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Research Article

Lack of STAT6 Attenuates Inflammation andDrives Protection against Early Steps ofColitis-Associated Colon CancerSonia A. Leon-Cabrera1,2, Emmanuel Molina-Guzman1, Yael G. Delgado-Ramirez1,Armando V�azquez-Sandoval1, Yadira Ledesma-Soto1, Carlos G. P�erez-Plasencia1,Yolanda I. Chirino1, Norma L. Delgado-Buenrostro1, Miriam Rodríguez-Sosa1,

Felipe Vaca-Paniagua1,3, Federico �Avila-Moreno1, Emma B. Gutierrez-Cirlos1,Luis E. Arias-Romero1, and Luis I. Terrazas1,3

Abstract

Colitis-associated colon cancer (CAC) is one of the mostcommon malignant neoplasms and a leading cause of death.The immunologic factors associated with CAC development arenot completely understood. Signal transducer and activator oftranscription 6 (STAT6) is part of an important signalingpathway for modulating intestinal immune function andhomeostasis. However, the role of STAT6 in colon cancerprogression is unclear. Following CAC induction in wild-type(WT) and STAT6-deficient mice (STAT6–/–), we found that 70%of STAT6–/– mice were tumor-free after 8 weeks, whereas 100%of WT mice developed tumors. STAT6–/– mice displayed fewerand smaller colorectal tumors than WT mice; this reducedtumorigenicity was associated with decreased proliferationand increased apoptosis in the colonic mucosa in the earlysteps of tumor progression. STAT6–/– mice also exhibited

reduced inflammation, diminished concentrations COX2 andnuclear b-catenin protein in the colon, and decreased mRNAexpression of IL17A and TNFa, but increased IL10 expressionwhen compared with WT mice. Impaired mucosal expressionof CCL9, CCL25, and CXCR2 was also observed. In addition,the number of circulating CD11bþLy6ChiCCR2þ monocytesand CD11bþLy6ClowLy6Gþ granulocytes was both decreasedin a STAT6-dependent manner. Finally, WT mice receiving aSTAT6 inhibitor in vivo confirmed a significant reduction intumor load as well as less intense signs of CAC. Our resultsdemonstrate that STAT6 is critical in the early steps of CACdevelopment for modulating inflammatory responses and con-trolling cell recruitment and proliferation. Thus, STAT6 mayrepresent a promising target for CAC treatment. Cancer ImmunolRes; 5(5); 385–96. �2017 AACR.

IntroductionChronic inflammation is widely associated with increased

susceptibility to developing colorectal cancer (1). Patients withinflammatory bowel diseases such as Crohn's disease andulcerative colitis (UC), which are characterized by prolongedinflammation of the intestine, have an increased risk of devel-oping colorectal cancer (2). Epidemiologic studies indicatethat treatment with anti-inflammatory drugs could prevent or

delay colorectal cancer, suggesting the involvement of inflam-matory pathways in tumor progression (3). In a geneticallysusceptible host, epithelial barrier breakdown could result inmagnified responses to microbial products, thus leading tochronic inflammation and tumorigenesis. Furthermore, pro-inflammatory cytokines produced in response to commensalbacteria can create a microenvironment that enhances epithe-lial cell proliferation, angiogenesis, and increased susceptibil-ity to mutations (1).

JAK/STAT is an important signaling pathway for modulatingintestinal immune function (4). Signal transducer and activatorof transcription 6 (STAT6) proteins are activated by phosphor-ylation of Janus kinase (JAK) family of proteins in response tothe binding of interleukin (IL) 4 or 13 to their common type IIIL4 receptor (IL4R; ref. 5). When STAT6 is phosphorylated byJAK, STAT6 homodimers are formed and translocate to thenucleus to initiate the transcription of IL4, IL13, and otherresponsive genes (5, 6).

In addition to its pivotal role in IL4 and IL13 signaling,STAT6 participates in epithelial malignancies (7, 8). STAT6 isalso constitutively stimulated in a number of cancer types,including colon (9) and prostate cancer (10), mediastinal largeB-cell lymphoma (10), and Hodgkin's lymphoma (11). Typi-cally, STAT6–/– mice have defects in Th2 polarization (12) and

1Unidad de Biomedicina, Facultad de Estudios Superiores-Iztacala, UniversidadNacional Aut�onoma de M�exico, Edo. De M�exico, Mexico. 2Carrera de M�edicoCirujano, Facultad de Estudios Superiores Iztacala, Universidad NacionalAut�onoma de M�exico, Edo. De M�exico, Mexico. 3Laboratorio Nacional en Salud,Facultad de Estudios Superiores-Iztacala, Universidad Nacional Aut�onoma deM�exico, Mexico.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Luis I. Terrazas, Facultad de Estudios Superiores Izta-cala, UNAM. Av. De los Barrios 1, Tlalnepantla, Estado de Mexico. Mexico 54090.Phone: 52-55-5623-1333, ext. 39773; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-16-0168

�2017 American Association for Cancer Research.

