High Expression of IL-13 Receptor a2 in Colorectal Cancer ... · for this receptor in cell adhesion, migration, invasion, and metastatic colonization of colorectal cancer cells. A

Microenvironment and Immunology

High Expression of IL-13 Receptor a2 in Colorectal CancerIs Associated with Invasion, Liver Metastasis, and PoorPrognosis

Rodrigo Barderas1, Rub�en A. Bartolom�e1,María Jes�us Fernandez-Ace~nero2, Sofia Torres1, and J. Ignacio Casal1

AbstractAutocrine secretion of cytokines bymetastatic colorectal cancer cells and their role during invasion and liver

homing has been poorly characterized. In this study, we used cytokine arrays to analyze the secretomes ofpoorly and highly metastatic colorectal cancer cells. Compared with poorly metastatic cancer cells, highlymetastatic cells expressed increased levels of the immunosuppressive cytokines interleukin (IL)-4 and IL-13 inaddition to increased surface expression of the high affinity IL-13 receptor IL-13Ra2, suggesting that IL-13Ra2mediates IL-13 effects in colorectal cancer cells. Silencing of IL-13Ra2 in highly metastatic cells led to adecrease in adhesion capacity in vitro and a reduction in liver homing and increased survival in vivo, revealing arole for this receptor in cell adhesion, migration, invasion, andmetastatic colonization. In support of this, IL-13signaling activated the oncogenic signaling molecules phosphoinositide 3-kinase, AKT, and SRC in highlymetastatic cells. Clinically, high expression of IL-13Ra2 was associated with later stages of disease progressionand poor outcome in patients with colorectal cancer. Our findings therefore support a critical role for IL-13Ra2expression in colon cancer invasion and metastasis. Cancer Res; 72(11); 2780–90. �2012 AACR.

IntroductionMetastasis is the final step of the malignant transformation,

being responsible for the fatal outcome in patients with cancer.However, metastasis is a complex process that involves anumber of different and sequential steps. Cells must detachfrom the primary tumor, migrate, gain access to blood orlymphatic vessel, and colonize a new target organ (1). There-fore, metastasis cannot be considered as a single process,but as a collection of different events, all of them exhibitingdifferent molecular traits. Migration, invasion, and survival arefundamental aspects of the process.

Dissemination and metastasis of cancer cells depend onextensive interactions within the tumor microenvironment(2–3). A number of these interactions are regulated throughchemokines and their receptors, which govern many differentaspects of the malignant phenotype. So, it is critical to char-acterize chemokines and their receptors expression in meta-

static cells. Chemokine-mediated inflammation has beenshown to play an important role in tumor biology by influ-encing tumor growth, invasion, and metastasis (4). Chemo-kines were initially described as regulators of leukocyte traf-ficking and recruitment in inflammation sites. Infiltration ofwhite blood cells, mostly tumor-associated macrophages,and the presence of proinflammatory molecules as interleukin(IL)-8 and IL-6 are typical features of cancer-related inflam-mation. Cytokines and chemokines are produced by multiplecell types, including fibroblasts, endothelial, and epithelialcells (5), including cancer cells, which also express theirG-protein coupled receptors. These G-proteins activate a num-ber of signaling pathways such as phospholipase C (PLC)-b,phosphoinositide 3-kinase (PI3K), and the mitogen—activatedprotein kinase (MAPK) cascade, which play a critical role inadhesion, migration, and survival of cancer cells (6).

Concomitant with these effects, cytokines and chemokinesalso confer cancer cells with the ability to adhere to fibronectinand collagen IV, allowing the formation of micrometastases(7). Therefore, chemokine networks control critical steps of theadhesion-invasion-metastasis cascade, providing the neces-sary signals that facilitate cancer cell survival and growth indistant sites (3). Little is known about the role played by theautocrine secretion of chemokines by tumor epithelial cellsin cancer-related inflammation, which might regulate therecruitment and maturation of macrophages and other effec-tor cells. We used the well-known KM12 cell model for thestudy of late metastatic events in colorectal cancer, includingliver colonization and survival of the tumor cells in the newenvironment. KM12C and KM12SM epithelial cells differ onlyin theirmetastatic properties (8). KM12SM, a highlymetastatic

Authors' Affiliations: 1Functional Proteomics, Department of Cellular andMolecularMedicine, Centro de InvestigacionesBiol�ogicas (CIB-CSIC); and2Department of Pathology, Fundaci�on Jim�enez Díaz, Madrid, Spain

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

R. Barderas and R.A. Bartolom�e contributed equally to this work.

Corresponding Author: J. Ignacio Casal, Functional Proteomics Labora-tory, Centro de Investigaciones Biol�ogicas (CIB-CSIC), Ramiro de Maeztu9, Madrid 28040, Spain. Phone: 34-918373112; Fax: 34-915360432;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-4090

�2012 American Association for Cancer Research.

