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Research Articles

Lack of STAT6 Attenuates Inflammation and Drives Protection against Early Steps of Colitis-Associated Colon Cancer

Sonia A. Leon-Cabrera, Emmanuel Molina-Guzman, Yael G. Delgado-Ramirez, Armando Vázquez-Sandoval, Yadira Ledesma-Soto, Carlos G. Pérez-Plasencia, Yolanda I. Chirino, Norma L. Delgado-Buenrostro, Miriam Rodríguez-Sosa, Felipe Vaca-Paniagua, Federico Ávila-Moreno, Emma B. Gutierrez-Cirlos, Luis E. Arias-Romero and Luis I. Terrazas
DOI: 10.1158/2326-6066.CIR-16-0168 Published May 2017

Abstract

Colitis-associated colon cancer (CAC) is one of the most common malignant neoplasms and a leading cause of death. The immunologic factors associated with CAC development are not completely understood. Signal transducer and activator of transcription 6 (STAT6) is part of an important signaling pathway for modulating intestinal immune function and homeostasis. However, the role of STAT6 in colon cancer progression 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 fewer and smaller colorectal tumors than WT mice; this reduced tumorigenicity was associated with decreased proliferation and increased apoptosis in the colonic mucosa in the early steps of tumor progression. STAT6–/– mice also exhibited reduced inflammation, diminished concentrations COX2 and nuclear β-catenin protein in the colon, and decreased mRNA expression of IL17A and TNFα, but increased IL10 expression when compared with WT mice. Impaired mucosal expression of CCL9, CCL25, and CXCR2 was also observed. In addition, the number of circulating CD11b+Ly6ChiCCR2+ monocytes and CD11b+Ly6ClowLy6G+ granulocytes was both decreased in a STAT6-dependent manner. Finally, WT mice receiving a STAT6 inhibitor in vivo confirmed a significant reduction in tumor load as well as less intense signs of CAC. Our results demonstrate that STAT6 is critical in the early steps of CAC development for modulating inflammatory responses and controlling cell recruitment and proliferation. Thus, STAT6 may represent a promising target for CAC treatment. Cancer Immunol Res; 5(5); 385–96. ©2017 AACR.

Introduction

Chronic inflammation is widely associated with increased susceptibility to developing colorectal cancer (1). Patients with inflammatory bowel diseases such as Crohn's disease and ulcerative colitis (UC), which are characterized by prolonged inflammation of the intestine, have an increased risk of developing colorectal cancer (2). Epidemiologic studies indicate that treatment with anti-inflammatory drugs could prevent or delay colorectal cancer, suggesting the involvement of inflammatory pathways in tumor progression (3). In a genetically susceptible host, epithelial barrier breakdown could result in magnified responses to microbial products, thus leading to chronic inflammation and tumorigenesis. Furthermore, pro-inflammatory cytokines produced in response to commensal bacteria can create a microenvironment that enhances epithelial cell proliferation, angiogenesis, and increased susceptibility to mutations (1).

JAK/STAT is an important signaling pathway for modulating intestinal immune function (4). Signal transducer and activator of transcription 6 (STAT6) proteins are activated by phosphorylation of Janus kinase (JAK) family of proteins in response to the binding of interleukin (IL) 4 or 13 to their common type II IL4 receptor (IL4R; ref. 5). When STAT6 is phosphorylated by JAK, STAT6 homodimers are formed and translocate to the nucleus to initiate the transcription of IL4, IL13, and other responsive genes (5, 6).

In addition to its pivotal role in IL4 and IL13 signaling, STAT6 participates in epithelial malignancies (7, 8). STAT6 is also constitutively stimulated in a number of cancer types, including colon (9) and prostate cancer (10), mediastinal large B-cell lymphoma (10), and Hodgkin's lymphoma (11). Typically, STAT6–/– mice have defects in Th2 polarization (12) and are able to reject or delay primary mammary tumor growth, prevent the recurrence of primary tumors, and/or reject established, spontaneous metastatic disease in mammary carcinoma (7). STAT6–/– mice also show enhanced immunity against tumors, due to the increased differentiation of CD4 T cells toward a Th1 phenotype (12, 13). However, the role of STAT6 in colon carcinogenesis is less clear. STAT6–/– mice are sensitive to DSS-induced colitis, exhibit chronic architectural changes, and produce more IFNγ in this type of injury (14). Additionally, a more severe colonic inflammatory response in DNBS colitis in STAT6–/– mice has been reported (15). In contrast, in oxazolone-induced colitis, a murine model of colitis with pathologic and immunologic features similar to UC, colitis was attenuated in STAT6–/– mice accompanied by reduced claudin-2 induction, suggesting an important role for STAT6 in epithelial barrier function (16). Despite evidence suggesting that 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 developing colitis-associated colon cancer (CAC) using the azoxymethane (AOM)/dextran sodium sulfate (DSS) model, examining the dependence of colonic inflammation, epithelial cell proliferation and apoptosis, and monocytic-granulocytic recruitment on STAT6 signaling. Our results suggest a critical role for STAT6 in the early events of CAC development in promoting the survival of colonic epithelial cells, perhaps by modulating early inflammatory responses that could be detrimental for tumor development.

