IL13 Receptor a2 Signaling Requires a Scaffold Protein ...Pathways in Colon Cancer Metastasis Ruben A. Bartolome1, Irene García-Palmero1, Sofía Torres1, María Lopez-Lucendo2, Irina

Microenvironment and Immunology

IL13 Receptor a2 Signaling Requires a ScaffoldProtein, FAM120A, to Activate the FAK and PI3KPathways in Colon Cancer MetastasisRub�en A. Bartolom�e1, Irene García-Palmero1, Sofía Torres1, María L�opez-Lucendo2,Irina V. Balyasnikova3, and J. Ignacio Casal1

Abstract

IL13 signaling through its receptor IL13Ra2 plays a criticalrole in colon cancer invasion and liver metastasis, but themechanistic features of this process are obscure. In this study,we identified a scaffold protein, FAM120A (C9ORF10), as asignaling partner in this process. FAM120A was overexpressedin human colon cancer cell lines and 55% of human coloncancer specimens. IL13Ra2-FAM120A coimmunoprecipitationexperiments revealed further signaling network associationsthat could regulate the activity of IL13Ra2, including FAK, SRC,PI3K, G-protein–coupled receptors, and TRAIL receptors. Inaddition, FAM120A associated with kinesins andmotor proteins

involved in cargo movement along microtubules. IL13Ra2-trig-gered activation of the FAK and PI3K/AKT/mTOR pathways wasmediated by FAM120A, which also recruited PI3K and func-tioned as a scaffold protein to enable phosphorylation andactivation of PI3K by Src family kinases. FAM120A silencingabolished IL13-induced cell migration, invasion, and survival.Finally, antibody blockade of IL13Ra2 or FAM120A silencingprecluded liver colonization in nude mice or metastasis. Inconclusion, we identified FAM120A in the IL13/IL13Ra2 signal-ing pathway as a key mediator of invasion and liver metastasisin colon cancer. Cancer Res; 75(12); 2434–44. �2015 AACR.

IntroductionInterleukin 13 (IL13) has been associated to different pathologic

conditions (asthma, autoimmune diseases, ulcerative colitis, andothers; ref. 1). More recently, we demonstrated that IL13 signalingthrough IL13 receptor subunita-2 (IL13Ra2) plays a critical role incolon cancer invasion and liver metastasis (2). We described theoverexpression of IL13Ra2 in 66% of tumor samples frompatientswith colon cancer,whichwas associated to late stagesofprogression(metastasis in lymph nodes or liver) and poor outcome of patientswith colorectal cancer (2). IL13Ra2 is overexpressed in a variety ofhuman tumor types, such as colon, glioblastoma, renal cell carci-noma, pancreatic, melanoma, head and neck, mesothelioma, andovarian, where it has been proposed as biomarker and potentialtherapeutic target (2–10). In glioblastoma multiforme, IL13Ra2expression occurs in 75% of tumors and is associated with poorprognosis (3). A similar situation occurs for head and neck squa-mous cell carcinoma (8) and ovarian cancer (9).

IL13 binding to IL13Ra2 induced a significant increase in celladhesion, migration, and invasion capacity of colorectal cancercells (2). Cell signaling was independent of type II IL4 receptorand IL13Ra1 receptor as KM12 cells did not express thesealternative IL13 receptors on the cell surface (2). Moreover,silencing of IL13Ra2 increased mice survival and provoked aclear reduction in liver colonization in mouse xenograft models(2). Although initially was considered a decoy receptor (11),there are multiple evidences that IL13Ra2 is functional andinduces the activation of several pathways and the transcriptionfactor AP-1, inducing the expression of TGFb (2, 12, 13).IL13Ra2 cytoplasmic domain is very short, consisting of only14 amino acids residues, which does not contain any recog-nizable motif and makes difficult its interaction with otherproteins. Nevertheless, this receptor participates in signal trans-duction, triggering the activation of several signaling proteins,such as PI3K and Src kinases (2, 12, 13). Little was known aboutthe signaling mechanisms of IL13 through IL13Ra2 in metas-tasis and cancer progression.

Here, we have identified the molecular partners of IL13Ra2and the mechanisms of signal transduction by using immuno-precipitation experiments combined with a proteomicapproach. We identified the adaptor FAM120A as the scaffoldprotein necessary for IL13Ra2-mediated signaling. FAM120A,also known as c9orf10 or OSSA (oxidative stress-associated Srcactivator), was required for the activation and recruitment ofFAK, Src, PI3K, and most of the proteins involved in IL13Ra2signaling, providing an overall picture of IL13 signaling incolorectal cancer cells and its relevance in liver metastasis.FAM120A and/or IL13Ra2 targeting abolished liver coloniza-tion in a mouse model.

