RRAD Promotes EGFR-Mediated STAT3 Activation and Induces Temozolomide Resistance of Malignant Glioblastoma

Published OnlineFirst October 13, 2014; DOI: 10.1158/1535-7163.MCT-14-0244

Molecular Cancer Cancer Biology and Signal Transduction Therapeutics

Seon-Yong Yeom1, Do-Hyun Nam2, and Chaehwa Park1

Abstract Glioblastoma multiforme (GBM) is an extremely aggressive brain cancer with a median survival of less than 2 years. GBM is characterized by abnormal activation of receptor tyrosine kinase and constitutively activated STAT3. Although EGFR phosphorylation and STAT3 activation are essential for the maintenance of GBM cancer stem cells, the molecular mechanism underlying endosome-mediated STAT3 activation is not fully understood. In the current study, we showed that GTP-binding protein RRAD (RAS associated with diabetes, RAD) physically associates with EGFR, and EEA1, enhancing the stability and endosome-associated nuclear translocation of EGFR. Functionally, RRAD contributes to the activation of STAT3 and expression of the stem cell factors OCT4, NANOG, and SOX2, thereby enhancing self-renewing ability, tumor sphere formation, EMT, and in vivo tumorigenesis. Most importantly, RRAD contributes to poor survival in patients with GBM. RRAD expression is correlated with temozolomide resistance, and, conversely, depletion of RRAD leads to sensitization of highly temozolomide-resistant GBM cells. Our data collectively support a novel function of RRAD in STAT3 activation and provide evidence that RRAD acts as a positive regulator in the EGFR signaling pathway. These results demonstrate a critical role for RRAD in GBM tumorigenesis and provide a rationale for the development of pharmacologic inhibitors of RRAD in GBM. Mol Cancer Ther; 13(12); 3049–61. 2014 AACR.

Introduction conditions (5, 6). Therefore, the stem-like function is Glioblastoma multiforme (GBM) is an extremely more appropriate to distinguish cancer stem cells. The aggressive common adult brain tumor. The current stan- functional properties of cancer stem cells include the dard therapy for GBM includes surgery, radiation, and ability to form spheres, self-renew and differentiate, and treatment with temozolomide. Despite improvements survive drug toxicity. GBM is characterized by abnormal in the standard treatment regimens, the survival rate activation of receptor tyrosine kinase (RTK) signaling for GBM is only about 9.8%, and most patients eventually pathways, and constitutively activated STAT3 is frequent- experience recurrence (1). Cancer stem cells, a cellular ly coexpressed with EGFR in high-grade gliomas (7). subpopulation with sustained self-renewal and differen- STAT3 activation is known to be essential for the main- tiation potential, are responsible for tumor initiation, tenance of GBM cancer stem cells (8). The cytoplasmic propagation, recurrence, and treatment resistance (2–4). STAT3 protein is recruited to activated receptors and Expression patterns of surface markers, including CD133, subsequently phosphorylated at Tyr705 by the receptor CD15, CD44, L1CAM, CD49f, A3B5, and EGFR collective- kinase or an associated kinase. EGFR can serve as a ly support the existence of highly tumorigenic cancer stem scaffold for trafﬁcking of STAT3 (9). Phosphorylated cells. However, surface markers display variable expres- STAT3 colocalizes with receptor–ligand complexes on the sion according to cell-cycle status or environmental endosome and is transported from the plasma membrane to the perinuclear region (10). Data from the current study support a novel function of RRAD (RAS associated with 1Research Institute for Future Medicine, Samsung Medical Center, Sung- diabetes, RAD) in STAT3 activation and GBM malignan- kyunkwan University School of Medicine, Seoul, Korea. 2Department of cy, induced through physical interactions of RRAD with Neurosurgery, Samsung Medical Center, Sungkyunkwan University EGFR/STAT3/EEA1 and endosome-mediated nuclear School of Medicine, Seoul, Korea. translocation of EGFR. Note: Supplementary data for this article are available at Molecular Cancer RRAD is a Ras-related GTPase encoded by a gene Therapeutics Online (http://mct.aacrjournals.org/). located at human chromosome 16q22, which is initially Corresponding Author: Chaehwa Park, Research Institute for Future Medicine, Samsung Medical Center, Sungkyunkwan University School of identiﬁed by subtractive hybridization and selectively Medicine, 50 Irwon-dong, Seoul 135-710, Korea. Phone: 822-3410-3458; overexpressed in type II diabetic muscle as compared E-mail: [email protected] with muscle of nondiabetic or type I diabetic indivi- doi: 10.1158/1535-7163.MCT-14-0244 duals (11). RRAD differs from the other Ras-related 2014 American Association for Cancer Research. GTPases in a number of properties, including lack

