il4 receptor ilr4a regulates metastatic colonization by ... · molecular and cellular pathobiology...

Molecular and Cellular Pathobiology

IL4 Receptor ILR4a Regulates Metastatic Colonization byMammary Tumors through Multiple Signaling Pathways

Katherine T. Venmar, Kathy J. Carter, Daniel G. Hwang, E. Ashley Dozier, and Barbara Fingleton

AbstractIL4, a cytokine produced mainly by immune cells, may promote the growth of epithelial tumors by

mediating increased proliferation and survival. Here, we show that the type II IL4 receptor (IL4R) is expressedand activated in human breast cancer and mouse models of breast cancer. In metastatic mouse breast cancercells, RNAi-mediated silencing of IL4Ra, a component of the IL4R, was sufficient to attenuate growth atmetastatic sites. Similar results were obtained with control tumor cells in IL4-deficient mice. Decreasedmetastatic capacity of IL4Ra "knockdown" cells was attributed, in part, to reductions in proliferation andsurvival of breast cancer cells. In addition, we observed an overall increase in immune infiltrates within IL4Raknockdown tumors, indicating that enhanced clearance of knockdown tumor cells could also contribute tothe reduction in knockdown tumor size. Pharmacologic investigations suggested that IL4-induced cancercell colonization was mediated, in part, by activation of Erk1/2, Akt, and mTOR. Reduced levels of pAkt andpErk1/2 in IL4Ra knockdown tumor metastases were associated with limited outgrowth, supporting rolesfor Akt and Erk activation in mediating the tumor-promoting effects of IL4Ra. Collectively, our results offer apreclinical proof-of-concept for targeting IL4/IL4Ra signaling as a therapeutic strategy to limit breast cancermetastasis. Cancer Res; 74(16); 4329–40. �2014 AACR.

IntroductionBreast cancer is still the second leading cause of cancer-

related deaths among women in the United States (1). Thisis largely due to the high mortality rate in metastaticdisease. Cytokines and chemokines in the tumor microen-vironment are known to promote breast cancer progressionand metastasis (2). Significantly, IL4, a Th2 cytokine, isupregulated in the microenvironment of breast carcino-mas (3), and the IL4 receptor (IL4R) is overexpressed bybreast cancer cells themselves (4). In the immune system,IL4 is produced predominantly by activated T cells, mastcells, basophils, and eosinophils, and is known to regulatethe survival, proliferation, and differentiation of B and Tlymphocytes through IL4R (5). Therefore, it is plausible thatupregulated epithelial IL4R could promote breast tumorgrowth.The IL4/IL4R interaction has been shown to enhance the

proliferation and survival of breast cancer cells in vitro (6, 7),and the survival of other epithelial cancer cell types in vivo (4).

Still, there are no data about the influence of IL4R expressionon the proliferation and survival of breast cancer cells in vivo, oron metastatic tumor growth in general. Furthermore, activa-tion of signaling pathways downstream of the IL4/IL4R inter-action that mediate enhanced tumor growth remains largelyunresolved in breast cancer cells that overexpress a differentform of the IL4R than immune cells.

Lymphoid cells express the type I IL4R composed of thecommon g-chain (gc) and the IL4Ra chain (5). Heterodi-merization is necessary for receptor functionality (8). Non-lymphoid cells, including breast cancer cells, express thetype II IL4R, composed of the IL4Ra and IL13 receptoralpha 1 (IL13Ra1) chains. IL13 is the only other cytokineknown to bind and activate type II IL4Rs. However, IL4binds with higher affinity and is the prototypical ligand forthe IL4R (2).

In lymphoid cells, the IL4/type I IL4R interaction can resultin the activation of two primary pathways: (i) the insulinreceptor substrate protein (IRS)/PI3K/Akt pathway and (ii)the JAK/Stat6 pathway (5). Activated Akt (9) and Stat6(5, 10, 11) are known regulators of proliferation and survival.However, IL4Ra signaling from the type II IL4Rmay differ fromthe type I receptor. Demonstrative of this concept, the acti-vation of Akt in response to IL4 seems to be context and cell-type dependent (5, 12), and IL4 has also been shown to activateErk in several nonhematopoietic cell types (5). Here, weinvestigate whether IL4Ra, a key component of the IL4R, canpromote the metastatic colonization and outgrowth of mam-mary tumor cells, and explore mechanisms of IL4/IL4R-induced colonization ability in vitro.

Department of Cancer Biology, Vanderbilt University Medical Center,Nashville, Tennessee.

