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STAT2 is an essential transcription factor in the type I interferon (IFN-α/β) signal transduction pathway and known for its role in mediating antiviral immunity and cell growth inhibition. Unlike other members of the STAT family, IFNs are the only cytokines known to date that can activate STAT2. Given the inflammatory and antiproliferative dual nature of IFNs, we hypothesized that STAT2 prevents inflammation-induced colorectal and skin carcinogenesis by altering the inflammatory immune response. Contrary to our hypothesis, deletion of STAT2 inhibited AOM/DSS-induced colorectal carcinogenesis as measured by prolonged survival, lower adenoma incidence, smaller polyps, and less chronic inflammation. STAT2 deficiency also inhibited DMBA/TPA-induced skin carcinogenesis as indicated by reduced papilloma multiplicity. A potential mechanism by which STAT2 promotes carcinogenesis is through activation of pro-inflammatory mediators. Deletion of STAT2 decreased AOM/DSS-induced expression and release of pro-inflammatory mediators such as interleukin 6 and CCL2 and decreased interleukin-6 release from skin carcinoma cells, which then decreased STAT3 activation. Our findings identify STAT2 as a novel contributor to colorectal and skin carcinogenesis that may act to increase the gene expression and secretion of pro-inflammatory mediators, which in turn activate the oncogenic STAT3 signaling pathway.
Local chronic inflammation is believed to be a contributing component in the malignant transformation of the intestinal and skin epithelium (1). Patients suffering from chronic inflammatory bowel disease (IBD), such as ulcerative colitis (UC) and Crohn’s disease are at a higher risk of developing colorectal cancer (2). However, the molecular pathogenesis of colorectal cancer is poorly understood. Local chronic inflammation can alter the homeostasis of the intestinal microenvironment via persistent recruitment of immune cells and by prolonging the activation of NFκB and IL1-R signaling pathways (3–6). Inflammatory cytokines such as TNF-α, IL-1, IL-6 and IFN-γ create a microenvironment that promotes colorectal and skin carcinogenesis by enhancing cell proliferation and angiogenesis (7–12).
The Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathway is an additional signal transduction pathway important in regulating pro- and anti-inflammatory responses. Activation of this pathway is closely linked to IBD. Among the cytokines that can activate the JAK/STAT pathway, IFN-γ plays an indispensable role in experimental inflammatory bowel disease and spontaneous development of colorectal carcinomas in mice (10, 13). In contrast, IFN-α/β is protective in dextran sodium sulfate (DSS)-induced colitis (14) and when administered to ulcerative colitis patients (15).
STAT2 is a necessary transcription factor in the IFN-α/β signaling pathway (16). Unlike other members of the STAT family that are activated by multiple growth factors and cytokines, thus far only IFN-α/β activates STAT2 (17). IFN-α/β exerts anti-proliferative, immune-modulatory, and antiviral effects via a complex consisting of STAT1, STAT2 and IRF9, which enters the nucleus and binds in the promoter region of IFN-stimulated genes (18). Studies using STAT2 deficient cell lines and mice demonstrated that STAT2 is necessary for the antiviral, apoptotic, and cell growth inhibitory effects of IFN-α/β (19–21). Yet the role of STAT2 in inflammation and tumorigenesis is largely unknown. STAT2−/− mice expressing an IFN-α transgene in the central nervous system died prematurely with neuroblastomas and had excessive production of the pro-inflammatory cytokine IFN-γ (22). Lymphocytes from colons of UC and Crohn’s disease patients had decreased STAT2 protein levels (23). Thus, we hypothesized that STAT2 prevents carcinogenesis by modulating the inflammatory immune response.
We utilized STAT2−/− mice in two-stage models of inflammation-induced colorectal and skin carcinogenesis and showed that, contrary to our hypothesis, STAT2 was required for promotion of colorectal and skin carcinogenesis. A potential mechanism is via a STAT2 dependent increase in the gene expression and secretion of pro-inflammatory mediators like IL-6, which then activate the oncogenic STAT3 signaling pathway.