1Department of Cellular and Molecular Medicine, Centro de Investiga-ciones Biol�ogicas (CIB-CSIC), Madrid, Spain. 2Proteomics Facility,Centro de Investigaciones Biol�ogicas (CIB-CSIC), Madrid, Spain. 3TheBrain Tumor Center, University of Chicago, Chicago, Illinois.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: J. Ignacio Casal, Functional Proteomics Laboratory,Centro de Investigaciones Biol�ogicas (CIB-CSIC), Ramiro de Maeztu, 9, 28040Madrid. Spain. Phone: 34-91-8373112; Fax: 34-91-5360432; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-14-3650

�2015 American Association for Cancer Research.

CancerResearch

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Materials and MethodsCell culture and reagents

KM12C and KM12SM human colon cancer cells were obtainedfrom I. Fidler's laboratory (MD Anderson Cancer Center. Hous-ton, TX). These cell lines were authenticated by short tandemrepeat analysis. Other cell lines were obtained directly from theATCC. These cell lines were passaged fewer than 6 months afterpurchase for all the experiments. All cell lines were cultured inDMEM (Gibco-Life Technologies) containing 10% FCS (Sigma-Aldrich) and antibiotics at 37�C in a 5% CO2-humidifiedatmosphere.

IL13was used at 10ng/mL andpurchased fromPeproTech. PP2(used at 3 mmol/L), PP3 (3 mmol/L), and UO126 (5 mmol/L)inhibitors were from Calbiochem. LY294002 (25 mmol/L) wasfromSigma-Aldrich and FAK inhibitor 14 (10mmol/L) fromSantaCruz Biotechnology. All antibodies used in this article are listed inSupplementary Table S1.

siRNAs transfectionsFor transient transfections, siRNAs targeting specifically

FAM120A (SASI-Hs01-00149752, Sigma-Aldrich) or controlsiRNAs were transfected with JetPrime (Polyplus Transfection)according to the manufacturer's instructions.

Western blot analysis and immunoprecipitation assaysCells were detached, washed, and lysed with protease and

phosphatase inhibitors in lysis buffer (1% Igepal, 100 mmol/LNaCl, 2 mmol/L MgCl2, 10% Glycerol in 50 mmol/L Tris-HCl).Protein extracts were separated in SDS-PAGE, transferred to nitro-cellulose membranes, which were incubated with primary anti-bodies (Supplementary Table S1) followed by incubation witheither HRP-anti-mouse IgG (Thermo Scientific) or HRP-anti-rab-bit IgG (Sigma-Aldrich). Specific reactive proteins were visualizedwith SuperSignal West Pico Chemiluminescent Substrate(Thermo Scientific).

For immunoprecipitation, cells were lysed and 500 mg of celllysate were incubated with the indicated antibodies. The immu-nocomplex was captured by adding 100 mL of Protein G-sephar-ose beads (Sigma-Aldrich). After washing, samples were resus-pended in 2� Laemmli buffer, boiled for 5 minutes, centrifugedand subsequently loaded onto SDS-PAGE gels, which were ana-lyzed by Western blot analysis. As a control, we incubated thelysates with an unrelated IgG coupled to Sepharose beads todiscard unspecific proteins.

Mass spectrometry of immunoprecipitated proteinsFor proteomic analysis, 10 mg of cell lysates were immuno-

precipitated as before, and the proteinswere loaded in SDS-PAGE,whichwere divided in three slices for in-gel digestionwith trypsin.Peptides were trapped onto a C18-A1 2 cm precolumn (ThermoScientific) and separated on a Biosphere C18 column (Nano-Separations) using a flow rate of 250 nL/minute in a 100-minutegradient from 0% to 95% Buffer B (0.1% formic acid in aceto-nitrile) on a nanoEasy HPLC coupled to a nanoelectrospay ionsource (Proxeon). Mass spectra were acquired in a linear ion trapquadrupole (LTQ) Orbitrap Velos in data-dependent mode withan automatic switch between mass spectrometry and MS/MSscans using a top 15 method. Full scans were acquired in theOrbitrapwith amass rangeof 400 to1,200Th, a target valueof 106

ions and a resolution of 3� 104 (atm/z 400). The 15most intense

ions were submitted to collision-induced dissociation in the LTQusing normalized collision energy of 35%and a target value of 104

ions. Dynamic exclusion was enabled with a repeat count of oneand exclusion duration of 30 seconds. Mass spectra were searchedusing SEQUEST search enginewithProteomeDiscoverer (ThermoScientific) against the Uniprot Database. Search parametersincluded precursor and fragment mass tolerances of 10 p.p.m.and 0.8 Da, respectively, a maximum of two missed cleavagesallowed, a fixed modification of carbamidomethyl cysteine and avariable modification of methionine oxidation. Identified pep-tides were validated using Percolator algorithm with a q-valuethreshold � 0.01.

