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http://dx.doi.org/10.10014-4827/& 2014 E

Abbreviations: ARArrest Specific 1; GDsoluble form of GAS

nCorresponding autE-mail address: j1 Instituto de Fisi

Research Article

A soluble form of GAS1 inhibits tumor growth andangiogenesis in a triple negative breast cancer model

Adriana Jiméneza, Adolfo López-Ornelasa,1, Enrique Estudillob,Lorenza González-Mariscalb, Rosa O. Gonzálezc, José Segoviab,n

aDepartamento de Farmacología, Centro de Investigación y de Estudios Avanzados del IPN, México D.F., MexicobDepartamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del IPN,Av. IPN # 2508, México D.F. 07300, MexicocDepartamento de Matemáticas, Universidad Autónoma Metropolitana-Iztapalapa, México D.F., Mexico

a r t i c l e i n f o r m a t i o n

Article Chronology:

Received 12 March 2014Received in revised form4 June 2014Accepted 22 June 2014Available online 30 June 2014

Keywords:

Growth Arrest Specific 1

tGAS1Breast cancerArteminERK1/2Angiogenesis

016/j.yexcr.2014.06.016lsevier Inc. All rights reser

TN, Artemin; EGFP, enhaNF, glial derived neurotr1; VE-Cadherin, vascularhor. Fax: þ52 55 5747 3754segovia@fisio.cinvestav.mxología Celular-Neurocienci

a b s t r a c t

We previously demonstrated the capacity of GAS1 (Growth Arrest Specific 1) to inhibit thegrowth of gliomas by blocking the GDNF–RET signaling pathway. Here, we show that a solubleform of GAS1 (tGAS1), decreases the number of viable MDA MB 231 human breast cancer cells,acting in both autocrine and paracrine manners when secreted from producing cells. Moreover,tGAS1 inhibits the growth of tumors implanted in female nu/nu mice through a RET-independentmechanism which involves interfering with the Artemin (ARTN)-GFRα3-(GDNF Family Receptoralpha 3) mediated intracellular signaling and the activation of ERK. In addition, we observed thatthe presence of tGAS1 reduces the vascularization of implanted tumors, by preventing themigration of endothelial cells. The present results support a potential adjuvant role for tGAS1 inthe treatment of breast cancer, by detaining tumor growth and inhibiting angiogenesis.

& 2014 Elsevier Inc. All rights reserved.

Introduction

Breast cancer is the most common cancer in women worldwide[1]. In breast cancer multiple molecular alterations are presentincluding the constitutive activity of the estrogen receptor (ER),and the progesterone receptor (PR), the amplification of

ved.

nced green fluorescent pophic factor; GFRα, GDNFendothelial cadherin; VE.(José Segovia).as, Universidad Nacional A

epidermal growth factor receptor 2 (HER2), and the amplificationand mutations of the p53 and PTEN genes, whose loss of functionpromotes the unconstrained activation of AKT, moreover triplenegative breast cancer (ER, PR, HER2, negative) is a subtypecharacterized by its high aggressiveness [2–4]. Breast tumors giverise to metastasis that develop mainly in lung, liver, brain and

rotein; ERK, extracellular-signal regulated kinase; GAS1, Growthfamily receptor alpha; RET, rearranged during transfection; tGAS1,GF, vascular endothelial growth factor

utónoma de México, México D.F., México.

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bone [5]. Since angiogenesis plays a central role in tumor growthand metastasis, to inhibit this process appears as an encouragingstrategy for cancer treatment and the prevention of metastasis. Inthis regard, promising results have been obtained in renal cellcarcinoma and breast cancer clinical trials after treatment withBevacizumab, a monoclonal antibody against vascular endothelialgrowth factor A (VEGF-A) [6].GAS1 is a 37 kDa protein that presents structural homology

with the receptors for the GDNF-family of ligands (GFRαs) [7],a family of co-receptors that bind GDNF-family ligands (GFLs) andform a complex with RET to promote cell survival and prolifera-tion through the activation of the MAPK and PI3K/AKT signalingpathways [8,9]. In contrast, the expression of GAS1 inhibits DNAsynthesis [10], and its overexpression reduces the proliferation ofbladder, lung and fibrosarcoma cancer cell lines [11]. Furthermore,GAS1 induces apoptosis of primary gliomas and neuroblastomacells by inhibiting the GDNF pathway and inducing the activationof caspases 9 and 3, through a process that involves decreasedphosphorylation of RET at Tyr 1062, the reduced phosphorylationof AKT, the translocation of Bad to the mitochondria and therelease of cytochrome-c to the cytosol [12–16].The apoptotic effect of GAS1 has been employed in experi-

mental gene therapy for the treatment of gliomas showing thatGAS1, and a truncated and secreted form of GAS1 (tGAS1), inhibittumor growth [17,18]. Interestingly, breast metastatic tumorshave low GAS1 levels and its knockdown increases lung metas-tasis [19]; whereas the expression of RET and GFRα1 is associatedwith breast cancer progression [20–22] and Artemin (ARTN),a ligand of the GDNF family that binds to GFRα3, is present inthese tumors and participates in cell proliferation, invasion andangiogenesis [23–25].This information prompted us to study whether GAS1 could

