Signal Transduction

EGFR Signals through a DOCK180-MLK3 Axis toDrive Glioblastoma Cell InvasionSean A. Misek1, Jian Chen1,2, Laura Schroeder1,3, Chotirat Rattanasinchai1,3,Ashley Sample1,2, Jann N. Sarkaria4, and Kathleen A. Gallo1,3

Abstract

A hallmark of glioblastoma (GBM) tumors is their highlyinvasive behavior. Tumor dissemination into surroundingbrain tissue is responsible for incomplete surgical resection,and subsequent tumor recurrence. Identification of targets thatcontrol GBM cell dissemination is critical for developing effec-tive therapies to treat GBM. A majority of GBM tumors havedysregulated EGFR signaling, due most frequently to EGFRamplification or the presence of a constitutively active EGFRvIIImutant. Mixed lineage kinase 3 (MLK3) is a mitogen-activatedprotein kinase kinase kinase (MAP3K) that can activate mul-tiple MAPK pathways. In this study, evidence is provided thatMLK3 is essential for GBM cell migration and invasion, and thatan MLK inhibitor blocks EGF-induced migration and invasion.

MLK3 silencing or MLK inhibition blocks EGF-induced JNKactivation, suggesting that MLK3-JNK signaling promotes inva-sion of GBM cells. Mechanistically, it is demonstrated thatDOCK180, a RAC1 guanine nucleotide exchange factor (GEF)overexpressed in invasive GBM cells, activates the MLK3-JNKsignaling axis in a RAC1-dependent manner. In summary, thisinvestigation identifies an EGFR–DOCK180–RAC1–MLK3–JNK signaling axis that drives glioblastoma cell migration anddissemination.

Implications: On the basis of these findings, MLK3 emerges asa potential therapeutic target for the treatment of glioblastoma.Mol Cancer Res; 15(8); 1085–95. �2017 AACR.

IntroductionGlioblastoma (GBM) tumors are the deadliest of adult brain

tumors. Invasion of GBM tumor cells into the surrounding brainleads to tumor relapse, and a dismal patient prognosis. Even withrecent treatment advances in surgery, radiotherapy, antiangio-genic therapies, and temozolomide regimens, the average survivaltime of patients with GBM is typically 12 to 15 months (1).Substantial evidence now suggests that angiogenesis inhibitorsand radiotherapy may actually promote tumor invasion (2). Thesignaling pathways required for GBM cell migration and invasionare not fully delineated. Characterization of the mechanisms thatdrive GBM cell dissemination may lead to the identification ofnovel therapeutic targets.

Dysregulation of EGFR signaling is a major defining character-istic of the classical GBM subtype (3), and EGFR signaling has aclearly defined role in GBM tumor progression. This is commonlydue to either amplification of the EGFR gene or the presence of aconstitutively active, ligand independent, mutant EGFRvIII lack-ing amino acids 6–273 of its extracellular domain (4). Despite theimportance of EGFR inGBM, direct targeting of EGFR in GBMhasbeen largely unsuccessful, in part due to de novo or acquiredresistance to EGFR-targeted therapies. Some explanations for lackof efficacy of EGFR inhibitor therapy include intratumoral het-erogeneity, poor binding of inhibitors to EGFRvIII (5), andcompensatory upregulation of c-MET or other receptor tyrosinekinases (RTK; ref. 6). An alternative strategy to targeting EGFRdirectly may be to identify and target key signaling nodes down-stream of EGFR.

In addition to its role in proliferation, EGFR activation is alsoimportant in GBM cell migration (7, 8). EGFR and other RTKssignal tomultiple Rho family GTPases that control focal adhesiondynamics, cytoskeletal remodeling, and gene transcription duringmigration and invasion. Several studies support a critical role forRAC1 in GBMmigration and invasion. For instance, Rac guaninenucleotide exchange factors (GEFs), including Ect2, Vav3, Trio,and SWAP-70, which activate Rac1, are upregulated in gliomasand contribute to cell migration (9). Dedicator of cytokinesis 1(DOCK1), also known as DOCK180, is a Rac GEF (10).DOCK180 associates with a cofactor Engulfment and cellmotility1 (ELMO1), which may contribute to the proper localization ofDOCK180. DOCK180 is overexpressed at the invasive front ofhuman GBM tumors, and promotes migration and invasion ofGBM cells through activation of RAC1 (11). Despite the role forRac1 in GBM migration and invasion, it has been difficult todevelop nontoxic, small molecules that directly target Rac. Target-ing critical Rac effector proteins might provide an alternativesolution to this problem.

1Department of Physiology, Michigan State University, East Lansing, Michigan.2Department of Biochemistry and Molecular Biology, Michigan State University,East Lansing, Michigan. 3Department of Cell and Molecular Biology Program,Michigan State University, East Lansing, Michigan. 4Department of RadiationOncology, Mayo Clinic, Rochester, Minnesota.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

S.A. Misek and J. Chen contributed equally to this article.

Current address for J. Chen: Department of Medicine and Pharmacology andCancer Biology, Duke University Medical Center, Durham, North Carolina; andcurrent address for A. Sample, Department ofMedicine, Section of Dermatology,University of Chicago, Chicago, Illinois.