STAT2-deficient (STAT2−/−) mice were generated and kindly provided by Dr. Christian Schindler (Columbia University, NY) (24). Strain-matched wild type (WT) mice were purchased from the Animal Production Area of the National Cancer Institute-Frederick (Frederick, MD). All mice were on a 129SvJ genetic background. Mice were bred in our own animal facility and housed under specific helicobacter pathogen-free environment. These studies were performed in accordance to the NCI-Frederick ACUC guidelines.
To examine the effect of STAT2 on tumor incidence and multiplicity, 10 wild type and 11 STAT2−/− female mice were intraperitoneally injected at 6 weeks of age with the carcinogen azoxymethane (AOM) at 10mg/kg body weight. One week later, the mice were started on the first of three 21-day DSS cycles, consisting of receiving for 5 days the tumor promoter dextran sodium sulfate (DSS) at 3% in the drinking water followed by 16 days of receiving regular water. Body weights of mice were measured weekly. Mice showing signs of morbidity were sacrificed. The remaining mice were sacrificed 16 wk after AOM induction and 58 days after removal from DSS in the water. Colons were removed and flushed with PBS buffer and cut longitudinally. The entire colons from all 21 mice were examined by experienced histotechnicians using a dissecting microscope and all visible lesions were counted and measured, fixed in 10% neutral buffered formalin (NBF), and embedded in paraffin. A board certified pathologist examined H&E-stained tissue sections containing the largest and the smallest proliferative lesion of each mouse (32% of the 129 colon lesions) to confirm the specificity of macroscopic count and to evaluate tumor progression according to Boivin et al. (25).
To examine the early stages of colorectal carcinogenesis, the same protocol was used except mice were sacrificed after 5 days within the first DSS cycle. Age-matched wild-type and STAT2 −/− female mice not subjected to the colorectal carcinogenesis protocol were used as normal controls. Colon tissue sections were either paraffin embedded for immuno-histochemistry, snap frozen for RNA analysis, or used for protein analysis or colon tissue culture.
The effect of STAT2 on proliferation and cell crypt structure in early stages of colorectal carcinogenesis was measured by detection of Ki67 antigen in colon tissue sections of 2 mice per group. The effect of STAT2 on STAT3 activation was measured by detection of tyrosine 705 phosphorylated STAT3 in adenoma-containing colonic tissue collected from WT and STAT2−/− mice 16 wks after AOM induction. Embedded colon sections were deparaffinized, rehydrated, and antigen were retrieved in a pressure cooker for 4 min in 0.01M citric acid, pH 6.0. Sections were then cooled for 25 min followed by pretreatment with 1.5% hydrogen peroxide dissolved in methanol for 10 min. After blocking with 10% normal goat serum, sections were incubated with anti-Ki67 antibody (1:10,000, Vector Labs, CA) or with anti-phospho STAT3-Y705 (1:50, Cell Signaling, Danvers, MA) for 1 h at room temperature. After incubation with biotin conjugated secondary antibody and streptavidin-HRP, signals of Ki67 and phospho-STAT3 stained nuclei were detected by using Vector Elite Kit (Vector Labs, CA).
RNA of colon tissue sections was quantified from 4 individual mice per AOM/DSS treated group and 2 individual mice per untreated group. RNA was isolated from colon tissue section using a combination of the Mini-Beadbeater (BioSpec, OK) to disrupt and homogenize samples and the RNeasy Mini Kit (Qiagen, CA). All samples were DNase-treated during the on-column RNA isolation steps to eliminate genomic DNA contamination. The RNA Cleanup kit from (Qiagen) was used to purify RNA samples if needed. Total RNA (2 μg) was reverse transcribed using the SuperArray RT2 First Strand Kit (C-03) (SABiosciences, MD). The resulting cDNAs were analyzed by quantitative PCR using the Mouse Signal Transduction and the Stress and Toxicity RT2 Profiler PCR Arrays (SABiosciences, MD). PCR was performed on BioRad iQ5 cycler (BioRad, CA) according to the manufacturer’s instructions.