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of characteristic domains such as prenylation motifs, 7 days, and protein extracted for additional experiments a GTP-binding domain, and NH2- and COOH-terminal or dissociated with Accutase (Invitrogen). extensions (12). RRAD participates in CaMKII signaling cascade, where it interacts with calmodulin, calmodu- Plasmids, transfection, and antibodies lin-dependent protein kinase II, and tropomyosin Full-length RRAD was cloned from HeLa mRNA for (13, 14). Flag-tagged cloning into pCMVTag2B (Clontech). To Ras signaling activates the downstream effectors establish stable cell lines overexpressing RRAD (C5 RAF, MEK and ERK, and other signaling modules, and C9), LN229 cells were transfected with a control vector including PI3K–mTOR–Akt, TIAM1–Rac, Ral, and or that containing Flag-tagged RRAD (21). Successfully PLC–PKC. Therefore, there is a strong rationale for the transfected cells were selected, and overexpression use of the Ras signaling pathway for developing ther- of RRAD confirmed with immunoblotting using an anti- apeutic interventions. Clinical trials are ongoing to Flag antibody. Expression of RRAD was in the order: evaluate the efficacy of Ras–RAF inhibitors in multiple C5>C9>vector. The 21-nucleotide-long siRNAs corre- cancer types. In malignant gliomas, however, somatic sponding to RRAD (siRRAD#1 sense, 50-GCAAGUU- mutations of Ras or RAF are very rare (15–17). Primary CAUUGAGACAUCUU-30; antisense 50-GAUGUCU- GBM tumors are reported to express significantly lower CAAUGAACUUGCUU-30; siRRAD#2, sense 50-GGACG- levels of K-Ras and H-Ras transcripts, compared with GAGAAGAGGCAUCAUU-30; antisense 50-UGAUGC- normal brain tissues, and all tissues do not express CUCUUCUCCGUCCUU-30) and control siRNA (siC) detectable levels of Ras proteins (18). K-Ras/H-Ras were purchased from Dharmacon (Thermo Scientific). expression levels are not associated with survival in STAT3-CA (Flag-tag) plasmid was obtained from the GBM cohort (18). Interestingly, inhibition of RAF Addgene. Cells were transfected with siRNA using Effec- and STAT3 has a cumulative prognostic impact in tene(Qiagen).AntibodiesagainstEGFR,STAT3,a-tubulin, human GBM, signifying an additive effect of the two PARP, b-actin, and Twist (Santa Cruz Biotechnology), and independent signaling pathways (19). Vimentin (BD Biosciences) were purchased. Antibodies Previously, we and others showed that RRAD expres- against RRAD, OCT4, NANOG, and SOX2 (Abcam) and sion is positively correlated with malignant progression p-STAT3 Y705, p-EGFR Y845, GFAP, Flag, and N-cadherin (20–22). The roles of RRAD are attributed to its ability to (Cell Signaling Technology) were additionally used. inhibit tumor suppressors, nm23 or GCIP, via direct Horseradishperoxidase–conjugatedsecondaryantibodies interactions (20, 21). However, these findings do not fully were obtained from Santa Cruz Biotechnology. Reactive explain how RRAD enhances tumor initiation and treat- proteins were visualized using the Thermo ECL Kit. ment resistance. Particularly, the possible role of RRAD in STAT3 activation is yet to be established. In this study, we Cell fractionation evaluated RRAD expression in GBM, and addressed a For subcellular fractionation, cytosol and nuclear frac- positive correlation between RRAD level and tumor tions were prepared as described previously (9). Cells malignancy. RRAD promotes malignant progression and were lysed in lysis buffer (20 mmol/L HEPES, pH 7.0, 10 enhances resistance to temozolomide via endosome- mmol/L KCl, 2 mmol/L MgCl2, 0.5% Nonidet P-40, mediated EGFR/STAT3 signaling. protease inhibitor mixture). Recovered nuclei were lysed in hypertonic buffer (150 mmol/L NaCl, 1 mmol/L Materials and Methods EDTA, 20 mmol/L Tris, pH 8.0, 0.5% Nonidet P-40, protease inhibitor mixture), and the nuclear fraction col- Cell culture and reagents lected after centrifugation at maximum speed. Human GBM cell lines (U87-MG, U138-MG, U251, LN229, A172, and DBTRG-05MG) were purchased from Immunoprecipitation the ATCC and no further authentication was done. Adher- For immunoprecipitation, LN229 cells transfected with ent cells were maintained in DMEM with heat-inactivated pCMVTaq2B-RRAD were washed with cold PBS and lysed 10% FBS, penicillin, and streptomycin (Gibco BRL). in buffer (20 mmol/L HEPES, pH 7.0, 150 mmol/L NaCl, 1 Tumor spheres were cultured in serum-free DMEM/ mmol/L EDTA, 2 mmol/L b-glycerophosphate, 1% Triton F12 (Invitrogen) supplemented with basic fibroblast X-100, 10% glycerol, 1 mmol/L phenylmethylsulfonyl fluo- growth factor (20 ng/mL; Invitrogen), EGF (20 ng/mL; ride, and 1 protease inhibitor cocktail). Anti-EGFR anti- BD Biosciences), and N2 supplement (1 ; Invitrogen). body was incubated with cell lysates overnight at 4 C. Temozolomide, cycloheximide, and Dynasore were Immune complexes were pulled down by the addition of obtained from Sigma. Cells were placed in an incubator protein A/G agarose. After washing with lysis buffer, under conditions of 37 C with 5% CO2. immune precipitates were analyzed via SDS-PAGE and immunoblotting. Sphere formation Cells were resuspended in DMEM/F12 containing 20 GST pull-down assay ng/mL EGF, bFGF, and N2 supplement (1) as the stem RRAD cDNA was cloned in-frame into pGEX4T-1 cell–permissive medium. Spheres were collected after 5 to (Amersham) to produce the GST fusion protein, GST-

