cpeb1 regulates the expression of mtdh/aeg-1 and ...fabp7 nm_001446.3 gliomas chakravarti et al....

Chromatin, Gene, and RNA Regulation

CPEB1 Regulates the Expression of MTDH/AEG-1 andGlioblastoma Cell Migration

Dawn M. Kochanek and David G. Wells

AbstractCytoplasmic polyadenylation element-binding protein 1 (CPEB1) is an mRNA-binding protein present in both

neurons and glia. CPEB1 is capable of both repressing mRNA translation and activating it depending upon itsphosphorylation state. CPEB1-boundmRNAs are held in translational dormancy until CPEB1 is phosphorylated,leading to the cytoplasmic polyadenylation of the bound mRNA that triggers translation. Here, we show thatCPEB1 can bind to and regulate translation of the mRNA-encoding metadherin (MTDH, also known as AEG-1and Lyric) in the rat glioblastoma cell lineCNS1.MTDH/AEG-1 is being revealed as a critical signalingmolecule intumor progression, playing roles in invasion, metastasis, and chemoresistance. By using a mutant of CPEB1 thatcannot be phosphorylated (thereby holding target mRNAs in translational arrest), we show that inhibiting CPEB1-mediated translation blocks MTDH/AEG-1 expression in vitro and inhibits glioblastomas tumor growth in vivo.CPEB1-mediated translation is likely to impact several signaling pathways thatmaypromote tumor progression, butwe present evidence suggesting a role in directed cell migration in glioblastoma cells. In addition, reporter mRNAcontaining CPEB1-binding sites is transported to the leading edge of migrating cells and translated, whereas thesamemRNAwith point mutations in the binding sites is synthesized perinuclearly. Our findings show that CPEB1is hyperactive in rat glioblastoma cells and is regulating an important cohort ofmRNAswhose increased translation isfueling the progression of tumor proliferation and dispersal in the brain. Thus, targeting CPEB1-mediated mRNAtranslation might be a sound therapeutic approach. Mol Cancer Res; 11(2); 149–60. �2012 AACR.

IntroductionAltered protein synthesis is a hallmark of cancer withmany

genes having documented aberrant transcription rates.However, we were interested in what was happening to theposttranscriptional mechanisms controlling mRNA transla-tion in transformed cells, specifically glioblastomas. ThemRNA-binding protein cytoplasmic polyadenylation ele-ment-binding protein 1 (CPEB1) is present in both neuronsand astrocytes, where it regulates the transport and transla-tion of a distinct set of mRNAs (1–3). CPEB1 binds tomRNAs that contain a cis-element in their 30-untranslatedregion (UTR) called a cytoplasmic polyadenylation element(CPE; ref. 4). Once bound to the CPE, CPEB1 initiallysilences mRNA translation but can activate translationfollowing phosphorylation (4, 5). In astrocytes, CPEB1 isphosphorylated by Aurora kinase A and confirmed CPEB1

targets include b-catenin and cyclin B1 mRNA (3, 6), bothproteins and the kinase have been implicated in cancer. Todetermine a functional role for CPEB1 in glioblastoma, weused amutant version of CPEB1 thatmaintains the ability tobind specific mRNA, but does not contain the phosphor-ylation site necessary for activation, thus inhibiting transla-tion of bound mRNA.The mutant CPEB1 protein we termed DN-CPEB1

(dominant negative-CPEB1) because it binds specifically toCPEB1 targets, blocking the endogenous CPEB1 frombinding, and reduces the translation of bound mRNA(1, 3, 7). Our rationale for using this approach was pred-icated on our goal of turning off CPEB1-mediated mRNAtranslation. By using siRNA, or other means to knockdownthe expression of CPEB1 protein, the result would be toliberate bound mRNA from repression, thus in essenceactivating translation of CPEB1 targets (6). If we overexpressCPEB1 protein, we cannot be sure what fraction of theexpressed CPEB1 is phosphorylated, again complicating theinterpretation as the translation of some bound mRNAwould be dormant and some activated. However, by expres-sing the phospho-mutant, we can attain the greatest certaintythat we are actually inhibiting CPEB1 function.In astrocytes, CPEB1 was first described as a regulator of

b-catenin mRNA and DN-CPEB1 expression blocked cellsfrom migrating in a traditional scratch assay in vitro (3). Leftunanswered in that study was whether DN-CPEB1 expres-sion blocked all movement or rather altered the cells ability

Authors' Affiliation: Department of Molecular, Cellular & DevelopmentalBiology, Yale University, New Haven, Connecticut

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

Corresponding Author:David G.Wells, Department of Molecular, Cellular& Developmental Biology, 260 Whitney Ave, KBT 338, Box 208103, NewHaven, CT 06520. Phone: 203-432-3481; Fax: 203-432-8999; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-12-0498

�2012 American Association for Cancer Research.

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Published OnlineFirst January 29, 2013; DOI: 10.1158/1541-7786.MCR-12-0498

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to move in a directed or progressive manner. Directional cellmigration requires cell polarity allowing for protrusion at theleading edge and retraction at the rear. Indeed, movement islargely driven by actin polymerization and acto-myosincontraction at the leading edge of a cell (8). Much of theasymmetric localization of organelles and proteins requiredfor cell migration are dependent upon microtubules (9).Perhaps surprisingly, one component that is asymmetricallylocalized and required for directional migration is mRNA (9,10). In fact, local protein synthesis may be an importantfeature in establishing cellular asymmetry during cellulardevelopment and migration (10, 11).By examining the 30-UTR of mRNAs encoding proteins

misregulated in glioblastoma, we identified a number thatcould potentially be targets for CPEB1 regulation (see Table1). Among that group, we selected MTDH/AEG-1 forfurther analysis based on 2 properties. The first was itsability to interact and activate the b-catenin pathway(12); and second, the emerging evidence that MTDH/AEG-1 may coordinate the activity of several signalingpathways all leading to the progression of tumor growthand spread in a number of different tumor types (includingglioblastoma; ref. 13–17). If CPEB1 is regulating the syn-thesis of MTDH/AEG-1, it would suggest that CPEB1 iscoordinating the synthesis of proteins that are functionallyrelated (e.g., b-catenin and MTDH/AEG-1), and thattargeting CPEB1-mediated protein synthesis might be use-ful in many types of cancer not just glioblastoma.