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are able to reject or delay primary mammary tumor growth,prevent the recurrence of primary tumors, and/or reject estab-lished, spontaneous metastatic disease in mammary carcinoma(7). STAT6–/– mice also show enhanced immunity againsttumors, due to the increased differentiation of CD4 T cellstoward a Th1 phenotype (12, 13). However, the role of STAT6in colon carcinogenesis is less clear. STAT6–/– mice are sensitiveto DSS-induced colitis, exhibit chronic architectural changes,and produce more IFNg in this type of injury (14). Addition-ally, a more severe colonic inflammatory response in DNBScolitis in STAT6–/– mice has been reported (15). In contrast, inoxazolone-induced colitis, a murine model of colitis withpathologic and immunologic features similar to UC, colitiswas attenuated in STAT6–/– mice accompanied by reducedclaudin-2 induction, suggesting an important role for STAT6in epithelial barrier function (16). Despite evidence suggestingthat STAT6 plays key roles in intestinal biological processes,the role that STAT6 may play in intestinal tumorigenesis,particularly in colon cancer, preceded by chronic inflammation,remains unclear.

In the present study, we evaluated the role of STAT6 in devel-oping colitis-associated colon cancer (CAC) using the azoxy-methane (AOM)/dextran sodium sulfate (DSS) model, examin-ing the dependence of colonic inflammation, epithelial cellproliferation and apoptosis, and monocytic-granulocytic recruit-ment on STAT6 signaling. Our results suggest a critical role forSTAT6 in the early events of CAC development in promotingthe survival of colonic epithelial cells, perhaps by modulatingearly inflammatory responses that could be detrimental for tumordevelopment.

Materials and MethodsMice

Six-week-old female STAT6–/–and STAT6þ/þ (WT) mice on aBALB/c genetic background were originally purchased from TheJackson Laboratory Animal Resources Center andmaintained in apathogen-free environment at the FES-Iztacala, UniversidadNacional Aut�onoma de M�exico (UNAM) animal facilities. Theanimals were fed Purina Diet 5015 (Purina) and water ad libitum.All experimental procedures were approved by the EthicalCommittees of the UNAM, according to the University AnimalCare and Use Committee.

Murine model of colitis-associated colorectal cancerThe CAC model was carried out as described previously

(17). Briefly, WT and STAT6–/– mice received an intraperitoneal(i.p.) injection containing 12.5 mg/kg azoxymethane (AOM;Sigma). Five days later, 2% dextran sodium sulfate (DSS, MW:35,000–50,000, MP Biomedicals. Solon) was dissolved in theanimals' drinking water for 7 days. Afterwards, the mice receivedregular drinking water for 14 days. This experimental series wasrepeated twice. To examine early and late transformative stepsin CAC, the mice were killed on days 20, 40, and 68 after AOMinjection. Throughout the experiment, the mice were monitoredweekly for body weight, stool consistency, and the presence ofblood in the rectumor stools. The disease activity score (DAI) wascalculated as the sum of the diarrhea score plus the bloodystool score as follows: 0 ¼ normal stool and normal-coloredstool, 1 ¼ mildly soft stool and brown stool, 2 ¼ very soft stooland reddish stool, and 3 ¼ watery stool and bloody stool.

During animal necropsy, the colon was removed, weighed,and submitted to macroscopic inspection. Immediately, thecolonic tissue was either fixed in 100% ethanol and embeddedin paraffin for histopathologic analysis or snap frozen in liquidnitrogen for RNA and protein extraction.

Histologic analysisCollected tissues from WT and STAT6–/–mice were fixed in

100% ethanol and embedded in paraffin for posterior 5-mmcross-sectioning. The tissue sections were stained with hema-toxylin and eosin (H&E; for pathologic evaluation) or Alcianblue (for acidic polysaccharides). For immunohistochemicalanalysis, the sections were deparaffinized in xylene and thenrehydrated with graded alcohols and processed as reportedpreviously (18). The sections were incubated overnight at 4�Cwith the respective primary antibodies diluted in 1X PBS (anti-COX2, 1:100, GeneTex; anti-b-catenin, 1:100, GeneTex; anti-Ki67, 1:100, Biolegend) and then developed following theconventional technique. The slides were analyzed using anAxioVert.A1 image capture optical microscope (Carl ZeissMicroscopy GmbH). Tissue microphotographs were capturedusing an AxioCam MRc and ZEN lite 2011 software v.1.0.1.0.Quantification of COX2þ, b-cateninþ, and Ki67þ cells wasperformed using ImageJ software v.4.9 by counting cells in10 high-powered fields from each mouse.

TUNEL stainingApoptosis was detected using the In Situ Cell Death Detection

Fluorescein Kit (Roche), analyzing samples with a ZeissVert A1conventional epifluorescence microscope and a LEICA TCS SP2confocal microscope (the analyzed area in each sample was2.8 mm2, and 20 fields of 50 mm2 were evaluated).

RNA extraction and RT-PCRTissues were first disrupted in a tissue homogenizer (Bullet

Blender; Next Advance). Total RNA from colon tissue wasextracted using TRIzol reagent (Invitrogen) following the man-ufacturer's instructions. The RNA concentration was determin-ed by measuring the absorbance at 260 nm. One microgramof RNA was used for first-strand cDNA synthesis with RevertAidH Minus First Strand cDNA Synthesis Kit (Thermo FisherScientific). The primer sequences and cycling conditions arelisted in Supplementary Table S1.