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cell line, was derived from the poorly metastatic cell lineKM12C, through successive passages in nude mice. A previousproteomic characterization of these 2 colon cancer cell linesshowed a preference for expression of homing molecules inKM12SMcells (9).Moreover, a decrease in the expression levelsof enzymes from the glycolysis, pentose phosphate pathway,and amino acid transporters was observed.To characterize the metastatic process in colorectal can-

cer, we analyzed the cytokine/chemokine profiles releasedby colorectal metastatic cancer KM12SM cells using anti-body microarrays. These microarrays constitute a powerfultool to get a complete overview of cytokine profiles andpathways alterations in cancer (10). We observed highersecretion of IL-13, which was associated to a more abundantexpression of their receptor IL-13Ra2 in the highly metastaticKM12SM cells. Silencing of IL-13Ra2 revealed a major rolefor this receptor in cell adhesion, migration, invasion, andmetastatic colonization of colorectal cancer cells. A clearincrease of expression of IL-13Ra2 was observed in late-stagehuman colon cancer tissues, showing a correlation betweenIL-13Ra2 expression and poor outcome of patients withcolon cancer metastasis.

Materials and MethodsCell cultureKM12C and KM12SM human colon cancer cells were

obtained directly from I. Fidler's laboratory (MD AndersonCancer Center, Houston, TX). KM12 cells were expanded in thelaboratory to prepare a large batch of working aliquots thatwere stored in liquid nitrogen. For each experiment, cells werethawed and kept in culture for a maximum of 10 passages.These 2 cell lines were not authenticated in our laboratory.Parental cells, and their derivatives, were cultured in Dulbec-co's Modified Eagle's Medium (DMEM; Gibco-Life Techno-logies) containing 10% fetal calf serum and antibiotics at 37�Cin a 5% CO2-humidified atmosphere. Other cell lines werepurchased directly from the American Type Culture Collection(ATCC) and cultured as recommended. All these cell lineswere authenticated by ATCC and were passaged fewer than6 months after purchase for all the experiments.

Human protein cytokine arrayConditioned medium from each KM12 cell type was col-

lected after 24 and 48 hours in serum-free medium andincubated with arrays containing 79 human cytokine specificantibodies as described (10). Then, the membranes werescanned and analyzed using Redfin, a 2D-gel image analysissoftware (Ludesi). Relative cytokine intensities were normal-ized in comparison to control spots on the same membrane.Ratios between both cell lines were calculated for the differentcytokines at the 3 experiments. Individual quantification ofIL-4 and IL-13 was carried out by specific ELISA kits (RayBio-tech; see Supplementary Methods).

Stable transformation of KM12 cells, cell adhesion, andinvasion assayspLKO.1 vectors containing 5 different short hairpin

RNAs (shRNA) for IL-13Ra2 were purchased from Open

Biosystems. As shRNA control vectors, we used a scrambledshRNA and an empty pLKO.1 vector obtained from AddGene(11, 12). Stable transformed cells were obtained by lentiviralinfection. Briefly, HEK293T cells were transfected withpLKO.1 vectors and the packaging vectors pCMV-rev,pMDLg-pRRE, and pNGVL-VSVG with jetPRIME Transfec-tion Reagent (Polyplus). After incubating the cells for 12 to15 hours in serum-free medium, medium was replaced withDMEM containing 10% FBS and antibiotics. The day after,conditioned medium containing lentiviral particles wascentrifuged, diluted 1:2 to 1:10 in DMEM containing 10%FBS and antibiotics and directly added to KM12C andKM12SM cells. After 3 days of incubation, infected KM12Cand KM12SM CRC cells were selected using 1 mg/mL puro-mycin (Sigma) during 2 to 3 weeks. Then, cells were culturedwith medium containing 0.5 mg/mL puromycin.

Cell adhesion and invasion using Matrigel assays werecarried out according to previously published procedures(13; see Supplementary Methods for a full description). Inhi-bitors LY294002 and PP2 were from Sigma and UO126 wasfrom Calbiochem. For apoptosis and proliferation assays seeSupplementary Methods.

Western blottingThe following antibodies were used in the study. Anti-IL-

4Ra, anti-IL-13Ra1, and anti-a2 integrin were from Abcam,whereas anti-IL-13Ra2 was from R&D Systems. AKT, p-AKT,p-ERK1/2, p-JNK, STAT3, p-STAT3, STAT6, and p-STAT6 werefrom Cell Signalling. Extracellular signal–regulated kinase(ERK)1/2, c-jun-NH2-kinase (JNK), and RhoGDI were fromSanta Cruz Biotechnology. Tubulin was from Sigma. Anti-b1integrin was a gift from Dr F. S�anchez-Madrid (H. de laPrincesa, Madrid, Spain).

For Western blotting, KM12C and KM12SM cells werewashed twice with chilled PBS and homogenized in 0.05%SDS. Protein extracts were sonicated 3 times for 30 seconds onice, and clarified at 10,000� g at 4�C. Protein extracts were runin 10% SDS-PAGE and immunoblotting was carried out fol-lowing standard procedures.