IHC analysisParaffin samples were obtained from 119 patients diagnosed

and treated of colorectal adenocarcinoma between 2001 and2014 in Fundaci�on Jim�enez Díaz (Madrid, Spain). Clinicopatho-logical data are shown in Supplementary Table S2. Each samplewas deparaffinated for antigen retrieval using citrate sodiumbuffer at pH 6.0 for 20 minutes and subsequent incubation withthe primary antibody against FAM120A. The reaction wasrevealed using DAB as chromogen and hematoxylin for counter-staining. In all cases, sections fromnormal colonicmucosa distantfrom the tumor site were used as negative controls.

Wound healingCells were cultured to confluence in Matrigel-coated plates

(0.4 mg/mm2). A 1-mm wide wound was done across the mono-layer. Cells were incubated in serum-free medium containingIL13, inhibitors, and antibodies. Pictures were taken either imme-diately (0 hour) or after 48 hours in culture at 37�Cafter the injury.Migration was quantified as a percentage of wound closure.

Invasion assaysFor Matrigel invasion assays, 6� 104 KM12C or KM12SM cells

were resuspended in invasion medium (serum-free DMEM con-taining 0.4% BSA) and loaded onto 8 mm pore-size filters coatedwith 35 mL ofMatrigel (1:3 dilution; BD Biosciences) in Transwellplates (Costar) in presence of inhibitors or antibodies. The lowercompartment of the invasion chamber was filled with mediumcontaining IL13 or with medium alone. After 22 hours at 37�C,noninvading cells were removed from the upper surface of thefilter, and cells that migrated through the filter were fixed with 4%paraformaldehyde (Sigma-Aldrich), stained with crystal violet,and counted under a microscope.

Survival assaysCellswere incubatedwith1mmol/LH2O2 for 16hourswithout

serum in the presence or absence of IL13, antibodies or inhibitors.Cells were detached and incubated with FITC labeled-Annexin V(Miltenyi Biotec Inc.) and propidium iodide according to themanufacturer's instructions, and analyzed by cytofluorometry(Coulter Epics XL).

Flow cytometryCells were detached with 2 mmol/L EDTA in PBS, incubated at

4�C with primary antibodies (10 mg/mL) for 30 minutes, washedand incubated with Alexa 488 labeled-secondary antibodies(Dako). Fluorescence was analyzed in a Coulter Epics XLcytofluorometer.

IL13Ra2 Signaling in Colorectal Cancer Metastasis

www.aacrjournals.org Cancer Res; 75(12) June 15, 2015 2435




ImmunofluorescenceKM12SM cells were cultured onto Matrigel-coated coverslips,

treated with or without IL13 (10 ng/mL) for 5 minutes, and fixedwith 1%paraformaldehyde in PBS containing 0.1%Triton X-100.Cells werewashed three timeswith PBS and incubated 30minuteswith primary antibodies (Supplementary Table S1), washed againand incubated 25minuteswith secondary antibodies labeledwithAlexa-468 or Alexa-568, and40,6-diamidino-2-phenylindole (Life

Technologies). Then, samples were mounted with MountingFluorescence Medium (Dako) and images were captured usinga TCS-SP5-AOBS confocal microscope (LEICA) with a 100� oilimmersion objective.

Metastasis experiments in nude miceThe Ethical Committee of the Consejo Superior de Investiga-

ciones Científicas (Madrid, Spain) approved the protocols used

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Figure 1.FAM120A associates with IL13Ra2. A, classification of IL13Ra2-coimmunoprecipitated proteins detected by proteomics assays in KM12SM cells accordingto their location (left) and function (right). B, protein interaction network for IL13Ra2. C, verification of FAM120A–IL13Ra2 interaction by immunoprecipitation andWestern blot analysis. D, expression of FAM120A in the indicated human colorectal cancer cell lines by Western blotting. E, by IHC, 55% of patients withcolon cancer (n¼ 119) expressed FAM120A in tumor samples [25% showed low/moderate expression (FAM120Alo) and 30% high expression (FAM120Ahi)]. IL13Ra2was positive in 65% of patient samples (left). Representative images of FAM120A expression intensity in colorectal cancer samples and adjacent normalcolon tissue of three patients obtained by IHC (right). F, patients with tumors coexpressing IL13Ra2 and high levels of FAM120A showed an increased risk ofrecurrence at 18 months (� , P < 0.05).

Bartolom�e et al.