Materials and MethodsCell culture preparationAstrocytes were isolated as previously described (3). Brief-

ly, astrocytes were isolated from postnatal day 1mice pups of

either sex, treated with trypsin, physically dissociated, andplated at approximately 100,000 cells/mL on 60-mm tissueculture dishes, glass bottom tissue culture dishes, 6-wellplates or 18-mm glass coverslips (MatTek, Carolina Biolo-gicals). Astrocytes were maintained in growth mediumcontaining minimum essential medium (MEM) with glu-tamine (Invitrogen), 10% horse serum (Atlanta Biologicals),glucose (1 mg/mL), pyruvate (1 mmol/L), and penicillin/streptomycin (100 mg/mL). For neuron-free cultures, cellswere plated in 75-mm flasks and grown for 1 to 2 weeksbefore trypsinization and replating on tissue culture dishes.CNS-1 cells, a gift from Rick Matthews [State University ofNew York (SUNY), Upstate Medical University, Syracuse,NY], were cultured as previously described (3). Woundswere carried out by scratching the confluent monolayerswith a 0.1 to 10 mL plastic pipette tip. Human U87astrocytoma cells were cultured in growth medium contain-ing Dulbecco's modified Eagle's medium (DMEM; Invitro-gen) supplemented with 10% FBS (Invitrogen) and peni-cillin/streptomycin (100 mg/mL).

DN-CPEB1-GFP expressionCNS-1 cells were transfected using Lipofectamine 2000

(Invitrogen). Briefly, 0.5 mg of plasmid DNA was mixedwith 3 mL of transfection reagent and mixed in 200 mL ofOptimem (Invitrogen). DNA and reagent were incubatedfor up to 6 hours at room temperature before addition to theCNS-1 cells. Cells were transfected in antibiotic-free MEM(Invitrogen) for 4 hours before the transfection mixture wasreplaced with fresh growth medium. Constructs used fortransfection encoded either enhanced GFP (EGFP) or anEGFP-tagged truncated CPEB1 protein that does not con-tain the phosphorylation site needed for activation but does

Table 1. CPE-containing mRNAs mis-regulated in various stages of glioblastoma

Protein name Accession # Mis-regulated in Reference

Adenosine deaminase NM_015840.3 Grade III Chumbalkar et al. 2005Cadherin related tumor suppressor NM_005245.3 GBMs (secondary) Schwartz et al. 2005Calpactin 1 light chain NM_174650.1 GBM Hobbs et al. 2003Centrosome associated protein 350 NM_014810.2 GBMs (primary) Schwartz et al. 2005Copine1 NM_003915.5 Gliomas Chakravarti et al. 2001Cyclin B1� NM_001238.2 Gliomas Holtkamp et al. 2005, Kim et al. 2011�

Dihydropteridine reductase NM_000320.2 Grade II Schwartz et al. 2004FABP5 NM_001444.2 Grade III Hobbs et al. 2003FABP7� NM_001446.3 Gliomas Chakravarti et al. 2001, Gerstner et al. 2012�

Glutamate dehydrogenase 1 NM_005271.3 Grades l&ll Hiratsuka et al. 2003Metadherin (AEG-1)�� NM_178812.3 GBM Emdad et al. 2006Nucleolar GTP binding protein 1 NM_012341.2 GBM & Grade III Hiratsuka et al. 2003PTEN� NM_000314.4 GBM Odreman et al. 2005, Alexandrov et al. 2012�

Tropomodulin 2 NM_014548.3 Grade III Chumbalkar et al. 2005Tubulin specific chaperone A NM_004607.2 GBM Hobbs et al. 2003Tumor protein p53� NM 000546.5 GBM Odreman et al. 2005, Burns et al. 2011�

�Validated CPEB1 targets.��Validated in this manuscript.

Kochanek and Wells

Mol Cancer Res; 11(2) February 2013 Molecular Cancer Research150




contain the mRNA-binding domain (DN-CPEB1-GFP).Cloning of the DN-CPEB1-GFP construct into pEGFP-C1(BD Biosciences) has been previously described (3).

Immunoprecipitation and DN-CPEB1-GFP pull-downTotal protein lysate was isolated from CNS1 cells, or

CNS1 cells that had been transfected with DN-CPEB1-GFP, using the miRVana PARIS Kit (Life Technologies).This lysate was then mixed with rabbit anti-CPEB1 (1:100)or anti-GFP (1:100; Invitrogen) for 30 minutes before theaddition of 50mLProteinAMAGbeads (GEHealthcare) for30 minutes. Beads were pelleted, washed 3 times, and RNAwas isolated by ethanol precipitation using miRVana PARISkit. RNAwas resuspended inwater and treatedwithDNase I(50 U/mL; Ambion) for 15 minutes at 37�C followed byreverse transcription (RT) and PCR analysis to detectmRNA-encoding metadherin (MTDH) and glial fibrillaryacidic protein (GFAP) mRNAs. Primer sequences were asfollows: MTDH/AEG-1: (forward) 50-gcggcggccacctattc-30and (reverse) 50-cgggctccttcgcttttt-30; GFAP: (forward) 50-agtgcccggctggaggtg-30 and (reverse) 50-cgagggcgagggcgatgc-caccta-30.

Western blot analysisCultured cortical astrocytes or CNS1 cells were grown to

confluence in 60-mm culture dishes and lysed in a 1% SDSsolution containing 2mmol/L EDTA, 2mmol/L EGTA, 10mmol/L Tris pH 7.5, and 1mmol/L sodium orthovanadate.Following harvesting of the lysates, proteins were separatedon a 12% SDS-PAGE gel, transferred to a polyvinylidenedifluoride (PVDF) membrane (Ambion) and probed withantibodies against CPEB1 (1:1,000), phosphorylatedCPEB1 (pCPEB1; 1:1,000), metadherin (1:1,000; Milli-pore), and b-actin (1:5,000; Sigma). Blots were developedusing enhanced chemiluminescence (ECL; Pierce) and ana-lyzed for densitometry using Image J (U.S. NIH). Anti-CPEB1 and pCPEB1 antibodies were made for us byYenZym Antibodies, LLC. The CPEB1-GST used in char-acterizing our antibodies was obtained from Abnova.