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RRAD, for the GST pull-down assay. GST and GST-RRAD Transwell migration assay fusion proteins were immobilized on glutathione-Sephar- Cells (1 105) were loaded into the top chamber of ose beads (Amersham) and incubated with U251 lysates Transwell plates (8 mm pore size; Corning Costar). FBS overnight. Beads were washed four times, and bound (10%) was used as a chemoattractant in the bottom proteins eluted with SDS loading buffer containing chamber. After incubation for 24 hours, cells in the 5% b-mercaptoethanol, followed by SDS-PAGE and bottom chamber were fixed and stained with 0.005% immunoblotting. (w/v) crystal violet. The number of migrated cells was quantified by counting those in five random fields of Cell growth assessment and soft-agar colony each membrane. formation assay To assess cell numbers, an equal volume of 0.4% (w/v) Evaluation of tumorigenicity Trypan blue was added to each cell suspension, and To examine the effect of RRAD on tumor formation, viability determined on the basis of the ability of live cells adherent cells (3 106) or tumor spheres (3 103) were to exclude the vital dye. Viable cells were counted using a implanted subcutaneously into 6-week-old male BALB/c hemocytometer. To examine anchorage-independent nude mice and tumor growth monitored using calipers. growth, cells (1 104) were suspended in 0.4% top agar Tumor volume was calculated using the formula: (a b2) over a bottom layer of 0.8% base agar in 6-well plates. The 0.5, in which a is the long axis and b the short axis in solidified soft agar was overlaid with DMEM containing millimeters. 10% FBS, and the medium changed every 4 to 5 days. Colonies were visualized by staining with 0.005% (w/v) Patient datasets and data analysis crystal violet and colonies (defined as groups containing a All glioma patient data were publicly available in the minimum of 50 cells) counted under a phase-contrast deidentified form and obtained from the NCI Repository microscope. for the Molecular Brain Neoplasia Data (REMBRANDT) database. Differences between the groups were analyzed P Phospho-RTK antibody array with the log-rank value. Graphs were created using Cells were plated and lysed in NP-40 lysis buffer (1% Rembrandt data for Affymetrix probes 204803 with the NP-40, 10% glycerol, 20 mmol/L Tris-HCL, pH 8, 137 highest geometric mean intensity and associated survival mmol/L NaCl, 2 mmol/L EDTA, 1 mmol/L sodium data. Up (high)- and downregulation (low) among glioma orthovanadate, and protease inhibitors). Cell lysates specimens refers to a 2-fold (or greater) change in RRAD (250 mg) were incubated overnight on RTK array mem- expression, compared with specimens from patients with branes (ARY-001, R&D Systems). After binding RTKs on nonglioma. the membrane, unbound molecules were washed away. A pan-anti-phospho-tyrosine antibody conjugated to horse- Statistical analysis radish peroxidase was used to detect phosphorylated Data in the graphs represent mean SD of values from tyrosine on activated receptors with the ECL method. at least three independent measurements. To determine the differences in mean values, Student t test was applied. Clonogenic survival assay Intergroup comparisons were made with the paired two t Cells were seeded in 6-well plates (500 cells/well) and samples test. Differences were considered significant at P < exposed to temozolomide (10–300 mmol/L) for 48 hours, 0.05. followed by further observation for 7 to 14 days. Cell densities or colonies were assessed after crystal violet Results staining. Colonies of more than 50 cells were counted. RRAD expression is upregulated in glioblastoma and The dye was subsequently extracted with 10% acetic acid associated with poor survival and absorbance determined using spectrophotometry Previously, we showed that RRAD enhances malignant (570 nm). progression in prostate cancer (21). Accordingly, we examined whether the RRAD protein is upregulated in the GBM Immunocytochemistry cell lines, including U87-MG, U138-MG, U251, LN229, A- Cells were fixed with 4% paraformaldehyde and per- 172, and DBTRG-05MG. Notably, cellular RRAD protein meabilized with 0.1% Triton X-100 in PBS. After fixation, levels were upregulated in malignant cells, compared with cells were incubated with the appropriate primary anti- normal control (Fig. 1A, P < 0.05). We further validated bodies in a solution of PBS with 3% BSA at 4C overnight. RRAD overexpression in human glioma specimens. As Anti-EGFR mouse polyclonal (1:500) and anti-Flag rabbit shown in Fig. 1B, RRAD expression levels were enhanced polyclonal (1:200) antibodies were employed for analysis. in 6 of 6 tumor samples, compared with normal tissue Staining was visualized using anti-mouse Alexa Fluor 488 controls (tumor vs. N1; P < 0.05). All glioma patient data and anti-rabbit Alexa Fluor 594 (Molecular Probes). were publicly available in the deidentified form and Nuclei were counterstained using 40,6-diamidino-2-phe- obtained from the NCI Repository for the Molecular Brain nylindole (DAPI). Stained cells were visualized under a Neoplasia Data (REMBRANDT) database. Using the data- fluorescence microscope (Carl Zeiss, LSM 700). base, we evaluated the survival rates of patients according

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A C a

High vs. low, P < 0.0001 High vs. intermediate, P < 0.0001 Low vs. intermediate, P = 0.2458

RRAD

β-Actin RRAD/actin (fold) Probability of survival

0 1,000 2,000 3,000 4,000 b Follow-up (days)

P = 0.0020 B P = 0.0002

P > 0.5

RRAD 0.89 0.93 1.25 1.32 β-Actin RRAD/actin (fold) N1 N2 N3 T1 T2 T3 T4 T5 T6 N1 N2 N3 T1 T2 T3 T4 T5 T6

Figure 1. Elevated RRAD expression is associated with poor survival of patients with glioma. RRAD protein expression in GBM cell lines (A) and primary brain tumors (B). Freshly frozen specimens of brain tumors (T) showed increased RRAD expression, compared with corresponding normal tissues (N). Actin was used as the loading control. Left, a Western blotting experiment; right, bands on the Western blot analysis were quantiﬁed by densitometry and the data are presented in histogram format. , P < 0.05 (vs. normal N1). C, the upregulation of RRAD in glioma is associated with a decreased lifespan in patients. a, Kaplan–Meier survival curves. Patients with GBM were divided into three groups based on RRAD mRNA expression as described in Materials and Methods. High RRAD expression correlates with poor survival. All glioma patient data were publicly available in the deidentiﬁed form and obtained from the NCI Repository for the Molecular Brain Neoplasia Data (REMBRANDT) database. b, relative mRNA expression levels of RRAD for lower grade glioma and GBM. The numbers indicate mean values and the lines represent mean with SE.

to their RRAD mRNA levels (Fig. 1C). Specifically, data To address whether RRAD expression is correlated with were analyzed to determine the survival of 343 patients genetic alteration, we analyzed mRNA expression in high with intermediate, low or high RRAD expression. Overall, RRAD (36 cases) and low RRAD (73 cases) glioma samples. 37 patients displayed more than 2-fold RRAD upregula- Our results disclosed several genes preferentially upregu- tion, 233 patients displayed intermediate RRAD expres- lated in high RRAD population (Supplementary Fig. S1). sion, and 73 had more than 2-fold downregulation. Median These genes included genes that increase cell proliferation survival of patients with upregulated, intermediate, and (CHI3L1, PTX3, ANXA1), invasion (PDPN, LOX), metas- downregulated expression in glioma specimens was 297, tasis (NNMT), inflammation (CCL2), and mesenchymal 540, and 606 days, respectively. We observed significant signature (TIMP1, IL13RA2). To identify a putative group differences in terms of survival in high RRAD versus in which RRAD has a strong impact on survival, we divided intermediate RRAD (P < 0.0001) and high RRAD versus patients into four percentiles and analyzed survival for the low RRAD populations (P < 0.0001). No significant differ- four independent groups. As shown in Supplementary Fig. ences were evident in terms of survival between low S2, RRAD expression >75th percentile was correlated with RRAD and intermediate RRAD populations (P ¼ lower survival (P ¼ 0.0027). Collectively, the data show that 0.2458). To understand whether the increased RRAD high expression of RRAD is significantly associated with expression was associated with any particular glioma GBM and predicts a poor prognosis of glioma. subtype, we compared the expression of RRAD among oligodendroglioma (n ¼ 49), astrocytoma (n ¼ 104), mixed RRAD enhances EGFR protein stability and EGFR- (n ¼ 6), and GBM (n ¼ 185). Expression of RRAD was induced STAT3 activation significantly higher in GBM, compared with other lower Glioblastoma is characterized by the abnormal activa- grade glioma (Fig. 1C, b and Supplementary Fig. S1). tion of RTK signaling pathways (7). To identify RRAD-