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

Corresponding Author: Barbara Fingleton, Vanderbilt University MedicalCenter, 771 PRB, 2220 Pierce Avenue, Nashville, TN 37233-6480. Phone:615-936-5877; Fax: 615-936-2912; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-0093

�2014 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst June 19, 2014; DOI: 10.1158/0008-5472.CAN-14-0093

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Materials and MethodsCell lines and culture

The 4T1 cell line was acquired from Dr. Albert Reynolds atVanderbilt University (Nashville, TN). PyVT-R221a cells wereisolated from a polyoma middle T oncoprotein (PyMT) tumoras previously described (13). The R221a line was authenticatedby expression of the polyoma antigen, and themorphology andgrowth rates of both cell lines were consistently monitored.Cell lines were maintained in DMEM (Invitrogen) containing10% FBS (Atlanta Biologicals) at 37�C with 5% CO2. Missionlentiviral shRNA particles (Sigma) targeting two differentregions of murine IL4Ra (NM_010557) along with controlnontarget particles were used to infect 4T1 cells. Controlnontarget and specificmurine IL4Ra-targeted shRNA lentiviralparticles (Santa Cruz Biotechnologies) were used to infectR221a cells. Postinfection, shRNA-expressing cells were select-edwith puromycin dihydrochloride (Sigma). The percentage ofIL4Ra protein knockdown was calculated by densitometry inAdobe Photoshop following Western blot analysis.

Reagents and antibodiesMurine IL4 was purchased from BD Biosciences. The small-

molecule inhibitors, U1026 (EMD Millipore), Ly294002, andrapamycin (both Sigma) were dissolved in DMSO (Sigma) anddiluted with culture medium to the appropriate concentrationforWestern blot analyses and clonogenic assays. The followingantibodies were used to probe Western blots: murine IL4Ra,Stat6, Erk2, and b-actin from Santa Cruz Biotechnologies, anti-pStat6 (pY641) from Invitrogen, pErk1/2 (Thr202/Tyr204), Akt,pAkt (ser473), pAkt (Thr308), mTOR, pmTOR (ser2481),pmTOR (ser2448), IRS1, and pIRS1 (ser612) from Cell SignalingTechnology. An IL4Ra-Fc chimeric protein (R&D Systems) andisotype human IgG1k control antibody (SouthernBiotech) wereused in clonogenic assays.

Commercial arraysA human tissue array containing 16 stage IIIa/IIIb and 34

stage IIa/IIb primary breast carcinomas from patients withlymph nodemetastases, cat#BR10010a, was purchased fromU.S. Biomax. The array was probed with antibodies for humanIL4Ra (Santa Cruz Biotechnologies) and pStat6 (pY641; Sig-ma). Samples were scored categorically as positive or negativefor IL4Ra and pStat6. Conditioned media (serum free) fromR221a or 4T1 combined sh-control or IL4Ra knockdown cloneswere added to an antibody array to detect 62 murine secretedfactors (RayBiotech Inc. #AAM-CYT-3-4) per manufacturer'sinstructions. Protein expression was quantified by densitom-etry in ImageJ relative to positive controls.

Analysis of murine tumor modelsAnimal procedures were conducted in accordance with

Guidelines for the Care and Use of Laboratory Animals fol-lowing approval by the Institutional Animal Care and UseCommittee. Female age-matched BALB/c, FVB/N, and BALB/cIL4�/�mice (BALB/c-Il4tm2Nnt/J; Jackson Laboratories) wereused. Two individual clones for each cell line (R221a or 4T1)were mixed to generate combined sh-control or IL4Ra knock-

down cells preinjection. Additional assays with 4T1 cells wereperformed using one knockdown clone, which recapitulatedfindings with combined clones. At necropsy, lungs were inflat-ed with Bouin's solution (Ricca Chemical Company) to facil-itate surface tumor counts. Formalin-fixed, paraffin-embeddedtissue sections from lungs and livers were stained with hema-toxylin and eosin (H&E) to assess tumor burden. Tissuesections were also stained with primary antibodies for IL4Ra(Santa Cruz Biotechnologies), Ki67 (Abcam), cleaved caspase-3(Cell Signaling Technology), Ly6B.2, CD3-e, or F4/80 (AbDSerotec). In addition, lung sections were probed for pStat6(pY641; Sigma), pErk1/2 (Santa Cruz Biotechnologies), andpAkt (ser473; Cell Signaling Technology). Archival paraffin-embedded PyVT and orthotopic 4T1 and R221a sh-controlmammary tumors were stained for IL4Ra and pStat6 andanalyzed similarly to the human tissue array. Biotinylatedsecondary antibodies were obtained fromVector Laboratories.Images were white balanced for presentation using AdobePhotoshop.