Cancer Res; 75(12) June 15, 2015 Cancer Research2436




for experimental work withmice. Swiss nudemice (Charles River;n ¼ 10 per condition) were inoculated in spleen with 106

KM12SM cells in 0.1mL PBS. After 22 hours, mice were subjectedto removal of the spleen. Mice were daily inspected for signs ofdisease, such as abdominal distension, locomotive deficit, ortumor detectable by palpation. When signs were visible, micewere euthanized, subjected to necropsy, and inspected for metas-tasis in liver.

For liver colonization assessment, mice were inoculated asbefore and euthanized at various times after cells inoculation(2, 8, 24, and 56 hours). RNAwas isolated from liver using TRIzol(Invitrogen), retrotranscribed and 0.3 mg cDNA subjected to PCRwith Taq DNA polymerase (Invitrogen) to amplify humanGAPDH as previously described (14). As a control, a 20 cycles-amplification of murine b-actin was performed.

Statistical analysesData were analyzed by one-way ANOVA followed by the

Tukey–Kramer multiple comparison test. The significance of thedifference for survival curves was estimated with the log-rank test,whereas the differences in early recurrencewere analyzed by the c2

test. In all analyses, the minimum acceptable level of significancewas P < 0.05.

ResultsIdentification of protein partners for IL13Ra2

To characterize the protein interaction network of IL13Ra2, celllysates of the highly metastatic cell line KM12SM were immuno-precipitated using anti-IL13Ra2monoclonal antibody coupled toSepharose beads. An irrelevant antibody was used as a control.Precipitated proteins were fractionated by SDS-PAGE followed bymass spectrometry analysis. We identified 158 proteins coimmu-noprecipitated exclusively with anti-IL13Ra2 antibody, usingPercolatorwith q value�0.01 (Fig. 1A). Proteinswithin a locationbiologically inaccessible for amembrane protein (such as nuclear,mitochondrial, and peroxisomal) or those proteins involved inirrelevant protein-binding functions as ribosomal proteins, cha-perons, and common background proteins (15) were removedfrom further analysis. At the end, five proteins (SupplementaryTable S3) were selected as the best candidates for specific inter-action with IL13Ra2. In addition, we identified other 22 proteinsinvolved in vesicle trafficking from Golgi and endoplasmaticreticulum to cell membrane (Supplementary Table S4). One ofthe five proteins, FAM120A (Family with sequence similarity120A), a scaffold protein, was related to the Src family kinases(16), which were already known to be involved in IL13Ra2signaling (2). Amongst the other four identified proteins wereEPHB4, a receptor tyrosine kinase that mediates ephrin signaling;CMTM6, an unknown transmembrane protein; and PDZD8 andACTB proteins, which are involved in cytoskeletal organization(Fig. 1B).

The association of FAM120A with IL13Ra2 was confirmed bycoimmunoprecipitation in four human colorectal cancer cell linesand further detection by Western blot analysis (Figs. 1C and 2D).Expression of FAM120A was demonstrated in a panel of ninehuman colorectal cancer cell lines. All of them were positive,except Caco2 (Fig. 1D). To study the clinical relevance ofFAM120A, we carried out immunochemical analysis of 119paired colorectal cancer patient samples. We observed cyto-plasmic staining of FAM120A [ranging from low/moderate

(FAM120Alo) to intense (FAM120Ahi)] in 55% of tumor samples,whereas adjacent normal tissue samples showed a very weakexpression (Fig. 1E). Patients with tumors expressing high levelsof both, IL13Ra2 and FAM120A, showed a significantly increasedrisk of recurrence at 18 months (Fig. 1F) compared with the restof patients. These results indicate a significant overexpressionof FAM120A in colon cancer, and the association of such over-expression with a higher risk of early recurrence in combinationwith IL13Ra2.

Identification of FAM120A protein networksTo identify protein networks containing FAM120A, KM12SM

lysates were subjected to immunoprecipitation using anti-FAM120A antibodies followed by mass spectrometry. Afterdiscard proteins coimmunoprecipitated with control beads andthose placed in inaccessible place for a cytosolic protein, 222proteins were found to interact with FAM120A (Fig. 2A). Asbefore, we removed 131 proteins based on their protein-bind-ing functions (i.e., ribosomal, chaperones, etc.). From theremaining 91 proteins, 48 were involved in protein transport.This large number of proteins involved in vesicle trafficking, notcommonly associated with cytosolic proteins, suggests an addi-tional role for FAM120A in protein transport (SupplementaryTable S5).