Metastasis experiments in nude miceSwiss nude mice (Charles River) were used for intrasplenic

or intratail inoculation. The Ethical Committee of the ConsejoSuperior de Investigaciones Científicas (Madrid, Spain)approved the protocols used for experimental work with mice.Mice were injected with 106 KM12 cells in 0.1 mL PBS. Micewere daily inspected for signs of disease, such as abdominaldistension, locomotive deficit, or tumor detectable by palpa-tion.Metastasis nodes in liverwere determinedby counting thenumber of visible nodules in dissected livers.

Immunohistochemical analysisA total of 80 patients diagnosed and treated of colorectal

adenocarcinoma between 2001 and 2006 in Fundaci�on Jim�enezDíaz (Madrid, Spain)were used for the study.Wehave reviewedthe clinical records of the patients to determine tumor stage atthe time of diagnosis and outcome (Supplementary Table S1).Hematoxylin & eosin–stained sections of the colectomy

Role of IL-13Ra2 in Colorectal Cancer Metastasis

www.aacrjournals.org Cancer Res; 72(11) June 1, 2012 2781




specimens were reviewed to select representative areas of thetumor to carry out immunohistochemical detection of IL-13and IL-13Ra2. The working dilution was 1:100 for IL-13 and1:500 for IL-13Ra2. IL-13Ra2 immunohistochemistry was car-ried out on a tissue microarray (TMA) using an automatedsystem for immunostaining (Dako Autostainer), with antigenretrieval at high pH. The immunostained sections were coun-terstainedwith hematoxylin and the intensity of themembraneand cytoplasmic staining was graded as absent, weak, mod-erate, or intense, although for subsequent statistical analysisthe cases were reclassified as positive (either moderate orintense) or negative (either complete absence of positivity orweak staining similar to control areas of normal colonicmucosa). In all cases, sections from normal colonic mucosadistant from the tumor site were used as negative controls.

Statistical analysesData from the patients were recorded in an excel file.

Descriptive statistics included mean (and SD) for quantitativeGaussian variables and percentages for qualitative ones. Bivar-iant analysis was made with one-way ANOVA followed byTukey–Kramer multiple comparison test for quantitative vari-ables and c2 test for qualitative ones. In both analyses, theminimum acceptable level of significance was P < 0.05. Survivalcurves were plotted with Kaplan–Meier technique and com-pared with the log-rank test. The level of significance for survivalwas also settled at a P value less than 0.05.

ResultsChemokine profiling in the secretome of KM12 cells

We investigated the production and release of 79 differentchemokines, growth factors, and other immunomodulators in

conditioned culture media of colorectal cancer KM12 cells byusing a chemokine array. Results after 24 and 48 hours ofculture are shown in Fig. 1A. Several chemokines, proangio-genic, and growth factors were highly expressed in both celllines, among them GROa (CXCL1), IL-8 (CXCL8), Rantes(CCL5), macrophage inflammatory protein (MIP)-1b (CCL4),eotaxin-2 (CCL24), angiogenin, MIF, osteoprotegerin, or insu-lin-like growth factor–binding protein 2 (IGFBP2; Fig. 1B).Regarding differential expression, TIMP2, IL-4, and IL-13 wereupregulated in the highly metastatic cells (Fig. 1A and B). IL-4and IL-13 differences were confirmed by using a specificELISA, with fold-changes between 1.5 and 2.13, in the presenceor absence of serum, respectively (Fig. 1C).

Most of the biologic effects of IL-4 and IL-13 are mediatedby STAT transcription factors (14). We analyzed KM12 cellsby Western blotting using antibodies specific for total andphosphorylated forms of STAT3 and STAT6 (Fig. 1D). Totaland activated STAT6 were more abundant in KM12SMcells, with no significant differences in STAT3. High levels ofSTAT6 in colon cancer cells have been associated to highinvasiveness/metastasis (15). These results support a potentialaction of IL-4 and IL-13 on KM12 cells.

IL-13Ra2mediates IL-13 action on KM12 cells: effects oncell adhesion

IL-4 and IL-13 usually regulate biologic activity through thecommon type II receptor IL-4Ra/IL-13Ra1 complex (16),which mediates signal transduction through the JAK/STAT6pathway (17). In addition, IL-13 binds a high-affinity receptorcalled IL-13Ra2 (18), which was initially assigned as a decoyreceptor (19). We tested KM12 cells for the presence of thesereceptors. IL-4Ra was not detected on the surface of KM12

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Figure 1. Expression ofchemokines, growth factors, andimmunomodulators in conditionedmedium from KM12SM andKM12C cells using cytokine arrays.A, representative image of cytokineantibody arrays results afterscreening of conditioned mediumfrom KM12 cells. The experimentwas carried out in triplicate.B, cytokines highly expressed inKM12 cells at 48 hours. Bar graphwas calculated for each cytokinewith the median value in arbitraryunits of 3 independent assays.Inset, signal intensity for IL-4 andIL-13 obtained from 3 independentarrays. C, expression ratios for IL-4and IL-13 in conditionedmediumofKM12 cells in the presence orabsence of serum at 48 hours afterELISA quantification. D, STATsignaling alterations in KM12 cellswere tested by immunoblotting.Experiments were carried out intriplicate and quantified bydensitometry.