Statistical analysisAll statistical analyses were performed using GraphPad

Prism software. For comparison of two conditions, data wereanalyzed using the parametric unpaired Student t test, or thenonparametric Mann–Whitney. Otherwise, ANOVA or Krus-kal–Wallis with posttest was used. In graphs, error barsrepresent the SD, and P values are indicated by asterisks(� , �0.05; ��, �0.01; and ��, �0.001).

ResultsIL4Ra expression and activation in human breast cancerand murine mammary cancer models

To examine the relative abundance and activation of IL4Rain human breast cancers, we first immunostained a humantissue array consisting of 50 primary node-positive breastcancer samples for IL4Ra and downstream phosphorylationof STAT6 (pSTAT6). A total of 82% of the samples were IL4Rapositive, and 42% were positive for both IL4Ra and pSTAT6(Fig. 1A). The majority of carcinomas were classifiable byHER2 status (38 HER2 positive and 12 HER2 negative, of whichfive were triple negative). However, sample numbers were toolow to make statistically significant correlations betweensubtype and IL4Ra or pSTAT6 positivity. Mammary tumorsfrom the PyMT oncogene transgenic mouse, and tumorsresulting from orthotopic injection of R221a and 4T1 murinemammary cancer cells, were also positive for IL4Ra and pStat6(Fig. 1B). Thus, human breast cancers and our murine modelsboth show IL4R expression and activation.

IL4Ra promotes the growth of murine mammary tumormetastases in the lung and liver

Because the IL4/IL4R interaction is species specific, onlymurine mammary tumor cell lines could be used to studyfunctional roles of IL4R signaling in mouse models. The 4T1line was derived from a spontaneous mammary cancer in aBALB/c mouse (14). The R221A line came from a mammarytumor in an MMTV-PyVT transgenic mouse on the FVB/N

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background (13). Using commercially available lentiviral par-ticles carrying shRNA, we generated two clones representingdifferent knockdown sequences for the 4T1 line. A separatemix of IL4Ra-targeted shRNA sequences was used to maketwo different clones of R221A cells. Corresponding sh-controllines made by expression of nontargeting shRNA sequenceswere also generated. Western blot analysis confirmed a reduc-tion (62%–90%) in IL4Ra protein expression (Fig. 2A), and inStat6 activation in R221a and 4T1 IL4Ra knockdown clonescompared with sh-controls.Combined sh-control or IL4Ra knockdown clones for each

cell line (R221a or 4T1) were injected into the tail vein or spleenof age-matched female mice to examine colonization of lungand liver, respectively. At endpoint, the number of surfacetumors on the lungs of tail vein injected mice was significantlyreduced in tumors originating from IL4Ra knockdown clonesfor both cell lines (Supplementary Fig. S1A). In addition, grossliver weights of mice receiving splenic injections of R221aIL4Ra knockdowncloneswere significantly reduced comparedwith sh-controls, but no significant difference in liver weightwas seenwith the 4T1 line (Supplementary Fig. S1B). Followinggross examination, lungs and livers were serially sectioned forfurther analysis. IHC analysis confirmed that functional knock-down of IL4Ra was retained in vivo, as IL4Ra expression (Fig.2C) and nuclear pStat6 levels (Fig. 2D) were significantlyreduced in IL4Ra knockdown lung tumors compared with

sh-control for both cell lines. Serial lung (Supplementary Fig.S1C) and liver (Supplementary Fig. S1D) sections were alsostained with H&E, and used to calculate tumor burden. In boththe R221a and 4T1 cell lines, reduced IL4Ra expressionresulted in a significant decrease in metastatic lung and livertumor burden (Fig. 3A and B).

We previously determined that neither R221a nor 4T1cells produce detectable levels of IL4 by Luminex analysis(data not shown), suggesting that the cellular source of IL4in vivo is likely the host. To determine the contribution ofhost IL4 to mammary cancer metastasis to the lung, as wellas the significance of the IL4/IL4R interaction in vivo, wild-type (WT) or IL4�/� (IL4 KO) BALB/c mice were injectedvia tail vein with either combined sh-control or IL4Raknockdown 4T1 clones. Supporting the role of IL4 in pro-moting metastatic tumor growth, the number of lung surfacetumors (Fig. 3C) and the lung tumor burden (Fig. 3D) atendpoint were significantly decreased in IL4 KO mice com-pared with WT mice receiving sh-control cells. These resultssuggest that the IL4/IL4Ra interaction is a strong promoterof lung metastatic tumor growth in vivo.

