown of ATF4 and combination of radiation lead todecreased proliferation and colony formation

Although the prodeath functions of PERK are activatedduring prolonged ER stress, prosurvival signaling is also knownto occur via ATF4 (17). Translation of ATF4 is promoted byphosphorylation of eIF2a and results in upregulation of genesinvolved in mitigation of oxidative stress (18). To determinewhether IR also induces ATF4, we treated D54 with IR (3 or6 Gy) and analyzed for ATF4 levels by Western immunoblot

analyses. We found that ATF4 levels increased in a dose-dependent manner 24 hours after IR (Fig. 3A). To determinethe role of ATF4 in proliferation and colony formation wesilenced ATF4 using siRNA (Fig. 3B). Knockdown of ATF4 inD54 prior to IR with 3 Gy resulted in a 23% decrease inproliferation (P < 0.0001; Fig. 3C) and 12 % decrease in colonyformation (P < 0.05; Fig. 3D) when compared with IR alone.To assess the role of ATF4 in apoptosis, we analyzed PARPcleavage by flow cytometry. D54 cells following the ATF4knockdown, when treated with 3 Gy IR, showed a 2.3-foldincrease in PARP cleavage when compared with IR alone(Fig. 3E). These results suggest that ATF4 could play an impor-tant role in glioma cell viability during the radiation response.

DiscussionEarlier, we found that radiation can induce ER stress and

downstream signaling associated with the ERSR (8). Inductionof ER stress appears to be linked to changes in ROS balancesecondary to irradiation. Our interest in the PERK pathway ofthe ER stress response stems from its role in mediating oxidativestress responses in various cell models (17, 18). Reactive oxygenspecies primarily mediate the effects of radiation on cell physi-ology. We therefore hypothesized that PERK signaling mayplay a role in the radiation response. In cancers such as GBM,

Figure 3.

Knockdown of ATF4-decreasedproliferation and colony formation. A,D54 cells were treated with 3 Gy or6 Gy and harvested 24 hours postIR.Shown is the immunoblot analysis ofATF4 expression in D54. B, D54 cellswere treated with ATF4 siRNA. Shownis the downregulation of ATF4 mRNAexpression following siRNAtreatment. C, D54 cells were treatedwith ATF4 siRNA followed by 3 Gy IR.Shown are the cell viability determined96 hours postIRs with PrestoBluecell viability reagent. D, D54 cellswere treated with ATF4 siRNA andirradiated with 3 Gy. Cells wereallowed to grow 7–10 days andcolonies comprised of 50 or more cellswere scored. Shown are the colonyformation assays of D54 cells after theATF4 knockdown. E, D54 cells weretreated with ATF4 siRNA andirradiated with 3 Gy and were stained96h later with anti-cleaved PARP-PEantibody and analyzed using flowcytometry. Shown are the cleaved-PARP assays in D54. In all graphs, datashown are the means � SD (n ¼ 5).� , P < 0.05; �� , P < 0.0001.






which involve radiation as an essential component of therapy,studying PERK signaling may reveal molecular targets for drugdevelopment.

First, we studied PERK activation by measuring the abun-dance of phospho-eIF2a. Our observation that radiation-induced phosphorylation of eIF2a, together with our findingthat PERK activity is required for this phosphorylation, indi-cates that radiation may be activating the ER stress response inGBM. Activation of PERK signaling by radiation has beenobserved in vascular endothelial cells following high dose(15 Gy) IR (9). The fact that lower doses of radiation (3 Gyand 6 Gy) could activate this pathway in GBM suggeststhat there may be differential sensitivity of GBM cells to theeffects of radiation. We speculate that GBM cells may be lessadapted to managing oxidative stress, and may require induc-tion of PERK signaling to mitigate radiation-induced oxidativedamage.