Colon cell lysates were prepared from 3 individual mice per group. Colons were flushed in cold PBS supplemented with 30 U/ml penicillin, 30 μg/ml streptomycin (Invitrogen, CA), cut longitudinally, and then cut into pieces of 200–300mg. Individual colon pieces were placed in a 12-well flat tissue culture plate and covered with 2 mL of serum-free RPMI medium supplemented with high doses of penicillin and streptomycin. Colon tissue was incubated at 37°C for 24 h and then digested for 2 h at 37°C in a shaking bath in a solution of 1X PBS containing 10% fetal calf serum, 15 mM Hepes, 5mM EDTA, antibiotics, collagenase (50X), and DNase (100X). Collected cells were filtered, washed and lysed in ice cold lysis buffer previously described (20). After centrifugation at 4°C for 10 min, lysate was collected and proteins were resolved by SDS-PAGE (Invitrogen, CA). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane, which was blocked for 30 min with blocker™ casein in TBS (Pierce, IL) and then blotted with phospho-STAT1 Y701, (BD Biosciences Pharmingen, CA), Cox2 (Cayman Chemical, MI), IRF-1 (M-20), STAT2 Santa Cruz Biotechnology, CA), and Actin (AbCam, MA). Following incubation with HRP-conjugated secondary antibody, membranes were washed and developed by chemiluminescence using the ECL Western blotting system (Pierce, IL). Total protein concentrations were measured by a standard Bio-Rad Bradford protein assay.
Cytokine release in early stages of colorectal carcinogenesis was quantified in mouse colons. Colons were flushed in cold PBS supplemented with 30 U/ml penicillin, 30 μg/ml streptomycin (Invitrogen, CA), cut longitudinally, and then cut into pieces of 200–300mg. Individual colon pieces were placed in a 12-well flat tissue culture plate and covered with 2 mL of serum-free RPMI medium supplemented with penicillin and streptomycin. Culture plates were incubated at 37°C for 24 h. Supernatants were collected, centrifuged at 14,000 rpm at 4°C for 5 min, and stored at −80°C. Cytokine and chemokine concentrations in the supernatant were measured using the mouse inflammation cytometric bead array (CBA) kit (BD Biosciences) according to the manufacturer’s instructions.
The biological implication of STAT2 in papilloma incidence and multiplicity was assessed by applying 102 μg of 9,10-Dimethyl-1,2-Benzanthracene (DMBA) in 0.2 ml acetone to the shaved dorsal skin area of wild type and STAT2−/− female mice (n=10/group) at 7 wk of age. Starting 2 wk after initiation, 6 μg of the tumor promoter 12-O-tetradecanocylphorbol-13-acetate (TPA) in acetone was applied to the same skin area twice weekly for 20 wks. Mice were visually examined weekly for skin papilloma and squamous cell carcinoma number and size. Papillomas were counted when lesions were palpable. Only tumors that reached at least 2mm in diameter and had been present for at least 2 wks were included in the analysis. Mice with papillomas that had progressed to carcinoma were euthanized. Carcinomas were harvested to establish tumor cell lines.