inhibit tumor growth and metastasis in a murine breast cancermodel. By delivering tGAS1 using lentiviral vectors, we showedthat the ectopic expression of tGAS1 inhibits breast tumor growth.Interestingly, MDA MB 231 breast cancer cells do not expressGDNF, RET nor GFRα1, however they express ARTN [23] and herewe demonstrate that they also express GFRα3 and that tGAS1inhibits the activation of ERK in a RET-independent manner andinterfering with the ARTN-induced pathway through which tGAS1induces cancer cells into quiescence. Furthermore, conditionedmedium containing tGAS1 inhibits the migration of endothelialcells in vitro and treatment with tGAS1 reduces the vasculariza-tion of MDA MB 231 tumors evaluated by immunohistochemistryfor VE-cadherin, through a mechanism not yet identified.

Methods

Construction of expression vectors

The coding sequence of human GAS1 was amplified by PCR andinserted into the pENTR/DTOPO vector (Invitrogen) to create thepENTR/DTOPO-GAS1 vector. pENTR/DTOPO-GAS1 was recombinedwith the pLenti6.3/TO/V5-DEST vector (ViraPower HiPerformGateway Expression T -Rex System, Invitrogen) to create thepLenti6.3/TO/V5-GAS1. To obtain pENTR/DTOPO-tGAS1, the GAS1coding sequence was amplified by PCR ending at arginine 315,before the GPI anchor consensus sequence [17], and recombinedwith the pLenti6.3/TO/V5-DEST vector to generate the pLenti6.3/

TO/V5-tGAS1 clone. Likewise, using the coding sequence of theenhanced green fluorescent protein (EGFP) the pLenti6.3/TO/V5-EGFP vector was constructed. This vector was used as a control.

Lentiviral production

Lentiviral vectors pLenti6.3/TO/V5-GAS1, pLenti6.3/TO/V5-tGAS1,pLenti6.3/TO/V5-EGFP and pLenti3.3/TR (tetracycline repressorelement) were produced according to the manufacturer's protocol(ViraPower HiPerform Gateway Expression T -Rex System, Invitrogen).Briefly, pLenti6.3/TO/V5-GAS1, pLenti6.3/TO/V5-tGAS1, pLenti6.3/TO/V5-EGFP or pLenti3.3/TR vectors were transfected to HEK293FT cellsusing Lipofectamine 2000 (Invitrogen) and after 48 h culture mediacontaining the lentiviruses were collected, centrifuged, filtered andthe viral title was subsequently determined.

Generation of MDA clones with inducible expression ofEGFP, GAS1 and tGAS1

The triple negative and metastatic MDA MB 231 human breastcancer cells were first infected with the tetracycline repressorelement (TR) virus obtained from the transfection of HEK293FT cellswith the pLenti3.3/TR vector and selected using antibiotic resistancewith 500 μg/mL of geneticin. Afterwards, cells were infected withEGFP, GAS1, or tGAS1 virus obtained from the transfection ofHEK293FT cells with the pLenti6.3/TO/V5-EGFP, pLenti6.3/TO/V5-GAS1 or pLenti6.3/TO/V5-tGAS1, vectors and selected by antibioticresistance with 5 μg/mL of blasticidin to obtain MDA–EGFP, MDA–GAS1 and MDA–tGAS1 clones respectively. On other hand, to obtainMDA MB 231 cells constitutively expressing EGFP, MDA-WT cellswere infected only with pLenti6.3/TO/V5-EGFP lentivirus andselected with 5 μg/mL of blasticidin.

Cell viability assays

To determine cell viability, 30,000 cells per well were plated foreach condition, and 72 h after exposure to the culture mediumwith or without tetracycline (2 μg/mL) viable cells were countedusing the trypan blue exclusion technique.

Effect of tGAS1 conditioned medium

MDA–tGAS1 and MDA-WT cells were plated in p60 culture dishesand exposed to culture medium with or without tetracycline(2 μg/mL) for 72 h. Then, medium from each condition werecollected, centrifuged, filtered and added to independent MDA-WT cells and after 72 h in culture, viable cells were counted bythe trypan blue exclusion technique.

Immunoprecipitation

MDA–tGAS1 media in the absence or in the presence of tetra-cycline for 72 h were collected, centrifuged, filtered, and thenclarified for 3 h with protein A agarose (Roche) at 2 to 8 1C. Mediawere centrifuged and transferred to fresh tubes for the addition ofhuman GAS1 antibody (ProScience, Poway, CA [17,18]) or NMDAε1antibody (Santa Cruz), as a control of immunoprecipitation of anunrelated protein and incubated for 1 h. Then protein A agarosewas added and the mixture was incubated overnight at 2 to 8 1C.After centrifugation, the medium was filtered and added to MDA-

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WT cells to determine cell viability. Protein A agarose wasprocessed according to the manufacturer's protocol and subse-quently samples were electrophoresed and western blot analysisof the immunoprecipitates were performed.