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Cancer Res; 72(11) June 1, 2012 Cancer Research2782




cells by flow cytometry (Supplementary Fig. S1A) nor intra-cellular by Western blotting (Supplementary Fig. S1D). IL-13Ra1was not accessible on the cell surface (SupplementaryFig. S1B), although some intracellular expression was detectedusing permeabilized cells in flow cytometry (SupplementaryFig. S1C) or Western blotting (Supplementary Fig. S1E). Incontrast, a higher surface expression of IL-13Ra2was observedon the surface of KM12SM than in KM12C cells by flowcytometry, 73.9% versus 33.9% (Fig. 2A). Western blot analysesconfirmed the increased expression of IL-13Ra2 in KM12SMcells respect to KM12C, as well as a high expression in othermetastatic colon cancer cells such as SW48 or HT29 (Fig. 2B).Because the IL-4Ra/IL-13Ra1 heterodimer for IL-13 signalingwas not available on the cell surface, we hypothesized that IL-13Ra2 could mediate IL-13 action in KM12 cells.To examine the potential effect of IL-13Ra2 on colorectal

cancer metastasis, IL-13Ra2 was silenced on KM12C andKM12SM cells by preparing stable shRNA transfectants. Thelack of expression of IL-13Ra2 was confirmed by Western

blotting (Fig. 2C) and semiquantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) (data not shown). Bothanalyses showed that less than 10% of the original proteinexpression levels were detected after knockdown withshRNA#23. This silencing did not affect the expression of IL-13Ra1, which showed similar intracellular levels (Supplemen-tary Fig. S1E). To analyze the effect of IL-13Ra2 silencing onautocrine IL-13 production we quantified the levels of IL-13 byELISA. We observed a decrease in the expression of IL-13 in IL-13Ra2–silenced cells. The reduction was more dramatic forKM12SM cells (Supplementary Fig. S2).

Then, we analyzed the adhesive properties of the cell linesbefore and after IL-13Ra2 silencing using Matrigel. In basalconditions, adhesion capacity of scrambled KM12SM over-expressing IL-13Ra2 duplicated that of KM12C cells. Thedecrease in adhesion capacity after IL-13Ra2 silencing wasparticularly significant for KM12SMcells (>50%) and negligiblefor KM12C cells (Fig. 2D). Addition of IL-13 increased signif-icantly cell adhesion in scrambled control cells, but not in the

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Figure 2. IL-13Ra2 is overexpressed onKM12SMcells and increases cell adhesion. A, flow cytometric analysis of KM12C andKM12SMcells. The percentageof positive cell is shown inside each panel. B, twenty-five micrograms of protein from lysates of the indicated colorectal carcinoma cell lines were resolved inPAGE and subjected to Western blotting using anti-IL-13Ra2 or, as a control, anti-RhoGDI antibodies. C, KM12C and KM12SM cells were infected withretroviral vectors containing different shRNAs targeting IL-13Ra2, scrambled shRNA, or empty vectors, and IL-13Ra2 expression was assessed byWestern blotting. Bands were quantifiedwithMultiGauge software. Tubulin was used as loading control. D, cell adhesion toMatrigel of IL-13Ra2–silenced orcontrol KM12 cells, pretreated for 5 hours with or without IL-13 plus the indicated antibodies. Adhesion was significantly upregulated by incubationwith IL-13 (��, P < 0.01; ��, P < 0.001) and significantly decreased by incubation with anti-IL-13Ra2 (DD, P < 0.01; DDD, P < 0.001). Data represent themean � SD of 3 independent experiments.






silenced cells. The use of anti-IL-13Ra2 antibody blocked celladhesion for IL-13–treated scrambled cells, being most rele-vant for KM12SM (Fig. 2D). Still, antibody blocking was not aseffective as IL-13Ra2 silencing. These results suggest that IL-13Ra2 also contributes to constitutive cell adhesion in theabsence of IL-13. As a control, IL-13Ra1–silenced cells (Sup-plementary Fig. S3A) did not show alterations in cell adhesion(Supplementary Fig. S3B).

IL-13Ra2 expression promotes cell migration andinvasion in KM12 cells through PI3K and SRC activation

To determine the effect on cell migration, we used wound-healing assays and different amounts of IL-13 for 22 hours. IL-13 was sufficient to promote migration in KM12 cells, with anoptimum at 10 ng/mL (Fig. 3A). KM12SM cells displayed twicemore migratory capacity than KM12C cells in presence ofserum, which might contain IL-13 and other promigratoryfactors. KM12 cells showed a much lower cell migration in

wound healing after IL-13Ra2 silencing (Fig. 3B). Addition ofIL-13 caused a significant increase in the migration speed ofscrambled cells (Fig. 3C). In contrast, silenced cells wereinsensitive to IL-13. This inhibition was also confirmed by theuse of anti-IL-13Ra2 antibodies. To study the pathwaysinvolved in the increase of cell migration we tested differentinhibitors. The increase in migration induced by IL-13 wasstrongly reduced by LY294002, a PI3K inhibitor, partiallyreduced by PP2 (SRC inhibitor), and not affected by UO126(MEK1/2 inhibitor) in KM12 cells. The inhibition was morepronounced on KM12SM cells.