ELISAMesenteric lymphoid cells from mice were plated in 96-well

plates coated with anti CD3/CD28 (BioLegend)-antibodies(2 mg/mL). After 24 hours, supernatants were analyzed for thepresence of IL10, IL17A, and TNFa in all samples using a mouseIL10 and TNFa ELISA kit (Peprotech Mexico) and mouse IL17AELISA kit (BioLegend).

Flow cytometryFor flow cytometry circulating blood was obtained during

animal necropsy. The cells were washed with 1� PBS and blockedusing antibodies to CD16/CD32. The cells were simultaneouslystained with antibodies to CD11b, Ly6C, Ly6G, CD4, CD8(BioLegend) and CCR-2 (R&D Systems) for 30 minutes at 4�C.The cells were washed twice and analyzed using the FACSCalibursystem and Cell Quest software (Becton Dickinson).

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STAT6 inhibition in vivoBALB/c mice were induced for CAC as described above and

when initializing the first DSS cycle received every third dayintraperitoneally 10 mg/kg of AS1517499 (Axon Medchem)reconstituted in dimethyl sulfoxide (DMSO) and PBS at thetime points indicated. Control mice received the same volumeof vehicle alone. Mice were sacrificed one week after the first(day 20), second (day 40), and third (day 68) DSS-cycle.Tissues and tumors counts were processed as described above.The effectiveness of this treatment was tested by measuringSTAT6 phosphorylation in colon tissue by Western blot.

Immunoblot analysisColon tissues were lysed in RIPA buffer supplemented with

proteinase and phosphatase inhibitor cocktail (Roche), and cen-trifuged for 10 minutes at 14,000 rpm. Supernatants were col-lected, run on SDS gels, and transferred onto membranes. Themembranes were blocked and probed with antibodies to phos-pho-STAT6 Y641 (Santa Cruz Biotechnology), STAT6 (M-20;Santa Cruz Biotechnology), and b-actin (BioLegend).

Statistical analysisData were analyzed by one-way analysis of variance followed

by Tukey's multiple comparisons test or unpaired two-tailed ttests depending on the number of groups using GraphPadPrism 5 (San Diego). All statistical tests were performed con-sidering 95% confidence intervals. The data are expressed as themean � SE. �, P < 0.05; ��, P < 0.01.

ResultsSTAT6-deficient animals less susceptible to AOM/DSS-induced CAC

Previous studies have shown that STAT6 is involved inintestinal epithelial homeostasis and its activity is correlatedwith colon cancer cell growth in vitro (19, 20), yet the role ofSTAT6 has not been extensively studied in the early steps ofCAC. To examine the role of STAT6 in vivo, we used a well-established model of CAC (21). We evaluated CAC progressionat 20, 40 and 68 days in WT and STAT6–/–mice. We monitoredstool consistency and changes in body weight and tumordevelopment. As expected, WT-treated mice rapidly showedpiloerection and clinical signs of the disease (DAI) throughoutthe experiment (Fig. 1A). STAT6–/– mice did not have diarrheaor rectal bleeding during the course of treatment comparedwith similarly treated WT animals (Fig. 1A). During the courseof AOM/DSS administration, WT mice lost weight comparedwith the baseline weight (Fig. 1B). Nevertheless, STAT6–/–

animals had progressive weight gain over time that was notsignificantly affected by AOM/DSS administration. Two and 8weeks after treatment, the weight in WT mice was significantlydecreased compared with STAT6–/– animals (Fig. 1B). Consis-tent with this, the polypoid lesions and the early signs of thedisease appeared in WT mice at 40 days of treatment, whereasSTAT6–/– mice showed mild symptoms and no tumors (Fig.1C). At this time, 70% of WT mice had increased numbers oftumors and an increased tumor load, whereas all STAT6–/–

animals remained tumor-free (Fig. 1C). At necropsy on day68, all WT animals displayed reddish polypoid tumors in themedial and distal zones of the colon, and macroscopic damageand pathologic alterations were also observed in these zones

(Fig. 1D). At this time, only 30% of the STAT6–/– mice devel-oped tumors (Fig. 1C), and they were scarce and small (Fig.1D–F). Additionally, the weight of the excised colons from thececum to the rectum of the treated mice showed an increase ondays 40 and 68 in WT animals, but no increase was observed inSTAT6–/– mice (Fig. 1G). These results indicate that STAT6deficiency not only decreased tumor promotion but also slo-wed tumor progression.