Materials and Methods

Mice

Six-week-old female STAT6–/–and STAT6+/+ (WT) mice on a BALB/c genetic background were originally purchased from The Jackson Laboratory Animal Resources Center and maintained in a pathogen-free environment at the FES-Iztacala, Universidad Nacional Autónoma de México (UNAM) animal facilities. The animals were fed Purina Diet 5015 (Purina) and water ad libitum. All experimental procedures were approved by the Ethical Committees of the UNAM, according to the University Animal Care and Use Committee.

Murine model of colitis-associated colorectal cancer

The 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 the animals' drinking water for 7 days. Afterwards, the mice received regular drinking water for 14 days. This experimental series was repeated twice. To examine early and late transformative steps in CAC, the mice were killed on days 20, 40, and 68 after AOM injection. Throughout the experiment, the mice were monitored weekly for body weight, stool consistency, and the presence of blood in the rectum or stools. The disease activity score (DAI) was calculated as the sum of the diarrhea score plus the bloody stool score as follows: 0 = normal stool and normal-colored stool, 1 = mildly soft stool and brown stool, 2 = very soft stool and reddish stool, and 3 = watery stool and bloody stool.

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

Histologic analysis

Collected tissues from WT and STAT6–/–mice were fixed in 100% ethanol and embedded in paraffin for posterior 5-μm cross-sectioning. The tissue sections were stained with hematoxylin and eosin (H&E; for pathologic evaluation) or Alcian blue (for acidic polysaccharides). For immunohistochemical analysis, the sections were deparaffinized in xylene and then rehydrated with graded alcohols and processed as reported previously (18). The sections were incubated overnight at 4°C with the respective primary antibodies diluted in 1X PBS (anti-COX2, 1:100, GeneTex; anti-β-catenin, 1:100, GeneTex; anti-Ki67, 1:100, Biolegend) and then developed following the conventional technique. The slides were analyzed using an AxioVert.A1 image capture optical microscope (Carl Zeiss Microscopy GmbH). Tissue microphotographs were captured using an AxioCam MRc and ZEN lite 2011 software v.1.0.1.0. Quantification of COX2+, β-catenin+, and Ki67+ cells was performed using ImageJ software v.4.9 by counting cells in 10 high-powered fields from each mouse.

TUNEL staining

Apoptosis was detected using the In Situ Cell Death Detection Fluorescein Kit (Roche), analyzing samples with a ZeissVert A1 conventional epifluorescence microscope and a LEICA TCS SP2 confocal microscope (the analyzed area in each sample was 2.8 mm2, and 20 fields of 50 mm2 were evaluated).

RNA extraction and RT-PCR

Tissues were first disrupted in a tissue homogenizer (Bullet Blender; Next Advance). Total RNA from colon tissue was extracted using TRIzol reagent (Invitrogen) following the manufacturer's instructions. The RNA concentration was determined by measuring the absorbance at 260 nm. One microgram of RNA was used for first-strand cDNA synthesis with RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). The primer sequences and cycling conditions are listed in Supplementary Table S1.

ELISA

Mesenteric lymphoid cells from mice were plated in 96-well plates coated with anti CD3/CD28 (BioLegend)-antibodies (2 μg/mL). After 24 hours, supernatants were analyzed for the presence of IL10, IL17A, and TNFα in all samples using a mouse IL10 and TNFα ELISA kit (Peprotech Mexico) and mouse IL17A ELISA kit (BioLegend).

Flow cytometry

For flow cytometry circulating blood was obtained during animal necropsy. The cells were washed with 1× PBS and blocked using antibodies to CD16/CD32. The cells were simultaneously stained 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 FACSCalibur system and Cell Quest software (Becton Dickinson).