Corresponding Author: Kathleen A. Gallo, Michigan State University, 567Wilson Road, East Lansing, MI 48824. Phone: 517-884-5156; Fax: 517-355-5125; E-mail: gallok@msu.edu

doi: 10.1158/1541-7786.MCR-16-0318

�2017 American Association for Cancer Research.

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Mixed lineage kinase 3 (MLK3) is a MAP3K which activatesmultiple MAPK pathways, including JNK, and, in some settings,P38 and ERK (12–14). The Rho family GTPases, Cdc42 and Rac,play critical roles in activating MLK3. MLK3 is a multi-domainprotein, consisting of an N-terminal SH3 domain, followed by aserine/threonine kinase domain, leucine zipper region, a Cdc42/Rac-interactive binding (CRIB) motif and a large, proline-rich C-terminal region. MLK3 is maintained in an auto-inhibited, closedconformation through an interaction between its N-terminal SH3domain and a proline-containing sequence located between thezipper andCRIBmotifs (14). Binding of active, GTP-bound RAC1or CDC42 through the CRIB motif of MLK3 (14), disrupts SH3-mediated autoinhibition, leading to zipper-regulated dimeriza-tion, trans-autophosphorylation, and, ultimately, downstreamMAPK activation (15–17).

Work by our lab and others has demonstrated an importantrole for MLK3, and downstream MAPK pathway activation, incancer cell migration and invasion (18–20). Our previous studydemonstrates that MLK3 is essential for breast cancer metastasis(19). Mechanistically, we found that MLK3 silencing preventsfocal adhesion turnover, and inhibits migration, by modulatingJNK-paxillin signaling in triple-negative breast cancer. Baseduponthese findings, and the established role for DOCK180 in GBMinvasion, we hypothesized that DOCK180 may signal throughMLK3 in GBM cell migration and invasion.

Herein, we provide evidence that EGFR activates theMLK3-JNKsignaling axis to drive GBM cell migration. We demonstrate thatDOCK180 activates MLK3-JNK signaling in a RAC1-dependentmanner and provide evidence that DOCK180 forms a complexwithMLK3. TheMLK inhibitor CEP-1347blocks dissemination ofGBM cells from three-dimensional (3D) tumor spheroids. Takentogether, these data suggest that an EGFR–DOCK180–RAC1–MLK3–JNK signaling axis promotes glioblastoma cell migrationand invasion, and that MLK inhibitors could be an effectivetherapeutic strategy to treat GBM.

Materials and MethodsCell lines and reagents

Human GBM cell lines U87MG, LN229, and LN18 cells werepurchased fromATCC, and authenticated by STR and amelogeninprofiling at the time of experiments. HEK-293T cell line was fromATCC. Short-term cell cultures from the EGFRvIII-positive PDXline GBM6were generated as previously described (21). U87MG-EGFRvIII cells were a gift from Dr. Frank Furnari (UCSD, SanDiego, CA). NHA cells (22) were provided byDr. Shi-Yuan Cheng(Northwestern University, Chicago, IL) with permission of Dr.Russell Pieper (UCSF, San Francisco, CA). All cell lines werecultured in DMEM medium containing 10% FBS and 1% peni-cillin–streptomycin. Rabbit anti-JNK (C-17), ERK (C-16), andp38 (C-20); and mouse anti-DOCK 180 (H-4), and Myc (9E10)antibodies were from Santa Cruz Biotechnology. Goat anti-ELMO1 was from Abcam. Rabbit anti-MLK3 antibody was fromEpitomics or homemade (23). Mouse anti-p-JNK (4668; G9), p-ERK (E10) and p-p38 (28B10) were from Cell Signaling Tech-nologies. Mouse anti-Flag and actin; and rabbit anti-HA antibodywere from Sigma. Secondary antibodies IRDye800 (mouse) orIRDye680 (rabbit) were from LI-COR Biosciences. Alexa Fluorantibodies (mouse or rabbit) were from Invitrogen. CEP-1347was generously provided by Cephalon, Inc., a wholly ownedsubsidiary of Teva Pharmaceuticals. SP600125was obtained from

Calbiochem. Matrigel was from Becton Dickinson. PureCol(bovine collagen type I)was purchased fromAdvancedBiomatrix.

Plasmid transfection and RNA interferenceThe plasmid pCXN2-Flag-DOCK180 was from Dr. M. Mat-

suda (Kyoto University, Kyoto, Japan); plasmids, pCG-Myc-DOCK180 and pCG-myc-ELMO1, were from Dr. J. Skowronski,(Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Racexpression plasmids, pcDNA-HA-RAC1, pcDNA-HA-Rac-T17N,and pcDNA-Myc-RAC1-G12V, were acquired from the GuthriecDNA Resource Center (Bloomsberg, PA). The pRK- Flag-MLK3construct has been previously described (14). HEK-293T,U87MG, LN229, and LN18 cells were transfected using Lipo-fectamine 2000 according to the manufacturer's instructions(Invitrogen). MLK3 siRNA (19) and control siRNA were fromDharmacon. DOCK180 siRNA (sc-35207) was from Santa CruzBiotechnology (11). Forty-eight hours posttransfection, cellswere subjected to migration assays or lysates were harvested forimmunoblotting.