The remaining 43 proteins associated with FAM120A (Supple-mentary Table S6) were subjected to GO analysis (Fig. 2B).FAM120A interactors were involved in actin cytoskeleton dynam-ics, cell signaling, and apoptosis and cell adhesion. According toSTRING interaction analysis and data mining, FAM120A formspart of different protein complexes, such as focal adhesion com-plexes, G-protein–coupled receptors, and TNFRSF10B receptor(also known as TRAILR2; Fig. 2C). A selection of the proteinscoimmunoprecipitated with FAM120A, together with other pro-teins involved in signaling, cell adhesion, cell death, and actincytoskeleton dynamics (FAK, Src, HCK, RAC, RHOA, PI3K, AKT,RAF1, ERK1/2, and CASP8) was confirmed for association withFAM120A by immunoprecipitation and Western blot analysis(Fig. 2D). No association was found between the adaptor proteinFAM120A and IL13Ra1or Ras, in agreement with data obtainedby mass spectrometry.

To study subcellular localization of IL13Ra2 and FAM120A,wecarried out confocal microscopy analysis (Fig. 2E). IL13Ra2showed an intracellular distribution, with some surface staining,whereas FAM120A staining was cytosolic and associated tomem-brane. In addition, we tested AKT as surrogate for signalinginteraction. There was a clear colocalization of IL13Ra2 withFAM120A and AKT, showing a slight increase for AKT after 5minutes of IL13 treatment.

Together, these results confirm the colocalization of IL13Ra2with FAM120A and other signaling molecules.

IL13 signaling through IL13Ra2 requires FAM120AWe explored the kinetics of IL13 signaling through IL13Ra2 in

KM12SM cells. IL13 induced the activation of mTOR, FAK, Srcfamily kinases, AKT, and ERK1/2, along with a decrease of p53levels (Fig. 3A). Then, we studied the interaction of cell signalingproteins with FAM120A upon treatment with IL13 (Fig. 3B). FAK,PI3K, Src, Fyn, AKT, and p53 showed a quick association withFAM120A at 5 minutes after treatment with IL13 and furtherdissociation at 60 minutes. ERK1/2 MAP kinases showed alsoassociation to FAM120A after 5minutes, but remained associated






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for longer times. Although RHOA decreased, RAC increased withtime, supporting the high invasive capacity of these cells. Incontrast, IL13Ra2 remained permanently associated withFAM120A.

To study the role of FAM120A in IL13 signal transduction incolon cancer, KM12SM cells were FAM120A silenced with specificsiRNAs or treated with scrambled siRNAs (Fig. 3C). Silencingcaused a loss of mTOR, FAK, and AKT activation after IL13

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Figure 3.Signaling triggered by IL13 is partlycontrolled by FAM120A. A, KM12SMcells were exposed to IL13 for theindicated times, lysed, and theextracts were resolved in SDS-PAGEand analyzed with the indicatedantibodies by Western blot analysis.RHOGDI was used as loading control.B, Western blot analysis showing theresults of KM12SM cells treated withIL13 for 0, 5, and 60 minutes andsubjected to immunoprecipitationusing anti-FAM120A or controlantibodies. C, Western blot analysisshowing the results of KM12SM cellstransfected with FAM120A or controlsiRNAs. D, knocked down KM12SMcells or control cells were exposed toIL13 for the indicated times, lysed, andanalyzed as in A. E, Western blotanalysis showing the results of cellstreated with mock- and Src-inhibitors,PP3 and PP2, respectively, and IL13for the indicated times.

Figure 2.Proteins coimmunoprecipitated with FAM120A. A, classification of FAM120A-coimmunoprecipitated proteins detected by proteomics assays in KM12SMcells. B, functionof proteins that interactwith FAM120A. C, representationof FAM120A-coimmunoprecipitated proteins according todifferent functional clusters (cellprotrusion, GPCR, and death receptor). D, confirmation of FAM120A-coimmunoprecipitated proteins by immunoprecipitation and Western blot analysis. E,KM12SM cells treated or not with IL13 were used to determine IL13Ra2, FAM120A, and AKT localization by immunofluorescent confocal microscopy. IL13Ra2 showedmembrane and intracellular expression with a clear colocalization with FAM120A and AKT.






treatment. In contrast, activation of Src family kinases andERK1/2were not affected by FAM120A silencing (Fig. 3D). To examine theeffect of Src in IL13 signaling, we used the PP2 inhibitor inKM12SM cells treated with IL13. PP2 caused a marked inhibitionof mTOR, AKT, and ERK1/2, but not FAK (Fig. 3E). In contrast,PP3, a mock inhibitor, did not affect Src pathway activation.Together, these results indicate that IL13 signaling requires Srcactivation for AKT/PI3K activation together with FAM120A.How-ever, Src activation was FAM120A independent.