Then, we tested the ability of the cells for invasion acrossMatrigel (Fig. 3D). In medium alone, the invasion was low, buttwice higher for KM12SM cells. Addition of IL-13 caused alarge increase of invasion in the control cells, but not in thesilenced cells, which showed between 6 and 10 times lessinvasion capacity, similar to basal levels. As before, the use ofPI3K inhibitor or anti-IL-13Ra2 decreased the invasive

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Figure 3. IL-13 increases cellmigration and invasion through IL-13Ra2. A, cultures were incubatedin presence of the indicatedconcentrations of IL-13. Migrationspeed of the cells was calculatedas the distance covered in 48hours. B, wound-healing assay ofKM12C and KM12SM cellsincubated in medium alone or with10% serum. Photographs weretaken 48 hours after scratching.C, KM12 cells were incubated inpresence or absence of IL-13(10 ng/mL) with the indicatedantibodies and inhibitors. D,invasion across Matrigel of theKM12 cell lines treated asindicated. Data represent themean� SD of 3 independentexperiments. For increase inmigration (�, P < 0.05; ��, P < 0.01;��, P < 0.001) and for reduction(D, P < 0.05; DD, P < 0.01; DDD,P < 0.001). E, KM12 cells werestarved in medium, treated with IL-13 (10 ng/mL) or 10% serum for 5hours and lysed. The extracts wereanalyzed byWestern blotting usingthe indicated antibodies. �,observed changes in expression.

Barderas et al.





capacity of the scrambled cells to basal level. SRC inhibitor wasalso quite effective to decrease the invasive capacity ofKM12SM, but not so much on KM12C cells. No effect wasobserved for MAP–ERK kinase (MEK1/2 inhibitor). As a con-trol, IL-13Ra1 silencing in KM12SM cells did not alter cellinvasion capacity (Supplementary Fig. S3C).To confirm the role of IL-13Ra2 in cell migration and

invasion, we generated KM12 cells overexpressing this recep-tor, which were tested in migration and invasion assays. BothKM12C and KM12SM transfectants showed an increase inmigration and invasion in response to IL-13 compared withcontrol transfectants (Supplementary Fig. S4A and S4B).Collectively, these results support that IL-13 action was medi-ated by IL-13Ra2 for migration and invasion in colorectalcancer metastasis.

Pathways activation in IL-13Ra2–mediated cell invasionTo study the mechanism of action of IL-13Ra2 in cell

invasion, we characterized the levels of activated SRC, FAK,AKT, ERK, JNK, and STAT6 in response to IL-13 or serum(Fig. 3E). Expression of p-SRC and p-AKT was more abun-dant in KM12SM cells treated with serum than with IL-13,whereas only p-AKT, but no p-SRC, was observed in KM12Ccells. These differences in p-SRC effect between KM12SMand KM12C cells might explain previous differences inmigration and invasion induced by the SRC inhibitor. The

presence of a double band with different intensities in p-SRCbetween serum and IL-13–treated cells suggests that adifferent member from the SRC family might be implicatedin KM12SM cells, as the antibody recognizes phosphoryla-tion in the C-terminal tyrosine of 6 members of this family.The decrease of p-AKT in silenced KM12SM cells treatedwith IL-13 confirmed the relevance of PI3K and suggests apotential effect on survival. MAPK signaling was not affectedby IL-13Ra2 silencing as no differences were observed forERK1/2 or JNK. Activation of p-STAT6 was observed inKM12SM cells with serum, but not when cells were treatedonly with IL-13 (Fig. 3E). Therefore, IL-13–triggered activa-tion through IL-13Ra2 is STAT6 independent. Other factorsmust activate STAT6 in serum-cultured KM12SM cells.

Silencing of IL-13Ra2 expression inKM12 cells decreasessurvival, tumorigenesis, and proliferation

The activation of the PI3K pathway has important biologiceffects on cell survival and proliferation (20). To test whetherIL-13 signaling via IL-13a2 can modulate cell survival, KM12cells were subjected to apoptosis assays. In response tooxidative stress induced by hydrogen peroxide, scrambledKM12C and KM12SM cells showed similar levels of apopto-sis (Fig. 4A). The addition of IL-13 caused a moderate effectin promoting survival in these oxidative stress conditions.This effect was dependent on IL-13Ra2, as the addition of

Figure 4. IL-13Ra2 promotes cellsurvival and proliferation. A, cellswere incubated with H2O2 for 16hours in presence of medium aloneor with IL-13, and in presence orabsence of LY294002 inhibitor oranti-IL-13Ra2 antibodies, andsubjected to apoptosis detectionassays. B, colony formation assay insoft agar with the indicated cell lines.C, proliferation and cell viability wasdetermined by MTT assays after 24to 48 hours of incubation in mediumwith or without IL-13 (10 ng/mL) andanti-IL-13Ra2 antibody. Opticaldensity was significantly increasedby addition of IL-13 (�, P < 0.05;��, P < 0.01) and significantlyinhibited by anti-IL-13Ra2(D, P < 0.05; DD, P < 0.01).

