The aggressive nature and inherent therapeutic resistance of glioblastoma multiforme (GBM) has rendered the median survival of afflicted patients to 14 months. Therefore, it is imperative to understand the molecular biology of GBM to provide new treatment options to overcome this disease. It has been demonstrated that the protein kinase R–like endoplasmic reticulum kinase (PERK) pathway is an important regulator of the endoplasmic reticulum (ER) stress response. PERK signaling has been observed in other model systems after radiation; however, less is known in the context of GBM, which is frequently treated with radiation-based therapies. To investigate the significance of PERK, we studied activation of the PERK–eIF2α–ATF4 pathway in GBM after ionizing radiation (IR). By inhibiting PERK, it was determined that ionizing radiation (IR)-induced PERK activity led to eIF2α phosphorylation. IR enhanced the prodeath component of PERK signaling in cells treated with Sal003, an inhibitor of phospho-eIF2α phosphatase. Mechanistically, ATF4 mediated the prosurvival activity during the radiation response. The data support the notion that induction of ER stress signaling by radiation contributes to adaptive survival mechanisms during radiotherapy. The data also support a potential role for the PERK/eIF2α/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 of radiation therapy.

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

This article is featured in Highlights of This Issue, p. 1445

Glioblastoma multiforme (GBM), the most common primary malignant brain tumor in adults, is characterized by a high propensity for invasion and proliferation although being resistant to therapy (1). Although slight improvements in GBM therapy outcomes were achieved with the advent of temozolomide and radiotherapy, the 5-year survival rate remains under 10% (2). In efforts to improve therapy for patients afflicted with GBM, oncologists have sought to identify novel molecular targets involved in promoting the viability and therapeutic resistance. One approach to therapy for GBM involves targeting elements of the endoplasmic reticulum stress response (ERSR). The ERSR has been increasingly shown to be highly deregulated in cancer, where prosurvival aspects of the stress response are preferentially activated and involved in oncogenesis (3). In GBM, upregulation of a downstream prosurvival mediator of the ERSR, GRP78, has been linked to tumor grade, temozolomide resistance, and prognosis (4–6). The ERSR is a broad signaling network involving three major pathways: activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and protein kinase R–like endoplasmic reticulum kinase (PERK; ref. 7).

During ER stress, ATF6 translocates to the Golgi where it is cleaved by proteases (7). The cleaved ATF6 cytosolic fragment can then act as a transcription factor and drive the synthesis of ER chaperones, including GRP78 (8). IRE1 splices Xbox binding protein1 (XBP1) mRNA to yield XBP1-S, which then upregulates transcription of genes involved in protein degradation (7, 8). PERK phosphorylates initiation factor 2α (eIF2α) and transiently halts the global protein translation although upregulating ATF4 translation (7). ATF4 then triggers expression of genes involved in amino acids metabolism and suppression of oxidative stress (8). However, during prolonged ER stress, ATF4 promotes CHOP expression, which is involved in promoting ER stress–induced cell death (7). Characterization of the physiologic roles of ATF6, IRE1, and PERK in the radiation response may lead to the identification of novel molecular targets to improve therapeutic efficacy.

Previously, we have shown that radiation can induce ER stress and downstream signaling associated with the ERSR (8). Studies have shown that PERK signaling is also induced by radiation in endothelial cells (9). In this study, we found that radiation activates the PERK pathway in GBM. We show that radiation induction of VEGF-A, a recently identified target of ATF4 (10), occurs in GBM and can be attenuated by inhibition of PERK. We also found that radiation can sensitize GBM to chemical inducers of ER stress in a PERK-dependent manner. Furthermore, we found that knockdown of ATF4 in combination with radiation led to reduced proliferation and colony formation. This study supports a potential role for the PERK/eIF2α/ATF4 axis in modulating cell viability in irradiated GBM.