STAT6 inhibition in vivo

BALB/c mice were induced for CAC as described above and when initializing the first DSS cycle received every third day intraperitoneally 10 mg/kg of AS1517499 (Axon Medchem) reconstituted in dimethyl sulfoxide (DMSO) and PBS at the time points indicated. Control mice received the same volume of 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 measuring STAT6 phosphorylation in colon tissue by Western blot.

Immunoblot analysis

Colon tissues were lysed in RIPA buffer supplemented with proteinase and phosphatase inhibitor cocktail (Roche), and centrifuged for 10 minutes at 14,000 rpm. Supernatants were collected, run on SDS gels, and transferred onto membranes. The membranes were blocked and probed with antibodies to phospho-STAT6 Y641 (Santa Cruz Biotechnology), STAT6 (M-20; Santa Cruz Biotechnology), and β-actin (BioLegend).

Statistical analysis

Data were analyzed by one-way analysis of variance followed by Tukey's multiple comparisons test or unpaired two-tailed t tests depending on the number of groups using GraphPad Prism 5 (San Diego). All statistical tests were performed considering 95% confidence intervals. The data are expressed as the mean ± SE. *, P < 0.05; **, P < 0.01.

Results

STAT6-deficient animals less susceptible to AOM/DSS-induced CAC

Previous studies have shown that STAT6 is involved in intestinal epithelial homeostasis and its activity is correlated with colon cancer cell growth in vitro (19, 20), yet the role of STAT6 has not been extensively studied in the early steps of CAC. To examine the role of STAT6 in vivo, we used a well-established model of CAC (21). We evaluated CAC progression at 20, 40 and 68 days in WT and STAT6–/–mice. We monitored stool consistency and changes in body weight and tumor development. As expected, WT-treated mice rapidly showed piloerection and clinical signs of the disease (DAI) throughout the experiment (Fig. 1A). STAT6–/– mice did not have diarrhea or rectal bleeding during the course of treatment compared with similarly treated WT animals (Fig. 1A). During the course of AOM/DSS administration, WT mice lost weight compared with the baseline weight (Fig. 1B). Nevertheless, STAT6–/– animals had progressive weight gain over time that was not significantly affected by AOM/DSS administration. Two and 8 weeks after treatment, the weight in WT mice was significantly decreased compared with STAT6–/– animals (Fig. 1B). Consistent with this, the polypoid lesions and the early signs of the disease appeared in WT mice at 40 days of treatment, whereas STAT6–/– mice showed mild symptoms and no tumors (Fig. 1C). At this time, 70% of WT mice had increased numbers of tumors and an increased tumor load, whereas all STAT6–/– animals remained tumor-free (Fig. 1C). At necropsy on day 68, all WT animals displayed reddish polypoid tumors in the medial and distal zones of the colon, and macroscopic damage and pathologic alterations were also observed in these zones (Fig. 1D). At this time, only 30% of the STAT6–/– mice developed tumors (Fig. 1C), and they were scarce and small (Fig. 1D–F). Additionally, the weight of the excised colons from the cecum to the rectum of the treated mice showed an increase on days 40 and 68 in WT animals, but no increase was observed in STAT6–/– mice (Fig. 1G). These results indicate that STAT6 deficiency not only decreased tumor promotion but also slowed tumor progression.

Figure 1.
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/kg AOM 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 the baseline weight in WT and STAT6–/– mice. C, Percentage of tumor-free mice during AOM-DSS treatment in WT and STAT6–/– animals. D, Representative photographs of colons from WT and STAT6–/– mice on day 68 after the AOM/DSS tumor induction protocol. WT mice displayed shortened and edematous colons with nodular and polypoid tumors compared with STAT6–/– mice. E, Number and F, size of tumors in WT and STAT6–/– mice on day 68 after AOM/DSS treatment. G, Colons from WT and STAT6–/– mice were obtained during animal necropsy and weighed. The data are expressed as the mean ± SE from 5 mice per group and are representative of three independent experiments. **, P < 0.01.