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the antibody or the silencing of the receptor abolished theincrement in survival (Fig. 4A). The presence of LY294002, aPI3K inhibitor, inhibited this increase in survival inducedby IL-13 through IL-13Ra2. These results indicate thatIL-13 also affects the survival program in KM12 colorectalcancer cells through PI3K-AKT activation mediated by theIL-13Ra2 receptor. This moderate reduction in cell apopto-sis may play a role during metastasis, facilitating survivalof metastatic cells.

Then, we tested the effect of IL-13Ra2 silencing ontumorigenesis through the ability to form colonies in softagar. KM12SM displayed a higher capacity to proliferate inan anchorage-independent environment than KM12C(Fig. 4B). However, IL-13Ra2–silenced cell lines showed asignificant reduction in colony formation, indicating a cru-cial role of this receptor in tumorigenesis. Regarding pro-liferation, we observed only a minor effect in cells treatedwith IL-13 (Fig. 4C). This IL-13 effect was blocked afterincubation with anti-IL-13Ra2 antibodies (Fig. 4C). Theseresults suggest that IL-13 signaling via IL-13Ra2 does notplay a major role in cell proliferation in colorectal cancercells.

Silencing of IL-13Ra2 in KM12SM cells provokes adecrease in liver homing and an increase in mousesurvival

As a final approach to verify the role of IL-13Ra2 in coloncancer invasion and metastasis, we carried out intrasplenicinjections of control and silenced KM12 cells to analyze theirability and speed to generate metastasis in liver. IL-13Ra2–silenced KM12SM cells induced longer survival of mice thancontrol KM12SM cells (P < 0.05; Fig. 5A). This prolongedsurvival was due to the slower growth of the tumor in themice inoculated with IL-13Ra2–silenced KM12SM cells andlower ability to colonize liver and cause metastasis. Postmor-tem analysis showed that tumors at primary inoculation sitereached similar sizes ranging between 0.7 and 1 cm3 in controland silenced KM12SM cells. Poorly metastatic KM12C cellsdid not cause metastasis, and only 30% of inoculated micedeveloped a tumor in spleen 180 days after inoculations.Therefore, IL-13Ra2–silenced KM12SM cells lost, in a signi-ficant way, the ability to colonize the liver, as few mice showedmacroscopic metastasis after liver dissection (Fig. 5B).

To corroborate the ability of KM12 cells for liver homing, wecollected the livers 24 hours after spleen injection and carriedout a PCR for amplification of human glyceraldehyde-3-phos-phate dehydrogenase (GAPDH; Fig. 5C). Whereas humanGAPDH could be detected in livers from mice inoculatedwith control cells (especially KM12SM), the PCR amplificationresulted in a barely detectable band in mice inoculatedwith silenced cells. To examine IL-13Ra2 expression inKM12SM cells as well as their invasive properties after in vivopassage, cells were isolated from the tumors and cultured untilconfluence. IL-13Ra2 expression remained very low (Fig. 5D)and their invasiveness across Matrigel was not altered byin vivo passage (Fig. 5E). Collectively, these data confirm thecapacity of IL-13Ra2 to mediate homing and liver metastasisin colon cancer.

IL-13Ra2 overexpression in human patients isassociated to late stages and lower overall survival

To investigate the relevance of our results in human coloncancer, we decided to study the levels of expression of IL-13 andIL-13Ra2 in tumor and adjacent normal tissue samples. Weobserved IL-13 expression mainly associated to human epi-thelial colon cancer cells, with weak or no IL-13 expression inthe stroma of the tumors (Fig. 6A). For statistical analysis ofIL-13Ra2 expression, we used a TMA with representativesections of tumor and normal colonic mucosa from 80 pati-ents diagnosed and treated of colorectal adenocarcinoma andfollowed on the long term (>5 years). The series was retro-spectively selected. IL-13Ra2 expression was not detected in27 cases (33.7%) and was present with either moderate orhigh intensity in the remaining 53 cases (66.3%; Fig. 6B). Weakor no staining was observed in all control normal samples.We found a statistically significant association betweenIL-13Ra2 expression and tumor progression (T-stage), withhigher expression in T3 or T4 tumors as compared with T1or T2 (P¼ 0.013), lymph node involvement (higher expressionin tumors with lymph node involvement, P ¼ 0.013), andmetastasis at the moment of diagnosis (P ¼ 0.038; Fig. 6C).We found no significant association with histologic grade ofthe tumor, but all high grade and colloid tumors showedintense IL-13Ra2 expression, as opposed to well-differentiatedtumors, that were mostly negative. Finally, we wanted to knowwhether there was an association between IL-13Ra2 expres-sion and survival of patients with colon cancer. Survivalanalysis showed a clear association with poor prognosisin terms of lower overall survival for patients with highIL-13Ra2 expression (P ¼ 0.03; Fig. 6D).