Cell cultures and chemicals

The human glioblastoma cell line D54 was a gift from Dr. Yancey Gillespie (University of Alabama at Birmingham, Birmingham, AL). Human glioblastoma cell line LN827 was a gift from Dr. Joshua Rubin (Washington University in St. Louis, St. Louis, MO). Mouse glioblastoma cell line GL261 was obtained from NCI. Human embryonic kidney 293T cells were obtained from ATCC. D54 and GL261 cells were cultured in DMEM/F12 media containing 10% FBS and 1% penicillin–streptomycin. LN827 cells were cultured in DMEM containing 10% FBS and 1% penicillin–streptomycin. 293T cells were cultured in DMEM low-glucose media containing 10% FBS and 1% penicillin–streptomycin. All cell cultures were grown in a humidified incubator at 37°C with 5% CO2. 2-Deoxy-glucose (2DG) was purchased from Sigma. GSK2606414 and Sal003 were purchased from EMD Millipore. Radiation of cells was performed at a dose rate of 2.5 Gy/minute with RS2000 160 kV X-ray Irradiator using a 0.3-mm copper filter (Rad Source Technologies).

Lentiviral transduction

The pLenti-CHOP-mCHerry plasmid was a gift from Dr. Fumihiko Urano (Washington University in St. Louis, St. Louis, MO). Lentiviruses were generated by cotransfecting pLenti-CHOP-mCherry, the envelope plasmid (pCMV-VSV-G), and the packaging plasmid (pCMV-dR8.2) into 293T cells using Fugene 6. The target cells were incubated with virus supernatant for 18 hours, after which they were allowed to recover for 24 hours before selection with 2 μg/mL puromycin for 72 hours. Puromycin-resistant cells were then used in downstream assays.

Transfection of siRNA

Silencer-select predesigned siRNAs against ATF4 and nontargeting control siRNA were purchased from Life Technologies/Ambion. Lipofectamine RNAiMax transfection reagent (Life Technologies/Ambion) was used to deliver siRNAs, according to manufacturer's protocol. Gene silencing was confirmed 48 hours after transfection by qRT-PCR.

Quantitative real-time PCR analysis

One microgram of RNA was used to produce cDNA with High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems). Primer sequences were as follows: PERK primers 5′-ACGATGAGACAGAGTTGCGAC-3′ (forward), 5′-ATCCAAGGCAGCAATTCTCCC-3′ (reverse); VEGF-A primers 5′-AGGGCAGAATCATCACGAAGT-3′ (forward), 5′-AGGGTCTCGATTGGATGGCA-3′ (reverse); actin primers 5′-CATGTACGTTGCTATCCAGGC-3′ (forward), 5′-CTCCTTAATGTCACGCACGAT-3′ (reverse); GAPDH primers 5′-GGAGCGAGATCCCTCCAAAAT-3′ (forward), 5′-GGCTGTTGTCATACTTCTCATGG-3′ (reverse). The ΔΔCt method for quantitation of relative gene expression was used to determine the mean expression of each target gene normalized to the geometric mean of actin and GAPDH.

Flow cytometry

Cells (D54) were transfected with ATF4 specific and nontargeting control siRNA and radiated with 3 Gy. Ninety-six hours after IR, cells (1 × 105) were collected and stained with Cleaved-PARP PE antibody (BD Biosciences) according to the manufacturer's protocol. Stained cells were analyzed by flow cytometry. Similarly, cells transduced with CHOP-mCherry were treated with specified doses of radiation prior to collection after 96 hours for analysis by flow cytometry.

Western immunoblot analysis

Protein extracts (40 μg) were analyzed using antibodies for the detection of phospho-eIF2α, total-eIF2α (Cell Signaling Technology), and ATF4 (ProteinTech). Antibody against tubulin (Sigma) or GAPDH (Cell Signaling Technology) was used to normalize protein loading in each lane. Blots were imaged with ChemiDoc-MP Imaging System (Bio-Rad), and analyzed with Image Lab Software (Bio-Rad).

Colony formation assays

Cells were seeded at defined cell densities depending on the radiation dose and allowed to attach overnight. Cells were then irradiated with 0 or 3 Gy. After incubating for 7–10 days, plates were stained with 0.5% crystal violet. Colonies comprised of 50 cells or more were counted as a colony. The colony counts were normalized to plating efficiency and represented as a surviving fraction relative to control (sham/nontargeting siRNA).

Cell proliferation assays

Glioma cells (3,000 per/well) were seeded in 96-well plates 48 hours after siRNA transfection. Cells were irradiated with 3 Gy and allowed to grow for 96 hours. Proliferation was determined by reading the optical density by adding 10 μL of PrestoBlue cell viability reagent (Life Technologies) to each well. The cells with presto blue were incubated at 37°C for 15 minutes; the absorbance was measured at 560ex/590em with a microplate reader.