Skin carcinoma cells lines were established from carcinomas induced on WT (LD791) and STAT2−/− (AG1279) mice by adapting a protocol used for establishing melanoma cell lines (26). Briefly, freshly excised tumors were minced into small pieces and allowed to adhere to collagen coated plates in MEL 2% medium (80% MCDB 131 medium, 20% L-15 medium, 2% FBS, 5μg/mL bovine insulin, 15μg/ml Bovine Pituitary Extract, 5ng/mL EGF and 1.68 mM, 10U/mL penicillin, 10 μg/mL streptomycin and 2.2 g of GlutaMax™). Once monolayers were obtained, cells were trypsinized and passaged onto regular plastic dishes with 10% FCS containing DMEM medium supplemented with antibiotics and GlutaMax. The AG1279 skin tumor cell line was later stably reconstituted with human STAT2. A flag tagged human STAT2 construct in pCDNA3 mammalian expression vector whose expression is under the control of a CMV promoter was kindly provided by C. Horvath, Northwestern University, Evanston IL). Cells were transfected using Lipofectamine 2000 (Invitrogen) in combination with either 5 μg of a pcDNA3 empty vector (negative control) or pcDNA3 encoding STAT2. Following an overnight incubation, cells were placed under selection with 10μg/mL puromycin (Sigma). STAT2 and tyrosine phosphorylated STAT3 expression of the tumor cell lines was assessed by Western blot analysis as previously described. Additionally, cells were treated or not with exogenous murine IL-6 (Peprotech, Inc., NJ) and analyzed for phospho-STAT3 Y705. Actin was used to monitor for equal protein loading. IL-6 and CCL2/MCP-1 concentrations were measured using the mouse inflammation cytometric bead array (CBA) kit (BD Biosciences) according to the manufacturer’s instructions. The measurements were repeated twice with 3 replications each.
Student’s t-test was used to compare body weight, RNA, and protein data of 2 groups and generalized least squares means were used to compare data of more than 2 groups. A logistic rank test was used to compare survival curves. Fisher’s exact test was used to compare in all mice, including those that had to be prematurely sacrificed, tumor incidences and first appearance of skin papilloma number. The non-parametric Wilcoxon’s rank sum test was used to compare tumor multiplicity. For PCR array data analysis, the ΔΔCt method was used and each gene fold-change was calculated as difference in gene expression between naive and AOM/DSS colon samples. Data were normalized against 5 housekeeping genes that were included in the PCR array by subtracting the average Ct value for five housekeeping genes from the average Ct value of gene of interest (i.e., calculating a ΔΔCt). ΔΔCt were compared across groups using t-tests. Due to the large number of comparisons made in our analyses, differences in measurements of gene expression between groups was considered statistically significant when P < 0.005. All tests were two-sided except for tumor multiplicity. Significance was declared at P ≤ 0.05 and trends toward significance were declared at P ≤ 0.10. Unadjusted means and standard deviations (SD) are presented.
Deletion of STAT2 protected the mice from the colon irritant DSS. STAT2−/− mice lost less body weight after one DSS cycle (at 16 days, 5% vs. 16% in wild type (WT) mice; P = 0.0004; Fig. 1A). We observed in STAT2−/− mouse colons less inflammatory infiltrate after the first DSS cycle, less crypt destruction, and less uncontrolled proliferation with Ki67 staining in cells within well-oriented crypts (Fig. 1B). These changes are characteristic of an attenuated inflammatory response.
STAT2 deletion also decreased tumor multiplicity and size in AOM/DSS treated mice. STAT2−/− mice developed 1.6-fold fewer tumors than WT mice (6.4 vs. 4.1 tumors/mouse; P = 0.05; Fig. 2A) and on average the tumors were smaller (Fig. 2B). Furthermore, a loss of STAT2 inhibited tumor progression. In comparison to WT mice, fewer STAT2−/− mice had histologically confirmed adenomas (18 vs. 80%; P = 0.009), a similar number of STAT2−/− mice had dysplastic lesions called gastrointestinal intraepithelial neoplasia (GIN; 36 vs. 30%), and more STAT2−/− mice had hyperplastic gut associated lymphoid tissue lesions (GALT; 91 vs. 50%; P = 0.06; Fig. 2C and Supplementary Fig S1). In addition, numerically fewer STAT2−/− mice showed inflammation around the tumors than WT mice (27% vs. 50%; Fig. 2C and Supplementary Fig S1). No adenocarcinoma or flat lesions were observed. We did not observe consistent and distinct morphological differences between STAT2−/− mice and WT mice in normal or tumor tissue of the same grade (Supplementary Fig S1). Consistent with the inhibited tumor development and progression, STAT2 deficiency enhanced survival. STAT2−/− mice showed increased long-term survival at the end of the 16 week study in which all of these mice (n=11) remained viable while only 40% of WT mice (4 out of 10) survived (P = 0.003; Fig. 2D). Collectively, these results suggest that STAT2 plays an active role in tumor development and tumor progression.