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

On days 20 and 40, WT mice showed an increased number of inflammatory cells infiltrating the lamina propria and submucosa compared with STAT6–/– mice (Fig. 2A). A histologic study at day 68 revealed extensive chronic inflammation confined to the lamina propria of WT animals (Fig. 2A). This histologic finding indicates the presence of well-differentiated adenocarcinomas, with glands covered by atypical epithelial cells with large and dysplastic nuclei as well as numerous mitotic structures. In contrast, the colonic adenomas of STAT6-deficient mice exhibited mild dysplastic changes (Fig. 2A). The increased inflammation in the colon tissue of WT mice also correlated with a remarkable decrease in goblet cell numbers (Fig. 2B). Colonic tissue from STAT6–/– mice displayed only a scant leukocyte infiltrate with no changes in the number of goblet cells (Fig. 2B).

Figure 2.
Figure 2.

WT mice exhibit more severe histopathologic alterations than STAT6–/– mice following AOM/DSS-induced CAC. A, Representative H&E-stained colonic sections 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 was more pronounced in the distal colon compared with the proximal colon. B, Alcian blue stain of colon tissue (top) from WT and STAT6–/– animals for visualizing 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 region in five fields in four different slides per animal. Data are expressed as mean ± SE from 5 mice per group and are representative of three independent experiments. **, P < 0.01

β-Catenin signaling is a critical pathway in gastrointestinal tumorigenesis (1), and increased expression of nuclear β-catenin has been reported in CAC during AOM/DSS regimens (17). Both cytoplasmic and nuclear β-catenin staining were clearly observed in 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 significantly less β-catenin expression throughout the administration of AOM/DSS (Fig. 3A). Furthermore, COX-2, an important inflammatory mediator implicated in colorectal inflammation and cancer, was elevated in colonic tumors of WT animals during early and late stages of CAC development compared with STAT6–/– mice (Fig. 3B). In contrast, COX-2 expression was not increased in the colons of STAT6–/– mice on days 20 and 40; however, a significant increase in COX-2 staining was observed on day 68 in these animals (Fig. 3B).

Figure 3.
Figure 3.

β-catenin and COX-2 expression is delayed in the colons of STAT6–/– animals. Immunohistochemical stain and quantification for (A) β-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 tumor cells positive for β-catenin (A) and COX-2 (B) was conducted as described in the Materials and Methods section. The data are expressed as the mean ± SE from 5 mice per group and are representative of three independent experiments. Statistical significance was determined by one-way ANOVA with Tukey test *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

STAT6 deficiency increased apoptosis and reduced cell proliferation in early CAC

Previous studies have suggested that STAT6 may play a critical role in controlling cell proliferation in breast cancer (22), but its role in colon cancer is less understood. To compare mucosal homeostasis between STAT6–/– mice and WT mice, we examined epithelial cell proliferation by the expression of Ki67 protein and apoptosis by TUNEL staining in the colon. Interestingly, we observed significantly increased numbers of Ki67+ cells in the colons of WT mice compared with STAT6–/– mice during the early and late steps of tumor development (days 40 and 68; Fig. 4A and C). In addition, we observed increased numbers of TUNEL+ cells in the colons of STAT6–/– mice in the very early stages of CAC development (20 days; Fig. 4B and D) and not changes were observed in the late stages (data not shown), indicating the importance of STAT6 signaling during the initial steps of colon tumorigenesis. These data suggest that a significantly increased number of cells in WT tumors are actively proliferating compared with STAT6–/– tumors, but apoptosis is active during the early stages of STAT6–/– animals.

Figure 4.
Figure 4.

Increased apoptosis and reduced epithelial cell proliferation in STAT6–/– mice during early CAC development. Evaluation of (A) cell proliferation by Ki67 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) Ki67+ cells was conducted as described in the Materials and Methods section. D, Fluorescence quantification in colon tissue of TUNEL+ cells. The data are presented as the percentage of mean fluorescence, which was expressed as arbitrary units. The data are expressed as the mean ± SE from 5 mice per group in three independent experiments. *, P < 0.05; **, P < 0.01.