Preparation of cellular lysates, immunoblotting, andimmunoprecipitations

Cells were lysed as previously described (24). Total cellularlysateswere kept on ice for 10minutes, sonicated for 1minute andcleared by centrifugation at 14,000 rpm for 15 minutes. Proteinconcentration of the cleared supernatant was determined by theBradford assay (Bio-rad). Proteins in the cleared cellular lysateswere resolved by SDS-PAGE and transferred to Immobilon-FLtransfer membranes (Millipore). Membranes were blocked inOdyssey blocking buffer (LI-COR Biosciences) for 30 minutes,followed by incubation with primary antibodies overnight at 4�Cand then incubation with secondary antibodies at room temper-ature for 1 hour. After washing in TBST buffer (50 mmol/L Tris,150mmol/L NaCl, 0.05% Tween-20, pH 7.4) three times, immu-noblots were developed by chemiluminescence with X-ray film orvisualized by fluorescence using the LI-COR Odyssey infraredscanner (LI-COR Biosciences).

For co-immunoprecipitations, cleared cellular lysates (800 mg)were incubated with the appropriate antibodies at 4�C overnight,followed by incubation with 20 mL protein A-agarose beads(Invitrogen) for an additional 2–4 hours. For fragment co-IPs,20 mL protein A-agarose beads were preincubated with 1 mL ofMyc-Tag antibody for 30 minutes at room temperature. Beadswere washed once in lysis buffer, and incubated overnight with800 mg of lysate. After washing with HNTG buffer (20 mmol/LHEPES (pH 7.5), 150 mmol/L NaCl, 0.1% (w/v) Triton X-100,10% (w/v) glycerol) 3 times and PBS once, beads were pelletedand dissolved in boiling 1.5� SDS sample buffer and subjected toimmunoblotting using appropriate antibodies. All experimentswere repeated at least 3 times.

Proximity ligation assayThe proximity ligation assay was performed as described in the

manufacturer's protocol (Sigma) utilizing DOCK180 (sc-514080) and MLK3 (homemade) antibodies. Antibody labelingwas performed with the DUO92004 (anti-mouse) andDUO92002 (anti-rabbit) kits fromSigma. TheDUO92008 (TexasRed) kit from Sigma was used for detection. Experiments wereperformed in 8-well chamber slides (Corning), with 40 mL totalvolume per well. Experiments with isotype control antibodyserved as a negative control. Following detection, slides were

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mounted in mounting media containing DAPI and imaged usingan EVOS FLmicroscope (Thermo Fisher Scientific) with Texas RedandDAPIfilter cubes. Imageswere acquired at 40�magnification.Three independent experiments were performed.

Transwell migration assayTranswell migration assays were performed as previously

described using Boyden chambers (8-mm pore size, CorningCostar). GBM cells were serum-deprived overnight, dissociatedwith trypsin-EDTA, and resuspended as single cells in serum-free medium. Cells (5 � 104 in 400 mL) in serum-free DMEMmedium were introduced into the top chamber. The bottomchamber contained DMEM medium, supplemented with 5%FBS as the chemoattractant. Cells were allowed to migrate for 6hours, fixed, and then stained with crystal violet. After removalof nonmigratory cells from the top chamber with a cottonswab, migrated cells on the bottom of the Transwell membranewere counted in five random fields under a light microscope(magnification, 100�). All Transwell experiments were repeat-ed at least three times.

Wound-healing assayConfluent monolayers of LN229 cells were scratched using a

200 mL pipette tip. Floating cells were removed and mitomycin C(1 mg/mL) was added to fresh medium to inhibit proliferation.Wound recovery was monitored by phase contrast microscopyover time. Wound width was quantified with ImageJ software.Percentage closure of the wound was expressed as previouslydescribed (24). Three independent experiments were performed,each in triplicate wells.

Three-dimensional Matrigel cultureLN229 cells were dissociated with trypsin and seeded as

single cells on solidified Matrigel in triplicate wells of the 8-well chamber slide (Falcon) and covered by overlay medium(DMEM supplemented with 10% FBS, 1% streptomycin/pen-icillin and 3% Matrigel). Two days after seeding, the cells weretreated with vehicle (DMSO) or CEP-1347 (400 nmol/L);culture medium was replaced with fresh medium containingvehicle or CEP-1347 every 2-3 days for 21 days. Three-dimen-sional structures were stained with 1 mg/mL 40,6-diamidino-2-phenylindole (DAPI) for 15 minutes and visualized using anOlympus Fluoview confocal microscope in the MSU Center forAdvanced Microscopy. Three independent experiments wereperformed. Over 100 structures were imaged and analyzed foreach experimental condition.

Three-dimensional invasion assayTumor spheroids were generated using the hanging dropmeth-

od. Briefly, 20,000 cells in 20 mLmediumwere seeded as drops ona 60-mm dish. After inversion of the dish, multicellular spheroidswere allowed to form for 16–24 hours. Spheroids were thentransferred to a 0.75% agar-coated plate and cultured in completemedium for 24–72 hours. Spheroids (one per well) were thenembedded in Collagen type I matrix (2.4mg/mL) in FBS-contain-ing medium or in serum-free medium � 100 ng/mL EGF in 96-well plates and allowed todisseminate. Spheroidswere imagedbyphase contrast immediately after embedding in collagen (t ¼ 0);and at end point, samples were incubated with the vital stainCalcein-AM (5 mmol/L) for 30 minutes, images were acquiredusing a fluorescence microscope (Nikon Eclipse TE2000-U), andextent of invasion was quantified by counting the number of

disseminated cells from each spheroid. All 3D collagen invasionassays were repeated at least 3 times, with at least 3 spheroidsimaged per biological replicate.