IL4Ra-associated survival and proliferation contributeto metastatic colonization and outgrowth

The metastatic seeding and outgrowth ability of R221aand 4T1 clones were estimated by quantifying tumor

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Figure 1. IL4R expression andactivation in humanbreast cancersand murine models. A, left,representative images of a humantissue microarray of breastcarcinoma samples (n ¼ 50)stained by IHC for IL4Ra orpSTAT6. Right, summary ofcategorical (positive/negative)tissue microarray staining results.B, representative images of IL4Raand pStat6 immunostaining inmurine mammary tumors (n ¼ 3each). Scale bar, 100 mm. Inset,negative staining controls andone �40 image (scale bar, 10 mm)of pStat6 in an R221a tumor.

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number and area, respectively, from the H&E-stained lungand liver sections (Supplementary Fig. S1B and S1C). Theability of R221a IL4Ra knockdown clones to seed tumorswas significantly reduced in both the lung (Fig. 4A) and liver(Fig. 4B) compared with sh-control clones. Although thesame was true for 4T1 IL4Ra knockdown clones comparedwith sh-controls in the lung (Fig. 4A), the decrease in seedingability in the liver was not significant (Fig. 4B). However, inboth cell lines, reduced IL4Ra expression resulted in asignificant decrease in lung (Fig. 4C) and liver tumor out-growth (Fig. 4D).

In addition to quantifying tumor number and size, lungand liver metastases were stained for cleaved caspase-3

(apoptosis marker) and Ki67 (proliferation marker) to fur-ther examine the role of IL4Ra in promoting the survival andoutgrowth of metastatic tumors (Supplementary Fig. S2).Differences in cleaved caspase-3 in the lungs of mice receiv-ing either R221a or 4T1 IL4Ra knockdown cells by tail veininjection were not statistically significant (Fig. 4E). In R221atumors, this was likely due to the lack of assessable stainingwithin very small knockdown tumors. Quantification ofcleaved caspase-3 staining in liver metastases revealed asignificant increase in apoptosis in IL4Ra knockdowntumors of R221a, but not 4T1 recipient mice comparedwith sh-control (Fig. 4F). Ki67 staining results were moreconsistent in that mice receiving either R221a or 4T1 IL4Ra

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Figure 2. Functional knockdown ofIL4Ra expression in vitro and invivo. A andB,Western blot analysisillustrating the percent knockdownof IL4Raprotein expression (A) andStat6 activation in response to 20ng/mL IL4 in individual R221aand 4T1 control (ctl) and IL4Raknockdown (KD) clones (B). C andD, representative images andquantification of IHC staining forIL4Ra (�40, scale bar, 10 mm; C) orpStat6 (�20, scale bar, 100 mm; D)in R221a and 4T1 sh-control andIL4Ra knockdown lungmetastases (n ¼ 4–6). Inset,negative staining controls.

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knockdown cells exhibited a significant reduction in prolif-eration in both lung (Fig. 4G) and liver metastases (Fig. 4H).These data suggest that IL4Ra promotes the growth ofmurine mammary cancer cells at metastatic tumor sitesthrough enhanced survival and colonization ability andincreased proliferation for outgrowth.

Changes in immune infiltration contribute to IL4Ra-enhanced metastasisIt is possible that increases in infiltrating immune cells

could aid in the clearance of IL4Ra knockdown tumor cellswith reduced survival ability, thus decreasing metastatictumor size. We therefore quantified infiltrating immunecells in R221a and 4T1 sh-control and IL4Ra knockdownlung and liver metastases by IHC staining of neutrophils(Ly6B.2 positive), macrophages (F4/80 positive), and T lym-phocytes (CD3 positive; R221a: Supplementary Fig. S3; 4T1:Supplementary Fig. S4). There was an upward trend orsignificant increase in the number of each of these cell typesin R221a and 4T1 lung and liver IL4Ra knockdown meta-stases compared with sh-control (R221a: Fig. 5A–C; 4T1:Supplementary Fig. S5).Higher levels of immune infiltrates within knockdown

tumors could result from an increased expression of proin-flammatory cytokines. To examine this, a murine cytokinearray was used to test conditioned media from combinedR221a or 4T1 sh-control or IL4Ra knockdown clones for the