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

To evaluate immunologic markers in the tumor microenvironment in the absence of STAT6 signaling, we analyzed TNFα, IL17A, and IL10 mRNA levels by RT-PCR in the colon tissue of WT and STAT6–/– mice in the early and late stages of CAC development. In addition to decreased cell proliferation and inflammation, we detected decreased IL17A expression in whole colon homogenates from STAT6–/– mice on day 20 of AOM/DSS treatment compared with that in WT mice (Fig. 5A). A similar trend was observed with TNFα production, where the WT mice had increased levels of TNFα on days 40 and 68 after the 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, TNFα, and IL10 production and observed earlier and higher IL17 production in WT mice, whereas STAT6–/– animals produced little of this pro-inflammatory cytokine at early times and sustained this low level until day 40 after CAC induction (Fig. 5D). Nonetheless, no significant increase of TNFα was detected at any time (data not shown), but IL10 production was augmented in STAT6–/– mice in early stages (Fig. 5E). On the other hand, the chemokines CCL9, CCL25, and the chemokine receptor CXCR2 have been reported to play an important role in the recruitment of inflammatory cells, as well as in the invasion and metastasis of tumor cells (23, 24). Here, we found a significant reduction in the expression of CCL9, CCL25, and CXCR2 in whole colon homogenates from STAT6–/– mice on day 68 of AOM/DSS treatment compared with WT mice (Fig. 5F–H), suggesting important changes in cellular recruitment in the absence of STAT6. However, we did not find any changes of these chemokine expressions during early CAC development (data not shown).

Figure 5.
Figure 5.

Interleukin and chemokine detection during CAC development. A, IL17A, B, TNFα and C, IL10 cytokine mRNA expression in the colonic tissues of WT and STAT6–/– mice on days 20, 40, and 68 after AOM/DSS treatment. D, IL17A and E, IL10 concentrations in supernatants of mesenteric lymphoid cells stimulated with antibodies to CD3/CD28 (2 μg/mL) for 24 hours from WT and STAT6–/– mice on days 20, 40, and 68 after AOM/DSS treatment were 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 from 5 mice per group and are representative of three independent experiments. *, P < 0.05.

Circulating CD11b+Ly6ChiCCR2+ and CD11b+Ly6ClowLy6G+ cells

To determine the possible cellular mechanisms involved in the decrease of tumor growth in the absence of STAT6, we next examined various tumor-associated immune cell populations involved in tumor growth in WT and STAT6–/– mice during AOM/DSS regimens. Ly6C monocytes differ in their expression of a major chemokine receptor, CCR2. CCR2 is responsible for the recruitment of Ly6Chi monocytes to peripheral sites of inflammation (25). Flow cytometric analysis of circulating monocyte populations revealed enhanced accumulation of CD11b+Ly6ChiCCR2+ cells in WT mice during advanced stages of tumor development (day 68; Fig. 6A), in contrast, STAT6–/– mice displayed lower percentages of these circulating cells. Similarly, circulating granulocytic cells (CD11b+Ly6ClowLy6G+) were also significantly reduced in STAT6–/– mice as CAC progresses, whereas WT mice were able to maintain a sustained percentage of granulocytic cells (Fig. 6B).

Figure 6.
Figure 6.

STAT6 deficiency reduces the percentages of circulating inflammatory monocytes and granulocytes during CAC. Representative flow cytometry dot plots for CD11b+ living cells isolated from the circulation of WT and STAT6–/– mice. Flow cytometric analysis was performed with Ly6G and Ly6C markers expressed in the cell surface of circulating cells. A, Representative dot plots and graph display the proportion of CD11b+Ly6ChiCCR2+ monocytes gated on CD11b+ populations living cells isolated from the circulation of WT and STAT6–/– mice on day 68 after AOM administration. B, Representative dot plots and graph display CD11b+Ly6ClowLy6G+ cells from STAT6–/– mice compared with WT animals on days 20, 40, and 68 after AOM/DSS tumor induction protocol. C, Percentage of CD8+ and CD4+ cells gated on CD3+ living cells isolated from the circulation of WT and STAT6–/– mice on day 68 after AOM/DSS administration. The data are representative of two independent experiments that included n = 4–5 mice/group. Values are expressed as the mean ± SE. *, P < 0.05.

Conversely, no changes were observed in CD8+ and CD4+ circulating subpopulations between WT and STAT6–/– tumor-bearing mice (Fig. 6C).

In vivo STAT6 inhibition decreases tumor development during induced CAC

To further test the role of STAT6 in vivo during the early steps of CAC development, we performed an assay in which STAT6 activity was inhibited by treatment with AS 1517499, a compound widely used for specific STAT6 inhibition in both in vivo and in vitro assays (26, 27). AS 1517499 was administered i.p. because the first DSS cycle to WT animals (Fig. 7A). An early inhibition of STAT6 during CAC development reduced by half the signs of the disease reported as DAI score at day 40 after CAC induction (Fig. 7B). Similarly, animals receiving STAT6 inhibitor did not show significant changes in body weight, whereas vehicle-treated mice displayed body weight changes at early times after CAC induction (Fig. 7C). Moreover, the percentage of mice free of tumors expected at day 40 was reduced in mice receiving AS 1517499, this observation was associated with a significant reduction in tumor load at day 68 (Fig. 7D and E).