Statistical analysisResults are expressed as the mean � SEM, and at least three

independent experiments were performed unless otherwise not-ed. Unless indicated otherwise, statistical comparisons are basedon Student t test (for 2 conditions) or ANOVA (for 3 or moreconditions) and P < 0.05 is considered statistically significant.

ResultsMLK3 is required for glioblastoma cell migration

MLK3 expression was detected by immunoblotting in apanel of human GBM cell lines, as well as in normal humanastrocytes (NHA). As shown in Fig. 1A, multiple GBM celllines express MLK3. We selected two of these, LN18 andLN229, for interrogation of the role of MLK3 in migration.In a short-term (6 hours) transwell migration assays, MLK3silencing in both cell lines reduces migration by approximate-ly 60% (Fig. 1B and C).

MLK inhibition reduces invasion of aggressive GBMcells in a 3D matrix

Although gliomas rarely metastasize to distant organs, theyreadily invade and disseminate into surrounding brain tissue,making complete surgical resection challenging and contributingto disease recurrence. To better model GBM dissemination, weperformed 3D assays to assess growth and invasion into extra-cellular matrices (ECM). Matrigel contains laminin and collagenIV, key ECM components found along the brain vasculature (25).When plated as single cells in 3D Matrigel for 21 days, LN229glioblastoma cells form invasive spheroid structures with numer-ous protrusions (Fig. 1D, top left image) and disseminated cells(Fig. 1D, bottom left image), whereas continuous treatment withthe MLK inhibitor CEP-1347 results in the formation of smoothspherical structures lacking invasive features and disseminatedcells (Fig. 1D, right). The vehicle-control structures are larger thanthe structures that formed in the presence of CEP-1347, suggestingthat the MLK inhibitor impacts proliferation as well as invasion.Consistent with previous findings that CEP-1347 treatment leadsto cell-cycle arrest in some breast cancer cells (26), we find thatCEP-1347 reduces the viability of several GBM cell lines in two-dimensional (2D) culture (Supplementary Fig. S1). To betterseparate the effects of CEP-1347 on migration from its effects oncell viability, we utilized short-term 3D invasion assays of pre-formed GBM spheroids in collagen. U87MG cells, selected due totheir efficient formation of tumor spheroids by the hanging dropmethod, were transplanted into collagen matrices and allowed todisseminate for 72 hours. U87MG spheroids show extensiveinvasion and dissemination (Fig. 1E, left), which is effectivelyblocked by treatment with CEP-1347 (Fig. 1E, right).

MLK and JNK inhibitors block EGF-inducedmigration and invasion

EGFR signaling is aberrantly elevated in a majority of GBMtumors, and contributes to cell migration. Treatment with EGFincreased migration by 1.75-fold in LN229 cells in a woundhealing assay; and this migration was blocked by cotreatmentwith CEP-1347 (Fig. 2A). Likewise, in a 3D invasion assay,

EGFR and DOCK180 Signal via MLK3 in GBM Invasion

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Figure 1.

Silencing or inhibition of MLK3 impairs migration and invasion of aggressive GBM cells. A, Human glioblastoma cell lines or immortalized NHA cells were lysed, andrelativeMLK3 levelswere assessedby immunoblotting using anMLK3 antibody.b-Actin serves as a loading control.B, LN18 cells and (C) LN229 cellswere transfectedwith control or MLK3 siRNA (40 nmol/L) for 24 hours, followed by serumdeprivation overnight. Transwellmigration assayswere performed as described inMaterialsand Methods. Migrated cells were counted in 5 random fields and normalized to cell number in the control group (100%). Inset immunoblot images showMLK3 knockdown efficiency with b-actin serving as a loading control. D, LN229 cells (5,000) were seeded in 3D Matrigel culture as described in Materials andMethods. After 2 days, the cultureswere treatedwith DMSOor CEP-1347 (400 nmol/L). Fresh growthmedium containing DMSOor CEP-1347was replaced every 2–3days for 21 days, and, after staining with DAPI, representative bright field (10� magnification; top) and confocal DAPI (20� magnification; bottom) images wereobtained. Scale bar, 100mm. Total number of invasive protrusions from the largest cross section of each structurewas quantified. Statistical analyseswere performedusing a two-tailed t test and statistical significance is indicated as ����P < 0.0001. E, 3D collagen invasion assays were performed using U87MG cells. Briefly,tumor spheroids generated through the hanging drop method were embedded into 3D collagen and allowed to disseminate in serum-free medium � 400 nmol/LCEP-1347 for 72 hours (n > 10 spheroids per group were analyzed). Representative images of Calcein AM–stained structures are shown. Bright field images of theidentical spheroid taken immediately after seeding (t ¼ 0) are shown in inset. Scale bar, 50 mm; magnification, 10�. Fluorescence images were acquired and thenumber of invaded cells per spheroid was quantified as the number of cells that had dissociated from the original spheroid, which is outlined inwhite. Statistics wereperformed using a two-tailed t test and statistical significance is indicated as ���� , P < 0.0001. Number of experimental replicates is described in Materials andMethods.