Statistical analysis

Where indicated, statistical analyses were performed using the Student t test and one-way or two-way ANOVA. Bonferroni's multiple comparisons test was applied where necessary. These analyses were performed in Prism 6 (GraphPad Software), and statistical significance was indicated in each graph where appropriate.

Irradiation of glioma cells induces phosphorylation of eIF2α

PERK-mediated phosphorylation of eIF2α occurs during the ER stress response (11). Thus, to begin our study of PERK signaling in the radiation response, we investigated the effect of radiation on the phosphorylation status of eIF2α. We observed that the levels of phospho-eIF2α were elevated in a dose-dependent fashion in D54 and GL261 cell lines 24 hours post 3 Gy and 6 Gy IR (Fig. 1A). These observations support the IR induction of the eIF2α component of the ERSR.

Figure 1.

Radiation induction of p-eIF2a and VEGF-A is downstream of PERK. A, D54 and GL261 cells were irradiated with 3 Gy or 6 Gy and harvested 24 hours after IR. Shown are the immunoblot, showing eIF2α phosphorylation in D54 and GL261 cells lines. B, Cells were incubated with 1 μmol/L PERKi for 1.5 hours before 6 Gy IR and harvested 24 hours after IR. Shown are the immunoblots of eIF2α phosphorylation in D54 and LN827 cells. C, D54 cells were incubated with 1 μmol/L PERKi for 1.5 hours before 6 Gy IR, and RNA was analyzed 24 hours after IR. Shown are the qRT-PCR analysis of PERK and VEGF-A gene expression in D54 cells. In all graphs, data shown are the means ± SD (n = 4). ****, P < 0.0001,

Figure 1.

Radiation induction of p-eIF2a and VEGF-A is downstream of PERK. A, D54 and GL261 cells were irradiated with 3 Gy or 6 Gy and harvested 24 hours after IR. Shown are the immunoblot, showing eIF2α phosphorylation in D54 and GL261 cells lines. B, Cells were incubated with 1 μmol/L PERKi for 1.5 hours before 6 Gy IR and harvested 24 hours after IR. Shown are the immunoblots of eIF2α phosphorylation in D54 and LN827 cells. C, D54 cells were incubated with 1 μmol/L PERKi for 1.5 hours before 6 Gy IR, and RNA was analyzed 24 hours after IR. Shown are the qRT-PCR analysis of PERK and VEGF-A gene expression in D54 cells. In all graphs, data shown are the means ± SD (n = 4). ****, P < 0.0001,

Close modal

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

Phosphorylation of eIF2α is downstream of several other kinases, including controlled nonrepressed kinase (CGN2), heme-regulated eIF2α kinase (HRI), and protein kinase R (PKR; ref. 12). To determine whether PERK is involved in radiation-induced eIF2α phosphorylation, we used an inhibitor of PERK, GSK2606414 (PERKi). PERKi specifically inhibits PERK over other eIF2α kinases with an IC50 of 0.4 nmol/L (13). PERKi has been used in animal models to study Alzheimer's disease (14) and Parkinson disease (15). PERKi was found to be neuroprototective with minimal systemic neurotoxicity (15). We treated D54 cells with 1 μmol/L PERKi prior to 6 Gy IR and measured phospho-eIF2α levels 24 hours after IR. Pretreatment of D54 and LN827 cells with PERKi resulted in attenuation of baseline phosphorylation of eIF2α, and abrogation of radiation-induced phosphorylation of eIF2α (Fig. 1B). VEGF-A is a recently identified downstream target of the PERK/ATF4 axis (16). We examined the impact of PERK inhibition on VEGF-A RNA expression by qRT-PCR. We found that radiation-induced PERK 4.1-fold and VEGF-A 3.9-fold compared with untreated control (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 in radiation-induced eIF2α phosphorylation and associated downstream signaling.