In order to discover possible mechanisms for how the STAT2 transcription factor regulates inflammation-induced colorectal carcinogenesis, we performed pathway-focused RT-PCR array analysis using the Signal Transduction and Stress and Toxicity arrays with colons from 2 untreated and 4 AOM/DSS treated WT and STAT2−/− mice per group. Ablation of the STAT2 gene was associated with a slight change in gene expression (< 3 fold) of only a few genes (8 out 136) (Fig. 3A, B). As expected, AOM/DSS treatment induced the expression of multiple genes in the WT mice and deletion of STAT2 attenuated some of these changes. STAT2 deletion predominantly attenuated gene expression that was induced by AOM/DSS treatment and had less of an effect on gene expression that decreased when compared to WT mice. Closer examination shows that genes whose expressions were attenuated by STAT2 deletion are predominantly in the JAK/STAT and NFκB pathway in addition to genes involved in inflammation and angiogenesis (MMP7 and MMP10) (Fig. 3C, D). Our data suggest that STAT2 deletion attenuates the activation of inflammatory chemokines (Ccl2, Ccl3, Ccl4, Cxcl9, and Cxcl10) and cytokines (IL1a, IL1b and IL6) genes, which showed greater than 5 fold induction in the WT mice. Our data also suggest that STAT2 deletion attenuates the activation of the pro-carcinogenic JAK/STAT (Cxcl9, MMP10, Nos2) and NFkB (Cxcl1, IL1a, Nos2) pathways in response to a carcinogenic stimulus.
Consistent with the literature (23), STAT2 deficiency attenuated the DSS-induced increase in STAT1 activity (phosphorylation) and IRF-1 expression. Colon tissue of STAT2−/− mice had after the first DSS cycle a smaller induction of Cox-2 and IRF-1 and no increase in STAT1 activation compared to colon tissue of WT mice (Fig. 4A). These observations suggest that STAT2 deletion attenuates the activation of the anti-proliferative and immunomodulatory IFN-α/β signaling pathway to a carcinogenic stimulus.
In agreement with Fig 3, STAT2 deletion also attenuated the DSS-induced increase in the secretion of pro-inflammatory mediators. Supernatant of STAT2−/− mouse colon tissue had after the first DSS cycle 2.5-fold lower IL-6 (P = 0.04) and 4.5-fold lower Ccl2/MCP-1 (P = 0.01) concentrations than supernatant of WT mouse colon tissue, while no statistical differences in the concentrations of TNF-α and IFN-γ were noted (Fig. 4B). Concentrations of IL-10 and IL-12 were below detection levels (data not shown).
STAT3 is an oncogenic transcription factor activated by IL-6. STAT3 activation was evaluated in adenomas of WT and STAT2−/− mice (Fig. 4C). Constitutive STAT3 activation was detected in adenomatous tissue of WT and STAT2−/− mice; albeit at lower levels in the STAT2−/− mice. The uninvolved normal colonic epithelium of WT and STAT2−/− mice showed undetectable levels of activated STAT3 (data not shown). These data suggest that STAT2 deficiency attenuates and modulates the inflammatory response to a pro-carcinogenic stimulus via inhibition of STAT3 signaling.
Similar to our results in the AOM/DSS colorectal model, STAT2 deletion attenuated DMBA/TPA induced skin carcinogenesis. The onset of papilloma in the STAT2−/− group was delayed by two weeks compared to the WT group (week 12 vs. week 10, respectively; 0 vs. 40% tumor incidence in week 11, P = 0.03; Fig. 5A). While all of the mice eventually developed papillomas, STAT2−/− mice had throughout the study fewer papillomas than the WT mice with statistically significant differences starting at week 23 (4.3 vs. 7 papillomas per mouse; Fig. 5B). The carcinoma incidence and papilloma morphology did not differ between STAT2−/− and WT mice (data not shown).