ImmunocytochemistryAstrocytes or CNS1 cells were grown to confluence on 18-

mm glass coverslips and fixed in 4% paraformaldehyde. Thecells were permeabilized, then blocked in 10% horse serumin 1� PBS for 1 hour before incubation with rabbit anti-metadherin (1:50; Millipore) overnight at 4�C. Following arigorous wash, Alexa Fluor 568 (1:1,000; Invitrogen) wasadded at room temperature for 1 hour. Coverslips were thenwashed and mounted onto slides with ProLong Gold Anti-fade Reagent with 40,6-diamidino-2-phenylindole (DAPI;Invitrogen). Images were acquired with a Nikon EclipseE800 fluorescent microscope equipped with a Spot camera.Images were processed and labeled using Canvas 12 (ACDSystems of America).

Stereotaxic injectionsConfluent CNS-1 cells were transfected as described

earlier with either the EGFP or DN-CPEB1-GFP con-

structs. Twelve to 16 hours after transfection, the cells werewashed in 1� PBS, trypsinized, resuspended in growthmedium, and spun at 1,000� g for 5 minutes. The pelletedcells were resuspended in 1 mL of injection buffer (1� PBS,1 mg/mL calcium chloride, 1 mg/mL magnesium chloride,and 0.01% glucose), dissociated and brought up to a finalvolume of 10 mL. Washes were repeated 2 more times andthe cells were resuspended at a final concentration of 1.0 �105 cells per mL. Adult male Lewis rats were anesthetizedwith 90 mg/kg ketathesia/10 mg/kg xylazine and thenstereotaxically inoculated with a single injection of 1.5 �105 CNS-1 cells (1.5 mL) using a 10-mL Hamilton syringeinto the central striatum. Ten days after injection, a trans-cardial perfusion was conducted. Following the perfusion,brains were postfixed in 4% paraformaldehyde in 1� PBSfor 24 hours. All procedures were approved before imple-mentation by Yale University Institutional Animal Care andUse Committee.

Quantification of tumor spread and volumeTo quantify tumor volume, paraformaldehyde-fixed

brains were serial sectioned into 50-mm thick slices. Serialsections were crystal violet–stained and tumors were imagedunder a �1 objective using brightfield microscopy. Theimages were then aligned and stacked using ImageJ (U.S.NIH) and the resulting stacks were then subjected to3-dimensional (3D) tumor volume reconstruction (mm3)using the software Imaris (Bitplane Scientific Software). Toquantify tumor spread, the injection site was identified andthis section and another 100-mm caudal to the site wereimaged using fluorescence microscopy. Using ImageJ (U.S.NIH), the images were then divided into 10-mm medial–lateral bins, with the center of the injection site assigned bin0 to 10 mm. The number of GFP-expressing cells in eachbin was counted and expressed as a percentage of the 0 to10 mm bin to normalize for total fluorescent cells. All tumoranalysis was conducted blind to condition.

CPEB1-mCherry expressionThe full-lengthmouse CPEB1 open reading frame (ORF)

was cloned into themammalian expression vector pReceiver-M56 Expression Clone with a C-mCherry tag (GeneCo-poeia). Astrocytes and CNS1 cells were grown to 60%confluency then transfected with the CPEB1-mCherryconstruct (250 ng). At the time of transfection, cells weretreated with 200 nmol/L leptomycin B (LMB) or leftuntreated. At various time points, posttransfection/treat-ment cells were fixed, mounted, and imaged using fluores-cent microscopy.

Migration assaysAstrocytes and CNS-1 cells were cultured as described

earlier. Cells were plated on 1 mg/mL poly-D-lysine–coatedglass bottom dishes or 18-mm glass coverslips. Cells wereLipofectamine 2000 (Invitrogen) transfected with theappropriate construct, and approximately 16 hours aftertransfection, cultures were scratched and cell migration wasassessed via confocal microscopy (Zeiss LSM 510).

CPEB1-Mediated Translation Regulates Glioblastoma Growth

www.aacrjournals.org Mol Cancer Res; 11(2) February 2013 151




Quantification of directed cell migrationCNS-1 cells were transfected as described earlier with

either the DN-CPEB1-GFP or control GFP constructs, 12to 16 hours after transfection a wound was introduced.Using live cell confocal microscopy, transfected cells alongthe wound edge were located and imaged every 30 secondsfor 45 minutes. The individual trajectories of the cells werethen normalized to a starting point and tracked usingMetaMorph software (Molecular Devices).

Reporter construct generationConstructs pSL-MS2-12x and pMS2-GFPwere gifts from

Robert Singer (Albert Einstein Medical College, Bronx,NY). The CPE sequences used for the reporter were basedon the b-catenin 30-UTR, a well-characterized target ofCPEB1 (3, 7). Approximately, 250 nucleotides of the900-nucleotide b-catenin, 30-UTR was amplified frommouse cDNA as previously described (7). Constructs usedfor transfection encoded either the wild-type or mutatedCPE sequences digested out of pEGFP-C1 at the BglII andBamH1 sites (3) and subcloned in frame into theBamH1 siteof pBluescriptII KSþ (Clontech). The 30-UTR's were thendigested out of pBluescriptII KSþ at theNot1 and Xho1 sitesand cloned in frame into pcDNA3 (Invitrogen). The 12MS2-binding domains and multiple cloning site (MCS)were digested out of pSL-MS2-12x and cloned in frameinto the 30-UTR containing pcDNA3 at the BamH1 andXho1 sites. The MS2 protein, GFP, and nuclear localizationsignal (NLS) were digested out of pMS2-GFP and clonedinto pcDNA3 at theHindIII andXho1 sites. The destabilizedEGFP (dEGFP) was PCR-amplified out of pZSGreen1-DR(Clontech) using a forward primer designed to add in aKozak sequence and the Kpn1 restriction site: 50-AAAAGG-TACCATGGCCCAGTCCAAG-30. To the reverse primer,we added a farnesylation sequence and the BamH1 restric-tion site: 50-AAAAGGATCCCTAGAGAGCACACACT-TGCAGCTCATGCAGCCGGGGCCACTCTCATCAG-GAGGGTTCAGCTTAGATCTGAGTCCGGACACA-TTGATCCTAGC-30. The PCR product was purified thenligated in frame into pcDNA3 at theKpn1 and BamH1 sites.The CPE-containing and CPE-mutated 30-UTR's weredigested out of pcDNA3 (mentioned earlier) and cloned inframe into the Not1 and Xho1 sites.