IR potentiates ER stress which reduced proliferation in a PERK-dependent manner

PERK is a well-characterized switch between survival and death during persistent ER stress and is known to mediate cell death through induction of CCAAT/-enhancer–binding protein homologous protein (CHOP; ref. 11). Because we observed radiation-induced activation of the PERK and phospho eIF2α, we hypothesized that IR could affect the sensitivity of cells to chemical ER stressors. To test this hypothesis, we irradiated D54 and LN827 cells with PERK inhibitor (PERKi), ER stress inducer (2DG) and GADD34 phosphatase inhibitor (Sal003). We found that treatment with 5 mmol/L 2DG or 10 μmol/L Sal003 reduced cell viability by 23% and 33%, respectively, in D54, and 36% and 40%, respectively, in LN827 (Fig. 2A). Combination of 3 Gy IR and 5 mmol/L 2DG resulted in 49% and 51% attenuation in cell viability in D54 and LN827, respectively (Fig. 2A). Similarly, the combination of 3 Gy IR and 10 μmol/L Sal003 leads to a 48% and 51% decrease in cell viability in D54 and LN827, respectively (Fig. 2A). In both D54 and LN827, treatment with PERKi abrogated the additive effect of IR and 2DG or Sal003 on cell viability. To determine whether IR was enhancing prodeath signaling when combined with chemical induction of ER stress, we transduced D54 and LN827 with a CHOP-mCherry promoter reporter. IR (3 Gy) led to 1.13- and 1.2-fold increase in CHOP levels in D54 and LN827, respectively (Fig. 2B). Combining IR (3 Gy) with Sal003 lead to an increase of 6.8- and 1.8-fold expression of CHOP in D54 and LN827, respectively (Fig. 2B). These results indicate that IR can potentiate the effect of ER stressors on pro death signaling downstream of PERK.

Figure 2.

IR potentiates ER stress which reduced proliferation in a PERK-dependent manner. A, D54 and LN824 cells were treated with 1 μmol/L PERKi for 1 hour prior to 3 Gy IR. Twenty-four hours after IR, cells were treated with 5 mmol/L 2DG or 10 μmol/L Sal003. Shown is the cell viability of D54 and LN824, 72 hours postdrug treatment with PrestoBlue cell viability reagent. B, D54 and LN824 cells were treated with Sal003 for 1 hour prior to 3 Gy IR. The fluorescence (mCherry) was measured 96 hours postIR using flow cytometry. Shown are the CHOP promoter reporter assays of D54 and LN824 treated with Sal003.

Figure 2.

IR potentiates ER stress which reduced proliferation in a PERK-dependent manner. A, D54 and LN824 cells were treated with 1 μmol/L PERKi for 1 hour prior to 3 Gy IR. Twenty-four hours after IR, cells were treated with 5 mmol/L 2DG or 10 μmol/L Sal003. Shown is the cell viability of D54 and LN824, 72 hours postdrug treatment with PrestoBlue cell viability reagent. B, D54 and LN824 cells were treated with Sal003 for 1 hour prior to 3 Gy IR. The fluorescence (mCherry) was measured 96 hours postIR using flow cytometry. Shown are the CHOP promoter reporter assays of D54 and LN824 treated with Sal003.

Close modal

Knockdown of ATF4 and combination of radiation lead to decreased proliferation and colony formation

Although the prodeath functions of PERK are activated during prolonged ER stress, prosurvival signaling is also known to occur via ATF4 (17). Translation of ATF4 is promoted by phosphorylation of eIF2α and results in upregulation of genes involved in mitigation of oxidative stress (18). To determine whether IR also induces ATF4, we treated D54 with IR (3 or 6 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 determine the role of ATF4 in proliferation and colony formation we silenced ATF4 using siRNA (Fig. 3B). Knockdown of ATF4 in D54 prior to IR with 3 Gy resulted in a 23% decrease in proliferation (P < 0.0001; Fig. 3C) and 12 % decrease in colony formation (P < 0.05; Fig. 3D) when compared with IR alone. To assess the role of ATF4 in apoptosis, we analyzed PARP cleavage by flow cytometry. D54 cells following the ATF4 knockdown, when treated with 3 Gy IR, showed a 2.3-fold increase in PARP cleavage when compared with IR alone (Fig. 3E). These results suggest that ATF4 could play an important role in glioma cell viability during the radiation response.

Figure 3.