Activation of ERK1/2 by tGAS1 and Artemin co-treatment

MDA–tGAS1 cells were incubated with culture medium in thepresence or absence of tetracycline for 48 h to induce theexpression of tGAS1 or to repress it, respectively. Groups receivinghuman recombinant Artemin (10 ng/mL) (450-17, PEPROTECH)were incubated in the absence of tGAS1 or 24 h after of theaddition of tetracycline. Cells were harvested to extract protein toperform western blot analysis of ERK phosphorylation.

RT-PCR

Total RNA extraction was performed using the TRIzol Reagent(Invitrogen) according to the manufacturer's instructions. Subse-quently 5 μg of RNA was treated with DNAse (New EnglandBioLabs) and converted to cDNA using MMLV reverse transcrip-tase (Invitrogen) to perform the PCR. To amplify GAS1 and β-actinthe following primers were used 50ACG CAG GCC TCG AGC AGCTTG 3 0and 50CTG TGC CTG CTG CTG GCG ATG C 30, 50TGG CAC CACACC TTC TAC A 3 0and 50 TCA CGC ACG ATT TCC C 3 0, respectively.To amplify ARTN and GFRα3 primers previously reported fromKang et al. were used [23]. RET mRNA levels were determined aspreviously described [14].

Western blot analysis

Protein extraction was performed using a lysis buffer containingprotease inhibitors. 25 μg of protein per condition was run on SDS-PAGE gels and then transferred to PVDF membranes (BioRad) whichwere subsequently blocked for 1 h with a solution of 5% skim milk/BSA 1% in TBS/0.1% Tween [17]. Membranes were incubated overnightwith primary antibodies against human GAS1 (1:2000, ProScience,Poway, CA), human actin 1:5000 [26], GFP (1:1000, Millipore), ERK1/2and pERK1/2 (1:2000, Cell Signaling). Subsequent washes wereperformed with TBS/Tween 0.1% and then membranes were incu-bated for 1 h with anti-rabbit and anti-mouse secondary antibodiesdiluted 1:5000 (Invitrogen), followed by washes with TBS/0.1%Tween. Levels of pAKT and total AKT were determined as previouslydescribed [14]. The presence of proteins was revealed by chemilumi-nescence (Perkin-Elmer). The images were captured and analyzedusing a UVP BioImaging System and LabWorks Image Acquisition andAnalysis Software (UVP, Inc. Upland, CA).

Cell cycle analysis

MDA-WT, MDA–EGFP, MDA–GAS1 and MDA–tGAS1 cells wereincubated with culture medium in the presence or absence oftetracycline for 72 h. Then cells were harvested and fixed with70% ethanol overnight. After fixing, samples were washed withPBS and resuspended in a solution containing 20 μg/ml PI and200 μg/ml RNase (Sigma) in PBS/0.1% Triton X100 and incubatedfor 1 h at room temperature in the dark. Fluorescence wasmeasured using a FACS Calibur device (Becton Dickinson).

Immunofluorescence

MDA MB 231 cells were fixed with 4% paraformaldehyde (PFA),washed with PBS, permeabilized with PBS/0.2% Triton X-100 solutionfor 10min and blocked with PBS/1% BSA for 1 h. Then cells wereincubated overnight with anti-GAS1 antibody (1:500, ProScience,Poway, CA), followed by washes with PBS and incubation with1:500 Alexa Fluor 488 anti-rabbit secondary antibody (Invitrogen)and counterstained with DAPI (Vector). For VEGF immunofluores-cence, cells were blocked with PBS-0.01% Tween-20/3% BSA/2% goatserum for 30min and incubated overnight with anti-VEGF antibody(1:50, Santa Cruz) followed by incubation with 1:200 TRITC anti-rabbit secondary antibody (Vector) and counterstained with DAPI.The images were obtained with an Olympus BX51 epifluorescencemicroscope coupled to a CoolSNAP-Procf Color camera with theImage-Pro PLUS software (Media Cybernetics, Inc.)MDA MB 231 tumor slices were permeabilized with PBS/0.2%

Triton X-100 solution for 10 min and blocked with PBS/1% BSAand incubated overnight with anti-VE-cadherin antibody (1:50,Santa Cruz) followed by incubation with 1:100 TRITC anti-rabbitsecondary antibody and counterstained with DAPI (Vector).Images were obtained with a Leica SP8 Confocal microscope andLeica Application Suite Advanced Fluorescence (LAS AF, LeicaMicrosystems) software. To quantify the number of VE-cadherinpositive areas, complete fields of 10–12 slices per tumor (n¼4)were analyzed and every time a signal of VE-cadherin wasobserved, it was considered as a positive area and the percentageof positive areas/tumor was determined.