STAT6 is required for tumorigenesis in the AOM/DSS-inducedCAC model

On days 20 and 40, WT mice showed an increased number ofinflammatory cells infiltrating the lamina propria and submu-cosa compared with STAT6–/– mice (Fig. 2A). A histologic studyat day 68 revealed extensive chronic inflammation confined tothe lamina propria of WT animals (Fig. 2A). This histologicfinding indicates the presence of well-differentiated adenocar-cinomas, with glands covered by atypical epithelial cells withlarge and dysplastic nuclei as well as numerous mitotic struc-tures. In contrast, the colonic adenomas of STAT6-deficientmice exhibited mild dysplastic changes (Fig. 2A). The increasedinflammation in the colon tissue of WT mice also correlatedwith a remarkable decrease in goblet cell numbers (Fig. 2B).Colonic tissue from STAT6–/– mice displayed only a scantleukocyte infiltrate with no changes in the number of gobletcells (Fig. 2B).

b-Catenin signaling is a critical pathway in gastrointestinaltumorigenesis (1), and increased expression of nuclear b-cateninhas been reported in CAC during AOM/DSS regimens (17). Bothcytoplasmic and nuclear b-catenin staining were clearly observedin the intestines of WT mice during the early (20 and 40 days)and late stages (68 days) of tumor progression (Fig. 3A).However, colonic tissue from STAT6–/– mice displayed signifi-cantly less b-catenin expression throughout the administrationof AOM/DSS (Fig. 3A). Furthermore, COX-2, an importantinflammatory mediator implicated in colorectal inflammationand cancer, was elevated in colonic tumors of WT animalsduring early and late stages of CAC development comparedwith STAT6–/– mice (Fig. 3B). In contrast, COX-2 expression wasnot increased in the colons of STAT6–/– mice on days 20 and 40;however, a significant increase in COX-2 staining was observedon day 68 in these animals (Fig. 3B).

STAT6 deficiency increased apoptosis and reduced cellproliferation in early CAC

Previous studies have suggested that STAT6 may play acritical role in controlling cell proliferation in breast cancer(22), but its role in colon cancer is less understood. Tocompare mucosal homeostasis between STAT6–/– mice andWT mice, we examined epithelial cell proliferation by theexpression of Ki67 protein and apoptosis by TUNEL stainingin the colon. Interestingly, we observed significantly increasednumbers of Ki67þ cells in the colons of WT mice comparedwith STAT6–/– mice during the early and late steps of tumordevelopment (days 40 and 68; Fig. 4A and C). In addition, weobserved increased numbers of TUNELþ cells in the colons ofSTAT6–/– mice in the very early stages of CAC development(20 days; Fig. 4B and D) and not changes were observed in thelate stages (data not shown), indicating the importance ofSTAT6 signaling during the initial steps of colon tumorigenesis.These data suggest that a significantly increased number of

STAT6 Roles in Early-Stage Colitis-Associated Colon Cancer

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cells in WT tumors are actively proliferating compared withSTAT6–/– tumors, but apoptosis is active during the early stagesof STAT6–/– animals.

Fewer proinflammatory cytokines in early tumorigenesis incolons of STAT6–/– mice

To evaluate immunologic markers in the tumor microenvi-ronment in the absence of STAT6 signaling, we analyzed TNFa,IL17A, and IL10 mRNA levels by RT-PCR in the colon tissue ofWT and STAT6–/– mice in the early and late stages of CACdevelopment. In addition to decreased cell proliferation andinflammation, we detected decreased IL17A expression inwhole colon homogenates from STAT6–/– mice on day 20 ofAOM/DSS treatment compared with that in WT mice (Fig. 5A).A similar trend was observed with TNFa production, where theWT mice had increased levels of TNFa on days 40 and 68 afterthe initial AOM/DSS treatment compared with STAT6–/– mice(Fig. 5B). In contrast, we observed higher IL10 expression in the

colonic tissue of STAT6–/– mice than in WT mice on day 68(Fig. 5C). Additionally, we evaluated IL17A, TNFa, and IL10production and observed earlier and higher IL17 production inWT mice, whereas STAT6–/– animals produced little of this pro-inflammatory cytokine at early times and sustained this lowlevel until day 40 after CAC induction (Fig. 5D). Nonetheless,no significant increase of TNFa was detected at any time (datanot shown), but IL10 production was augmented in STAT6–/–

mice in early stages (Fig. 5E). On the other hand, the chemo-kines CCL9, CCL25, and the chemokine receptor CXCR2 havebeen reported to play an important role in the recruitment ofinflammatory cells, as well as in the invasion and metastasisof tumor cells (23, 24). Here, we found a significant reductionin the expression of CCL9, CCL25, and CXCR2 in whole colonhomogenates from STAT6–/– mice on day 68 of AOM/DSStreatment compared with WT mice (Fig. 5F–H), suggestingimportant changes in cellular recruitment in the absence ofSTAT6. However, we did not find any changes of these

Figure 1.

STAT6-deficient animals show decreased susceptibility to AOM/DSS-induced CAC. A, DAI on days 20, 40, and 68 following treatment with 12.5 mg/kgAOM injection and 2% DSS in drinking water per 7 days followed by 7 days of regular water. B, Percent change in body weight compared with thebaseline weight in WT and STAT6–/– mice. C, Percentage of tumor-free mice during AOM-DSS treatment in WT and STAT6–/– animals. D, Representativephotographs of colons from WT and STAT6–/– mice on day 68 after the AOM/DSS tumor induction protocol. WT mice displayed shortened andedematous colons with nodular and polypoid tumors compared with STAT6–/– mice. E, Number and F, size of tumors in WT and STAT6–/– mice on day68 after AOM/DSS treatment. G, Colons from WT and STAT6–/– mice were obtained during animal necropsy and weighed. The data are expressed as themean � SE from 5 mice per group and are representative of three independent experiments. �� , P < 0.01.