Visualizing mRNA localizationCNS-1 cells were cultured on 18-mm glass coverslips

(Carolina Biologicals) and then cotransfected as describedearlier with the MS2-GFP-NLS construct and either theCPE-containing or CPE-mutated MS2 constructs. Twelveto 16 hours after transfection, the cells were fixed with 4%paraformaldehyde in PBS, mounted onto slides using Pro-long Gold with DAPI (Invitrogen), and imaged underconfocal microscopy. Quantification of mRNA expressionwas determined by counting the number of transfected cellsin 3 fields per coverslip. Fields were visualized and selectedunder �63 microscopy. Blinded to condition, each trans-fected cell in the field was visualized under brightfield andfluorescence to determine the location of the mRNA.

mRNA expression was classified as either perinuclear orexpressed throughout the cell including in the periphery.

Quantification of fluorescencePrimary astrocytes were cultured as described earlier and

transfected with the farnyslated-dEGFP construct contain-ing either the CPE-containing or CPE-mutated reporter,and 12 to 16 hours after transfection a wound was intro-duced. One to 2 hours after the wound using live confocalmicroscopy under a �63 objective, transfected cells alongthe wound edge were located and imaged, photobleached,and subsequently imaged every 30 seconds after the bleach-ing for 15 minutes. To quantify fluorescence in the peri-nuclear region and at the peripheral edge of the cell, a regionof interest in both locations was defined in the phase image.The areas were then overlaid on the fluorescent image andthe mean pixel intensity was calculated using the T-stacks,intensity versus time monitor function plug-in on MacBio-photonics ImageJ (U.S. NIH). To correct for backgroundfluorescence, pixel intensity of the same size area wasmeasured immediately adjacent to the transfected cell andthis value was subtracted.

ResultsCPEB1 regulates MTDH/AEG-1 expressionTable 1 lists proteins that have been implicated in glio-

blastoma progression that we determined to contain a CPEsequence in their 30-UTR by sequence analysis. Some ofthese have previously been verified targets of CPEB1; here,we selected to examine MTDH/AEG-1 for the reasonsoutlined in the introduction. The mRNA encodingMTDH/AEG-1 contains a conserved CPE sequence in the30-UTR that is located within 100 nucleotides of thehexanucleotide sequence, which is the cleavage and poly-adenylation signal present just 50 to the poly(A) tail in nearlyall mRNA (Fig. 1A). To determine if CPEB1 binds toMTDH/AEG-1 mRNA in CNS1 glioblastoma cells, weimmunoprecipitated CPEB1 protein under conditions thatallowed us to isolate the bound mRNA. The RNA wasisolated and underwent RT-PCR using primers specific forMTDH/AEG-1 or the non-CPE–containing mRNAencoding GFAP. MTDH/AEG-1 mRNA is only detectedin the CPEB1 immunoprecipitation, not when a nonspecificantibody is used for immunoprecipitation. The interactionof MTDH/AEG-1 mRNA is specific in that the non-CPE–containing mRNA GFAP is not bound to CPEB1 protein(Fig. 1B). To confirm this interaction, we expressed a mut-ated version of CPEB1 that contains the mRNA-bindingdomain but not the activation (phosphorylation) domaintagged with GFP (DN-CPEB1-GFP) in CNS1 cells andimmunoprecipitated using an antibody specific for GFP(Fig. 1C). Again, the MTDG/AEG-1 mRNA specificallyinteracts with the DN-CPEB1-GFP protein. This result alsoindicates that the DN-CPEB1-GFP protein is capable ofcompeting with the endogenous CPEB1 for target mRNA.MTDH/AEG-1 immunoreactivity was detected at low

levels in primary rat astrocytes in culture, but seems greatly

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enhanced in the rat astrocyte-derived glioblastoma cell lineCNS1 (Fig. 2A). CNS-1 cells have been shown to expressGFAP, S100, and importantly, CPEB1 (3, 18). Indeed,when we quantified Western blot analyses of astrocytecultures and compared them with CNS1 cultures, theMTDH/AEG-1 level is significantly higher in CNS1 cells.Similar elevation ofMTDH/AEG-1 was also observed whenwe quantified Western blot analyses of astrocytes and com-pared them with the human astrocytoma cell line, U87 (Fig.2A). Because CPEB1 stimulates the translation of boundmRNA following phosphorylation at T171, if CPEB1 werepartly responsible for this increase in MTDH/AEG-1, onewould expect the level of pCPEB1 to be greater in CNS1cells indicating active translation of bound mRNA. Wecompared the pCPEB1 state of both astrocytes and CNS1cells using a phospho-specific antibody we raised against thissite (Supplementary Fig. S1A). Although the total level ofCPEB1 protein in astrocytes and CNS1 cells is about equal,pCPEB1 levels are significantly higher in CNS1 cells (Sup-plementary Fig. S1A), suggesting increased expression ofCPEB1-mediated targets. To determine if pCPEB1 is con-tributing to the increase in MTDH/AEG-1, we expressedDN-CPEB1-GFP in both CNS1 and U87 cells. When thisnonphosphorylatable CPEB1-mutant protein is expressed

the levels of MTDH/AEG-1 protein is drastically andsignificantly diminished, whereas the level of mRNA encod-ing MTDG/AEG-1 does not change (Fig. 2B). Finally, thisdecrease in MTDH/AEG-1 is detected specifically in cellsexpressing the DN-CPEB1 protein (Fig. 2C).