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treatment with EGF promoted invasion of U87MG cells, whichwas blocked by CEP-1347 as well as by the JNK inhibitorSP600125 (Fig. 2B). The overall size of spheroids did not appre-ciably change over the 24-hour period of treatment (data notshown). These findings are consistent with the idea of an EGFR–MLK–JNK signaling axis drivingGBMcellmigration and invasion.

MLK3 silencing and MLK inhibition blockEGFR-mediated JNK activation

As our data demonstrate that MLK and JNK inhibition blocksEGF-induced migration and invasion, we wanted to determinewhether MLK3 is required for EGF signaling to other MAPKs. EGFactivates JNK, p38, and ERK in LN18, LN229, and U87MG GBMcell lines (Fig. 3A). CEP-1347 treatment blocks EGF-induced JNKactivation, but not p38 or ERK activation. This suggests that MLKssignal primarily to JNK to regulate EGF-induced GBM cell migra-tion. CEP-1347 can inhibit multipleMLKs. To determine whetherMLK3, specifically, is required for JNK activation, MLK3 wassilenced in LN18, LN229, and U87MG GBM cells with siRNA.Reduction of MLK3 protein levels reduces EGF-induced JNK

activation (Fig. 3B), indicating thatMLK3 is required for signalingfrom EGF to JNK.

Because DOCK180 is a RAC1-specific GEF that drives gliomaprogression (11), and because Rac can activate MLK3 signalingto JNK (12, 17), we examined whether DOCK180 silencingcould affect JNK signaling in GBM cells. As shown in Fig. 3B,reduction of DOCK180 levels using siRNA reduces EGF-induced JNK activation in multiple GBM cell lines, parallelingthe effects observed upon MLK3 silencing. Thus, DOCK180 andMLK3 are both required for EGF-induced JNK activation. Thesedata, taken together, support the existence of an EGF–EGFR–DOCK180–MLK3–JNK signaling axis that drives invasion inGBM cells. Although CEP-1347 blocks EGF-induced JNK acti-vation in the context of wild-type EGFR, a substantial portionof GBMs harbor the constitutively active EGFRvIII variant. Totest whether CEP-1347 blocks signaling through EGFRvIII, weutilized U87MG cells stably expressing EGFRvIII. These cellsshow constitutive EGFRvIII activation, as demonstrated byimmunoblotting with a phospho-EGFR antibody, as well aselevated JNK and p38 activity, but similar ERK activity, when

Figure 2.

EGF-induced migration and invasionare blocked by inhibition of MLK3 andJNK. A, A wound was formed on aconfluent monolayer of LN229, andallowed to close for 24 hours in serum-freemedium� 100 ng/mL EGF,� 400nmol/L CEP-1347, as indicated.Wound width was quantified usingImageJ and is represented aspercentage wound closure relative tocontrol. Dotted lines indicateapproximate wound width at t ¼ 0.B, Preformed individual U87MGspheroids were seeded into a collagenmatrix (see Materials and Methods)�EGF (100 ng/mL), � CEP-1347(400 nmol/L) or the JNK inhibitorSP600125 (5 mmol/L), as indicated, inserum-free medium. After 24 hours,structures were stained with Calcein-AM. Epifluorescence images wereacquired and, for quantitation ofdissociated cells, brightness andcontrast were adjusted to reveal themaximal number of cells that haddisseminated from spheroid att¼ 24 hours. The total number of cellsthat had invaded away from spheroidwas quantified from at least 19spheroids per treatment group. Scalebar, 50 mm; magnification, 10�.Statistics were performed usingANOVA with a Dunn's multiplecomparisons test and statisticalsignificance compared with control isindicated as � , P < 0.05; �� , P < 0.01.Three or more biological replicateswere performed for each experiment.

EGFR and DOCK180 Signal via MLK3 in GBM Invasion

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compared with parental U87MG cells (Fig. 3C). CEP-1347blocks EGFRvIII-induced JNK activation, but has little effecton p38 or ERK activation. Collectively, these data suggestthat both wild-type EGFR and the oncogenic EGFRvIII promoteJNK activation through MLKs. Taken together with the effectsof SP600125 on EGF-induced invasion (Fig. 2B), these dataare consistent with the notion that both wild-type and onco-genic EGFR promote GBM invasion through an MLK–JNKsignaling axis.

DOCK180/ELMO1/Rac activate MLK3–JNK signalingAs DOCK180 and MLK3 are both required for EGFR-mediated

JNK activation in GBM cells (Fig. 3B), we hypothesized thatDOCK180, and its putative cofactor ELMO1, could activateMLK3. To determine whether DOCK180/ELMO1 can induceMLK3 activation, DOCK180, ELMO1, and MLK3 were co-expressed in HEK-293T cells and MLK3 and JNK activities wereassessed by immunoblotting with specific phospho-antibodies.As shown in Fig. 4A,MLK3, expressed alone, is only weakly active.Co-expression of MLK3 with either DOCK180 or ELMO1, weaklyactivates MLK3 and JNK. Co-expression of both DOCK180 andELMO1 with MLK3 robustly activates both MLK3 and JNK (Fig.4A). ELMO1 overexpression increases DOCK180 protein levels,presumably due to stabilization of DOCK180 (11), which likelyexplains the robust increase in MLK3 and JNK activation whenDOCK180 and ELMO1 are co-expressed. These results demon-strate that the Rac GEF, DOCK180, and its cofactor ELMO1 canactivate MLK3.