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A LN229-Vector B Vector RRAD

P = 0.006 EGF 0 10 20 60 0 10 20 60 (min) pEGFR 4 pEGFR(Y845) 3 pSTAT3(Y705) 2 LN229-RRAD RRAD

pEGFR 1 EGFR

p-EGFR expression β 0 -Actin Vec RRAD

C D 1.2 Vector =0

RRAD 1.0 Input IP:IgG IP:EGFR Vector RRAD 0.8 Vec RRAD Vec RRAD Vec RRAD CHX 0 3 6 9 0 3 6 9 (h) 0.6

RRAD EGFR 0.4

pEGFR RRAD 0.2 P = 0.0002 -Actin, vs. t vs. EGFR/ b -Actin, β-Actin 0.0 EGFR 0369 CHX (h)

Figure 2. RRAD mediates EGF-induced STAT3 activation in cancer cells. A, RRAD-induced upregulation of phosphorylated EGFR. RTK phosphorylation in LN229 cells expressing RRAD or control vector. Individual RTKs are spotted in duplicate and the identity of phospho-EGFR is indicated. Positive control spots are located at the corners of the human phospho-RTK array. Spot intensity was quantified by densitometry and the data are presented in histogram format. B, EGF-induced STAT3 activation is enhanced in the presence of RRAD. Serum-starved LN229 cells expressing RRAD or control vector were treated with 100 ng/mL EGF for 0 to 60 minutes, and whole-cell lysates immunoblotted with an antibody specifically recognizing the phospho-tyrosine at Y845 of EGFR, and phospho-STAT3 at Y705. Actin was used to show equal loading. C, RRAD physically associates with EGFR. Whole-cell extracts were immunoprecipitated with EGFR antibody and immunoblotted with a general antiphosphotyrosine, EGFR, and Flag (tagged for RRAD) antibody. D, RRAD increases EGFR protein stability. RRAD-overexpressing LN229 cells were treated with 20 mg/mL cycloheximide (CHX) for 0 to 9 hours. EGFR and RRAD protein levels were analyzed by immunoblotting. EGFR levels were normalized to Actin and compared with t ¼ 0. Data represent mean values SD of three independent experiments. associated RTK, we screened a human phospho-RTK EGFR to actin expression at 9 hours for LN229-RRAD vs. array using whole-cell lysates from vector-(LN229-Vec- LN229-Vector: 0.85 vs. 0.18; P ¼ 0.0002) cells. To rule out the tor) and RRAD-(LN229-RRAD)–transfected cells. Among possibility of clonal selection, experiments were repeated multiple RTKs, the spot intensity of phosphorylated using U87-MG after transient transfection as well as in the EGFR was the most significantly enhanced by RRAD context of RRAD targeting. As shown in Supplementary (Fig. 2A). Previous studies have reported that STAT3 Fig. S3, transient overexpression of RRAD promoted phos- activation in GBM is commonly induced by growth factor phorylation of EGFR, and conversely, depletion of RRAD receptors on the cell surface (23). EGFR signaling induces effectively reduced EGFR phosphorylation. STAT3 activation in GBM, and phosphorylation of EGFR at tyrosine 845 is essential for activating STAT3. Consis- RRAD enhances endosome-mediated EGFR tent with these findings, we showed that RRAD promotes translocation to the nucleus phosphorylation of EGFR and STAT3 (Fig. 2B). We did not RTKs, including IGFR, HGFR(c-Met), FGFR, VEGFR, observe significant differences in total EGFR expression and the EGFR family, have been shown to localize to the during phosphorylation by EGF. Moreover, RRAD phys- nucleus (23, 24). Cell surface EGFR translocates to the ically associates with EGFR, as observed from coimmuno- inner nuclear membrane through nuclear pore complexes, precipitation experiments (Fig. 2C). As RRAD does not which is mediated by importin-b and endosome (25, 26). exert kinase activity to phosphorylate EGFR, we assessed Phosphorylated STAT3 colocalizes with receptor–ligand for changes in the stability of EGFR protein by treating complexes on the endosome and is transported from the LN229-Vector and LN229-RRAD cells with cycloheximide plasma membrane to the perinuclear region (10). RRAD (Fig. 2D). To assess the turnover rate of EGFR, the protein associates with importin-b through three conserved NLS half-life was measured at various time points after cyclo- regions (27). Surprisingly, in our experiments, the mem- heximide treatment. The EGFR level declined more slowly brane-bound early endosomal marker, EEA1, also copre- in LN229-RRAD, compared with LN229-Vector (ratio of cipitated with RRAD (Fig. 3A).

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A B C

Cytosol Nucleus LN229-Vector LN229-RRAD DAPI DAPI EGFR

STAT3

EEA1 EGFR EGFR/RRAD EGFR/RRAD CBB stain RRAD(Flag) STAT3 Tubulin D GST-RRAD PARP EGFR/RRAD/DAPI EGFR/RRAD/DAPI Dy, Dynasore GST

D EGFR/EEA1 EGFR/EEA1/DAPI DAPI E LN229-Vector EEA1 Tubulin PARP Cytosol Nucleus LN229-RRAD

Figure 3. RRAD colocalizes with EGFR and enhances internalization. A, in vitro binding assays using GST-RRAD protein. Recombinant GST-RRAD protein was purified and conjugated to glutathione beads and incubated with cell lysates at 4C overnight. Glutathione bead-associated proteins were resolved using SDS-PAGE, and analyzed by immunoblotting with EGFR, STAT3, and EEA1 antibodies. GST recombinant proteins were verified with Coomassie Brilliant Blue (CBB) staining. B, LN229-Vector– and -RRAD–transfected cells maintained in serum-starved medium for 4 hours were treated with the dynamin inhibitor Dynasore to prevent receptor internalization. Subcellular fractionation and immunoblotting were performed to analyze EGFR, STAT3, and RRAD protein levels. Tubulin was used as a cytoplasmic marker and PARP as a nuclear marker. C, Vector (LN229-Vector)- and RRAD-expressing (LN229-RRAD) cells were immunostained for EGFR and RRAD, and analyzed using confocal microscopy. Cells were treated with EGF (100 ng/mL) for 20 minutes, and stained with a mouse polyclonal anti-EGFR antibody (green) and rabbit anti-Flag antibody (red). Colocalization of Flag-RRAD and EGFRin vesicle structures is shown in yellow. Original magnification is 800. Merged image of RRAD (red), EGFR (green), and DAPI-stained DNA (blue) clearly shows nuclear colocalization of EGFR with RRAD. D, RRAD is required for EGFR and EEA1 nuclear localization by endosomal sorting. LN229-Vector and LN229–RRAD cells were immunostained with EEA1 and EGFR and analyzed using confocal microscopy. Original magnification is 800. Merged image of EEA1 (red), EGFR (green), and DAPI-stained DNA (blue) clearly shows nuclear colocalization of EGFR with EEA1 in RRAD-expressing cells. E, LN229- Vector and -RRAD cells maintained in serum-starved medium for 4 hours were treated with Dynasore, followed by cellular fractionation. The EEA1 protein level was analyzed via immunoblotting.