Figure 7.
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 at day 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 inhibitor AS 1517499 on days 20 and 40 after AOM/DSS administration. G, Change in the phosphorylation of STAT6 after treatment with STAT6 inhibitor AS 1517499 determined by immunoblotting. Higher levels of phosphorylated STAT6 (pSTAT6) were observed in WT and WT+VHC mice on day 20 after AOM/DSS administration. The data are expressed as the mean ± SE from 5 mice per group and are representative of two independent experiments. a, (week 2 and 3 vs. week 0 in the WT group). *, P < 0.05; **, P < 0.001.

Histologically, we also observed a positive effect of STAT6 inhibition, given that mice receiving AS 1517499 displayed less colon tissue damage as well as a higher number of goblet cells in the colon (Fig. 7F). The effectiveness of in vivo STAT6 inhibition was reflected by a reduction in STAT6 phosphorylation in colon tissue from mice receiving AS 1517499 (WT+AS 1517499), compared with vehicle-treated (WT+VHC) or untreated-CAC mice (WT; Fig. 7G).

Discussion

In the tumor microenvironment, cytokines play a key role in cancer cell survival, proliferation, and metastasis by orchestrating critical signaling pathways. In the present study, we provide a link between STAT6 and colorectal tumorigenesis under chronic inflammatory conditions. In the absence of STAT6, we observed an important decrease in tumor formation and size as well as the number of tumors in a model of colitis-associated colon cancer, supporting a critical role for STAT6 in CAC development and progression. In our model, we detected a reduction in cell infiltration and the production of proinflammatory cytokines in the colons of STAT6–/– mice, which was accompanied by increased epithelial cell apoptosis and decreased proliferation, resulting in the abrogation of tumor progression. Notably, we established a dichotomous role for STAT6 in tumor development in (i) preventing epithelial cell apoptosis in the initial steps of transformation, and (ii) promoting the local pro-inflammatory response.

STAT6 has been reported as an essential signaling molecule for intestinal homeostasis that is involved in effector functions in mastocytosis, goblet cell hyperplasia, tissue eosinophilia, intestinal barrier remodeling and fibrosis, and tumor cell growth (5, 28, 29). An increase in epithelial STAT6 phosphorylation has been reported in the colons of patients with UC (8). This increase was related to enhance intestinal permeability by stimulating apoptosis and increasing the expression of claudin-2, a pore-forming tight junction protein (30). Using a STAT6 knockout mouse, Madden and colleagues (31) demonstrated that IL13 increases mucosal permeability in a STAT6-dependent fashion. Furthermore, administration of recombinant mouse IL4 increases the responsiveness of the intestinal epithelium to PGE2 and histamine in a mast cell- and STAT6-dependent fashion, demonstrating the importance of this signaling pathway in intestinal cell function (31). In our mouse model of AOM/DSS-induced CAC, mice with global deletion of STAT6 exhibited significantly reduced numbers of tumors, decreased inflammatory infiltrate, and maintenance of epithelial structure. Under these experimental conditions, STAT6 could favor an increase in mucosal permeability, thus enhancing the response of the intestinal epithelium to microbial products, which may result in chronic inflammation and tumorigenesis.

We observed a decrease in tumor development in STAT6–/– mice. Consistent with this finding, we also detected less inflammatory infiltrate and decreased expression of proinflammatory cytokines in the mucosa, accompanied by no changes in the number of goblet cells. In a mouse coinfection model with the helminth Heligmosomoides polygyrus and the gram-negative bacterium Citrobacter rodentium, STAT6–/– mice exhibited less intestinal inflammation in association with reduced infiltration of colonic lamina propria of alternatively activated macrophages (32). In addition, 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 of immune cells into the intestinal epithelium. In contrast, in the oxazolone (OXA)-induced colitis model, STAT6–/– mice showed crypt hyperplasia, crypt loss, cryptitis, and active infiltrates of neutrophils and mononuclear cells in the lamina propria compared with wild-type control mice (16). Furthermore, a report indicated that STAT6–/– mice are sensitive to acute DSS-induced colitis (14), which suggests that acute inflammatory responses are mainly associated with Th1 profiles (34). However, CAC development has been demonstrated to be a complex pathology where various 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 with the Wnt signaling pathway and nuclear β-catenin, as well as reduced mRNA expression of Lgr5 and c-Myc, both of which are primary Wnt/β-catenin target molecules. Reduction in these markers has been associated with the impaired mucosal expression of M2 macrophage-associated genes (35). β-Catenin is an important component of the Wnt signaling pathway. In fact, mutations in Wnt-associated protein-coding genes have been reported in premalignant lesions of the intestine (36). In line with these previous observations, our data demonstrate that STAT6–/– mice had significantly lower expression of β-catenin and COX2 when treated with AOM/DSS, suggesting a role for STAT6 signaling in tumor initiation during CAC.