expression of 62 secreted factors. Five factors associatedwith recruitment of immune cells (MIG, IL1a, Lix, Eotaxin,SCF, and MIP-3) were modestly upregulated by IL4Raknockdown clones in comparison with sh-control clonesfor both cell lines (Fig. 5D). Macrophage inflammatoryprotein-2 (MIP-2) was the only cytokine that increased morethan 2-fold in knockdown clones for both cell lines. Leptinreceptor has no known direct role in immune cell recruit-ment; however, this protein was also upregulated more than2-fold by both R221a and 4T1 knockdown clones. Thus,although it is possible that increased MIP-2 production fromIL4Ra knockdown clones may lead to enhanced leukocyterecruitment, we were unable to identify robust patterns ofcytokine production that differed between sh-control andIL4Ra knockdown cells.

IL4-activated Akt, Erk, and mTOR mediate colonizationability of mammary cancer cells

Our in vivo data establishes that the IL4R mediatesenhanced colonization of mammary cancer cells at meta-static sites, and that host IL4 promotes the growth of lungtumor metastases. Therefore, we used exogenous IL4 toelucidate signaling pathways that control IL4Ra-inducedcolonization ability in vitro. Demonstrative of functionalIL4/IL4Ra signaling, treatment of R221a and 4T1 mammarycancer cells with IL4 resulted in the activation of Stat6(Fig. 6A). Erk1/2 and Akt (ser473) were also phosphorylated

Figure 3. The IL4/IL4Ra interactionpromotes metastatic tumor growthin vivo. Quantification of tumorburden from H&E-stained lung (A)and liver (B) sections (three permouse) from mice receiving R221aor 4T1 IL4Ra knockdown (KD) orsh-control cells (lung, n¼4–6; liver,n ¼ 5–8). C and D, enumeration oflung surface tumors (C) andquantification of lung tumor burden(D) from H&E-stained serialsections fromWTor IL4�/� (IL4 KO)mice receiving either 4T1 sh-control or IL4Ra knockdownclones by tail vein injection(n ¼ 3–6).






in response to IL4 in both cell lines, whereas Akt (thr308) wasonly robustly phosphorylated in R221a cells (Fig. 6A). Con-sistent with IRS1 inactivation and degradation (15, 16), IRS1

(ser612) was also phosphorylated in response to IL4 in bothcell lines in a temporal manner, and total IRS1 levels initiallyincreased then rapidly decreased in the 4T1 cell line.

Figure 4. IL4Ra-associated survivaland proliferation contribute tometastatic tumor growth. A and B,enumeration of R221a or 4T1IL4Ra knockdown (KD) and sh-control lung (A) and liver (B)metastatic tumors per mouse(lung, n ¼ 4–6; liver, n ¼ 5–8) fromH&E-stained sections (three permouse). C and D, quantification ofindividual lung (C) or liver (D) tumorareas from the sameH&E sections.E and F, quantification of totalcleaved caspase-3–positive areaper total IL4Ra knockdown or sh-control lung (E) or liver (F) tumorarea for eachmouse (lung, n¼ 4–6;liver, n ¼ 5–8). G and H,quantification of the total numberof Ki67-positive cells per IL4Raknockdown or sh-control tumorarea for eachmouse lung (G) or liver(H; lung, n ¼ 3–6; liver, n ¼ 5–8).

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We next confirmed the specificity of the IL4/IL4Ra inter-action in promoting colonization ability in vitro. R221a or 4T1combined sh-control or IL4Ra knockdown cells were plated atclonal density in the presence or absence of IL4 with an IL4-blocking decoy receptor (IL4Ra-FC), or an IgG control. Theability of IL4Ra-FC to neutralize IL4-induced Stat6 activation

at the dose used in clonogenic assays was confirmed byWestern blot analysis (Fig. 6A). As expected, the significantincrease in colonization ability in response to IL4 was blockedby the addition of IL4Ra-FC in control clones, but neither IL4nor IL4Ra-FC had a significant effect on the colonizationability of IL4Ra knockdown clones for either cell line

Figure 5. IL4Ra expression isassociated with changes inimmune infiltration and cytokineproduction. Quantification ofneutrophils (A), macrophages (B),and lymphocytes (C) fromimmunostained lung and liversections of mice receiving R221aor IL4Ra knockdown (KD) or sh-control cells (n ¼ 4–7). D,quantitation of the fold-change inprotein levels from R221a and 4T1IL4Ra knockdown and sh-controlconditioned media, assessedusing a commercial cytokinearray and densitometry. Proteinsincreased in IL4Ra knockdownclones for both cells lines that havepotential to regulate immunerecruitment are bracketed.