In vitro migration of endothelial cells

Primary cell cultures of rat microvascular endothelium [27] wereplated to confluence in Boyden chambers and exposed to theconditioned media of MDA–tGAS1 cells with or without tetra-cycline (2 μg/mL). After 24 h of incubation, the endothelial cellscontained in the membrane of the Boyden chamber were fixedwith 4% PFA, permeabilized with 0.03% saponin and stainedwith1% toluidine blue. Cells that migrated to the basal side ofthe membrane under each condition were counted using a lightmicroscopy. The images were obtained with an Olympus BX51microscope coupled to a CoolSNAP-Procf Color camera with theImage-Pro PLUS software (Media Cybernetics, Inc.)

In vivo models of breast tumors

Experiments were performed according to current Mexican legisla-tion NOM-062-ZOO-1999 (SAGARPA) and in agreement with theGuide for the Care and Use of Laboratory Animals of the NationalInstitutes of Health (NIH) and internal (CINVESTAV) guidelines.Heterotopic xenotransplantation of breast cancer cells were per-formed inoculating 2�106 MDA MB 231 cells subcutaneously intothe flanks of the hind limbs of female nu/nu mice 6–8 weeks old.Once tumors reached a volume of approximately 50mm3, they weretreated intratumorally for 3 days (day 0, 2 and 4) with 2�106 vp(viral particles) of tGAS1, EGFP lentivirus or medium without serum.Tumor growth was monitored continuously and tumor volume wascalculated using the equation: V¼(Dmax)(Dmin)2/(2); where Dmax andDmin represent the large and small axis, respectively, 15 days afterinitiation of treatment, animals were sacrificed and tumors dissected,

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homogenized and processed for protein and RNA extraction withTRIzol Reagent (Invitrogen).Orthotopic xenografts of breast cancer were performed inocu-

lating subcutaneously 2�106 MDA MB 231 cells that constitu-tively express EGFP in the 4 T and 5 T mammary glands of 6 to 8week-old nu/nu female mice. Tumors of approximately 30 or50 mm3 were treated for 3 days (day 0, 2 and 4) with 2�106 vp ofthe tGAS1 lentivirus or medium without serum. Tumor growthwas monitored continuously, and 28 days after the initiation oftreatment animals were sacrificed and the tumors dissected todetermine the expression of VE-cadherin.

Statistical analysis

In vitro experiments that include cell viability, the effect of condi-tioned media, ERK1/2 phosphorylation and cell migration wereanalyzed using one way ANOVA and Duncan´s post hoc test. Theresults of cellular cycle and VE-cadherin expression were analyzedusing Student's t-Test. Data of tumor growth (volume), considered asa time series with independent observations, were analyzed using asplit parcel method grouping the data into two groups covering aprincipal group (the treatment) and a secondary group included intothe principal group (time). A nested model was run, according to thefollowing: Vol¼AþB(A)þCþAC, where A is the treatment factor, B isthe experimental unit (mouse) and C is time, here A and C areconsidered constant factors, B is declared a nested factor. Thefollowing tests (ANOVA and Duncan's) were performed with thevariation of tumor volume in the subunits (Error II). A Po0.05 wasconsidered significant in all cases. All tests were performed using theNCSS software.

Results

GAS1 and tGAS1 decrease the number of viable cells byinducing cell cycle arrest

After lentiviral infections, three cell lines were obtained with aTet-On system that in response to tetracycline (2 μg/mL)

Fig. 1 – Expression of GAS1 and tGAS1 in infected MDA MB 231 cetransgenes. A) Expression of the transgenes, detected at the mRNAimmunocytochemistry. WT: MDA MB 231 non-infected wild type cEGFP, GAS1 or tGAS1 lentivirus. T,: with tetracycline 2 μg/mL, W/T:captured at 40� /0.50, scale bar 50 μm.

ectopically express GAS1 (MDA–GAS1), tGAS1 (MDA–tGAS1) andEGFP (MDA–EGFP), as demonstrated by the expression of thetransgenic mRNA and protein (Fig. 1).

Our first goal was to determine whether GAS1 and tGAS1 couldaffect the viability of the MDA MB 231 human breast cancer cellline. We observed that after 72 h in the presence of tetracycline,there was a decrease in the number of MDA–GAS1 and MDA–tGAS1 viable cells but not of MDA–EGFP cells (Fig. 2A). In thisassay we only appreciate the effect in the number of viable cellsand not in the number of dead cells (positive for trypan blue),which suggests that the effect of GAS1 and tGAS1 is mostly causedby cell cycle arrest. To determine cell cycle, we performed flowcytometry assays which showed an increase in cell population onthe G0/G1 phase and a reduction in S and G2/M phases in MDA-GAS1 and MDA–tGAS1 cells treated with tetracycline (Fig. 2B,Supplementary Fig. 1). MDA MB 231 cells do not express GFRα1nor GDNF but express ARTN [23]. However, the presence ofGFRα3, the cognate receptor for ARTN, had not been previouslyreported in MDA MB 231 cells. By RT-PCR we found the presenceof GFRα3 mRNA in MDA MB 231 cells. This finding intimates thattGAS1 is interfering with the ARTN pathway in MDA MB 231 cells,possibly affecting the ARTN–GFRα3 interaction (Fig. 2C). We alsoobserved, as had been previously described [21], that MDA MB231 cells do not express RET (Supplementary Fig. 2).