To confirm that DOCK180/ELMO1-induced activation ofMLK3-JNK signaling is dependent on RAC1, we used a dom-inant negative approach. Consistent with data presented in Fig.

4A, co-expression of DOCK180 and ELMO1 with MLK3 acti-vates both MLK3 and JNK. Overexpression of wild-type RAC1(RAC1WT) also activates MLK3 and JNK in cells overexpressingDOCK180 and ELMO1. However, expression of the dominantnegative (DN) variant of RAC1 (RAC1T17N) diminishesDOCK180 and ELMO1-induced activation of MLK3 and JNK(Fig. 4B) consistent with a requirement for Rac1 in DOCK180/ELMO1-induced MLK3 and JNK activation. Finally, to confirmthat RAC1 is able to activate MLK3, a constitutively activevariant of RAC1 (RAC1G12V) and low levels of MLK3 wereco-expressed in HEK-293T cells. Expressing low levels of MLK3is necessary to minimize MLK3 homodimerization-inducedautoactivation. Co-expression of RAC1G12V potently activatesMLK3 and JNK (Fig. 4C). Taken together, these data provideevidence that DOCK180/ELMO1 activates MLK3 and JNK in aRAC1-dependent manner.

Interaction of DOCK180 and MLK3Many GEFs form larger multiprotein complexes, which can

include GTPase effector proteins (27). We therefore assessedwhether DOCK180 is able to interact with MLK3. Under con-ditions of ectopic expression, DOCK180 and ELMO1 alone, orin combination (Fig. 4D), are able to co-immunoprecipitatewith MLK3, supporting the notion that they may form a largerprotein complex in cells. Both DOCK180 and MLK3 are large,multi-domain proteins. To better understand the nature of thisinteraction, we carried out experiments to determine whichregions of MLK3 are critical for interaction with DOCK180. Theability of HA-tagged fragments of MLK3 to co-immunoprecip-itate with Myc-tagged DOCK180 was assessed. The N-terminalhalf (1-399) and the C-terminal region (592-847) of MLK3 fail

Figure 3.

MLK3 silencing orMLK inhibition blocks EGF-induced JNKactivation. A, LN18, LN229, and U87MG cells were serum-deprived overnight, pretreated �400 nmol/L CEP-1347for 1 hour followed by addition of 100 ng/mL EGF for 15minutes, and then lysed. Immunoblots of cellular lysateswith the indicated antibodies are shown. B, LN18, LN229,andU87MGcellswere transfectedwith 40nmol/L controlsiRNA (-), or siRNA targeting MLK3 (M) or DOCK180 (D).Cells were serum-deprived for 24 hours, andsubsequently treated with 100 ng/mL EGF for 15 minutes.Cellular lysates were analyzed by immunoblotting withthe indicated antibodies. C, Parental U87MG or U87MG-EGFRvIII cells were serum-deprived overnight, treated�400 nmol/L CEP-1347 for 1 hour, as indicated, and lysed.Cellular lysates were analyzed by immunoblotting usingthe indicated antibodies. Three or more biologicalreplicates were performed for each experiment.

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to co-immunoprecipitate with DOCK180, whereas the centralportion (400-591) of MLK3 efficiently co-immunoprecipitateswith DOCK180 (Fig. 4E). Interestingly, this fragment containsthe MLK3 CRIB motif as well as the critical Pro residue thatmediates MLK3 autoinhibition. We were unable to co-immu-

noprecipitate endogenous MLK3 and DOCK180, perhaps dueto lack of sensitivity or a transient nature of the interaction. Toprovide evidence for the existence of an endogenous complexcontaining DOCK180 and MLK3, we utilized the more sensitiveproximity ligation assay. As shown in Fig. 4F, a proximity

Figure 4.

DOCK180/ELMO1 promote activationof MLK3 and JNK. A, HEK-293T cellswere transiently transfected withexpression constructs for Flag-MLK3,Flag-DOCK180 and Myc-ELMO1, asindicated, for 24–48 hours. Cellularlysates were analyzed byimmunoblotting with the indicatedantibodies. B, HEK-293T cells weretransiently transfected with theindicated combination of epitope-tagged expression constructs for HA-RAC1 wild-type (WT), dominantnegative mutant HA-RAC1 (N17), Flag-MLK3, Flag-DOCK180 and Myc-ELMO1for 24–48 hours. Total cellular lysateswere subjected to immunoblottinganalysis using the appropriateantibodies. b-Actin was used as aloading control. C, HEK-293T cellswere seeded in 6-well plates andtransfected with Flag-MLK3 (10 ng) orMyc-RAC1G12V (2 mg) for 24 hours.Cellular lysates were probed with theindicated antibodies. D, HEK-293Tcells were transiently transfectedwith the constructs expressingFlag-MLK3, Myc-DOCK180 and Myc-ELMO1 for 24–48 hours. Equal portionsof the total cellular lysates wereimmunoprecipitated using Flag-conjugated agarose beads. Theimmunoprecipitates (IP) wereanalyzed by immunoblotting (IB) withthe indicated antibodies. E, HEK-293Tcells were transiently transfected withexpression plasmids encodingMyc-DOCK180, and HA-MLK3fragments. Co-IP was performed witha Myc-tag antibody, and the lysateswere analyzed by immunoblottingwith the indicated antibodies. F,Proximity ligation assay wasperformed as described in Materialsand Methods using LN18 cells witheither isotype control antibodies(top), or DOCK180 þ MLK3 antibody(bottom). Scale bar, 10 mm. Three ormore biological replicates wereperformed for each experiment.