(Supplementary Fig. S6B). We then used small-molecule inhi-bitors to test the contribution of IL4-actived Erk andAkt to IL4-induced colonization ability. U1026 was used to inhibit the

activity of Erk1/2 (17), and LY294002, a known PI3K inhibitor,was used to inhibit Akt phosphorylation at both sites (thr308and ser473; ref. 18). First, the effective dose of inhibitor that

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Figure 6. Inhibition of IL4-activatedStat6, Akt, and Erk. RepresentativeWestern blot analyses were completedusing whole-cell lysates fromcombined R221a or 4T1 sh-controlclones and 20 ng/mL IL4. A, timecourse analysis of effector proteinsactivated within 40 minutes of IL4treatment. B, U1026 (R221a,10 mmol/L; 4T1, 15 mmol/L) inhibitsIL4-activated pErk1/2, LY294002(R221a, 5 mmol/L; 4T1, 8 mmol/L)inhibits IL4-activated pAkt, andrapamycin (R221a, 5 nmol/L; 4T1,3 nmol/L) inhibits pmTOR.

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decreased colony formation by 50% (ED50) was determinedfor each cell line (data not shown). Before their use in clono-genic assays, selected drug doses were also confirmed toinhibit IL4-induced activation of pErk1/2 or pAkt (thr308/ser473), by Western blot analysis (Fig. 6B). Suggesting thatIL4-activated Erk1/2 promotes initial colonization, U1026treatment in both cell lines blocked IL4-induced colony for-mation (Fig. 7A). Treatment with LY294002 also blocked IL4-induced colony formation in the 4T1 cell line, but not the R221aline (Fig. 7B).A potential complication with LY294002 is that it also

inhibits mTOR at concentrations slightly higher than theIC50 of PI3K (19). We therefore also tested the effect ofrapamycin. In both cell lines, combined IL4 and rapamycintreatment significantly reduced colony formation comparedwith IL4 treatment alone (Fig. 7C). We then discovered byWestern blot analysis that rapamycin treatment resulted instrong inhibition of IL4-activated mTOR ser2481, but notmTOR ser2448, indicating that activated mTORc2 rather thanmTORc1 may be important in regulating IL4-induced colonyformation (Fig. 6B).To evaluate whether the signaling pathways identified in

vitro were relevant in vivo, we immunostained for pAkt(ser473), pErk1/2, and pmTOR (ser2481) in R221a and 4T1sh-control and IL4Ra knockdown lung metastases (Supple-mentary Fig. S7). The anti-pmTOR (ser2481) antibody selec-tively labeled mitotic cells with high intensity and did notprovide useful information.However, wewere able to confirmasignificant reduction in both pErk1/2 and pAkt (ser473) inIL4Ra knockdown R221a and 4T1 lung metastases comparedwith sh-controls (Fig. 7D and E).

DiscussionThe biologic effects of the IL4/IL4Ra interaction have

recently been extended from immune cells to epithelial cancercells expressing the type II IL4R (IL4Ra and IL13Ra1 chains).Previous studies have established the importance of thisinteraction in promoting protumorigenic phenotypes, includ-ing proliferation and survival ability in several types of epithe-lial cancers (20). However, themajority of these studies utilizedin vitro assays and subcutaneous xenograft models in nudemice. Here, we have established the relevance of IL4Ra expres-sion in human breast cancer and in two syngeneic murinemodels of breast cancer (Fig. 1). Usingmurinemodels, we havedefined a novel role of epithelial IL4Ra in promoting meta-static mammary tumor growth.Using R221a and 4T1 IL4Ra knockdown clones and WT

mice in experimental metastasis assays, we demonstratedthat reduced IL4Ra expression results in significantly atten-uated metastatic tumor burden in both the lung and the liver(Fig. 3). This reduced burden was also seen in the lungs ofIL4�/� mice injected with control cells, indicating that reduc-ing either the ligand or receptor can attenuate metastaticgrowth (Fig. 3C and D). Notably, IL4�/� mice injected withIL4Ra knockdown cells showed a greater reduction in tumorburden than either IL4�/� or IL4Ra knockdown alone. Thiscould be partially explained by incomplete knockdown of

IL4Ra, but may also reflect a role for the other type II IL4Rligand, IL13 in vivo. Decreased metastatic ability of IL4Raknockdown clones was attributed to (i) reduced tumor cellsurvival as determined by seeding ability (number of tumors)and an increase in apoptosis by cleaved caspase-3 positivity,and (ii) a reduction in proliferation determined by tumoroutgrowth ability (tumor size) and a decrease in Ki67 positivity(Fig. 4). In some cases, differences in these parameters werenot significant at endpoint, which may be attributable tochanges that occurred earlier in tumor development. In addi-tion, there are large differences in the growth rates of the celllines. The aggressive and highly metastatic 4T1 line is typicallyused to establish models representative of clinical stage fourbreast cancer, whereas R221a cells rarely metastasize sponta-neously, and may represent less aggressive breast cancers.This growth disparity is most evident in the H&E-stainedimages of the 4T1 livers (Supplementary Fig. S1D), where largetumors were difficult to delineate from each other.