Disruption of CPEB1 inhibits glioblastoma growth invivoTo determine if CPEB1 inhibition could affect tumor

growth in vivo, we expressed either the DN-CPEB1-GFP orGFP alone in CNS-1 cells before implanting them into thecentral striatum of adult Lewis rats. A total of 150,000CNS1cells were injected into the right hemisphere and animalswere perfused 10 days after implantation. Brains wereprocessed and serial sectioned. To assay for cell migration,we used fluorescencemicroscopy to image transfected cells inserial sections and determined spread away from the injec-tion site (Fig. 3A). We counted the number of GFP-expressing cells in 10-mm wide bins (medial-laterally frominjection site), normalizing each bin to the number of cells inthe 10-mm bin centered on the injection site. This methodnormalizes the counts and allows for comparison betweengroups. The cell density was significantly increased in regionsdistal to the injection site in the GFP-expressing controlscompared with the DN-CPEB1-GFP–expressing cells (Fig.3B). Brains were also processed, stained (Fig. 3C andD), andsubjected to 3D volume reconstruction. In agreement withthe cell spread data, tumor reconstructions revealed thatexpression of DN-CPEB1-GFP resulted in a significantreduction in total tumor volume compared with the controlGFP-expressing tumors (Fig. 3E and F). The decrease intumor size observed by inhibiting CPEB1 could also bevisualized in rostral–caudal spread of theDN-CPEB1-GFP–transfected tumors (Fig. 3G). When we quantified the totaltumor volume as a group, we found the DN-CPEB1-GFP–transfected tumors displayed a significant 45% decrease intotal volume compared with control GFP-expressing tumors(Fig. 3H; GFP, n ¼ 10; DN-CPEB1, n ¼ 10; P < 0.05;Student t test, two-tailed). Brains were also processed andimmunostained for pCPEB expression. Tumors transfectedwith the GFP alone showed increased pCPEB expression ascompared with adjacent healthy tissue (Supplementary Fig.S1B). Thus, following disruption ofCPEB1 function there isa significant inhibition of tumor volume in glioblastoma thatmay be due in part to a lack of cell migration away from theinjection site.

CPE-dependent local protein synthesis in astrocytesThe apparent lack of migration in vivo in cells expressing

DN-CPEB1-GFP was similar to the effect we observed incultured cells (3). Because directed cell migration requiresasymmetric localization of proteins, we examined if CPEB1-mediated protein synthesis could contribute to the locali-zation of proteins to the leading edge of migrating cells. Weasked if the presence of a CPE sequence in the 30-UTR candirect protein synthesis into the periphery of the cell byexpressing a GFP reporter construct in astrocytes. Astrocytesare much broader and flatter cells in vitro than CNS1 cells

Figure 1. CPEB1 protein interacts with the MTDH/AEG-1 mRNA. A,MTDH/AEG-1 mRNA contains a CPE sequence within 100 nucleotides(nt) of the hexanucleotide (HEX) sequence that is conserved wheresequence was available, including humans, non-human primates,bovines, and rodents. Shown are the sequences in humans and mice(accession# NM_178812, NM_026002). B, CPEB1 protein wasimmunoprecipitated (IP) from CNS1 cells in culture using an antibodyspecific for CPEB1 but not when a nonspecific immunoglobulin G (IgG)antibody was used. RNA was isolated from the immunoprecipitationand RT-PCR was conducted using primers specific for MTDH/AEG-1(MTDH) or GFAP (n ¼ 3). C, CNS1 cells were transfected with DN-CPEB1-GFP followed by immunoprecipitated using an antibodyspecific for GFP. RNA was then isolated and an RT-PCR was againconducted using primers specific for MTDH/AEG-1 or GFAP (n ¼ 3).WB, Western blot analysis.






and allow for greater spatial resolution when imaging. Thereporter encoded a farnesylation sequence fused to the C-terminus of a dEGFP to anchor the GFP into the membranenear the site of synthesis (19). This reporter contained eitherCPE sequences in the 30-UTR or the same 30-UTR but withthe CPE sequences mutated (Fig. 4A). Astrocytes weregrown to near confluency and cells were transfected witheither the CPE-containing or mutated construct. Sixteenhours after transfection, a wound was introduced. Using livecell confocal imaging, transfected cells on the leading edge ofthe wound were located, imaged, photobleached, and sub-sequently imaged every 30 seconds for the reappearance offluorescence (Fig. 4B). We quantified GFP expression byselecting 2 regions of interest: one at the peripheral edge ofthe cell and the other adjacent to the nucleus (Fig. 4B; blackboxes). Cells expressing the CPE-containing 30-UTRshowed expression of GFP both around the nucleus and atthe distal edge of the cell within minutes (Fig. 4B and C).However, cells expressing the mutated CPE construct had aclear delay in fluorescence recovery at the peripheral edge of

the cell (Fig. 4B and C). Thus, the presence of CPEsequences in the 30-UTR can localize protein synthesis tothe leading edge of migrating astrocytes.