Knockdown of ATF4-decreased proliferation and colony formation. A, D54 cells were treated with 3 Gy or 6 Gy and harvested 24 hours postIR. Shown is the immunoblot analysis of ATF4 expression in D54. B, D54 cells were treated with ATF4 siRNA. Shown is the downregulation of ATF4 mRNA expression following siRNA treatment. C, D54 cells were treated with ATF4 siRNA followed by 3 Gy IR. Shown are the cell viability determined 96 hours postIRs with PrestoBlue cell viability reagent. D, D54 cells were treated with ATF4 siRNA and irradiated with 3 Gy. Cells were allowed to grow 7–10 days and colonies comprised of 50 or more cells were scored. Shown are the colony formation assays of D54 cells after the ATF4 knockdown. E, D54 cells were treated with ATF4 siRNA and irradiated with 3 Gy and were stained 96h later with anti-cleaved PARP-PE antibody and analyzed using flow cytometry. Shown are the cleaved-PARP assays in D54. In all graphs, data shown are the means ± SD (n = 5). *, P < 0.05; ****, P < 0.0001.

Figure 3.

Knockdown of ATF4-decreased proliferation and colony formation. A, D54 cells were treated with 3 Gy or 6 Gy and harvested 24 hours postIR. Shown is the immunoblot analysis of ATF4 expression in D54. B, D54 cells were treated with ATF4 siRNA. Shown is the downregulation of ATF4 mRNA expression following siRNA treatment. C, D54 cells were treated with ATF4 siRNA followed by 3 Gy IR. Shown are the cell viability determined 96 hours postIRs with PrestoBlue cell viability reagent. D, D54 cells were treated with ATF4 siRNA and irradiated with 3 Gy. Cells were allowed to grow 7–10 days and colonies comprised of 50 or more cells were scored. Shown are the colony formation assays of D54 cells after the ATF4 knockdown. E, D54 cells were treated with ATF4 siRNA and irradiated with 3 Gy and were stained 96h later with anti-cleaved PARP-PE antibody and analyzed using flow cytometry. Shown are the cleaved-PARP assays in D54. In all graphs, data shown are the means ± SD (n = 5). *, P < 0.05; ****, P < 0.0001.

Close modal

Earlier, we found that radiation can induce ER stress and downstream signaling associated with the ERSR (8). Induction of ER stress appears to be linked to changes in ROS balance secondary to irradiation. Our interest in the PERK pathway of the ER stress response stems from its role in mediating oxidative stress responses in various cell models (17, 18). Reactive oxygen species primarily mediate the effects of radiation on cell physiology. We therefore hypothesized that PERK signaling may play a role in the radiation response. In cancers such as GBM, which involve radiation as an essential component of therapy, studying PERK signaling may reveal molecular targets for drug development.

First, we studied PERK activation by measuring the abundance of phospho-eIF2α. Our observation that radiation-induced phosphorylation of eIF2α, together with our finding that PERK activity is required for this phosphorylation, indicates that radiation may be activating the ER stress response in GBM. Activation of PERK signaling by radiation has been observed in vascular endothelial cells following high dose (15 Gy) IR (9). The fact that lower doses of radiation (3 Gy and 6 Gy) could activate this pathway in GBM suggests that there may be differential sensitivity of GBM cells to the effects of radiation. We speculate that GBM cells may be less adapted to managing oxidative stress, and may require induction of PERK signaling to mitigate radiation-induced oxidative damage.

PERK has been implicated in the regulation of angiogenesis via downstream binding of ATF4 to the VEGF-A promoter (16, 19). To evaluate whether PERK inhibition is sufficient to disrupt downstream signaling events, we analyzed expression of PERK and VEGF-A mRNAs. Data showing that IR induced both PERK and VEGF-A mRNAs, and that PERK inhibition abrogated this response, suggests that signaling pathways downstream of PERK are intact during the radiation response. Because PERK is known to promote cell death during chronic ER stress, we tested the hypothesis that radiation-induced PERK activity could modulate the sensitivity of GBM to agents that induce ER stress. We used 2DG to induce ER stress and found that inhibition of PERK partially rescued GBM cells from ER stress. A similar response was observed in irradiated GBM cells, suggesting that radiation-induced PERK activity may potentiate the effects of ER stress–inducing agents. This was further supported by our finding that inhibition of GADD34 phosphatase–enhanced CHOP transcription in irradiated GBM. Sal003, a GADD34 phosphatase inhibitor is a positive regulator of eIF2α, and functions by inhibiting dephosphorylation of eIF2α (20), thereby simulating persistent PERK activity. These results highlight the potential of utilizing the prodeath functions of PERK to enhance therapeutic efficacy.