EGFR and DOCK180 Signal via MLK3 in GBM Invasion

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ligation assay demonstrates that endogenous DOCK180 andMLK3 exist in a protein complex, or in close proximity, in situ inLN18 cells (Fig. 4F). Taken together, these data suggest thatDOCK180 and MLK3 form a protein complex in cells.

Overexpression ofMLK3 rescues themigratory defect caused byDOCK180 silencing

As DOCK180 drives the migration of GBM cells (11), andbecause our data indicate that DOCK180 can activate MLK3, wetested whether MLK3 overexpression could rescue migration inDOCK180-silenced GBM cells. DOCK180 knockdown in LN229(Fig. 5A) orU87MG(Fig. 5B) reducesmigration by approximately50%. In both cell lines, MLK3 overexpression rescues the migra-tory defect. It is important to note that overexpression of highlevels of MLK3 induces its auto-activation, irrespective ofupstream stimulus.

CEP-1347 inhibits invasion of EGFRvIII-positive GBMCells in a 3D matrix

The constitutively active EGFRvIII mutant is present in a largeportion of GBM tumors and is considered a major driver ofGBM progression (3). Using existing human GBM cell lines, wefound that treatment with CEP-1347 or MLK3 siRNA inhibitedEGF-induced migration and signaling (Figs. 2 and 3). AsEGFRvIII-positive tumors lose EGFRvIII expression upon pas-saging in long-term culture, immortalized EGFRvIII-positiveGBM cell lines are lacking. Therefore, we made use of U87MGcells engineered to stably express EGFRvIII (28) to demonstratethat MLK inhibition is sufficient to block JNK activation inEGFRvIII-expressing U87MG (Fig. 3C). To test the effects of anMLK inhibitor in a more clinically relevant model, a GBMpatient-derived xenograft (PDX) model was used, as serialpassage of PDX lines in mice has been shown to more faithfullyrecapitulate the human tumors from which they are derived(21). GBM6 was selected as the model for this study, as itexpresses the oncogenic EGFRvIII. The GBM6 line was propa-gated in immunodeficient mice, subjected to short-term culturein vitro, and spheroids were formed and embedded in 3Dcollagen. As demonstrated in Fig. 5C, CEP-1347 blocks the3D invasion of GBM6 spheroids. These data suggest that MLKinhibitors may be effective against EGFRvIII-mutant GBMtumors, though, more detailed in vivo studies are required toconclusively determine efficacy.

DiscussionGBM tumors are highly invasive, and GBM cells readily invade

into the surrounding brain tissue. Identifying the molecularmechanisms which control invasion may yield new therapeutictargets to treat GBM. In this study, we identify an important rolefor MLK3 in GBM migration and invasion. Our signaling experi-ments suggest that an EGFR-DOCK180/ELMO1-RAC1-MLK3-JNK signaling axis controls GBM migration and invasion (Fig.5D). Finally, we provide evidence that DOCK180 andMLK3 forma protein complex in cells.

GBM tumors are typically classified into one of four sub-types, based upon gene expression profiling (3). The classicalsubtype of GBM is characterized by hyperactivation of EGFRdue either to amplification of the wild-type EGFR gene, orthrough acquisition of the EGFRvIII mutation, which is ligand-independent and constitutively active. Thus, EGFR has emerged

as an important therapeutic target in GBM (29, 30). Despite theprevalence of EGFR alterations in GBM and their associationwith poor prognosis (31), EGFR-targeted therapies have beendisappointing in the clinical realm (29). Multiple mechanismsof EGFR inhibitor resistance in GBMs expressing wild-typeEGFR as well as EGFRvIII, have been uncovered (6). Forinstance, the "type I" EGFR kinase inhibitor, erlotinib, haspoor efficacy in GBMs expressing EGFRvIII, likely due to rapiddissociation of the drug from the mutant EGFRvIII (32, 33). Inaddition, EGFR inhibitor treatment can lead to elimination ofEGFRvIII-expressing clones, which later reemerge upon cessa-tion of inhibitor treatment (34). One enticing, alternativestrategy to overcome this therapeutic challenge of GBM tumorsdriven by EGFR activation is to target key signaling nodesdownstream of EGFR.