PERK has been implicated in the regulation of angiogenesisvia downstream binding of ATF4 to the VEGF-A promoter(16, 19). To evaluate whether PERK inhibition is sufficient todisrupt downstream signaling events, we analyzed expressionof PERK and VEGF-A mRNAs. Data showing that IR inducedboth PERK and VEGF-A mRNAs, and that PERK inhibitionabrogated this response, suggests that signaling pathwaysdownstream of PERK are intact during the radiation response.Because PERK is known to promote cell death during chronicER stress, we tested the hypothesis that radiation-inducedPERK activity could modulate the sensitivity of GBM to agentsthat induce ER stress. We used 2DG to induce ER stress andfound that inhibition of PERK partially rescued GBM cells fromER stress. A similar response was observed in irradiated GBMcells, suggesting that radiation-induced PERK activity maypotentiate the effects of ER stress–inducing agents. This wasfurther supported by our finding that inhibition of GADD34phosphatase–enhanced CHOP transcription in irradiated GBM.Sal003, a GADD34 phosphatase inhibitor is a positive regu-lator of eIF2a, and functions by inhibiting dephosphorylationof eIF2a (20), thereby simulating persistent PERK activity.These results highlight the potential of utilizing the prodeathfunctions of PERK to enhance therapeutic efficacy.

During the early phase of the ERSR, PERK activity can promotesurvival by inducing translational arrest, upregulating chaper-ones, and enhancing expression of antioxidant genes (11). Weexplored the prosurvival functions of PERK signaling by targetingATF4 with siRNA. We found that ATF4 knockdown had reducedproliferation and colony formation in GBM. This suggests thatATF4 is downstream of PERK, and that PERK activation may beimportant in promoting cell viability. The PERK–eIF2a–ATF4pathway has been shown to confer protection from oxidativestress bypromoting glutathione production (18). In the context ofthe radiation response, we postulate that the antioxidant aspect ofATF4 signaling may account for its observed influence on cellsurvival. It remains to be shown how the dynamics of ATF4

expression may lead to different effects on cell viability. Giventhe dual roles of PERK–eIF2a–ATF4 in cell death and survival, itis conceivable that transient induction of ATF4 during the radi-ation response may be sufficient to enhance survival. Inductionof ATF4 however, as observed during chemical induction ofER stress, promotes CHOP transcription that leads to reducedproliferation and colony formation. This model could explainPERK-dependent potentiation of cell death when radiation wascombined with 2DG. Furthermore, this model highlights thepotential for radiation to trigger adaptive signaling in GBM thatcould contribute to tumor recurrence.

In conclusion, the data from this study show that induction ofPERK signaling occurs in irradiated GBM and that the dualfunction of PERK as amediator of survival and deathmay providemultiple approaches to enhancing the efficacy of radiation ther-apy. Furthermore, this study supports the notion that inductionof ER stress–signaling by radiation may contribute to adaptivesurvival mechanisms during radiation therapy. Beyond the impli-cations discussed regarding potentiation of cell death and adap-tive survival, the observed influence of PERK on VEGF-A mayindicate a connection between therapeutic stress and a proangio-genic response. Further investigation is needed to characterizethe functional role of radiation-induced VEGF-A in GBM.

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

Authors' ContributionsConception and design: D.Y.A. Dadey, V. Kapoor, D. Thotala, D.E. HallahanDevelopment of methodology: D.Y.A. Dadey, V. Kapoor, D. Thotala,D.E. HallahanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D.Y.A. Dadey, V. Kapoor, A. KhudanyanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):D.Y.A. Dadey, V. Kapoor, A. Khudanyan, D. Thotala,D.E. HallahanWriting, review, and/or revision of the manuscript: D.Y.A. Dadey, V. Kapoor,A. Khudanyan, D. Thotala, D.E. HallahanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D. Thotala, D.E. HallahanStudy supervision: D. Thotala, D.E. Hallahan

AcknowledgmentsThis work was supported by grants R01CA140220-01, R01CA174966,

and R21CA170169-01.The authors would like to thank the Siteman Cancer Center and

Dr. Andrei Laszlo for valuable discussion, feedback and review of thismanuscript.

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

Received March 2, 2018; revised May 16, 2018; accepted June 20, 2018;published first July 10, 2018.

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2018;16:1447-1453. Published OnlineFirst July 10, 2018.Mol Cancer Res David Y.A. Dadey, Vaishali Kapoor, Arpine Khudanyan, et al. Promoting Radiation ResistancePERK Regulates Glioblastoma Sensitivity to ER Stress Although

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