During the early phase of the ERSR, PERK activity can promote survival by inducing translational arrest, upregulating chaperones, and enhancing expression of antioxidant genes (11). We explored the prosurvival functions of PERK signaling by targeting ATF4 with siRNA. We found that ATF4 knockdown had reduced proliferation and colony formation in GBM. This suggests that ATF4 is downstream of PERK, and that PERK activation may be important in promoting cell viability. The PERK–eIF2α–ATF4 pathway has been shown to confer protection from oxidative stress by promoting glutathione production (18). In the context of the radiation response, we postulate that the antioxidant aspect of ATF4 signaling may account for its observed influence on cell survival. It remains to be shown how the dynamics of ATF4 expression may lead to different effects on cell viability. Given the dual roles of PERK–eIF2α–ATF4 in cell death and survival, it is conceivable that transient induction of ATF4 during the radiation response may be sufficient to enhance survival. Induction of ATF4 however, as observed during chemical induction of ER stress, promotes CHOP transcription that leads to reduced proliferation and colony formation. This model could explain PERK-dependent potentiation of cell death when radiation was combined with 2DG. Furthermore, this model highlights the potential for radiation to trigger adaptive signaling in GBM that could contribute to tumor recurrence.

In conclusion, the data from this study show that induction of PERK signaling occurs in irradiated GBM and that the dual function of PERK as a mediator of survival and death may provide multiple approaches to enhancing the efficacy of radiation therapy. Furthermore, this study supports the notion that induction of ER stress–signaling by radiation may contribute to adaptive survival mechanisms during radiation therapy. Beyond the implications discussed regarding potentiation of cell death and adaptive survival, the observed influence of PERK on VEGF-A may indicate a connection between therapeutic stress and a proangiogenic response. Further investigation is needed to characterize the functional role of radiation-induced VEGF-A in GBM.

No potential conflicts of interest were disclosed.

Conception and design: D.Y.A. Dadey, V. Kapoor, D. Thotala, D.E. Hallahan

Development of methodology: D.Y.A. Dadey, V. Kapoor, D. Thotala, D.E. Hallahan

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D.Y.A. Dadey, V. Kapoor, A. Khudanyan

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D.Y.A. Dadey, V. Kapoor, A. Khudanyan, D. Thotala, D.E. Hallahan

Writing, review, and/or revision of the manuscript: D.Y.A. Dadey, V. Kapoor, A. Khudanyan, D. Thotala, D.E. Hallahan

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D. Thotala, D.E. Hallahan

Study supervision: D. Thotala, D.E. Hallahan

This 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 this manuscript.