CPE-dependent mRNA localizationThe lack of localized protein synthesis in the absence of the

CPE in Fig. 4 could either be misregulation of mRNAtranslation or mRNA localization. To assess if mRNA-containing CPE sequences are trafficked differently fromnon-CPE–containing mRNA, we used the MS2 RNAvisualization assay pioneered in the Singer laboratory(20). Here, the single-stranded mRNA-binding phage coatprotein MS2 is fused to a fluorescent reporter and expressedalong with mRNA engineered to contain MS2-binding sites(21). We generated 2 reporter constructs, one containing 12MS2-binding sites fused to the CPE-containing 30-UTRshown in Fig. 4, the other containing 12 MS2-binding sitesfused to the mutated CPE 30-UTR. A third constructencoded a GFP-tagged MS2 protein carrying a NLS tolocalize to the nucleus any MS2-GFP protein not bound

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MTDHFigure 2. CPEB1 can regulate theexpression of MTDH/AEG-1. A,MTDH is upregulated inglioblastoma cells. Astrocyte andCNS1 cultures were probed withanti-MTDH/AEG-1 antibodyshowing increaseimmunoreactivity in the CNS1cells. Astrocyte, CNS1, and U87cultures were analyzed byWesternblot analysis for MTDH/AEG-1expression and levels quantified.Loading was normalized to b-actinand the relative amount of MTDHexpressed in the glioblastomas linewas compared with astrocytes(n¼ 3; �, P < 0.05 by Student t test,two-tailed). BandC,CNS1cultureswere transfected with eitherGFP or DN-CPEB1-GFP andprocessed for MTDH/AEG-1immunoreactivity. Cells expressingGFP alone show robust MTDH/AEG-1 expression (arrows in left),whereas cells expressing DN-CPEB1-GFP show decreasedexpression (arrows in right) eventhough neighboringnontransfected cells show robustexpression. CNS1 and U87cultures expressing GFP or DN-CPEB1-GFP analyzed by Westernblot analysis for MTDH/AEG-1expression show a significantdecrease in MTDH/AEG-1expression in DN-CPEB-GFP–expressing cultures. Loading wasnormalized to b-actin (n ¼ 3;�,P < 0.05; ��,P < 0.005 by Studentt test, two-tailed). However, RT-PCR showed no change in MTDH/AEG-1 mRNA levels.

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to mRNA (Fig. 5A). To take advantage of a higher trans-fection efficiency and rapid migration rate, the constructswere cotransfected into CNS-1 cells (18). Transfectedmonolayers were subjected to a scratch assay and assessedfor mRNA localization 1 to 2 hours after the wound. Cellsthat were transfected with the CPE-containing constructdisplayed mRNA expression throughout the cell includingthe periphery. However, cells transfected with the mutatedCPE construct expressed the tagged mRNA in a perinuclearlocation only (Fig. 5B). 70% of cells transfected with theCPE-30-UTR construct expressed the MS2-tagged mRNAat the peripheral edge. While, only 28% of cells transfectedwith the mutated CPE-30-UTR visualized MS2-taggedmRNA outside of the perinuclear region (Fig. 5C). Thisdramatic nuclear localization of the CPE-lacking reportersuggests that CPEB1 may play a role in not only targeting

the mRNA to the periphery but shuttling the mRNAbetween the nucleus and the cytoplasm. To confirm inastrocytes that CPEB1 is shuttled in and out of the nucleus,we transfected cells with a construct containing the fulllength CPEB1 fused to mCherry. The cells were then leftuntreated or treated with the nuclear export inhibitorLMB. In the untreated cells, mCherry-tagged CPEB1 wasexpressed throughout the cytoplasm including the peri-pheral edge of the cell (Fig 5D). This localization ofmCherry-CPEB1 is identical to that of endogenous CPEB1(3). However, after treatment with LMB for 6 hours, theexpression of mCherry-tagged CPEB1 was completelyrestricted to the nucleus in both astrocytes and CNS1 cells(Fig. 5D). Interestingly, CNS1 cells exhibit a faster responseto LMB than astrocytes, localizing the majority of CPEB1to the nucleus after just 1 hour of treatment (Fig. 5D,

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Figure 3. Disruption of CPEB1 function retards glioblastoma growth in vivo. A, CNS-1 cells expressing either GFP or DN-CPEB1-GFP were stereotaxicallyimplanted into the central striatum of adult rats. Using fluorescence microscopy, we identified the injection site (dashed line). DN-CPEB1-GFP–expressingtumors failed to migrate out of the injection site, whereas control GFP-transfected tumors displayed a wide medial–lateral spread. B, quantification of tumormigration. The number ofGFP-expressing cells in 10mmlength binsmedial-laterallywere counted, eachbinwas then normalized to thenumber of cells in the 0to 10 mm bin surrounding the injection site to account for total cells in the injection site. The cell density spread was significantly increased in the GFP-expressing control tumors compared with tumors transfected with the DN-CPEB1-GFP (GFP, n ¼ 10; DN-CPEB1-GFP, n ¼ 10; ��, P < 0.001 by two-wayANOVA; post-Bonferroni). C–D,brainswere serial sectioned, crystal violet–stained, and subjected to3Dvolume reconstruction using the software Imaris. E–F,3D reconstruction of representative tumors showing the medial lateral spread. G, 3D reconstruction showing the rostral (R) caudal (C) expansion ofrepresentative tumors. H, DN-CPEB1-GFP expressing tumors were reduced in total volume by 45% compared with GFP-expressing tumors (GFP, n ¼ 10;DN-CPEB1, n ¼ 10; �, P < 0.05 by Student t test, two-tailed).






compare middle panels). These data suggest that CPEB1plays a role in the localization of CPE-containing mRNA inastrocytes and glioblastoma cells and that the nuclear shut-tling of CPEB1 inCNS1 cells is enhanced. Further evidence,along with the increase in pCPEB1, that the activity of thispathway is enhanced in glioblastoma cells.

CPEB1 regulates directed cell migrationIn our initial characterization of CPEB1 in astrocytes and

CNS1 cells, the expression ofDN-CPEB1-GFP resulted in afailure of expressing cells to migrate in a wound healing assayin vitro (3). While it was determined that cells transfectedwith the dominant negative CPEB1 were less likely tomigrate into the wound, we were not able to determine ifthe cells had lost their ability to migrate or if they lackeddirectional movement. To address this distinction, CNS1cells were transfected with either DN-CPEB1-GFP or GFPalone and visualized 1 to 2 hours after inducing a wound in

the cultures. Transfected cells along the edge of the woundwere located and imaged every 30 seconds for 45 minutes.When individual trajectories were normalized to a startingpoint, 72% of the GFP-transfected control cells migratedtoward the wound. In contrast, only 16% of the DN-CPEB1-GFP cells migrated toward the wound (Fig. 6A).When compared as a group, control cells displayed a cleartrajectory, steadily migrating into the wound.However, cellstransfected with the DN-CPEB1-GFP displayed movementbut this movement was random (Fig. 6B). We next quan-tified the amount of time the cells spentmigrating toward thewound. Over a 45-minute time course, cells transfected withthe control GFP spent 85% of the time moving withdirectionality toward the wound. In contrast, cells trans-fected with the DN-CPEB1-GFP spent 25%moving with apreference toward the wound (Fig. 6C). These resultssuggest that disruption of CPEB1 function results in a lossof directed cell migration.