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chemokine expressions during early CAC development (datanot shown).

Circulating CD11bþLy6ChiCCR2þ and CD11bþLy6ClowLy6Gþ

cellsTo determine the possible cellular mechanisms involved in

the decrease of tumor growth in the absence of STAT6, we nextexamined various tumor-associated immune cell populationsinvolved in tumor growth in WT and STAT6–/– mice duringAOM/DSS regimens. Ly6C monocytes differ in their expressionof a major chemokine receptor, CCR2. CCR2 is responsible forthe recruitment of Ly6Chi monocytes to peripheral sites ofinflammation (25). Flow cytometric analysis of circulatingmonocyte populations revealed enhanced accumulation ofCD11bþLy6ChiCCR2þ cells in WT mice during advanced stagesof tumor development (day 68; Fig. 6A), in contrast, STAT6–/–

mice displayed lower percentages of these circulating cells.Similarly, circulating granulocytic cells (CD11bþLy6ClowLy6Gþ)were also significantly reduced in STAT6–/– mice as CAC pro-

gresses, whereas WT mice were able to maintain a sustainedpercentage of granulocytic cells (Fig. 6B).

Conversely, no changes were observed in CD8þ and CD4þ

circulating subpopulations between WT and STAT6–/– tumor-bearing mice (Fig. 6C).

In vivo STAT6 inhibition decreases tumor developmentduring induced CAC

To further test the role of STAT6 in vivo during the early stepsof CAC development, we performed an assay in which STAT6activity was inhibited by treatment with AS 1517499, a com-pound widely used for specific STAT6 inhibition in both in vivoand in vitro assays (26, 27). AS 1517499 was administered i.p.because the first DSS cycle to WT animals (Fig. 7A). An earlyinhibition of STAT6 during CAC development reduced by halfthe signs of the disease reported as DAI score at day 40 afterCAC induction (Fig. 7B). Similarly, animals receiving STAT6inhibitor did not show significant changes in body weight,whereas vehicle-treated mice displayed body weight changes at

Figure 2.

WT mice exhibit more severe histopathologic alterations than STAT6–/– mice following AOM/DSS-induced CAC. A, Representative H&E-stained colonicsections from WT and STAT6–/– mice on days 20, 40, and 68 after AOM/DSS treatment. WT mice displayed extensive colonic mucosal erosion,ulceration, severe crypt damage, and massive infiltration of inflammatory cells into the colonic mucosa compared with STAT6–/– mice. This effect wasmore pronounced in the distal colon compared with the proximal colon. B, Alcian blue stain of colon tissue (top) from WT and STAT6–/– animals forvisualizing goblet cells on days 20, 40, and 68 after AOM/DSS treatment. Quantification of goblet cells at day 68 (bottom) from at least 20 crypts per regionin five fields in four different slides per animal. Data are expressed as mean � SE from 5 mice per group and are representative of three independentexperiments. ��, P < 0.01






early times after CAC induction (Fig. 7C). Moreover, the per-centage of mice free of tumors expected at day 40 was reducedin mice receiving AS 1517499, this observation was associatedwith a significant reduction in tumor load at day 68 (Fig. 7Dand E).

Histologically, we also observed a positive effect of STAT6inhibition, given that mice receiving AS 1517499 displayedless colon tissue damage as well as a higher number of gobletcells in the colon (Fig. 7F). The effectiveness of in vivo STAT6inhibition was reflected by a reduction in STAT6 phosphory-lation in colon tissue frommice receiving AS 1517499 (WTþAS1517499), compared with vehicle-treated (WTþVHC) oruntreated-CAC mice (WT; Fig. 7G).

Discussion

In the tumor microenvironment, cytokines play a key role incancer cell survival, proliferation, and metastasis by orchestratingcritical signaling pathways. In the present study, we provide a linkbetween STAT6 and colorectal tumorigenesis under chronicinflammatory conditions. In the absence of STAT6, we observedan important decrease in tumor formation and size as well as thenumber of tumors in a model of colitis-associated colon cancer,supporting a critical role for STAT6 in CAC development andprogression. In our model, we detected a reduction in cell infil-tration and the production of proinflammatory cytokines in thecolons of STAT6–/– mice, which was accompanied by increased

Figure 3.

b-catenin and COX-2 expression is delayed in the colons of STAT6–/– animals. Immunohistochemical stain and quantification for (A) b-catenin and (B)COX-2 in the colon tissue of WT and STAT6–/– mice on days 20, 40, and 68 of the AOM/DSS tumor induction protocol. Quantification of the tumorcells positive for b-catenin (A) and COX-2 (B) was conducted as described in the Materials and Methods section. The data are expressed as themean � SE from 5 mice per group and are representative of three independent experiments. Statistical significance was determined by one-wayANOVA with Tukey test � , P < 0.05; �� , P < 0.001; �� , P < 0.0001.