In addition to proliferation and survival, we examinedwhether immune infiltrates contributed to the reduction inknockdown tumor size. We saw increases in almost all leuko-cyte populations examined in both R221a and 4T1 knockdownmetastases (R221a: Fig. 4; 4T1 Supplementary Fig. S5). Thusour results overall suggest that more efficient clearance ofknockdown tumor cells could contribute to the reduction intumor size visualized at endpoint compared with sh-controltumors.

It is feasible that enhanced production of proinflammatorycytokines from IL4Ra knockdown cells could recruit immunecells to these tumors. However, out of 62 secreted proinflam-matory factors examined by cytokine array, only MIP-2 wasincreased more than 2-fold by IL4Ra knockdown cells incomparison with sh-controls for both cell lines (Fig. 5D).MIP-2 secreted from epithelial cells enhances neutrophil andlymphocyte recruitment (21). Leptin/leptin receptor signalingreportedly induces MIP-2 production in preneoplastic coloncells (22). Therefore, the increased expression of leptin (>1.5-fold) and its receptor (>2-fold) within IL4Ra knockdowntumors could also facilitate neutrophil and lymphocyte recruit-ment. Still, strong patterns of cytokine production from IL4Raknockdown cells were lacking, indicating that other mechan-isms may contribute to IL4Ra-induced survival and prolifer-ation ability at metastatic sites.

The Jak/Stat6 pathway is activated by the IL4/IL4Ra inter-action in lymphoid cells (5), and in several epithelial cancer celltypes, including breast cancer (Fig. 1; ref. 7). We were able toconfirm activation of IL4R in vitro via phosphorylation ofdownstream Stat6 in response to IL4 (Fig. 6A). Stat6 has beenshown to regulate prometastatic processes, including migra-tion and invasion (23) and enhanced survival and prolifera-tion in vitro (4, 7, 21), although one study showed that Stat6activation decreased between ductal carcinomas in situ andinvasive breast ductal carcinomas (24). That study did notexamine IL4Ra expression, or other IL4/IL4Ra-activated path-ways, including PI3K/Akt and MAPK/Erk, that may be impor-tant for the promotion of metastatic phenotypes in vivo.

Using the Mek inhibitor, U1026, we determined that IL4-activated Erk1/2 promotes the colonization ability of






Figure 7. The IL4/IL4Ra interactionpromotes colonization ability viaErk, Akt, and mTOR activation.R221a or 4T1-combined sh-control clones were seeded � IL4(R221a, 2 ng/mL; 4T1, 10 ng/mL)and � drug. A, U1026 (R221a, 10mmol/L; 4T1, 15 mmol/L) inhibitsIL4-induced colony formation. B,LY294002 inhibits colonyformation in 4T1 cells (8 mmol/L),but not R221a cells (5 mmol/L). C,rapamycin (R221a, 5 nmol/L; 4T1, 3nmol/L) inhibits IL4-inducedcolonyformation. D and E, quantificationof pErk1/2 (D) and pAkt (ser473) (E)immunostaining in R221a and4T1 IL4Ra knockdown (KD) andsh-control lung metastases. Thepercentage of positive area permouse (n ¼ 4–6) is shown.

Venmar et al.





mammary cancer cells in vitro (Fig. 7A). The PI3K/Akt pathwayinhibitor, LY294002, blocked IL4-activated Akt in both celllines, but only abrogated colony formation in the R221a line(Fig. 7B). The R221a cells express the middle T oncoprotein, astrong driver of constitutive PI3K/Akt and Src kinase activity,which would be unaffected by LY294002 treatment (25). Theseresults confirm previous reports that activation of Akt inresponse to IL4 is cell type/context dependent (5, 12).We next examined whether IL4-activated mTOR could