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Kochanek and Wells





DiscussionMetadherin/AEG-1 is upregulated in many types of

cancer including glioma, breast, prostate, and liver, and hasbeen linked to increased proliferation, migration, cell sur-vival, chemoresistance, and a poor clinical outcome (14, 22).Emerging evidence is suggesting, it acts to coordinate manysignaling pathways to impart these diverse effects. Forexample, MTDH/AEG-1 expression is enhanced by theactivation PI3K/Akt pathway via phosphorylation ofGSK3b

(23). GSK3b phosphorylation also stabilizes b-catenin pro-tein and MTDH/AEG-1 increases LEF-1/TCF1, the tran-scription factor that mediates Wnt/b-catenin signaling (12).In addition, MTDH/AEG-1 activates the transcriptionfactor NF-kB to promote the expression of many genesimplicated in tumor growth and metastasis (12). The find-ings presented in our current work show that the synthesis ofMTDH/AEG-1 can be regulated by the mRNA-bindingprotein CPEB1 in astrocytes and glioblastoma cells. Our

Figure 5. mRNA trafficking isregulated by the presence of a CPE.A, schematic describing theconstructs used in this approach.The mRNA reporter constructscontained 12 binding sites for thecoat protein of the bacterial phageMS2 and either CPE-containing 30-UTR or one in which the CPEsequences had been mutated. Athird construct contained the GFP-tagged MS2 protein along with aNLS. B, CNS-1 cells cotransfectedwith the GFP-MS2 construct andeither the CPE-containing (top) orCPE-mutated (bottom) reporterconstructs. Cells expressing theCPE-containing construct displayedlabeled mRNA throughout thenucleus and the periphery of the cell(arrows) in contrast to cellsexpressing the mutated CPEconstruct, which expressed thetaggedmRNA only in and around thenucleus. C, quantification of thelocation of the mRNA (wild-type,n¼87;mutated, n¼50; �,P<0.01byone-way ANOVA; post-Tukey). D,astrocyte and CNS1 cultures weretransfected with mCherry-taggedCPEB1 and expression wasobserved throughout the cytoplasm.Six hours after LMB treatment(20 nmol/L), expression of mCherry-tagged CPEB1 was restricted to thenucleus. However, CNS1 cells showa faster response to LMB, localizingthe majority of mCherry-taggedCPEB1 to the nucleus after 1 hour(middle).

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work suggests that CPEB1 may be regulating a cohort ofmRNAs thatwork together to fuel the progression of tumors.Alteration in protein expression has been subject to

intense investigation in glioblastoma. While many screens

have been applied to identify protein expression patterns inglioblastoma, mechanisms that involve translational regula-tion of a group of mRNAs are still being elucidated. Oneexample of such regulation is the RNA-binding proteinMusashi1 (MSI1). MSI1 induces glioma growth by regu-lating several proteins that promote cell proliferation andsurvival (24). Similarly, in the glioblastoma cell line ln827,the RNA-binding protein ZFP36 binds to and regulates theexpression of PIM-1, PIM3, andXIAP leading to a reductionin the cell lines viability and migration (25). We now addCPEB1 to the list ofmRNA-binding proteins thatmay play arole in tumor progression. Our results suggest that targetingthe CPEB1 pathway may be an effective way to limitglioblastoma cell migration and tumor progression in vivo.By inhibiting CPEB1 function, we observe a statistically

significant reduction in glioblastoma tumor spread andgrowth in vivo. This inhibition in tumor size is similar tothat which we described in vitro by blocking CPEB1function (3). Likely, contributing to this decrease in tumorprogression by DN-CPEB1-GFP is the inhibition ofMTDH/AEG-1 expression shown here as well as the inhi-bition of b-catenin synthesis showed in a previous study (3).Undoubtedly, expressing DN-CPEB1 will inhibit the syn-thesis of many CPE-containing mRNAs and neither the lackof migration nor the reduced tumor size in vivo can beattributed to merely inhibiting the synthesis of these 2proteins. Rather multiple proteins necessary for both migra-tion and growth are likely contributing to this effect. Forexample, CPEB1 regulates the expression of cyclin B1 inastrocytes, and blocking CPEB1 function prevents entranceinto mitosis, leading to an inhibition of proliferation (6).Furthermore, we have shown in hippocampal neurons thattissue plasminogen activator (tPA), a serine protease thatregulates the physiologic processes that necessitate tissueremodeling and cell migration, is regulated by CPEB1 (26).Along with b-catenin, MTDH, and cyclin B1, FABP7 isregulated by CPEB1 in astrocytes (27). Not surprisingly,FABP7 is one of multiple CPE-containing mRNAs that aremisregulated in various stages of glioblastoma (Table 1).In agreement with a role for CPEB in tumor growth, the

CPEB1 paralog CPEB4 regulates the translation of a groupof proteins necessary for pancreatic ductal adenocarcinomagrowth, invasion, and vascularization (28). Originallythought to differ in their mRNA targets (29), recent studieshave shown that CPEB4 does recognize and bind to some ofthe sameCPE targets as CPEB1, thoughwith a lower affinity(30, 31). For example, like CPEB1 in hippocampal neurons,CPEB4 has been shown to bind and regulate tPA in bothnormal and cancerous pancreatic tissue (28). Interestingly,several reports implicate a downregulation of CPEB1 incancer (32–34), whereas the results presented here suggestthat an increase in CPEB1-mediated translation may sup-port the progression of cancer. Because CPEB1 can bothinhibit and activate translation, reducing the levels ofCPEB1 protein would free mRNA from repression andhave the same result as an increase in phosphorylation ofCPEB1-–namely an increase in target protein expression.For example, the increase in NF-kB signaling and the