FAM120A is required for IL13-induced cellmigration, invasion,and survival

To characterize FAM120A in cell invasion and metastasisinduced by IL13, we tested two metastatic cell lines, KM12SMand SW620. After FAM120A silencing, KM12SM or SW620 cellsknocked down for FAM120A or scrambled were treated withIL13 alone or combined with Src, PI3K, FAK, andMEK inhibitors.As a control, we performed the experiments in the presence ofanti-IL13Ra2 or control antibodies competing with IL13 forIL13Ra2 binding (17). IL13 provoked a strong increase in themigratory and, in particular, invasive capacity of KM12SM andSW620 cells (Fig. 4A and B). This increase was abolished afterFAM120A silencing. In SW620, the silencing of FAM120A inhib-ited also basal migration, indicating that the presence of thisprotein is required for cell motility. Treatment with Src, PI3K and,at a minor extent, FAK inhibitors caused a similar effect toFAM120A silencing and similar to anti-IL13Ra2 blocking.Regarding cell survival, IL13 induced a significant increase inviable KM12SM cell number in control cells, but not inFAM120A-silenced cells. IL13-mediated increase in cell survivalwas inhibited by all the inhibitors and, more strongly, by PI3Kand FAK inhibitors (Fig. 4C). In SW620 cells, IL13 also promotedcell survival; but this effect wasmoremoderate, probably becauseSW620 cells had a strong basal resistance to oxidative stress.Collectively, these data indicate that FAM120A, Src, PI3K, andFAK are critical mediators in IL13-induced cell migration, inva-sion, and survival.

FAM120A controls IL13Ra2 location in cell membraneWe also investigated the role of FAM120A in protein trafficking

and location. By flow cytometry, KM12SM and KM12C cellsknocked down for FAM120A displayed lower levels of IL13Ra2in cell membrane (Fig. 5A). However, the total amount ofIL13Ra2 was identical in FAM120A-silenced or control cells byWestern blot analysis (Fig. 5B). These results suggest thatFAM120A participates in the traffic and location of IL13Ra2 incell surface and, therefore, in the functionality of this receptor. Toprovide further evidence, we verified the association of FAM120Awith some of the identified proteins involved in protein traffic,such as MYH14, COPE, or Rab1A/B GTPase by Western blotanalysis (Fig. 5C). In addition, treatment of KM12SM cells withanti-IL13Ra2 antibody showed that anti-IL13Ra2 promotedIL13Ra2 degradation (Fig. 5D), probably by a mechanism of cellinternalization mediated by lysosomes. This effect could contrib-ute to the blocking capacities of the anti-IL13Ra2 antibody.

FAM120Aplays a key role in livermetastasis of colorectal cancerFinally, we studied the effect of FAM120A in cancer metastasis

in vivo. FAM120A-silenced or control KM12SM cells, in the pres-ence of anti-IL13Ra2or control antibodies, were inoculated in thespleen of nude mice. Spleens were removed 24 hours after

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Figure 4.FAM120A controls IL13-triggered cell migration, invasion, and survival.A–C, KM12SM and SW620 cells were transfected with control or FAM120A-targeting siRNAs and subjected to wound-healing assays (A), cell invasionassays through Matrigel (B), and survival to oxidative stress inducedby H2O2 (C). In addition, KM12SM cells were treated with anti-IL13Ra2or control antibodies (5 mg/mL) and the indicated inhibitors. Cellmigration/invasion/survival was significantly increased by addition of IL13(� , P < 0.05; ��, P < 0.01; �� , P < 0.001) and decreased by treatment withthe indicated inhibitors or antibodies (^, P < 0.05; ^^, P < 0.01;^^^, P < 0.001). D, Western blot analysis showing the results of SW620cells transfected with FAM120A or control siRNAs.

Bartolom�e et al.





inoculation, to prevent the formation of tumors in spleen. About60% of mice inoculated with control cells and control antibodyshowed severe signs of disease and were euthanized (Fig. 6A).Subsequent necropsy revealed macroscopic metastasis in liver(Fig. 6B). In contrast, mice inoculated either with FAM120Aknocked down cells or treated with anti-IL13Ra2 antibodies didnot develop disease, except for a mouse inoculated withFAM120A knocked down cells, which developed a tumor inperitoneal cavity without liver metastasis (Fig. 6A). After 120days postinoculation, surviving mice did not show macroscopicmetastasis in liver (Fig. 6B).

In addition, time-assays were performed to analyze the behav-ior of tumor cells in the first 3 days after inoculation. For thatpurpose, mice were euthanized between 2 hours and 56 hoursafter inoculation and RNA was isolated from the livers. After PCRamplification, human GAPDH, as a surrogate marker, wasdetected 2 hours after inoculation in all livers at similar levels,independently of the treatment. This quick detection was prob-ably due to fluid flow and cell travelling via the portal circulationto the liver within minutes. After 8 hours, PCR amplificationshowed a marked reduction, except for control mice. Moreover,bands were barely discernible at 24 or 56 hours after inoculationfor cells transfected with FAM120A siRNAs or treated with anti-IL13Ra2 antibodies, except for control siRNAs and control anti-bodies (Fig. 6C). These results suggest that FAM120A andIL13Ra2 are required for IL13-mediated engraftment or coloni-zation of colorectal cancer cells, increasing tumor cell adhesionand survival in the liver after cell arrival.