EGFR activates multiple, oncogenic signaling pathways inGBM cells, including the MAPK and PI3K/Akt pathways. EGF hasbeen previously demonstrated to activate JNK in GBM cells (35);however, the intermediary proteins required for EGF-induced JNKactivation are not well-defined. In this study, we demonstrate thatDOCK180 and MLK3 are required for EGF-induced JNK activa-tion. The connection between DOCK180 and JNK has beenpreviously demonstrated in HEK 293T cells and in Drosophilasystems (36, 37); however, for the first time, we demonstrate thatthis is mediated by MLK3 in the context of glioblastoma. Fur-thermore, we demonstrate that this signaling pathway promotesmigration and invasion of GBM cells. U87MG cells stably expres-sing the EGFRvIII variant display high basal JNK activity (38),which we now show is reduced by an MLK inhibitor (Fig. 3C).More recently, JNK activity has been demonstrated to be impor-tant in GBM "stem cell" survival (39, 40). Taken together, theMLK-JNK signaling pathway may be a promising therapeutictarget for GBM.

In this study, we provide evidence for complex formationbetween DOCK180 and MLK3, both in co-immunoprecipita-tion experiments with ectopically expressed proteins as well asin proximity ligation assays with endogenous proteins in GBMcells. It is uncertain whether interaction between DOCK180 andMLK3 is direct, or whether it is mediated by another protein(s).Both DOCK180 and MLK3 are large, multi-domain proteins. Tobetter define the nature of the DOCK180–MLK3 interaction, weassessed the ability of DOCK180 to interact with expressedfragments of MLK3. The N-terminal fragment of MLK3, whichcontains the SH3 and kinase domains, as well as the C-terminalproline-rich region fail to interact with DOCK180. The fragmentof MLK3 containing amino acids 400-591, which is comprisedof the leucine zipper, the critical proline residue for SH3-mediated autoinhibition, as well as the CRIB motif, is compe-tent to interact with DOCK180 (Fig. 4E). It is possible thatDOCK1800s association with MLK3 could facilitate the associ-ation of DOCK180-activated Rac1 with MLK3. Comprehensivebiochemical studies will be required to fully determine thenature and constituents of the DOCK180–MLK3 proteincomplex.

In summary, our study reveals the existence of an EGFR–DOCK180/ELMO1–RAC1–MLK3–JNK signaling axis which pro-motes glioblastoma cell migration and invasion. We propose thatinhibitors blocking this pathway, in combination with existingtreatment modalities, may potentially improve outcomes inpatients with glioblastoma. Both DOCK180 and ELMO1 areoverexpressed in the invasive front of human glioblastomas and

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Figure 5.

Overexpression of MLK3 rescues the migratory defect caused by DOCK180 silencing and MLK inhibitor blocks 3D invasion of glioma spheroids. A, LN229 (B) U87MGcellswere transiently transfectedwith control siRNAorDOCK180 siRNA for 24 hours, followedby transfection of a Flag-MLK3 expression construct, or control vector,as indicated for an additional 16 hours. Cells (5 � 104) were subjected to a Transwell migration assay for 6 hours. Migrated cells were quantified and normalizedto the control group (100%). C, GBM6 spheroids were generated, transplanted into 3D collagen culture, and allowed to disseminate � 400 nmol/L CEP-1347for 72 hours. Phase contrast images were acquired immediately after seeding the spheroids into the collagen culture, and Calcein AM–stained spheroids wereimaged after 72 hours. Shown are representative images from 3 biological replicates. Scale bar, 50 mm; magnification, 10�. D, Proposed model of EGFR–DOCK180–RAC1–M-LK3–JNK signaling axis that leads to invasion of GBM cells. Statistics were performed using an ANOVA followed by Dunn multiple comparisons test andstatistical significance is indicated as �� P < 0.01. Three or more biological replicates were performed for each experiment.

EGFR and DOCK180 Signal via MLK3 in GBM Invasion

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are believed to be promising targets for invasive glioblastoma(11). However, GEFs and GTPases have thus far been challengingtherapeutic targets. Because MLK3 is a readily targetable Rac/Cdc42 effector protein, MLK inhibitors could be useful therapeu-tics in an array of Rac/Cdc42-driven tumors. In line with theidea of targeting MLKs in GBM, MLK4 was recently demonstratedto be important in GBM stem cell survival (41). CEP-1347progressed to phase II/III clinical trials for the treatment ofParkinson's disease (42). Given its demonstrated safety inhumans (43), it is possible that CEP-1347or otherMLK inhibitorscould be readily translated into clinical trials for the treatment ofglioblastoma.

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

Authors' ContributionsConception and design: S.A. Misek, J. Chen, C. Rattanasinchai, K.A. GalloDevelopment of methodology: S.A. Misek, J. Chen, L. Schroeder,C. Rattanasinchai

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.A.Misek, L. Schroeder, C. Rattanasinchai, A. Sample,J.N. SarkariaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.A. Misek, J. Chen, L. Schroeder, C. Rattanasinchai,K.A. GalloWriting, review, and/or revision of the manuscript: S.A. Misek, J. Chen,L. Schroeder, C. Rattanasinchai, J.N. Sarkaria, K.A. GalloAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K.A. GalloStudy supervision: K.A. Gallo

Grant SupportThis work is supported by an MSU CTSI grant (K.A. Gallo).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 20, 2016; revisedMarch 14, 2017; acceptedMay 5, 2017;published OnlineFirst May 9, 2017.

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