Several lines of evidence suggest that IL4 signaling may promote the survival and proliferation of cancer cells by down-regulating anti-apoptotic proteins, such as PED, cFLIP, Bcl-xL, and Bcl-2 (37), while favoring survivin expression (38, 39). In vitro studies in colorectal cancer cell lines show that cells with high STAT6 activity are resistant to apoptosis and aggressively metastasis compared with STAT6-defective cell phenotypes (20). In accordance with such studies, our results demonstrated an important increase in the apoptosis rate in STAT6–/– mice early in CAC development and decreased intestinal cell proliferation, as estimated by Ki-67 expression. Additionally, IL4 may enhance the proliferation rate of colon cancer cells (40), given that the upregulation of IL4 constitutes an important mechanism that protects tumorigenic colon cancer stem-like cells (CD133+) from apoptosis (41). Thus, the requirement for the STAT6 signaling pathway for the biological effects of IL4 and IL13 is a critical factor in CAC development, as has been suggested also for breast cancer (42), in which IL4Rα signaling enhances metastatic growth through the promotion of tumor cell survival and proliferation. Human and murine mammary cancer cells treated with IL4 increase proliferation, glucose consumption and lactate production, indicating that IL4 can support breast cancer growth (43). Given that other studies have demonstrated that STAT6 phosphorylation could take place through IL4Rα independent-mechanisms (44), this opens the possibility that in colon cancer, other mechanisms independent of the IL4/IL4Rα/STAT6 axis could take place. Here, our results demonstrated a significant inhibition in tumor development in STAT6–/– mice, as 70% of these animals were tumor-free in advanced stages of CAC, suggesting that the STAT6 signaling pathway is centrally involved in tumor initiation and progression.

To address the question of whether the differences in tumor development between STAT6–/– and WT mice were related to inflammatory monocyte recruitment, we examined the Ly6Chi population expressing chemokine receptor CCR2 in STAT6–/– mice during CAC development. CCR2 is responsible for the recruitment of Ly6Chi monocytes to peripheral sites of inflammation, and in the AOM/DSS model, CCR2 knockout mice exhibit less macrophage infiltration and lower tumor numbers (45). We observed decreases in the percentage of CD11b+Ly6ChiCCR2+ cells in STAT6–/– mice compared with wild-type animals, indicating that STAT6 is a critical mediator of the initiation and promotion of CAC. Moreover, STAT6–/– mice also displayed fewer circulating CD11b+Ly6ClowLy6G+ granulocytic cells, which have been reported to have suppressive activity, and a reduction in this cell population is associated with improvement of antitumor responses (46). In contrast, IL4Rα-deficient mice receiving syngeneic TS/A, 4T1, or CT26 tumor cells showed no differences in the systemic numbers and T cell suppressive capacity of CD11b+Ly6G−Ly6Chi and CD11b+Ly6G+Ly6Clow cells compared with wild-type animals, supporting the idea that IL4Rα does not alter the phenotype and number of these cells (47). Nevertheless, a caveat of that study involves the use of tumor cell lines, which exert tumorigenic mechanisms that may differ from those found in chronic inflammation-preceded colon cancer. Additionally, other important cell populations could be involved in protection against CAC in STAT6-deficient mice. In this sense, we observed an increase in the levels of IL10. This is an immune-regulatory cytokine that is important in the modulation of inflammatory responses during cancer (48). The absence of STAT6 may promote the recruitment of an IL10-producing cell population, as is the case for T regulatory cells (Tregs). Tregs are essential in maintaining tolerance and down-modulating immune responses by secreting IL10 upon activation (49). STAT6 can directly inhibit FOXP3 expression by repressing the Foxp3 gene (50). Thus, STAT6 activation could facilitate inflammation by hampering inducible Treg development. However, the role of STAT6 in FOXP3 protein regulation during CAC remains to be elucidated. Interestingly, we observed an important decrease in the mRNA expression of CCL9, CCL25, and CXCR2 in STAT6–/– mice during late stages of tumor progression. CCL9 is secreted by mouse and human colon cancer cells, and lack of CCL9 expression dramatically suppresses the outgrowth of disseminated tumors in the liver (24). Similarly, CXCR2 in the tumor microenvironment has been associated with colon cancer growth and progression (51), but in our model, this receptor was downregulated in the absence of STAT6.