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Figure 6. CPEB1 regulates directed cell migration. A, CNS-1 cells weretransfected with DN-CPEB1-GFP or control GFP constructs andsubjected to live cell confocal imaging. Individual cell trajectories werenormalized to a starting point (asterix) with thewound located to the right.GFP-transfected cells migrated toward the wound (solid lines), whereasthe DN-CPEB1-GFP–expressing cells showed no directional preference.B, grouped cell trajectories over a 45-minute time course illustrate thatGFP-expressing cells (as a group) steadily migrated into the wound,whereas the DN-CPEB1-GFP–expressing cells displayed randommovement and thus as a group show no directional preference. C,quantification of the amount of time the cells spent migrating toward thewound. (GFP, n ¼ 7; DN-CPEB1-GFP, n ¼ 6; ��, P < 0.005 by Studentt test, two-tailed).

Kochanek and Wells





decrease in p53 levels detected in CPEB1 knockout mouseembryo fibroblasts (MEF; refs. 32, 35) could be in partaccounted for by an increase in MTDH/AEG-1 expression(12) following the release of its mRNA from translationalrepression by CPEB1. In either case, inhibiting CPEB1-mediated mRNA translation, with the use of DN-CPEB1-GFP, clearly inhibits tumor growth.Increasingly, roles for localized mRNA translation in

specific cellular compartments are being established and themolecular mechanisms regulating mRNA trafficking andtranslation are now being elucidated (36, 37). One well-studied example of an element in the 30-UTR being respon-sible for cellular trafficking is the b-actin zipcode. In 1994,Kislauskis and colleagues showed that a 54-nucleotidesequence in the 30-UTR of b-actin was responsible for itsintracellular trafficking in chicken embryo fibroblasts andthey termed this sequence "zipcode" (38). It was laterreported that the zipcode was being bound by an mRNA-binding protein named zipcode-binding protein1 (ZBP1)and the interaction between the 2 was necessary for thelocalization and translation of the b-actin mRNA (39, 40).Here, we show that trafficking of a CPEB1-regulated

mRNA is dependent upon the presence of CPE sequencesin the30-UTR.The role ofCPE sequences in targetingmRNAhas been disputed in neurons, where it has been examined inthe context of regulating aCaMKII mRNA. In hippocampalneurons, a reporter RNA containing the wild-type CPEsequence fromaCaMKII is more efficiently transported thanone containing a mutated CPE (1). However, in this studyonly the distal 170 nucleotides of the approximately 3,000-nucleotide longaCamKII 30-UTR (�5%)was used, and thisreport was in stark contrast to a previous study, which showedthat the most proximal 94 nucleotides of the aCamKII 30-UTRwere essential for the mRNAs localization (41). Indeed,another group reported a different, 1,200-nucleotide stretchof the aCamKII 30-UTR is essential for mediating its den-dritic localization (42). It may in fact be that many regions ofthe 30-UTR contribute to mRNA localization; thus, for ourreporter constructs,weused a larger region (30%)of thenative30-UTR. We found the presence of the CPEs necessary tomediate mRNA localization and protein synthesis at theperiphery of the cell. Given that MTDH/AEG-1 has distinctfunctions if expressed in the nucleus or cell surface (14), thesedata suggest the possibility that local synthesis of MTDH/AEG-1 could play a role in protein localization, and thusfunction inside the cell.Nuclear shuttling of CPEB1 has been reported in both

Xenopus oocytes and HeLa cells in which nuclear binding of

CPEB1 to mRNA is thought to regulate the degree to whichthe mRNAs are expressed in the cytoplasm (43, 44). Severalfindings support our conclusion that the CPE is necessary forCPEB1-mediated localization and local synthesis in astro-cytes and glioblastoma cells. First, constructs in which theCPE sequences have been mutated failed to localize themRNA to the periphery (Fig. 5). Second, constructs inwhich the CPE had been mutated were no longer synthe-sized at the leading edge of migrating astrocytes (Fig. 4).Third, CPEB1 moves between nucleus and cytoplasm (Fig.5). Fourth, expression of a dominant negative CPEB1 causesthe loss of directed cell migration (Fig. 6); likely due to theloss of local synthesis of CPE containing mRNAs. Our datasuggest amodel in which CPE-containingmRNA are boundby CPEB1 in the nucleus and trafficked to the periphery ofthe cell and subsequently translated. In support of this,studies in hippocampal neurons have shown expression ofthe RNA-binding domain of CPEB1 (akin to our DN-CPEB1-GFP) is primarily localized to the cell body (1, 7)and that the CPEB1-RBDconstruct is no longer able to bindto dyenin and kinesin (1), the motor proteins thought to beresponsible for CPEB1 transport.Our findings describe a novel role for CPEB1-mediated

mRNA translation in tumor progression and suggest dis-rupting CPEB1-mediated protein synthesis may be a viabletherapeutic approach in many types of cancer.

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

Authors' ContributionsConception and design: D.M. Kochanek, D.G. WellsDevelopment of methodology: D.M. Kochanek, D.G. WellsAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): D.M. KochanekAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): D.M. KochanekWriting, review, and/or revision of the manuscript: D.M. Kochanek, D.G. WellsStudy supervision: D.G. Wells

AcknowledgmentsThe authors thank Dr. Rick Matthews (SUNY Upstate Medical College) for his

guidance throughout the project and his assistance with the in vivo tumor experiments.The authors also thank Dr. Rob Singer (Albert Einstein College of Medicine) for theMS2 constructs. This research was supported by NIH-National Institute of Neuro-logical Disorders and Stroke grant NS-064282-01 (to D.G. Wells).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received August 19, 2012; revised November 19, 2012; accepted December 5,2012; published OnlineFirst January 29, 2013.

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2013;11:149-160. Published OnlineFirst January 29, 2013.Mol Cancer Res Dawn M. Kochanek and David G. Wells Cell MigrationCPEB1 Regulates the Expression of MTDH/AEG-1 and Glioblastoma

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