To examine whether RRAD affects subcellular location Next, we examined the involvement of endosomal of EGFR, cell lysates were fractionated and subjected to sorting in RRAD-induced EGFR nuclear transport by immunoblot analysis. The results indicate that EGFR investigating whether inhibition of endocytosis can block translocates to the nucleus in the presence of RRAD (Fig. this process. Dynasore is a small molecule that prevents 3B). Immunoﬂuorescence staining was performed to fur- receptor internalization (28). RRAD promoted the nuclear ther investigate whether EGFR associates with RRAD in expression of EGFR, as shown with immunoblotting (Fig. vivo. EGFR, which is normally localized on the plasma 3B), which was abrogated with Dynasore treatment. After membrane in the absence of EGF, was frequently detected EGF stimulation, EGFR and EEA1 colocalized as nuclear in the nuclear envelope after EGF stimulation (25). Con- and perinuclear structures in the presence of RRAD (Fig. sistent with cell fractionation data, we observed promi- 3D). RRAD-induced upregulation of nuclear EEA1 was nent perinuclear and nuclear colocalization of RRAD with conﬁrmed by subcellular fractionation and immunoblot- endogenous EGFR in vesicle structures (Fig. 3C). ting (Fig. 3E). The data collectively suggest that RRAD

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enhances the endosome-mediated nuclear translocation independent colony-forming ability according to RRAD of EGFR. overexpression. As shown in Fig. 4B, overexpression of RRAD led to a marked increase in colony-forming ability RRAD expression is correlated with STAT3 in soft agar, whereas RRAD depletion in highly aggressive activation and malignancy-associated properties U251 cells diminished colony formation (Fig. 4C). To In GBM, aberrant STAT3 activation is correlated with clarify whether changes in colony formation are associ- tumor grade and clinical outcomes (29). Activated STAT3 ated with differences in proliferation and/or survival, enhances the expression of a number of downstream Trypan blue exclusion staining was performed. As shown genes and regulates multiple behaviors of tumor cells, in Supplementary Fig. S5, depletion of RRAD led to such as survival, growth, angiogenesis, and invasion. decreased proliferation and survival of GBM cells. In Malignant progression of cancer involves loss of cell–cell addition, Transwell migration was upregulated in cells interactions together with acquisition of migratory prop- with higher RRAD expression (Fig. 4D), but diminished erties, and is often associated with epithelial–mesenchy- upon RRAD depletion (Fig. 4E). To ascertain whether the mal transition (EMT; ref. 30). Upon overexpression of effect of RRAD is EGFR-dependent, the Transwell migra- RRAD, the p-STAT3 (Y705) level was increased, and tion assay was performed in the presence of the EGFR EMT-associated proteins, including N-cadherin, Twist, inhibitor, geﬁtinib. As shown in Fig. 4F, the EGFR inhib- and Vimentin, were upregulated (Fig. 4A). In parallel, itor effectively blocked RRAD-induced colony formation RRAD depletion reduced STAT3 phosphorylation and and migration, clearly indicating an EGFR-dependent expression of TWIST (Supplementary Fig. S4). To address effect of RRAD. Consistent with these results, the scratch the requirement for RRAD in maintaining the tumorigenic assay revealed that RRAD increases tumor cell motility capability of GBM cells, we examined the anchorage- (Supplementary Fig. S6). Taken together, our ﬁndings

A B C LN229-RRAD LN229-RRAD U251 siControl Vec C9 C5 vec C9 C5 Vector siC siR#1 siR#2 RRAD(Flag) RRAD RRAD pSTAT3(Y705) β-Actin β-Actin STAT3 siRRAD#1 C9 P = 0.0002 100 P < 0.0001 N-cadherin 150 P = 0.0002 80 E-cadherin 60 100 TWIST siRRAD#2 40 P = 0.001 C5 50 Vimentin

Colonies/well 20 β Colonies/well -Actin 0 0 Vec C9 C5 siC siR#1 siR#2

D E F vector P = 0.005 siControl P = 0.026 P = 0.016 P = 0.008 P = 0.026 P < 0.0001 P = 0.002

C9 siRRAD#1 P = 0.0003

C5 siRRAD#2 RRAD - + + RRAD - + + Vec C9 C5 siC siR#1 siR#2 EGFRi -- + EGFRi -- + LN229-RRAD U251 LN229-RRAD LN229-RRAD

Figure 4. RRAD upregulates soft agar colony formation and migration. A, RRAD-dependent increase in phosphorylated STAT3 (Y705), N-cadherin, TWIST, and Vimentin expression of RRAD stable clones established in LN229. Protein expression was assessed with immunoblotting. Soft agar colony formation in RRAD-transfected LN229 (B) or siRRAD-transfected U251 cells (C). Cells were plated (300 cells/well) in soft agar for 3 weeks and the colonies counted. Cell migration in RRAD-transfected LN229 (D) or siRRAD-transfected U251 cells (E). Cells that migrated through the membrane were stained with crystal violet and counted directly under a microscope. RRAD expression was analyzed with immunoblotting using actin as a loading control. Data represent mean values SD of three independent experiments. F, EGFR inhibitor (geﬁtinib, 10 mmol/L) effectively blocked RRAD-induced colony formation and migration of LN229 (C5).