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epithelial cell apoptosis and decreased proliferation, resulting inthe abrogation of tumor progression. Notably, we established adichotomous role for STAT6 in tumor development in (i) pre-venting epithelial cell apoptosis in the initial steps of transfor-mation, and (ii) promoting the local pro-inflammatory response.

STAT6 has been reported as an essential signaling molecule forintestinal homeostasis that is involved in effector functions inmastocytosis, goblet cell hyperplasia, tissue eosinophilia, intes-tinal barrier remodeling and fibrosis, and tumor cell growth (5,28, 29). An increase in epithelial STAT6phosphorylationhas beenreported in the colons of patients with UC (8). This increase wasrelated to enhance intestinal permeability by stimulating apopto-sis and increasing the expression of claudin-2, a pore-formingtight junction protein (30). Using a STAT6 knockout mouse,Madden and colleagues (31) demonstrated that IL13 increasesmucosal permeability in a STAT6-dependent fashion. Further-more, administration of recombinant mouse IL4 increases theresponsiveness of the intestinal epithelium to PGE2 and hista-mine in a mast cell- and STAT6-dependent fashion, demonstrat-ing the importance of this signaling pathway in intestinal cellfunction (31). In our mouse model of AOM/DSS-induced CAC,mice with global deletion of STAT6 exhibited significantlyreduced numbers of tumors, decreased inflammatory infiltrate,and maintenance of epithelial structure. Under these experimen-tal conditions, STAT6 could favor an increase in mucosal perme-ability, thus enhancing the response of the intestinal epitheliumtomicrobial products, whichmay result in chronic inflammationand tumorigenesis.

We observed a decrease in tumor development in STAT6–/–

mice. Consistent with this finding, we also detected less inflam-

matory infiltrate and decreased expression of proinflammatorycytokines in the mucosa, accompanied by no changes in thenumber of goblet cells. In a mouse coinfection model with thehelminth Heligmosomoides polygyrus and the gram-negative bacte-riumCitrobacter rodentium, STAT6–/–mice exhibited less intestinalinflammation in association with reduced infiltration of coloniclamina propria of alternatively activated macrophages (32). Inaddition, in murine colitis induced by the overexpression of IL4,STAT6 mediates inflammatory responses in the mouse gut (33),supporting the idea that STAT6 is critical for the recruitment ofimmune cells into the intestinal epithelium. In contrast, in theoxazolone (OXA)-induced colitis model, STAT6–/– mice showedcrypt hyperplasia, crypt loss, cryptitis, and active infiltrates ofneutrophils and mononuclear cells in the lamina propria com-pared with wild-type control mice (16). Furthermore, a reportindicated that STAT6–/– mice are sensitive to acute DSS-inducedcolitis (14), which suggests that acute inflammatory responses aremainly associated with Th1 profiles (34). However, CAC devel-opment has been demonstrated to be a complex pathology wherevarious chronic inflammatory processes may take place (1).

Cosín-Roger and colleagues (35) demonstrated that STAT6–/–

mice treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS)exhibit impaired mucosal production of proteins associated withthe Wnt signaling pathway and nuclear b-catenin, as well asreduced mRNA expression of Lgr5 and c-Myc, both of which areprimary Wnt/b-catenin target molecules. Reduction in these mar-kers has been associated with the impairedmucosal expression ofM2macrophage-associated genes (35). b-Catenin is an importantcomponent of the Wnt signaling pathway. In fact, mutations inWnt-associated protein-coding genes have been reported in

Figure 4.

Increased apoptosis and reduced epithelial cell proliferation in STAT6–/– mice during early CAC development. Evaluation of (A) cell proliferation byKi67 immunostaining and (B) apoptosis by TUNEL assay in the colonic tissues of WT and STAT6–/– mice at the indicated times during CAC development.The percentage of (C) Ki67þ cells was conducted as described in the Materials and Methods section. D, Fluorescence quantification in colon tissue ofTUNELþ cells. The data are presented as the percentage of mean fluorescence, which was expressed as arbitrary units. The data are expressed as themean � SE from 5 mice per group in three independent experiments. � , P < 0.05; �� , P < 0.01.






premalignant lesions of the intestine (36). In line with theseprevious observations, our data demonstrate that STAT6–/– micehad significantly lower expression of b-catenin and COX2 whentreated with AOM/DSS, suggesting a role for STAT6 signaling intumor initiation during CAC.

Several lines of evidence suggest that IL4 signaling may pro-mote the survival and proliferation of cancer cells by down-regulating anti-apoptotic proteins, such as PED, cFLIP, Bcl-xL,andBcl-2 (37),while favoring survivin expression (38, 39). In vitrostudies in colorectal cancer cell lines show that cells with highSTAT6 activity are resistant to apoptosis and aggressively metas-tasis compared with STAT6-defective cell phenotypes (20). In

accordance with such studies, our results demonstrated an impor-tant increase in the apoptosis rate in STAT6–/– mice early in CACdevelopment and decreased intestinal cell proliferation, as esti-mated by Ki-67 expression. Additionally, IL4 may enhance theproliferation rate of colon cancer cells (40), given that the upre-gulation of IL4 constitutes an important mechanism that protectstumorigenic colon cancer stem-like cells (CD133þ) from apopto-sis (41). Thus, the requirement for the STAT6 signaling pathwayfor the biological effects of IL4 and IL13 is a critical factor in CACdevelopment, as has been suggested also for breast cancer (42), inwhich IL4Ra signaling enhances metastatic growth through thepromotion of tumor cell survival and proliferation. Human and

Figure 5.