promote IL4-induced colony formation for two reasons: (i)Akt was phosphorylated at ser473 in both cell lines in responseto IL4 (Fig. 6A) and (ii) LY294002 is known to fit in the activecatalytic domain of mTOR and inhibit its activity at slightlyhigher concentrations than the IC50 for PI3K inhibition (19).Rapamycin, a potent mTORc1 inhibitor, has also been shownto inhibit ser2481 phosphorylation and mTORc2 (26). Use ofthis compound in vitro demonstrated that IL4-activatedmTORc2 (ser2481), but not mTORc1 (ser2448), is a potentpromoter of colonization ability in both the R221a and 4T1 celllines (Fig. 6B and Fig. 7C). Our findings are consistent with aprevious report demonstrating that mTORc2 but notmTORC1 mediates the survival of breast cancer cells (27).IRS proteins mediate the activation of several signaling

pathways in response to IL4 (27–29). IRS1 expression in breastcancers is often decreased in high-grade invasive tumorscompared with well-differentiated tumors (24, 30–32). Also,IRS1 in murine lung metastases is inactivated by serine phos-phorylation, and loss of IRS1 expression in murine mammarycancer cells led to increased lung metastases (32). Serinephosphorylation of IRS1 for inactivation and/or proteasomaldegradation is known to be mediated by several kinases,including mTOR, Akt, and Mapk (33), three effector proteinsactivated in response to IL4 (Fig. 6). In R221a and 4T1 cells,ser612 is phosphorylated in a time-dependentmanner after IL4(Fig. 6A). This may explain the eventual decrease of total IRS1protein levels in 4T1 cells (Fig. 6A). These results indicate thatIL4 signaling may induce the inactivation and proteasomaldegradation of IRS1, thus removing a potential metastasissuppressor.In conclusion, we have demonstrated for the first time that

the IL4/IL4Ra interaction promotes mammary metastatictumor growth, and that loss of IL4Ra in mammary cancercells results in reduced survival and proliferation anddecreased colonization and outgrowth at metastatic sites. Itis known that the Mapk/Erk, and PI3K/Akt/mTOR signaling

axes are potent drivers of breast cancer growth (26, 27, 34, 35).We have shown that Erk, Akt, and mTOR are downstreameffectors of the IL4Ra-associated prometastatic colonizationphenotype. In addition, suppression of IRS1 signaling may beanother mechanism by which IL4/IL4Ra promotes mammarycancer metastatic tumor growth. Overall, systemic inhibitionof IL4Ramay have promise as an anticancer therapy, providedthat any adverse effects on immune cells that signal throughthe type I IL4R could be minimized. Significantly, modified IL4ligands termed "superkines," are capable of specifically target-ing the type II IL4R, and are being tested for improving cytokinetherapy selectivity (36). We raise the possibility of using suchagents to target the type II IL4R onbreast cancer cells as a novelmeans to thwart metastatic tumor growth.

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

Authors' ContributionsConception and design: K.T. Venmar, B. FingletonDevelopment of methodology: K.T. Venmar, D.G. Hwang, E.A. DozierAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.T. Venmar, D.G. HwangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.T. Venmar, B. FingletonWriting, review, and/or revision of the manuscript: K.T. Venmar,B. FingletonAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K.T. Venmar, K.J. CarterStudy supervision: B. FingletonOther (conducted experiments and established cell lines): E.A. Dozier

AcknowledgmentsThe authors thank Drs. Sung Hoon Cho and Mark Boothby for Luminex

measurements, Dr. Joe Roland and the Epithelial Biology Core for access to theGelcount instrument and Leica SCN400 slide scanner, and Dr. Christine Lovly forconsultation and the IRS1 antibody.

Grant SupportThis work was supported by DOD award W81XWH-09-1-0446 and by R01

CA157781 (B. Fingleton). K.T. Venmar was supported by the Cellular, Biochemi-cal and Molecular Sciences training program (5T32GM008554). Use of theTranslational Pathology Shared Resource was supported by Cancer CenterSupport Grant #P30 CA068485. In addition, support was obtained from theNational Center for Research Resources, Grant UL1 RR024975-01, now at theNational Center for Advancing Translational Sciences, Grant 2 UL1 TR000445-06.

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 January 14, 2014; revised May 5, 2014; accepted May 29, 2014;published OnlineFirst June 19, 2014.

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Venmar et al.




2014;74:4329-4340. Published OnlineFirst June 19, 2014.Cancer Res Katherine T. Venmar, Kathy J. Carter, Daniel G. Hwang, et al. Tumors through Multiple Signaling Pathways

Regulates Metastatic Colonization by MammaryαIL4 Receptor ILR4

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