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indicate that RRAD enhances the malignant properties of stem cells (32). Expression analysis of EMT-regulating human GBM cells through STAT3 activation. (REX1 and SNAIL) and stemness-regulating transcription factor (OCT4, NANOG, and SOX2) levels disclosed upre- RRAD promotes self-renewal and sphere formation gulation of these factors with RRAD overexpression (Fig. of glioblastoma cells 5A). As tumor sphere formation is based on the unique In addition to invasion and metastasis, EMT is often properties of stem/progenitor cells to survive and grow in associated with self-renewal and tumor-initiating capabil- serum-free suspension, we performed the tumor sphere ity (31). EGFR and STAT3 activation are frequently impli- formation assay to examine whether RRAD enhances self- cated in the stem cell properties of GBM (7). Stem cell renewal of GBM. Our data showed that RRAD enhances transcription factors, such as SOX2, OCT4 and NANOG, sphere formation in a concentration-dependent manner are critical for maintaining self-renewal, proliferation, sur- (Fig. 5B). The limiting dilution sphere-forming assay dem- vival, and multilineage differentiation potential of GBM onstrated that RRAD-expressing LN229 (C5) cells generate

LN229-RRAD A B C U87-MG Vec C9 C5 a b siC siR#1 siR#2 RRAD NANOG RRAD P < 0.0001 vector OCT4 * GFAP SOX2 pSTAT3

REX1 RT-PCR C9 OCT4 SNAIL P = 0.001 * SOX2 GAPDH * P = 0.0001 TWIST Nanog C5 Vimentin OCT4 P = 0.004 β-Actin SOX2 Vec C9 C5 β-Actin LN229-RRAD Western blot P = 0.004 D E

a P = 0.0002 b P = 0.0001 U87-MG U251 P = 0.0002 P = 0.0001 siControl siControl

siRRAD - + + siRRAD#1 siRRAD#1 STAT3-CA - - + * *, P < 0.0001 RRAD * # , P = 0.01 pSTAT3 ** siC siR#1 #2 siC siR#1 #2 STAT3(Flag) siRRAD#2 siRRAD#2 * * RRAD ### # SOX2 β-Actin OCT4 U87-MG U251 β-Actin

Figure 5. RRAD is critical for the maintenance of stem-like properties. A, RRAD-dependent expression of OCT4, NANOG, and SOX2. Tumor spheres obtained from indicated stable clones were subjected to RT-PCR and Western blot analysis of stemness-regulating genes (OCT4, NANOG, and SOX2). B, RRAD level-dependent increase in tumor sphere formation. a, the sphere-forming activities of control vector and RRAD stable clones were determined after 5 to 7 days of culture. Right, images show the morphologic appearance of the cells under an inverted microscope. Left, the sphere-forming frequency (>50 mm spheres) of LN229 cells. b, limiting dilution sphere-forming assay. LN229 cells were plated into 96-well plates at various seeding densities (5–500 cells/well, 14 wells/condition). Data are presented as mean number SD of spheres per indicated number of inoculated cells. Limiting dilution analyses were performed using extreme limiting dilution analysis (http://bioinf.wehi.edu.au/software/elda). , P < 0.0001 (vs. vector). C, RRAD knockdown diminishes the expression of stemness- or EMT-regulating molecules. U87-MG sphere was transfected with either siRNA of RRAD or control siRNA. After 48 hours, cells were analyzed for p-STAT3 (Y705), GFAP, TWIST, Vimentin, OCT4, and SOX2 expression using immunoblotting. Actin was used to show equal loading. D, knockdown of RRAD inhibits tumor sphere formation. a, U87-MG and U251 cells (104 cells/well in 24-well plate) were treated with either siRRAD or siControl, and sphere-forming activities determined after 7 days of culture. Right, images show the morphologic appearance of cells under an inverted microscope. Left, sphere-forming frequency (> 50 mm spheres) of both U87-MG and U251 cells. Immunoblots reveal efﬁcient knockdown of RRAD. Scale bar, 100 mm. Data represent mean values SD of three independent experiments. b, limiting dilution sphere-forming assay. U87-MG cells were plated into 96-well plates at various seeding densities (5–500 cells/well, 14 wells/condition). Data are presented as mean number SD of spheres per indicated number of inoculated cells. , P < 0.0001 (vs. vector). E, STAT3 activation can rescue RRAD depletion-induced effects on self-renewal of U87-MG cells. The sphere-forming activity (>50 mm spheres) was determined after 7 days of culture. Tumor spheres obtained from indicated stable clones were subjected to Western blot analysis of indicated proteins.

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one tumor sphere in 12.4 cells, whereas vector-expressing constitutively active STAT3 (STAT3-CA) could rescue the cells generate one sphere in 64 cells (Fig. 5B). Moreover, phenotypes caused by RRAD knockdown. STAT3 activa- depletion of RRAD attenuated the expression of stem cell tion efficiently rescued the impaired sphere formation and transcription factors, OCT4 and SOX2, the lineage marker, downregulation of SOX2 and OCT4 (Fig. 5E). The results GFAP, and EMT proteins, TWIST and Vimentin, as shown collectively indicate that RRAD-induced STAT3 activa- in Fig. 5C. Previous studies have shown that GBM stem tion is critical for the self-renewal of GBM cells. þ cells are enriched with the CD44 cell population (33, 34). þ FACS analysis revealed that the percentage of CD44 cells RRAD depletion sensitizes temozolomide-resistant increases with RRAD expression, but is markedly reduced glioblastoma cells upon depletion of RRAD (Supplementary Fig. S7). Next, we examined whether the decrease in RRAD is Notably, RRAD knockdown diminished the ability of related to drug-induced cell growth inhibition. An assay GBM to form tumor spheres (Fig. 5D). As shown in Fig. of cell viability using Trypan blue staining revealed that 5D, the limiting dilution sphere-forming assay demon- siRRAD, but not control siRNA, specifically inhibits strated that RRAD-depleted U87-MG cells generate one growth in aggressive GBM cell lines, including U87-MG, tumor sphere in 55.2 (siRRAD#1) or 63.3 (siRRAD#2) cells, U138-MG, and U251 (Supplementary Fig. S5). Stem-like whereas control cells generate one sphere in 18.1 cells (Fig. properties of cancer cells have been implicated in resis- 5D). To confirm mechanistic link between RRAD and tance to chemotherapy and radiotherapy (35). The alky- STAT3, we examined whether ectopic expression of a lating agent, temozolomide, is frequently used to treat

A B Mock Vector RRAD(C9) RRAD(C5) LN229 100 100 LN229-TMZR 80 80 60 60 40 (%) 40 (%) 20 20 Relative ABS 570 Relative ABS 570 0 m 0 TMZ 0 30 100 300 ( mol/L) TMZ 0 10 30 100 300 (mmol/L)