DiscussionAlthough the KM12 cell model probably does not give a

complete picture of the spontaneous metastasis in humancolon cancer, actually is giving us excellent insights in the celladhesion, invasion, and colonization of the liver in metastasisand the criticalmolecules involved in these processes. Here, wehave described an important role for IL-13 and its receptor IL-13Ra2 in colorectal cancer invasion and metastasis. Thisconclusion was obtained from the following observations: (i)IL-13wasmore abundant in the secretome of highlymetastaticcells, (ii) IL-13Ra2 was overexpressed in highly metastaticKM12SM cells and other metastatic cell lines than poorlymetastatic KM12C cells, (iii) IL-13Ra2 silencing decreasedadhesion, invasion, and clonogenicity, (iv) IL-13Ra2 silencingsuppressed AKT activation and promoted apoptosis, (v) miceexperiments showed that removal of IL-13Ra2 reduced thehoming capacity in liver of KM12SM cells and increased thesurvival of inoculated mice, and (vi) human colon cancersamples showed a high expression of IL-13 and IL-13Ra2 incancer cells. IL-13Ra2 was mainly associated to T3 or T4tumors and to a lower overall survival. These results confirmedthat IL-13 and IL-13Ra2 expression were associated to colo-rectal cancer invasion and liver metastasis in cancer cells.

IL-13 has been previously associated to pathologicconditions such as asthma, autoimmune diseases, and

Barderas et al.





inflammatory conditions (21). It has been involved in T-help-er 2 cell (TH2) differentiation, STAT6-dependent M2 polar-ization, and TGF-b1 production and fibrosis (22, 23). Stim-ulation of mouse J774 cells with IL-4 or IL-13 in combinationwith TNF-a induced STAT6 phosphorylation (14) and abun-dant expression of IL-13Ra2 on the surface of the cells (23).IL-13 acts as an autocrine growth factor in pancreatic cancer

that promotes lymph node metastases (24) and is a majorregulator of M2 macrophages to suppress immune surveil-lance in metastasis (25). This counter-surveillance activityrequires the expression of IL-13Ra2 (26). IL-13Ra2 geneexpression was reported in pancreatic and breast cancermetastasis (27, 28). The almost absence of IL-4Ra andIL-13Ra1 in KM12 cells (this report) and other colon cancer

Figure 5. IL-13Ra2 promotes livermetastasis of KM12SM cells. A,Kaplan–Meier survival assay of nudemice inoculated with the indicatedKM12 cells. Survival of miceinoculated with IL-13Ra2–silencedKM12SM cells increasedsignificantly (�, P < 0.05) whencompared with those inoculatedwithscrambled cells. B, mice wereexamined for macroscopicmetastases in liver. Number ofmacroscopic metastases in liver wassignificantly reduced in miceinoculated with IL-13Ra2–silencedKM12SM cells (��, P < 0.01). ^,number of metastases in eachmouse. Average and SD are shown.C, nude mice inoculated as in A weresacrificed 24 hours after inoculation.RNA was isolated from the liver andsubjected to RT-PCR to amplifyhuman GAPDH (hGAPDH). Arepresentative experiment out of 3 isshown. Murine b-actin (mb-actin)was amplified as loading control. D,KM12 cells isolated from tumors bymechanical disaggregation werecultured to confluence and analyzedby Western blotting to confirmIL-13Ra2–silencing and tested forinvasion through Matrigel inpresence or absence of IL-13(10 ng/mL; E). Silenced KM12SMcells were included in the assay as acontrol.

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cells (29) indicates that IL-13 signaling occurs through theIL-13Ra2 receptor. Surprisingly, previous reports did notdetect IL-13Ra2 mRNA by RT-PCR in HT-29/B6 colon cancercells (30).

No signaling activity was initially described for IL-13Ra2due to its short cytoplasmic domain and the lack of JAK/STAT binding sequences (19). Therefore, IL-13Ra2 was con-sidered as a decoy receptor for IL-13 in mouse and humans(21). However, recent studies have showed that IL-13Ra2 isinternalized after IL-13 binding (31). It has been reportedthat IL-13Ra2 induces MAPK signal transduction in intes-tinal epithelial cells from ulcerative colitis or colorectalcancer (29) and pancreatic cancer (28). At low concentra-tions, IL-13Ra2 signaled through the MAPK pathway, but athigher concentrations worked as a decoy receptor (29). Incontrast, on metastatic KM12SM cells, IL-13 action is medi-ated through IL-13Ra2 independent from the expressionlevels, as denoted by the effect on both, KM12C and KM12SMcells.