Conditioned medium with tGAS1 decreases the number ofviable cells

We previously reported that conditioned medium with tGAS1decreased the viability of C6 glioblastoma cells due to both itsautocrine and paracrine effects [17,18]. To test whether condi-tioned medium from MDA–tGAS1 also decreased cell viability ofMDA wild type cells (MDA-WT), we induced the expression oftGAS1 in MDA–tGAS1 for 72 h and then administrated thisconditioned medium to independent cultures of MDA-WT cells.After 72 h of incubation with conditioned media we observed thatconditioned medium with tGAS1 significantly reduced the num-ber of MDA wild type cells (Fig. 3A, third column). To rule out the

lls. In this model, tetracycline induces the expression of thelevel by RT-PCR, B) protein level by western blot analysis and C)ells. EGFP, GAS1 and tGAS1 are MDA MB 231 cells infected withwithout tetracycline. Images of epifluorescence microscopy

Fig. 2 – GAS1 and tGAS1 overexpression decrease the number of viable cells by inducing cell cycle arrest. A) Incubation of MDA–GAS1 and MDA–tGAS1 with tetracycline decreases cell viability (n¼3 without tetracycline and n¼4 with tetracycline). B) Cell cycleanalysis shows an increased number of cells in G0/G1 in the presence of tetracycline, when compared to cells incubated withouttetracycline for MDA–GAS1 and MDA–tGAS1 cells (n¼3). In addition there is a reduction in the percentage of cells at S and G2/Mphases in MDA–GAS1 and MDA–tGAS1 incubated with tetracycline. C) MDA cells express mRNA for both ARTN and GFRα3. WT: MDAMB 231 wild type. EGFP, GAS1 or tGAS1: MDA MB 231 stable cells for EGFP, GAS1 or tGAS1. w/Tet: without tetracycline; Tet: withtetracycline. nnn Po0.001, ANOVA followed by Duncan's test in A; nPo0.05, Student's t-Test in B.

Fig. 3 – Conditioned media containing tGAS1 reduced cell viability. A) The viability of MDA WT cells was reduced after 72 hexposition to conditioned medium containing tGAS1 (tGAS1) when compared with MDA WT cells exposed for 72 h to conditionedmedium from which tGAS1 was immunoprecipitated (tGAS1/IP ) or control without tGAS1 (�tGAS1) (n¼4). B) Western blotanalysis of the immunoprecipitation of tGAS1 or NMDAε1 from MDA–tGAS1 medium in the presence or absence of tetracycline.NMDAε1: control immunoprecipitation done with a NMDAε1 antibody; w/11Ab: without primary antibody, þTet and �Tet: withand without tetracycline. One way ANOVA and Duncan post hoc nnPo0.01.

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possibility of having other factors being responsible for this effect,we depleted MDA–tGAS1 conditioned medium of tGAS1 byimmunoprecipitation with an anti-human GAS1 antibody andadministered it to MDA-WT (Fig. 3B). We observed no differencein cell viability with control medium, when tGAS1 was depletedby immunoprecipitation (Fig. 3A, second column), thus indicatingthat the reduction of viable cells is specifically due to tGAS1.

tGAS1 decreases ERK activation by interfering with ARTNsignaling

MDA MB 231 cells do not express GDNF nor RET, proteins associatedwith the tumor suppressor effect of GAS1 as we have previouslyshown [14–16]. However, MDA MB 231 cells express ARTN [23] andhere we demonstrate the expression of its receptor GFRα3. Bothmolecules are evolutionary related with GDNF and GFRα1, respec-tively [8]. These data suggest that in the present experiments weobserve a GAS1-mediated RET-independent mechanism of action. Wedecided to investigate whether the expression of tGAS1 preventedthe activation of ERK1/2 in the presence of ARTN in MDA–tGAS1 cells.The addition of recombinant ARTN significantly enhances ERK1/2phosphorylation, strikingly this increase is completely abolished inthe presence of tGAS1 (Fig. 4). We also observed a reduction of ERKphosphorylation in cells expressing tGAS1.These findings demonstratethat tGAS1 inhibits the effect of ARTN inducing the activation of theERK pathway and the proliferation of MDA MB 231 cells.

tGAS1 inhibits tumor growth in vivo

To test the potential of tGAS1 to inhibit tumor growth of MDA MB231 cells, we inoculated MDA-WT cells to create subcutaneoustumors in female nude mice. When tumors reached a volume ofapproximately 50mm3, we injected vehicle, or 2�106 vp of tGAS1 or

Fig. 4 – tGAS1 decreases ERK activation by interfering with ARTN siexpressing tGAS1 in the presence of ARTN. B) Densitometric analypresence of ARTN normalized to total ERK1/2 (n¼3). Control: cellsafter the administration of tetracycline, ARTN: cells without tGAS1tGAS1þARTN: cells expressing tGAS1 48 h after tetracycline adminisANOVA followed by Duncan's.