Reconstitution of STAT2 in an established STAT2−/− skin carcinoma cell line, as shown in Fig. 6A, increased activated STAT3 (Fig. 6B and 6C) and the secretion of IL-6 (Fig. 6D). STAT2−/− carcinoma cells had 3.5-fold lower IL-6 concentrations in the supernatant than STAT2 reconstituted cells, which in contrast had levels similar to those of the WT skin carcinoma (157 vs. 551 pg/mL; P = 0.0002; 515 pg/ml; Fig. 6D), while Ccl2/MCP-1 concentrations were not consistently affected (data not shown). Addition of exogenous IL-6 to STAT2−/− cells induced STAT3 tyrosine phosphorylation (Fig. 6C) thus suggesting that activation of STAT3 is not directly STAT2 dependent, but is mediated by IL-6. In summary, these observations suggest that STAT2 contributes to tumor development in inflammation-promoted carcinogenesis by increasing gene expression and release of pro-inflammatory mediators that in turn activate STAT3.
Our study examined the role of STAT2 in inflammation-promoted colorectal and skin carcinogenesis (24). STAT2−/− mice were protected from AOM/DSS-induced tumorigenesis as indicated by the decrease in colon tumor multiplicity, size, progression and improved long-term survival. During the early stages of colorectal carcinogenesis, lack of STAT2 resulted in less severe weight loss, less crypt destruction, and most notably, an attenuated increase in gene expression and secretion of pro-inflammatory mediators such as IL-6. We cannot exclude the possibility that AOM is metabolized differently in the STAT2−/− mice as this could have contributed to the colon tissue differences seen between the two strains of mice. However, these differences are unlikely because STAT2−/− mice develop tumors; albeit less (our data) while wild type 129/Sv do not develop tumors with AOM alone (27). Similarly, STAT2−/− mice were protected from DMBA/TPA induced skin papillomagenesis. During the late stages of skin carcinogenesis, STAT2 may have tumor promoting effects as overproduction of IL-6 by skin carcinoma cells that we established in our laboratory appeared to be STAT2 dependent. This is not surprising as production of IL-6 has been demonstrated previously to be required in skin carcinogenesis (28). Thus our findings reveal an unexpected and first described role of STAT2 as a potential promoter of skin and colon tumorigenesis that may act to increase gene expression and release of pro-inflammatory mediators, most notably IL-6.
The use of genetically engineered mouse (GEM) models of human cancer has provided new methods for studying cancer prevention (29). The creation of mice that are resistant to induced tumorigenesis not only validates the engineered target, but also allows the discovery of new molecular targets that are active in the early stages of tumor development. For instance, GEMs that express TAM67, a dominant negative inhibitor of AP-1, are resistant to chemical, UVB and virus-induced skin tumorigenesis (30, 31) and oncogene-induced breast cancer (29). For these studies, a small number of TAM67 regulated genes have been identified and validated as candidate targets for cancer prevention (32, 33). The observation that STAT2−/− mice are resistant to inflammation-induced colon and skin tumorigenesis, will allow us to identify new targets that are active in the early stages of colon and skin cancer, the first of which appears to be the pro-inflammatory cytokine, IL-6.
The range of pro-inflammatory cytokines and other soluble factors produced by activated innate immune cells can trigger not only tumor promotion, but also cell survival (1, 34–36). Activation of the JAK/STAT pathway induced IFN-γ is thought to drive inflammation and has been linked to colorectal carcinogenesis (10, 13). In a different study, STAT2−/− mice expressing an IFN-α transgene in the central nervous system produced excessive amounts of IFN-γ and developed neuroblastomas (22). In this model one could postulate that the protective effect of STAT2 deletion was hampered by the high levels of IFN-γ and; therefore, contributed to tumor development. In contrast, IFN-α/β activated pathways have been shown to elicit a protective effect not only during inflammation-induced colitis, but also during the early events of squamous skin carcinogenesis (14, 15, 37). Yet the mechanism of how this occurs remains largely unknown. One reasonable explanation could be that endogenously secreted IFN-α/β might antagonize stress and antigen-dependent effector T cell expansion through a STAT1/STAT2 dependent pathway (24, 38).