DiscussionIn this report, we describe a key role for FAM120A, also known

as OSSA or C9orf10 (16), in the signal transduction of IL13

through IL13Ra2 for the migration and invasion of colon cancercells. FAM120A was identified as a scaffold protein that interactswith IL13Ra2 using mass spectrometry and coimmunoprecipita-tion experiments. FAM120A is an abundant cytosolic protein thatshowed associationwith, at least, three protein networks involvedin focal adhesion, GPCR and TRAIL receptors. The expression ofFAM120A was required for the IL13-triggered Src and PI3K/AKT/mTOR signaling pathway activation (Fig. 7). Besides its scaffoldactivity, FAM120Awas associatedwithproteins involved inmotorproteins (kinesins) involved in cargo movement that could mod-ulate the presence of IL13Ra2 on the cell surface or its secretion(Fig. 7). Finally, the blocking of IL13Ra2 with specific antibodiesor the silencing of FAM120A strongly decreasedmetastatic growthand dissemination in colon cancer cells, providing two powerfulindependent strategies for metastasis control.

The binding of IL13 triggers the recruitment of several mole-cules to the IL13Ra2–FAM120A complex, such as FAK, PI3K, AKT,Src, p53, ERK1/2, and changes in the small GTPases associated tothe complex (RAC instead of RHOA). FAM120A recruits PI3K andworks as a scaffold protein for PI3K and the Src family kinases(SFK), enabling phosphorylation and activation of PI3K (16).Silencing of FAM120A impaired the activation of PI3K, AKT, andmTOR in the metastatic cells. However, it did not alter theactivation of SFKs as the activation levels, based on its phosphor-ylation status, are similar in FAM120A-silenced cells than incontrol siRNA transfected cells. The activation of AKT was depen-dent of Src kinase, confirming that FAM120A is the scaffoldrequired for Src-mediated PI3K activation, as previously reported(16). Along this line, a protector role to guard cells from oxidativestress-induced apoptosis by activation of SFKs was given toFAM120A (16). In addition, FAM120A could recruit FAK intothe IL13Ra2 complex to facilitate their mutual activation. Prob-ably, transient FAK dimerization leads to autophosphorylation in

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Figure 5.FAM120A regulates IL13Ra2 expression levels in cell membrane. A, flow cytometric-analysis showing the expression of IL13Ra2 in the surface of KM12Cand KM12SM cells FAM120A-silenced or control. B, Western blot analysis was performed to detect the lack of changes in total IL13Ra2 in the same cells afterFAM120A silencing. RHOGDI was used to assess total protein content. C, verification of FAM120A-interacting proteins involved in protein transport afterimmunoprecipitation and Western blot analysis. D, Western blot analysis showing the results of KM12SM cells treated with anti-IL13Ra2 or control antibody for theindicated times. A marked decrease of IL13Ra2 after antibody treatment was observed after 6 hours.






Tyr397, which increases the catalytic activity of FAK (18, 19). Thepresence of b1 integrin together with FAK in the FAM120Anetwork favors metastasis and cancer progression due to theactivation of the integrin signaling pathway that promotes tumorgrowth (20, 21). Activation of both pathways by IL13 in coloncancer cells required the presence of FAM120A.

Scaffold proteins modulate cell signaling by tethering individ-ual pathway components together and isolating different path-ways from one another. Scaffold proteins can establish connec-tions between pathways in order to distribute signals (22). Fur-thermore, many scaffold proteins interact with kinesin proteinsand are involved in cargo movement (23). In our proteomicanalysis, we identified a large number of proteins involved intransport, including kinesin proteins (KIF). Knocking downFAM120A decreased IL13Ra2 presence on cell surface, withoutaltering total IL13Ra2 expression. Therefore, FAM120A wouldplay a dual role: (i) IL13Ra2 transport and location and (ii)bringing together IL13Ra2 with FAK, PI3K, and SFKs. BecauseFAM120A modulates IL13Ra2 expression on cell surface,

FAM120A silencing would impair IL13Ra2 access to cell mem-brane, avoiding the interaction with IL13 and precluding signal-ing pathway activation.