Finally, inhibition of STAT6 by AS1517499 injection significantly inhibited colonic tumor formation in a manner similar to the inhibited tumor formation in STAT6–/– animals, supporting the idea that STAT6 may favor tumor initiation and progression by recruiting several immune cell types and/or influence the proliferative and invasive properties of cancer cells. However, it will be interesting to delineate at which point in the progression of CAC STAT6 inhibition would remain functional.

In conclusion, STAT6 signaling appears to be central to the initial steps of CAC development by modulating inflammatory responses and control mechanisms, such as immune cell recruitment and epithelial cell proliferation. STAT6 may represent a promising target for treating colitis-associated colorectal cancer in the near future.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: S.A. Leon-Cabrera, Y.G. Delgado-Ramirez, Y.I. Chirino, F. Ávila-Moreno, L.I. Terrazas

Development of methodology: S.A. Leon-Cabrera, Y.G. Delgado-Ramirez, Y. Ledesma-Soto, N.L. Delgado-Buenrostro, L.I. Terrazas

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S.A. Leon-Cabrera, E. Molina-Guzman, A. Vázquez-Sandoval, Y. Ledesma-Soto, N.L. Delgado-Buenrostro, L.E. Arias-Romero, L.I. Terrazas

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S.A. Leon-Cabrera, E. Molina-Guzman, Y.G. Delgado-Ramirez, A. Vázquez-Sandoval, E.B. Gutierrez-Cirlos,

Writing, review, and/or revision of the manuscript: S.A. Leon-Cabrera, C.G. Pérez-Plasencia, M. Rodríguez-Sosa, F. Vaca-Paniagua, F. Ávila-Moreno, E.B. Gutierrez-Cirlos, L.I. Terrazas

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y.G. Delgado-Ramirez, Y. Ledesma-Soto

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

Grant Support

Sonia A. Leon-Cabrera was supported by Programa de Apoyo a Proyectos de Investigación e Innovación tecnológica, PAPIIT, UNAM, grant numbers IA206216 and RA206216. Fellowship from L'Oreal-UNESCO-CONACYT for Women in Science. Luis I. Terrazas was supported by Consejo Nacional de Ciencia y Tecnología (CONACYT) grant number 280013 and Programa de Apoyo a Proyectos de Investigación e Innovación tecnológica, PAPIIT, UNAM grant number IN220316.

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

Acknowledgments

This work was supported by grants from Programa de Apoyo a Proyectos de Investigación e Innovación tecnológica, PAPIIT, UNAM, Grant number IA206216, RA206216, and IN220316. Consejo Nacional de Ciencia y Tecnología (CONACYT) Grant number 280013. S.A. Leon-Cabrera received a fellowship from L'Oreal-UNESCO-CONACYT for Women in Science. YDR is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and received a fellowship (606590) from CONACYT.

Footnotes

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

  • Received July 20, 2016.
  • Revision received October 5, 2016.
  • Accepted April 4, 2017.

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Lack of STAT6 Attenuates Inflammation and Drives Protection against Early Steps of Colitis-Associated Colon Cancer
Sonia A. Leon-Cabrera, Emmanuel Molina-Guzman, Yael G. Delgado-Ramirez, Armando Vázquez-Sandoval, Yadira Ledesma-Soto, Carlos G. Pérez-Plasencia, Yolanda I. Chirino, Norma L. Delgado-Buenrostro, Miriam Rodríguez-Sosa, Felipe Vaca-Paniagua, Federico Ávila-Moreno, Emma B. Gutierrez-Cirlos, Luis E. Arias-Romero and Luis I. Terrazas
Cancer Immunol Res May 1 2017 (5) (5) 385-396; DOI: 10.1158/2326-6066.CIR-16-0168
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