The molecular mechanism by which STAT2 deletion attenuates the increase in gene expression of pro-inflammatory mediators is unclear. Our data suggest that inactivation of the STAT2 signaling pathway confers protection in both early and later stages of colon carcinogenesis. A marked decrease in IL-6 production in the colons of STAT2−/− mice could in part explain the less pronounced inflammatory response seen in these animals. Although IL-6 is needed for the mucosal integrity of the colon, it also promotes the proliferation of premalignant cells and drives tumor formation in the early stages of carcinogenesis via activation of the STAT3 signaling pathway (36). In our study, an accumulation of GALT in areas of less progressed tumors after AOM/DSS treatment is seen when STAT2 is absent. In this case, STAT2 and/or IL-6 may be required for regulation of GALT production. Moreover, lesions that progress to adenoma carry activated STAT3 irrespective of STAT2 expression. One may speculate that the hyper-production of IL-6 seemingly coming from the inflammatory cells present in GALT is STAT2 dependent and that a strong IL-6 activated STAT3 signaling is what drives progression of the tumors into later stages of carcinogenesis.
Our results depict an autocrine IL-6/STAT3 signaling pathway as an important molecular target of STAT2 in colorectal and skin cells. IL-6 and STAT3 play a critical role in early and late stages of colorectal carcinogenesis (35). STAT3 null mice are resistant to skin carcinogenesis (39). Similar to STAT2−/− mice, IL-6−/− mice show decreased tumor multiplicity and size compared to WT mice after AOM/DSS treatment. However, unlike STAT2−/− mice, IL-6 deficient mice have more crypt cell destruction and more dramatic weight loss than control mice after DSS treatment demonstrating a need for basal IL-6 concentrations to maintain mucosal integrity. In our study, the increase in IL-6 gene expression and secretion was attenuated in STAT2−/− mice following a tumor promoting stimulus. This observation was recapitulated using a skin carcinoma cell line derived from a STAT2−/− mouse in which hyper-IL-6 secretion and constitutive STAT3 activation were seen after STAT2 reconstitution. A recent genomic analysis reported that both IL-6 and STAT2 are down-regulated by dietary flavonoids (40). The involvement of STAT2 in the production of pro-inflammatory mediators (Fig. 3) raises the question of whether STAT2/STAT3 heterodimers form to activate gene transcription. However, a previous study demonstrated that STAT2 does not associate with STAT3 (41).
In conclusion, our results quite unexpectedly link STAT2 to inflammation-associated colorectal and skin carcinogenesis. The model we propose is that STAT2 up-regulates IL-6 production, which in turn activates the STAT3 oncogenic pathway to drive tumor promotion. Nonetheless, additional studies in other animal models of cancer are warranted to define the role of STAT2 in skin and colon carcinogenesis.
Supplementary Figure S1. STAT2 deletion decreases tumor progression in AOM/DSS-induced colorectal carcinogenesis. Overview of H&E-stained sections of representative colons from Wild Type (left panel) and STAT2−/− mice (right panel) for normal tissue (A and E) and tissues with lesions (B-D and F-H). No differences were observed for normal tissue between Wild Type (A) and STAT2−/− (E). While lesions from Wild Type mice progressed to GIN and adenomas (B-D), lesions from STAT2−/− mice rarely progressed beyond hyperplasia to GALT (F-H).
Grant support: This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health under Contract No. N01-CO-12400. This project was also supported by Award Number K22CA095326 from the National Cancer Institute (A. Gamero).
This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
We thank Dr. Andres Klein-Szanto for his valuable assistance with immunohistochemical analysis.
A preliminary report has been presented at the International Cytokine Society and the International Society for Interferon and Cytokine Research in Montreal, Canada, October 12-16, 2008, and was published as an abstract in Cytokine 43(3):290.
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