Regarding other protein networks, FAM120A was also asso-ciated to TNFRSF10B (a proapoptotic death receptor for TRAIL)and caspase-8. The binding of TRAIL to TNFRSF10B leads tocaspase activation and initiation of apoptosis (24). Further inves-tigation is required to clarify whether IL13Ra2 contributes toresistance to apoptosis and survival by sequestering FAM120Afrom the TRAIL complex in addition to the activation ofPI3K. On the other hand, TRAIL signaling promotes a proinflam-matory pathway (25) that can establish a relationship with asimilar IL13 activity. Finally, FAM120A showed an associationwith GPR56, a G-protein–coupled receptor for type III collagen,which activates the RHOA pathway (26). RHOA overexpressionis usually associated with elevated lethality and aggressive pro-liferation (27, 28). Therefore, IL13 might have different roles,through FAM120A, in the activation of different pathways thatnot only promote cell migration and invasion, but also adhesion,

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Figure 6.FAM120A and IL13Ra2 are required forliver homing and metastasis. A,Kaplan—Meier survival results fornude mice inoculated in spleen withKM12SM cells previously transfectedwith FAM120A or control siRNAs andtreated with anti-IL13Ra2 or controlantibody at 5 mg/mL. Either silencingof FAM120A or treatment with anti-IL13Ra2 antibody significantlyincreased mice survival (� , P < 0.05;�� , P < 0.01). B, percentage of micewith macroscopic metastasis in liverwas significantly reduced in miceinoculated with FAM120A-silenced oranti-IL13Ra2–treated KM12SM cells(left; P < 0.01). Right, images of liversof mice treated as in A showingmetastasis. C, at the indicated times,RNA was isolated from livers ofinoculated mice and subjected toRT-PCR assays to detect humanGAPDH as surrogate of cellcolonization. Murine b-actin wasused as loading control.


Bartolom�e et al.




survival, andproliferation. These events facilitate the colonizationby tumor cells and the subsequent formation of metastasis. Theidentification of IL13Ra2 signaling should help to understand thesignaling events of IL13 inother cancers, asthma, ulcerative colitis,and other diseases.

In summary, FAM120A is a scaffold protein required for theproper IL13Ra2-trigger signaling, which leads to colon cancer cellactivation and liver metastasis. FAM120A modulates IL13Ra2location and transport between the cell membrane and intercel-lular compartments as well as the activation of different signalingpathways. Our results with ametastaticmousemodel suggest thatthe silencing of FAM120A is as effective as the treatmentwith anti-IL13Ra2 blocking antibodies (17) in preventing liver coloniza-tion by colorectal cancer cells and the formation of macroscopicliver metastases. These results support the use of FAM120A aspotential target for colon cancer therapy and reinforce the ther-apeutic value of IL13Ra2.

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

Authors' ContributionsConception and design: R.A. Bartolom�e, J.I. CasalDevelopment of methodology: R.A. Bartolom�e, I. García-Palmero, S. Torres,I.V. Balyasnikova

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R.A. Bartolom�e, I. García-Palmero, S. Torres,M. Lopez-Lucendo, I.V. BalyasnikovaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):R.A. Bartolom�e, I. García-Palmero, S. Torres, J.I. CasalWriting, review, and/or revision of the manuscript: R.A. Bartolom�e,I.V. Balyasnikova, J.I. CasalAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Lopez-Lucendo, J.I. CasalStudy supervision: J.I. Casal

Grant SupportR.A. Bartolom�e was supported by a contract to established research

groups of the Asociaci�on Espa~nola Contra el C�ancer (AECC). I. García-Palmero was supported by a contract from Comunidad de Madrid. S. Torreswas a recipient of a Juan de la Cierva fellowship. M. Lopez-Lucendo wassupported by a Proteored-ISCIII contract. This work was supported bygrants BIO2012-31023 from the Spanish Ministry of Economy and Com-petitiveness, grant to established research groups (AECC), grant S2011/BMD-2344/(Colomics2) from Comunidad de Madrid and ProteoRed-ISCIII platform.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedDecember 12, 2014; revisedMarch 30, 2015; accepted April 3, 2015;published OnlineFirst April 20, 2015.

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IL13Rα2

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ERK1/2

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FAK

MAPK2K2

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2015;75:2434-2444. Published OnlineFirst April 20, 2015.Cancer Res Rubén A. Bartolomé, Irene García-Palmero, Sofía Torres, et al.

Metastasisto Activate the FAK and PI3K Pathways in Colon Cancer 2 Signaling Requires a Scaffold Protein, FAM120A,αIL13 Receptor

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IL13 Receptor a2 Signaling Requires a Scaffold Protein ...Pathways in Colon Cancer Metastasis Ruben A. Bartolome1, Irene García-Palmero1, Sofía Torres1, María Lopez-Lucendo2, Irina

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