C D E LN229-TMZR LN229-TMZR RRAD siControl - + - - pSTAT3 siRRAD#1 + --- OCT4 siRRAD#2 --- + 80 SOX2 RRAD 60 P = 0.0004 β-Actin Western blot N-cadherin 40 P = 0.003 OCT4 TWIST 20 NANOG Vimentin Colonies (%) 0 SOX2 SOX2 TMZ 100 100 100 100 (mmol/L) - - - TWIST OCT4 siControl +

RT-PCR siRRAD#1 - - + - SNAIL b -Actin siRRAD#2 - -- + SLUG GAPDH

Figure 6. Depletion of RRAD sensitizes temozolomide-resistant GBM cells and decreases their stem-like properties. A, overexpression of RRAD enhances the survival of GBM (LN229-RRAD stable clone) cells treated with increasing doses of temozolomide over 48 hours. Crystal violet staining was used to estimate cell viability. All data are presented as percentages of the untreated control value. B, clonogenic survival assay of parental LN229 (LN229) and LN229 temozolomide-resistant (LN229-TMZR) cells. Temozolomide-resistant LN229 (TMZR) was established, as described in Materials and Methods. Clonogenic survival assays were performed by seeding 500 cells in 6-well plates, followed by treatment with temozolomide (30–300 mmol/L) for 48 hours, and further observed for 7 days. Survival rates were assessed using crystal violet staining of the colonies. C, parental LN229 and LN229-TMZR cells were subjected to RT-PCR and Western blot analysis for EMT- or stemness-regulating gene expression. GAPDH and actin were used to show equal loading. D, parental LN229 and LN229-TMZR cells transfected with siControl or siRRAD and subjected to immunoblot analysis for the indicated proteins. Actin was used to show equal loading. E, parental LN229 and LN229-TMZR cells transfected with siControl or siRRAD and subjected to clonogenic survival assays. Data are presented as mean values SD of three independent experiments. P < 0.005 versus siControl.

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patients with GBM. However, more than 90% of recurrent of stemness-regulating molecules (Fig. 6D) and survival of GBMs do not respond to repeated challenges with temo- temozolomide-resistant cells (LN229-TMZR, U87-MG, zolomide. To determine the precise role of RRAD in and A172) was dramatically reduced upon depletion of temozolomide resistance of GBM, we performed a clono- RRAD (Fig. 6E and Supplementary Fig. S9), indicating genic survival assay. Temozolomide resistance of GBM that RRAD expression is critical for maintaining stem cell increased in a RRAD expression–dependent manner properties and temozolomide resistance in GBM cells. (Figs. 6A). To select temozolomide-resistant GBM cells, LN229 cells were treated with high doses of TMZ (2 RRAD promotes in vivo tumorigenesis of mmol/L) for 24 hours and surviving live cells collected glioblastoma and is required for maintenance of after flow cytometer-assisted cell sorting, termed "temo- pluripotency zolomide-resistant" cells. TMZR cells showed marked We compared tumorigenesis of stable RRAD transfec- resistance to temozolomide, compared with parental cells tant LN229 and control cells after subcutaneous implan- (Fig. 6B). Specifically, the median inhibitory concentration tation of adherent monolayer cultured cells or tumor > m (IC50) of temozolomide was 300 mol/L in LN229-TMZR spheres into athymic nude mice. Our results revealed cells, whereas that for control LN229 cells was 10 mmol/L significantly higher tumor growth in RRAD- overexpres- (P ¼ 0.0096). RRAD expression was higher in LN229- sing monolayer cells (3 106) than those expressing vector TMZR, compared with parental LN229 cells in immunoblot controls over 4 weeks (Fig. 7A; P ¼ 0.0004). LN229-RRAD analysis (Fig. 6C). Previously, it was shown that stem-like sphere cells exhibited a high capacity to form tumors with properties of GBM cells contribute to chemoresistance to as low as 3 103 tumor spheres. Control cells formed temozolomide (36). In vitro tumor sphere formation assay tumors with an average size of 1 1mm3 over 4 weeks. In (Supplementary Fig. S8) showed that temozolomide-resis- contrast, RRAD-transfected spheres developed tumors tant LN229-TMZR can generate one tumor sphere in 22.1 that averaged 564 57 mm3 in size during the same cells, whereas LN229 can generate one sphere in 79.9 cells period (Fig. 7B; P ¼ 0.0002). (http://bioinf.wehi.edu.au/software/elda). In our experi- Human GBM cells from U87-MG display a predomi- ments, expression of EMT and/or stemness-regulating nant mesenchymal stem cell phenotype (37), express molecules was markedly increased in TMZR cells, com- typical markers, and are capable of adipogenic differen- pared with parental cells (Fig. 6C). Taken together, these tiation in vitro. Accordingly, we investigated whether results suggest that RRAD-expressing GBM cells are selec- RRAD knockdown suppresses these properties of GBM. tively resistant to temozolomide treatment. Upon cultivation of U87-MG cells in adipogenic media for We additionally examined whether depletion of RRAD three weeks, parental or siControl-transfected U87-MG controls the chemoresistance and stem-like properties of cells were stained with Oil-red O, indicating intracellular temozolomide-resistant GBM cells. Notably, expression lipid accumulation. However, no lipid droplets were

A 1,000 6 Monolayer (3×10 ) C Vector EGFR/STAT3/RRAD/EEA1 )

3 800 RRAD

600 Figure 7. RRAD promotes tumorigenesis of GBM. Effect of 400 RRAD expression on GBM growth EGFR/pSTAT3 in vivo. For each injection, stable P = 0.0004 LN229 cell clones were cultivated

Tumor size (mm Tumor 200 C as monolayer cells (3 106 cells; A) or as tumor spheres (3 103 cells; 0 OCT4, NANOG, SOX2 0 13 17 20 22 24 27 29 31 N B) implanted subcutaneously into TWIST, SNAIL, SLUG ﬂ Days after tumor innoculation the anks of athymic nu/nu mice. Five mice were used for each group B (bars, SEM). C, a model based on 1,000 Sphere (3×103) our studies illustrates that RRAD Vector activates STAT3 via endosome- ) 3 800 RRAD mediated EGFR translocation, GBM tumorigenesis thereby increasing the levels of 600 EMT-regulating (TWIST, SNAIL, and SLUG) and stemness- 400 regulating (OCT4, NANOG, and SOX2) genes.