EGFP lentivirus at days 0, 2 and 4 directly into tumors to overexpresstGAS1 or EGFP. The latter was used as control and tumor growth wasmonitored for 15 days after the beginning of treatment. Since day 4,we observed that tGAS1 treated mice showed smaller tumors whencompared to control mice (Fig. 5A). Tumors receiving tGAS1 do noteven reach a volume of 100 mm3, whereas control tumors reach avolume of 300 mm3 (Fig. 5B and C). Therefore these data indicate thattGAS1 suppresses tumor growth in vivo.

It has been documented that orthotopic xenografts replicatetumor development more reliably [28], thus we decided toinvestigate the efficacy of tGAS1 in tumors generated in themammary glands by inoculating MDA MB 231 cells that consti-tutively express EGFP. We initiated treatments at tumor volumesof 30 and 50 mm3, in order to represent earlier and advanceddisease conditions respectively. We found that under both con-ditions the administration of the tGAS1 lentivirus significantlydecreased tumor growth compared with controls. Since we haddemonstrated that lentivirus expressing an irrelevant protein hadno effect on tumor growth and it was the same as vehicle, forthese experiments the controls were injection of vehicle. Inter-estingly, in subjects in which the treatment began at an earlierstage tGAS1 completely halted tumor growth (Fig. 6).

tGAS1 decreases ERK activation in MDA MB 231 tumors

To understand the inhibitory effect of tGAS1 in tumor growth, weanalyzed the activation of ERK, since this pathway plays animportant role in G1/S cell cycle progression, and is deregulatedin breast cancer [29,30]. We detected, by western blot analysis asignificant reduction of phosphorylated ERK1/2 in the hetero-topically implanted tumors treated with tGAS1 lentivirus incomparison with the controls, indicating that tGAS1 inhibits theactivity of the ERK pathway in vivo (Fig. 7).

gnaling. A) Western blot analysis of ERK1/2 in MDA-tGAS1 cellssis of pERK1/2 in MDA–tGAS1 cells expressing tGAS1 in thewithout tGAS1 expression, tGAS1: cells expressing tGAS 48 hexpression plus 10 ng/mL of recombinant ARTN for 24 h, andtration plus 10 ng/mL of recombinant ARTN for 24 h. nnPo0.01,

Fig. 5 – tGAS1 inhibits the growth of tumors implanted subcutaneously in the hind limbs of nu/nu female mice. (A) Representativeimages of MDA WT tumors obtained from mice 15 days after of the first administration of tGAS1 and EGFP lentivirus or medium.(B) tGAS1 and EGFP are exclusively expressed in tumors treated with GAS1 and EGFP lentivirus, respectively (LvtGAS1, and LvtEGFP).(C) Growth of subcutaneous MDAWT tumors after the different treatments (n¼5). LvtGAS1, LvEGFP and medium: treatments withtGAS1 and EGFP lentivirus and culture media respectively. nnPo0.01 ANOVA followed by Duncan's.

Fig. 6 – tGAS1 reduces the growth of tumors implanted in the mammary glands of nu/nu mice, treatments started when tumorreached volumes of 30 or 50 mm3. (A) Representative images of orthotopic breast tumors undergoing different treatments in earlystage 28 days after the administration of tGAS1 lentivirus or media in mice treated in early stage (30 mm3). (B) Growth oforthotopical tumors in response to the different treatments in early stage (30 mm3, n¼4). (C) Growth of orthotopical tumors inresponse to the different treatments in advanced stage (50 mm3, n¼6). LvtGAS1 and Control: treatments with tGAS1 lentivirus andculture media respectively. Arrows indicate control tumors and arrow heads tumors treated with tGAS1 lentivirus. nPo0.05;nnnPo0.001, ANOVA followed by Duncan's.

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tGAS1 decreases tumor vascularization

In addition to the decrease of tumor size, treatment with tGAS1appeared to reduce the vascularization of the tumors (Figs. 4Aand 5A). To further explore this aspect, we identified bloodvessels by immunohistochemistry with an anti VE-cadherin antibody[31] and counted the number of VE-cadherin positive areas pertumor in each condition. Fewer positive areas for VE-cadherin in

tGAS1 tumors were detected in comparison with controls, indicatingthat tGAS1 treatment reduced the tumor blood supply (Fig. 8).

Conditioned medium containing t-GAS1 decreases themigration of endothelial cells

Since migration of endothelial cells is a key event for angiogenesiswe tested this process in vitro in response to conditioned tGAS1

Fig. 7 – Treatment with tGAS1 decreases the phosphorylation of ERK1/2 in breast tumors. A) Western blot analysis of ERK1/2 inMDA MB 231 heterotopic tumors treated with tGAS1 or EGFP lentivirus or culture media. B) Densitometric analysis of pERK1/2 inMDA MB 231 heterotopic tumors normalized to total ERK1/2 (n¼3). Control: tumors treated with culture media, EGFP and tGAS1:tumors treated with EGFP and tGAS1 lentivirus respectively. nnPo0.01, ANOVA followed by Duncan's.