Interleukin and chemokine detection during CAC development. A, IL17A, B, TNFa and C, IL10 cytokine mRNA expression in the colonic tissues of WTand STAT6–/– mice on days 20, 40, and 68 after AOM/DSS treatment. D, IL17A and E, IL10 concentrations in supernatants of mesenteric lymphoidcells stimulated with antibodies to CD3/CD28 (2 mg/mL) for 24 hours from WT and STAT6–/– mice on days 20, 40, and 68 after AOM/DSS treatmentwere measured by ELISA. F, CCL9, G, CCL25, and H, CXCR2 mRNA expression in the colon mucosa on day 68. The data are expressed as the mean � SE from5 mice per group and are representative of three independent experiments. � , P < 0.05.

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murine mammary cancer cells treated with IL4 increase prolifer-ation, glucose consumption and lactate production, indicatingthat IL4 can support breast cancer growth (43). Given that otherstudies have demonstrated that STAT6 phosphorylation couldtake place through IL4Ra independent-mechanisms (44), thisopens the possibility that in colon cancer, other mechanismsindependent of the IL4/IL4Ra/STAT6 axis could take place. Here,our results demonstrated a significant inhibition in tumor devel-opment in STAT6–/– mice, as 70% of these animals were tumor-free in advanced stages of CAC, suggesting that the STAT6 sig-naling pathway is centrally involved in tumor initiation andprogression.

To address the question of whether the differences in tumordevelopment between STAT6–/– and WT mice were related toinflammatory monocyte recruitment, we examined the Ly6Chi

population expressing chemokine receptor CCR2 in STAT6–/–

mice during CAC development. CCR2 is responsible for therecruitment of Ly6Chi monocytes to peripheral sites of inflam-mation, and in the AOM/DSS model, CCR2 knockout miceexhibit less macrophage infiltration and lower tumor numbers(45). We observed decreases in the percentage ofCD11bþLy6ChiCCR2þ cells in STAT6–/– mice compared withwild-type animals, indicating that STAT6 is a critical mediator ofthe initiation and promotion of CAC. Moreover, STAT6–/– micealso displayed fewer circulating CD11bþLy6ClowLy6Gþ granulo-cytic cells, which have been reported to have suppressive activity,and a reduction in this cell population is associatedwith improve-ment of antitumor responses (46). In contrast, IL4Ra-deficientmice receiving syngeneic TS/A, 4T1, or CT26 tumor cells showedno differences in the systemic numbers and T cell suppressive

capacity of CD11bþLy6G�Ly6Chi and CD11bþLy6GþLy6Clow

cells compared with wild-type animals, supporting the idea thatIL4Ra does not alter the phenotype and number of these cells(47). Nevertheless, a caveat of that study involves the use of tumorcell lines, which exert tumorigenic mechanisms that may differfrom those found in chronic inflammation-preceded colon can-cer. Additionally, other important cell populations could beinvolved in protection against CAC in STAT6-deficient mice. Inthis sense, we observed an increase in the levels of IL10. This is animmune-regulatory cytokine that is important in the modulationof inflammatory responses during cancer (48). The absence ofSTAT6 may promote the recruitment of an IL10-producing cellpopulation, as is the case for T regulatory cells (Tregs). Tregs areessential in maintaining tolerance and down-modulatingimmune responses by secreting IL10 upon activation (49). STAT6can directly inhibit FOXP3 expression by repressing the Foxp3gene (50). Thus, STAT6 activation could facilitate inflammationby hampering inducible Treg development. However, the role ofSTAT6 in FOXP3 protein regulation during CAC remains to beelucidated. Interestingly, we observed an important decrease inthe mRNA expression of CCL9, CCL25, and CXCR2 in STAT6–/–

mice during late stages of tumor progression. CCL9 is secreted bymouse andhuman colon cancer cells, and lack ofCCL9 expressiondramatically suppresses the outgrowth of disseminated tumors inthe liver (24). Similarly, CXCR2 in the tumor microenvironmenthas been associated with colon cancer growth and progression(51), but in our model, this receptor was downregulated in theabsence of STAT6.

Finally, inhibition of STAT6 by AS1517499 injection signifi-cantly inhibited colonic tumor formation in a manner similar to

Figure 6.