Tumor size (mm Tumor 200 P = 0.0002

0 0 13 17 20 22 24 27 29 31 Days after tumor innoculation

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observed in RRAD-depleted U87-MG cells (Supplemen- sistent with these findings. EMT is a unique process in tary Fig. S10). which epithelial cells undergo morphologic changes leading to increased motility and invasion (31, 43). The lack of effective GBM stem cell markers and het- Discussion erogeneity of GBM stem cells are limiting factors for the The hypothesis that within the heterogeneous tumor effective targeting of GBM (44, 45). Targeting of the mole- mass, cells with characteristics of cancer stem cells (CSC) cules responsible for temozolomide resistance is crucial to are responsible for tumor initiation, recurrence and resis- block GBM evasion of therapeutic responses. Given that tance to therapy is redirecting the therapeutic efforts RRAD promotes STAT3 signaling of GBM cells, it is pos- against cancer (38, 39). Hence, elucidation of the molec- sible that long-term exposure to RRAD induces a temozo- ular pathways involved in CSC control may be essential to lomide-resistant phenotype in GBM cells. To address this eradicate tumors. In the current study, we aimed to issue, we exposed GBM cells to temozolomide. Our data identify the targets responsible for resistance of GBM cells showed that temozolomide causes an increase in RRAD to temozolomide and maintenance of GBM stem-like cells. and ultimately leads to the formation of highly resistant Previously, we showed that RRAD overexpression is GBM tumors. Upregulation of RRAD expression mim- associated with chemoresistance of cancer cells and icked the effects of long-term exposure to temozolomide, RRAD knockdown sensitizes resistant cancer cells to whereas targeting RRAD reversed temozolomide resis- cytotoxic drugs (21, 22). RRAD has additionally been tance. Importantly, the increase in RRAD expression was associated with poor prognosis in breast cancer (14). apparent not only in vivo but also in human tumors in cases However, the exact function of RRAD in temozolomide of poor survival, indicating clinical relevance. resistanceof GBMiscurrentlyunknown.STAT3activation Overexpression of EGFR is associated with poor prog- is significantly correlated with stem-like GBM cells (8). To nosis of GBM (46). EGFR signaling has also been found to our knowledge, this is the first study to show that RRAD is mediate resistance to chemotherapy and radiotherapy capableof modulating endosome-mediated EGFR–STAT3 (47). Our results collectively showed that RRAD expres- signaling,therebypromotingstem-likefunctionandtemo- sion induces temozolomide resistance in GBM via zolomideresistanceofGBMcells.Moreover,RRADisover- enhancement of EGFR and STAT3 activation, as summa- expressed in GBM cell lines and tumor tissues and its rized in Fig. 7C. We further demonstrated that RRAD expression is inversely correlated with survival of patients inhibition attenuates acquired temozolomide resistance with GBM in publicly available clinical data. and stem-like properties in GBM, suggesting that RRAD We demonstrated that RRAD-expressing GBM cells inhibition constitutes an important strategy in the pre- harbor properties, including the ability to self-renew, vention of recurrence after GBM treatment. Previous generate tumor spheres, and grow tumors in vivo. Expres- studies by our group have shown that RRAD promotes sion of the stem cell transcription factors, OCT4, SOX2, the cell cycle through interactions with GCIP and conse- and NANOG decreased with the decrease in RRAD levels, quent inhibition of the GCIP-mediated decrease in cyclin suggesting a tight correlation between RRAD levels and D activity and Rb phosphorylation (21, 48). As RRAD self-renewal processes. OCT4, upregulated by RRAD, is depletion also renders GBM cells less proliferative, it responsible for the maintenance of stem cells (40, 41). appears that the RRAD pathway performs a dual function Thus, RRAD-induced STAT3 activation and OCT4 expres- in maintaining stem-like properties and accelerating pro- sion may facilitate the maintenance of GBM cells in a stem- liferation of the cycling subset in GBM. like state and contribute to EMT transition of GBM. Our data demonstrate that RRAD promotes endosome- We found that transient depletion of RRAD led to mediated STAT3 signaling, which is critical for the main- decreased STAT3 activation, implying that RRAD expres- tenance of stem-like cancer cells and temozolomide resis- sion is required for maintenance of the stem-like state. tance in GBM. Elucidation of the novel STAT3 activation Consequently, the capacity of GBM cells to form tumor pathway and identification of RRAD as a molecular target spheres was prevented, suggesting that RRAD-mediated are thus crucial findings that may facilitate sensitization of STAT3 activation is responsible for in vivo tumorigenesis. temozolomide-resistant GBM. These cellular changes were accompanied by downregulation of OCT4, SOX2, Vimentin, and TWIST. Therefore, Disclosure of Potential Conflicts of Interest we further investigated the mechanism by which RRAD No potential conflicts of interest were disclosed. maintains and/or promotes the stem-like properties of GBM. We propose that a RTK signaling pathway in GBM Authors' Contributions Conception and design: D.-H. Nam, C. Park cells is activated by RRAD, leading to phosphorylation Development of methodology: C. Park and activation of STAT3. Previously, it was reported that Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S.-Y. Yeom, C. Park constitutively activated STAT3 is frequently coexpressed Analysis and interpretation of data (e.g., statistical analysis, biostatis- with EGFR in high-grade gliomas (7), and EGFR coop- tics, computational analysis): S.-Y. Yeom, C. Park erates with STAT3 to facilitate EMT in human epithelial Writing, review, and/or revision of the manuscript: S.-Y. Yeom, C. Park Administrative, technical, or material support (i.e., reporting or orga- cancers (42). Our data showing that depletion of RRAD nizing data, constructing databases): C. Park promptly inhibits TWIST expression and EMT are con- Study supervision: D.-H. Nam, C. Park

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Acknowledgments The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked The authors thank Prof. D.-H. Nam (Sungkyunkwan University School advertisement of Medicine, Seoul, Korea) for providing human GBM specimens. in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant Support This work was supported by a grant from National Research Founda- Received March 21, 2014; revised August 12, 2014; accepted September tion of Korea (NRF-2011-0016973; to C. Park). 10, 2014; published OnlineFirst October 13, 2014.

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www.aacrjournals.org Mol Cancer Ther; 13(12) December 2014 3061

RRAD Promotes EGFR-Mediated STAT3 Activation and Induces Temozolomide Resistance of Malignant Glioblastoma

Seon-Yong Yeom, Do-Hyun Nam and Chaehwa Park

Mol Cancer Ther 2014;13:3049-3061. Published OnlineFirst October 13, 2014.

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