Fig. 8 – tGAS1 reduces the vascularization of tumors. A) Representative images of immunohistochemistry against VE-cadherin (red)in tumors. B) Percent of VE-cadherin positive areas per tumor in each condition (n¼4). tGAS1 and Control: treatments with tGAS1lentivirus and culture media respectively. Images of confocal microscopy captured at 40� /1.30, scale bars 50 μm in upper paneland 25 μm in lower panel. nPo0.05, Student's t-Test.

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medium. For this purpose, we plated endothelial cells in Boydenchambers with tGAS1 conditioned medium on the migration side.In the presence of medium from endothelial cells, tumor cellsshow a basal migration, but when incubated with tumor cellmedium without tGAS1, migration increases. In contrast, whentGAS1 conditioned medium was added, we observed a reductionof cell migration which is similar to the basal level (Fig. 9A and B).Employing the trypan blue assay we found that cell viability didnot change when compared to controls, thus indicating thattGAS1 inhibits endothelial cell migration, but does not inducecell death (Fig. 9C).Angiogenesis requires migration and proliferation of endothe-

lial cells. Since a crucial factor modulating these processes isVEGF, we next determined whether the level of VEGF was affectedby tGAS1. We observed very little difference in the amount of

VEGF between MDA–tGAS1 cells producing or not tGAS1 asdetermined by immuohistochemistry (Fig. 9D). Altogether thesedata suggest that tGAS1 inhibits endothelial cell migration by amechanism independent of VEGF.

Discussion

Breast cancer is the most frequent cancer in women worldwide,and although recent advances in the investigation of its molecularbasis have improved its treatment, new alternatives are needed.Here, we show that the expression of GAS1 and tGAS1 inhibits theproliferation of MDA MB 231 human breast cancer cells, retainingthem arrested in the G0/G1 phase (Fig. 2). This effect of GAS1is consistent with previous studies in NIH3T3 fibroblasts [10].

Fig. 9 – Conditioned medium containing tGAS1 decreases migration of endothelial cells by a mechanism independent of VEGF. (A)Endothelial cells exposed to tGAS1 conditioned medium reduce their migration, assessed by the Boyden chamber assay (n¼5). (B)Representative images of endothelial cells migration in Boyden chambers in the different conditions, (C) Conditioned media withtGAS1 has no effect on the viability of endothelial cells (n¼5). (D) VEGF immunocytochemistry on MDA tGAS1 cells with (þT) andwithout tetracycline incubation for 72 h and without primary antibody (w/Ab11). MDA–tGAS1 CM: conditioned media of 72 h fromMDA–tGAS1 cells, þtGAS1: conditioned medium with tetracycline, �tGAS1: conditioned medium without tetracycline, Control:conditioned media from endothelial cells. Images of light microscopy in B and epifluorescence microscopy in D captured at 40� /0.50, scale bar 50 μm. nnnPo0.001; ANOVA followed by Duncan's.

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Earlier reports indicated that the GPI anchor of GAS1 is notnecessary to induce its effects on cell growth arrest [32] and wehad previously demonstrated that a soluble form of GAS1 (tGAS1)impairs the viability of C6 glioblastoma cells and tumors derivedfrom these cells [17,18]. In this work, we demonstrated thecapacity of tGAS1 to induce cell arrest of breast cancer cells andto impede tumor growth in mice, thus suggesting that tGAS1 canbe used as an adjuvant therapy for the treatment of breast cancer.

The effect of GAS1 inhibiting glioma growth is caused by itscapacity to block the GDNF-signaling pathway involving theGFRα1-RET receptors and inducing apoptosis [14–16]. Interest-ingly the MDA MB 231 cells lack RET and GFRα1 [20,21] butexpress ARTN [23], a ligand of GDNF family that augments breasttumor growth and invasiveness. ARTN has been observed in othertypes of cancer such as endometrial, lung and pancreatic where itis related with proliferation and metastasis [23,33–35]. Never-theless the expression of its canonical receptor, GFRα3, remainedunknown. Here we report the expression of GFRα3 mRNA in MDAMB 231 cells (Fig. 2) suggesting that tGAS1 exerts its effect by

interfering with the interaction between GFRα3-ARTN since ourresults show an inhibition of tumor growth.Although RET is not present in MDA MB 231 cells, various

reports show that GFRα–GFL complexes can initiate signaling in aRET-independent manner through adhesion molecules such asNCAM inducing Fyn activation and causing inhibition of celladhesion [36,37]. There are also reports of the interaction ofGFRα–GFL complexes with integrins [38] which are involved inprocesses such as proliferation, growth, invasion, and cell survivalin cancer [39]. It has also been reported that blocking ARTN withneutralizing antibodies, completely inhibits ARTN-induced RETand ERK activation [40], which is in agreement with the reductionof ERK1/2 phosphorylation observed in MDA MB 231 cellsexpressing tGAS1 in the presence of ARTN, since these cells donot express RET, and thus the effect of tGAS1is mediated byinhibiting ERK1/2 (Fig. 4).ERK1/2 has an important participation in G1/S cell cycle

progression because the RAS/ERK/MAPK and RAS/PI3K pathwaysinactivate retinoblastoma (RB) blocking its growth inhibitory