STAT6 deficiency reduces the percentages of circulating inflammatory monocytes and granulocytes during CAC. Representative flow cytometry dot plotsfor CD11bþ living cells isolated from the circulation of WT and STAT6–/– mice. Flow cytometric analysis was performed with Ly6G and Ly6C markersexpressed in the cell surface of circulating cells. A, Representative dot plots and graph display the proportion of CD11bþLy6ChiCCR2þ monocytes gatedon CD11bþ populations living cells isolated from the circulation of WT and STAT6–/– mice on day 68 after AOM administration. B, Representative dotplots and graph display CD11bþLy6ClowLy6Gþ cells from STAT6–/– mice compared with WT animals on days 20, 40, and 68 after AOM/DSS tumorinduction protocol. C, Percentage of CD8þ and CD4þ cells gated on CD3þ living cells isolated from the circulation of WT and STAT6–/– mice on day 68after AOM/DSS administration. The data are representative of two independent experiments that included n ¼ 4–5 mice/group. Values are expressed asthe mean � SE. � , P < 0.05.






the inhibited tumor formation in STAT6–/– animals, supportingthe idea that STAT6may favor tumor initiation andprogressionbyrecruiting several immune cell types and/or influence the prolif-erative and invasive properties of cancer cells. However, it will beinteresting to delineate at which point in the progression of CACSTAT6 inhibition would remain functional.

In conclusion, STAT6 signaling appears to be central to theinitial steps of CAC development by modulating inflammatoryresponses and control mechanisms, such as immune cell recruit-ment and epithelial cell proliferation. STAT6 may represent apromising target for treating colitis-associated colorectal cancer inthe near future.

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

Authors' ContributionsConception anddesign: S.A. Leon-Cabrera, Y.G.Delgado-Ramirez, Y.I. Chirino,

F. �Avila-Moreno, L.I. Terrazas

Development of methodology: S.A. Leon-Cabrera, Y.G. Delgado-Ramirez,Y. Ledesma-Soto, N.L. Delgado-Buenrostro, L.I. TerrazasAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.A. Leon-Cabrera, E. Molina-Guzman, A. V�azquez-Sandoval, Y. Ledesma-Soto, N.L. Delgado-Buenrostro, L.E. Arias-Romero,L.I. TerrazasAnalysis and interpretation of data (e.g., statistical analysis, biostati-stics, computational analysis): S.A. Leon-Cabrera, E. Molina-Guzman,Y.G. Delgado-Ramirez, A. V�azquez-Sandoval, E.B. Gutierrez-Cirlos,Writing, review, and/or revision of the manuscript: S.A. Leon-Cabrera,

C.G. P�erez-Plasencia, M. Rodríguez-Sosa, F. Vaca-Paniagua, F. �Avila-Moreno,E.B. Gutierrez-Cirlos, L.I. TerrazasAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y.G. Delgado-Ramirez, Y. Ledesma-Soto

Study supervision: Y.G. Delgado-Ramirez, F. �Avila-Moreno, L.I. Terrazas

AcknowledgmentsThis work was supported by grants from Programa de Apoyo a Proyectos de

Investigaci�on e Innovaci�on tecnol�ogica, PAPIIT, UNAM, Grant numberIA206216, RA206216, and IN220316. Consejo Nacional de Ciencia y Tecno-logía (CONACYT) Grant number 280013. S.A. Leon-Cabrera received a

Figure 7.

In vivo STAT6 inhibition reduces colonic tumor load. A, WT mice were treated with the STAT6 inhibitor AS 1517499 or equivalent volume of vehicle(VHC) every third day during all AOM/DSS treatment and sacrificed at days 20, 40, and 68. B, DAI; C, Change of body weight; D, Number of tumors atday 68; E, Percentage of tumor-free mice; F, H&E (left) and Alcian blue (right) stained colonic sections from WT mice treated with the STAT6 inhibitorAS 1517499 on days 20 and 40 after AOM/DSS administration. G, Change in the phosphorylation of STAT6 after treatment with STAT6 inhibitor AS1517499 determined by immunoblotting. Higher levels of phosphorylated STAT6 (pSTAT6) were observed in WT andWTþVHC mice on day 20 after AOM/DSSadministration. The data are expressed as the mean � SE from 5 mice per group and are representative of two independent experiments. a, (week 2and 3 vs. week 0 in the WT group). � , P < 0.05; �� , P < 0.001.

Leon-Cabrera et al.





fellowship from L'Oreal-UNESCO-CONACYT for Women in Science. YDR isa doctoral student from Programa de Doctorado en Ciencias Biom�edicas,Universidad Nacional Aut�onoma de M�exico (UNAM) and received a fellow-ship (606590) from CONACYT.

Grant SupportSonia A. Leon-Cabrera was supported by Programa de Apoyo a Proyectos de

Investigaci�on e Innovaci�on tecnol�ogica, PAPIIT, UNAM, grant numbersIA206216 and RA206216. Fellowship from L'Oreal-UNESCO-CONACYT forWomen in Science. Luis I. Terrazas was supported by Consejo Nacional de

Ciencia y Tecnología (CONACYT) grant number 280013 and Programa deApoyo a Proyectos de Investigaci�on e Innovaci�on tecnol�ogica, PAPIIT, UNAMgrant number IN220316.

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.

Received July 20, 2016; revised October 5, 2016; accepted April 4, 2017;published OnlineFirst April 6, 2017.

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Leon-Cabrera et al.




2017;5:385-396. Published OnlineFirst April 6, 2017.Cancer Immunol Res Sonia A. Leon-Cabrera, Emmanuel Molina-Guzman, Yael G. Delgado-Ramirez, et al. against Early Steps of Colitis-Associated Colon CancerLack of STAT6 Attenuates Inflammation and Drives Protection

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