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

1.
Ramirez
YP
,
Weatherbee
JL
,
Wheelhouse
RT
,
Ross
AH
. 
Glioblastoma multiforme therapy and mechanisms of resistance
.
Pharmaceuticals
2013
;
6
:
1475
506
.
2.
Stupp
R
,
Hegi
ME
,
Mason
WP
,
van den Bent
MJ
,
Taphoorn
MJ
,
Janzer
RC
, et al
Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial
.
Lancet Oncol
2009
;
10
:
459
66
.
3.
Chevet
E
,
Hetz
C
,
Samali
A
. 
Endoplasmic reticulum stress-activated cell reprogramming in oncogenesis
.
Cancer Discov
2015
;
5
:
586
97
.
4.
Dong
D
,
Ni
M
,
Li
J
,
Xiong
S
,
Ye
W
,
Virrey
JJ
, et al
Critical role of the stress chaperone GRP78/BiP in tumor proliferation, survival, and tumor angiogenesis in transgene-induced mammary tumor development
.
Cancer Res
2008
;
68
:
498
505
.
5.
Pyrko
P
,
Schonthal
AH
,
Hofman
FM
,
Chen
TC
,
Lee
AS
. 
The unfolded protein response regulator GRP78/BiP as a novel target for increasing chemosensitivity in malignant gliomas
.
Cancer Res
2007
;
67
:
9809
16
.
6.
Zhang
LH
,
Yang
XL
,
Zhang
X
,
Cheng
JX
,
Zhang
W
. 
Association of elevated GRP78 expression with increased astrocytoma malignancy via Akt and ERK pathways
.
Brain Res
2011
;
1371
:
23
31
.
7.
Wang
M
,
Kaufman
RJ
. 
The impact of the endoplasmic reticulum protein-folding environment on cancer development
.
Nat Rev Cancer
2014
;
14
:
581
97
.
8.
Hetz
C
,
Mollereau
B
. 
Disturbance of endoplasmic reticulum proteostasis in neurodegenerative diseases
.
Nat Rev Neurosci
2014
;
15
:
233
49
.
9.
Dadey
DY
,
Kapoor
V
,
Khudanyan
A
,
Urano
F
,
Kim
AH
,
Thotala
D
, et al
The ATF6 pathway of the ER stress response contributes to enhanced viability in glioblastoma
.
Oncotarget
2016
;
7
:
2080
92
.
10.
Kim
EJ
,
Lee
YJ
,
Kang
S
,
Lim
YB
. 
Ionizing radiation activates PERK/eIF2alpha/ATF4 signaling via ER stress-independent pathway in human vascular endothelial cells
.
Int J Radiat Biol
2014
;
90
:
306
12
.
11.
Wang
Y
,
Alam
GN
,
Ning
Y
,
Visioli
F
,
Dong
Z
,
Nor
JE
, et al
The unfolded protein response induces the angiogenic switch in human tumor cells through the PERK/ATF4 pathway
.
Cancer Res
2012
;
72
:
5396
406
.
12.
Hetz
C
. 
The unfolded protein response: controlling cell fate decisions under ER stress and beyond
.
Nat Rev Mol Cell Biol
2012
;
13
:
89
102
.
13.
Donnelly
N
,
Gorman
AM
,
Gupta
S
,
Samali
A
. 
The eIF2alpha kinases: their structures and functions
.
Cell Mol Life Sci
2013
;
70
:
3493
511
.
14.
Axten
JM
,
Medina
JR
,
Feng
Y
,
Shu
A
,
Romeril
SP
,
Grant
SW
, et al
Discovery of 7-methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-1H-indol-5-yl)-7H-p yrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), a potent and selective first-in-class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)
.
J Med Chem
2012
;
55
:
7193
207
.
15.
Radford
H
,
Moreno
JA
,
Verity
N
,
Halliday
M
,
Mallucci
GR
. 
PERK inhibition prevents tau-mediated neurodegeneration in a mouse model of frontotemporal dementia
.
Acta Neuropathol
2015
;
130
:
633
42
.
16.
Mercado
G
,
Castillo
V
,
Soto
P
,
Lopez
N
,
Axten
JM
,
Sardi
SP
, et al
Targeting PERK signaling with the small molecule GSK2606414 prevents neurodegeneration in a model of Parkinson's disease
.
Neurobiol Dis
2018
;
112
:
136
48
.
17.
Wang
Y
,
Alam
GN
,
Ning
Y
,
Visioli
F
,
Dong
Z
,
Nör
JE
, et al
The unfolded protein response induces the angiogenic switch in human tumor cells through the PERK/ATF4 pathway
.
Cancer Res
2012
;
72
:
5396
406
.
18.
Avivar-Valderas
A
,
Salas
E
,
Bobrovnikova-Marjon
E
,
Diehl
JA
,
Nagi
C
,
Debnath
J
, et al
PERK integrates autophagy and oxidative stress responses to promote survival during extracellular matrix detachment
.
Mol Cell Biol
2011
;
31
:
3616
29
.
19.
Harding
HP
,
Zhang
Y
,
Zeng
H
,
Novoa
I
,
Lu
PD
,
Calfon
M
, et al
An integrated stress response regulates amino acid metabolism and resistance to oxidative stress
.
Mol Cell
2003
;
11
:
619
33
.
20.
Ghosh
R
,
Lipson
KL
,
Sargent
KE
,
Mercurio
AM
,
Hunt
JS
,
Ron
D
, et al
Transcriptional regulation of VEGF-A by the unfolded protein response pathway
.
PLoS One
2010
;
5
:
e9575
.
21.
Boyce
M
,
Bryant
KF
,
Jousse
C
,
Long
K
,
Harding
HP
,
Scheuner
D
, et al
A Selective Inhibitor of eIF2α dephosphorylation protects cells from ER stress
.
Science
2005
;
307
:
935
9
.