In murine macrophages, IL-13Ra2 mediates activatorprotein (AP-1)-dependent, STAT-6–independent signaling,resulting in inflammation and fibrosis in vivo (23). Previousreports in pancreatic cancer (28) and ulcerative colitis (29)showed STAT6-independent activation in IL-13Ra2–expres-sing cells, suggesting an inverse relationship between IL-13Ra2 expression and STAT6 activation. In colon cancermetastatic cells, signaling through IL-13Ra2 is also STAT6independent, and is not mediated by JNK or ERK. The lack ofERK activation could explain the minor effect of IL-13Ra2removal on cell proliferation and might explain why IL-13Ra2overexpression does not provide any competitive advantagein subcutaneously implanted tumors in immunodeficientmice (32).

In ourmodel, IL-13Ra2 signaling induced PI3K activation, asPI3K inhibitors blocked IL-13 effects on KM12 cells. SRCactivation was found exclusively on IL-13–treated KM12SMcells and might be related with the higher migration andinvasion of these cells. An alternative role for IL-13 in the

C

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Figure 6. IL-13Ra2 expression is associated to human colon cancer progression and poor survival of patients with colorectal cancer.A, immunohistochemical analysis of IL-13 expression in human CRC tissue showed that IL-13 expression occurs preferentially in CRC epithelial cells.B, immunohistochemical analysis of IL-13Ra2 expression in TMAs showing representative images of strong, moderate, or negative staining of differentcolon carcinomas and negative normal paired mucosa. Counterstaining was made with hematoxylin. Photographs were taken at �200 magnification.C, quantification of IL-13Ra2 expression by estimation of staining intensity was carried out as described in Materials and Methods. Eighty samples wereanalyzed. Positive and negative values were represented as bar graphs. P values were calculated with the c2 test. D, Kaplan–Meier analyses ofoverall survival of patients with cancer according to the expression of IL-13Ra2. Significant association of IL-13Ra2 expression with lower overallsurvival was found by comparing differences between curves with the log-rank test.

Barderas et al.





increase of intestinal epithelial permeability, mediated by thePI3K pathway without STAT6 involvement, has been reported(33). Ulcerative colitis, a well-known risk factor for coloncancer development, is probably driven by IL-13 (34). Also,oxazolone-induced colitis in mice is caused by IL-13–produc-ing natural killer T cells (35). Lack of epithelial barrier functionhas been reported for inflammatory bowel diseases (36, 37).This increase in permeability affected epithelial cells tightjunctions, induced epithelial apoptosis and cell restitutionarrest in ulcerative colitis (30). All these processes, whereIL-13 is a key effector, contribute to severe inflammation andcould constitute a connecting link between ulcerative colitis,colon inflammation, and colon cancer. In fact, we haveobserved a predominant association of IL-13 expression withcolon cancer epithelial cells.Immunohistochemical analysis of IL-13Ra2 expression in

a panel of metastatic human colon cancer samples showed aclear association with late stages in human cancer and pooroutcome of patients. This worst prognosis could be attrib-uted to the increased invasiveness and homing capacityof cells overexpressing IL-13Ra2. Liver colonization bymetastatic cells requires a different program of proteinexpression and signaling activation from early metastasis.Targeting some of the cytokines or regulatory moleculesinvolved in metastatic invasion and homing might be a

successful approach for decreasing metastasis recurrence incolorectal cancer.

In summary, we have shown a role for IL-13Ra2 in adhesion,invasion, survival, and colonization of highly metastatic coloncancer cells. Our results suggest that IL-13Ra2 does notfunction only as a decoy receptor of IL-13 in cancer cells butas signaling mediator. Moreover, the use of IL-13Ra2 expres-sion in patients with colon cancer as a prognostic biomarker inmetastatic colorectal cancer gives a pathophysiologic rele-vance to these findings and supports the interest of thismolecule as therapeutic target in colon cancer.

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

Grant SupportR. Barderas was a recipient of a JAE-DOC contract of the CSIC. R. Bartolome

was supported by a grant to established research groups of the Asociaci�onEspa~nola Contra el C�ancer (AECC). S. Torres was a recipient of a ProteoRedcontract. This researchwas supported by grants BIO2009-08818 from the SpanishMinistry of Science and Innovation, established research groups (AECC) andColomics 2 (CM) to J.I. Casal.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 19, 2011; revisedMarch 13, 2012; acceptedMarch 26, 2012;published OnlineFirst April 13, 2012.

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2012;72:2780-2790. Published OnlineFirst April 13, 2012.Cancer Res Rodrigo Barderas, Rubén A. Bartolomé, María Jesús Fernandez-Aceñero, et al. Associated with Invasion, Liver Metastasis, and Poor Prognosis

2 in Colorectal Cancer IsαHigh Expression of IL-13 Receptor

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High Expression of IL-13 Receptor a2 in Colorectal Cancer ... · for this receptor in cell adhesion, migration, invasion, and metastatic colonization of colorectal cancer cells. A

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