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activity. Furthermore, the inhibition of ERK1/2 down-regulatesBCL-2 proteins and promotes tumor cell death [29,41]. Recently,signaling pathways initiated by NRTN and ARTN have beenidentified in peripheral sensory neurons with or without theparticipation of RET. It has been reported that ARTN induces thesensitization of sensory neurons inducing the release of CGRP(calcitonin gene-related peptide) in both Ret-dependent and Ret-independent manners through NCAM causing the activation ofERK and AKT [42]. In this work we demonstrate that tGAS1inhibits the activation of ERK in a RET-independent mannerwithout affecting AKT activation (Fig. 4, Supplementary 3),suggesting that tGAS1 in the absence of RET induces cell cyclearrest and not apoptosis by interfering with ERK and not throughthe activation of AKT.In addition we tested the effect of tGAS1 in vivo. For this

purpose MDA MB 231 cells were inoculated into the flanks of thehind-limbs and mammary glands of immunocompromised miceto induce the development of tumors. The results show thattGAS1 significantly reduced tumor growth in both models (Figs. 5and 6). We also observed that treatment with tGAS1 in the earlystages of tumor (30 mm3) is more effective because in tumors ofthis size, tGAS1 completely halted tumor growth indicating thatthe therapeutic potential of tGAS1 is higher in the early phases ofthe disease (Fig. 6).Finally ERK signaling is deregulated in approximately one-third

of all human cancers including breast cancer where its hyper-activity plays a role in the progression of metastasis [30,43]. Basedon these results we investigated the activation of ERK1/2 in MDAMB 231 tumors and found a significant reduction of the phos-phorylation of ERK1/2 in tumors treated with tGAS1 lentivirus incomparison with the controls, in agreement with the effectobserved in the MDA-tGAS1 cells in the presence of ARTN(Figs. 7 and 4). This shows an important role for tGAS1 in ERKactivation, conceivably through the interaction with adhesionmolecules, such as those involved in the RET-independent signal-ing of GFRαs including NCAM and integrins, [36–38].Since control tumors appeared to be more vascularized, we

investigated whether tGAS1 could inhibit tumor-induced angio-genesis. For this purpose we identified blood vessels by immu-nohistochemistry against VE-Cadherin as reported by Labelle et al.in 2008, and found that tumors treated with tGAS1 have fewerpositive signals for VE-Cadherin (Fig. 8). Thus, suggesting thattreatment with tGAS1 provokes a reduction of blood supply to thetumors, limiting their growth and reducing their metastaticcapacity.In an effort to determine if the reduced formation of blood

vessels in the tumor was a direct effect of tGAS1, we performedin vitro migration assays, and observed that medium containingtGAS1 reduced the migration of endothelial cells (Fig. 9). On theother, hand it was reported that the expression of GAS1 is inducedby VE-cadherin and VEGF [44]. Our results show that tGAS1decreases the migratory capacity of endothelial cells but does notaffect their viability when compared with controls. Moreover, theexpression of tGAS1 in MDA MB 231 cells seems to induce a verylimited effect on VEGF expression. We propose that the reductionof angiogenesis induced by treatment with tGAS1, is due to amechanism that involves the inhibition of ARTN-induced signal-ing, since it has been reported that this ligand promotes angio-genesis in cancer cells negative for the estrogen receptor, likeMDA MB 231 cells [25].

In cancer, the generation of new blood vessels is a criticalprocess to produce metastasis, which occurs when cells of theprimary tumor access the bloodstream and travel to distant sitesin the body where they create colonies and generate new tumorswhich continue to proliferate [28]. In accordance with the effectof tGAS1 on angiogenesis and a previous report in which theknockdown of GAS1 increased lung metastasis [19], we proposethat tGAS1 reduces the generation of breast cancer metastasis,and it will be relevant to further evaluate the effect of tGAS1 inthe formation of breast cancer metastasis.

Conclusion

We had previously demonstrated the ability of GAS1 and tGAS1 toinhibit the growth of gliomas and neuroblastoma cells by pre-venting the activity of the GDNF–RET signaling pathway [14–18].In the present work, we extend these observations showing thattGAS1 can also inhibit breast tumor growth, by interfering withanother branch of the GDNF-family of ligands-GFRαs pathway,namely blocking the ARTN/ERK1/2 signaling in a RET-independent manner. These results increase the potential use ofGAS1 in the treatment of different tumors.

Competing interests

The authors declare no conflict of interest.

Acknowledgments

We want to thank Dr. Mónica Díaz Coránguez and Dolores MartínTapia for their assistance with the endothelial cell migrationassays; Paula Vergara for technical support and Rubén Sánchezfor laboratory assistance. This work was partially supported by aConacyt (Mexico) Grant 127357 (J.S).

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.yexcr.2014.06.016.

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