STAT2 contributes to promotion of colorectal and skin carcinogenesis

Abstract

Signal transducer and activator of transcription 2 (STAT2) is an essential transcription factor in the type I IFN (IFN-alpha/beta) 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 azoxymethane/dextran sodium sulfate-induced colorectal carcinogenesis as measured by prolonged survival, lower adenoma incidence, smaller polyps, and less chronic inflammation. STAT2 deficiency also inhibited 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-induced skin carcinogenesis as indicated by reduced papilloma multiplicity. A potential mechanism by which STAT2 promotes carcinogenesis is through activation of proinflammatory mediators. Deletion of STAT2 decreased azoxymethane/dextran sodium sulfate-induced expression and release of proinflammatory 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 proinflammatory mediators, which in turn activate the oncogenic STAT3 signaling pathway.

Figure 1

STAT2 deletion attenuates sensitivity to AOM/DSS induced colorectal carcinogenesis. Age matched wild type (n=10) and STAT2^−/− mice (n=11) were intraperitoneally injected with AOM followed by 3 cycles of 3% DSS in drinking water for 5 days followed by 16 days of normal drinking water and weighed 2–3 times weekly. (A) Attenuated weight loss (an indicator of colon inflammation) after first DSS cycle in STAT2^−/− mice. (B) Less crypt destruction and inflammatory infiltrate in STAT2^−/− mice after the first DSS cycle. Colon sections were stained for Ki67-positive cells within well-oriented crypts (stained brown) and counterstained with hemotoxylin (blue) to visualize the whole tissue at least two slides from each mouse (2 mice/group).

Figure 2

STAT2 deletion decreases colon tumor development and progression and improves long-term survival in response to AOM/DSS induced colorectal carcinogenesis. Age matched wild type (n=10) and STAT2^−/− mice (n=11) were intraperitoneally injected with AOM followed by 3 cycles of DSS. Number of colon lesions was counted using a dissecting microscope in all 21 mice. (A) Decreased number of polyps in STAT2^−/− mice. (B) Decreased size of tumors in STAT2^−/− mice. (C) Decreased tumor progression in STAT2^−/− mice. Histology of tumor sections of 2 representative tumors from each mouse was done. (D) Improved long-term survival in STAT2^−/− mice. *, P ≤ 0.05.

Figure 3

STAT2 deletion attenuates the pro-inflammatory gene response to AOM/DSS. RNA was isolated from colons of AOM/DSS treated mice (n=4) at the end of the first DSS treatment. Untreated mice (n=2) served as controls. RT-PCR focused pathway arrays, (A and C) Signal Transduction and (B and D) Stress and Toxicity were used for evaluating changes in gene expression of a subset of genes. Cluster maps (A and B) depict genes that varied significantly (P< 0.05) between one or more groups of mice. Green depicts lower expression and red depicts higher expression. Graphs (C and D) show a subset of genes that were induced >10 fold (C) and >5 fold (D) in the WT mice treated with AOM/DSS compared to the untreated WT mice (blue bars). Black bars show the difference between STAT2^−/− (KO) and WT untreated mice. Red bars show the effects of AOM/DSS on the KO mice relative to the untreated KO mice.

Figure 4

STAT2 deletion attenuates activation of the IFN-α/β signaling pathway and secretion of IL-6 and Ccl2/MCP-1 in response to AOM/DSS. (A) Colons were harvested and whole cell extracts were prepared from AOM/DSS treated and untreated individual mice (n=3) after 24 hr incubation in medium and resolved by SDS-PAGE analysis. Cox-2, IRF-1, phospho-STAT1 Y701 were detected by Western blot analysis. Actin was used to monitor for equal loading of protein. (B) Same as in (A) except supernatants of overnight incubated colons (n=4) were collected. Detection of inflammatory cytokines was measured by multiplex mouse inflammatory flow cytometric analysis. Values are presented as mean ± SE taken from individual mice. This experiment was performed twice, 4 mice/group. *, P < 0.05. (C) Sections with adenomatous tissue of representative colons from wild type (WT, left panel) and STAT2^−/− mice (right panel) were stained for phospho-STAT3 Y705. Immunostaining was scored on a 0–3 scale (3 being the highest intensity). WT adenomas were scored 2–3 whereas STAT2^−/− were scored 1–2.

Figure 4

STAT2 deletion attenuates activation of the IFN-α/β signaling pathway and secretion of IL-6 and Ccl2/MCP-1 in response to AOM/DSS. (A) Colons were harvested and whole cell extracts were prepared from AOM/DSS treated and untreated individual mice (n=3) after 24 hr incubation in medium and resolved by SDS-PAGE analysis. Cox-2, IRF-1, phospho-STAT1 Y701 were detected by Western blot analysis. Actin was used to monitor for equal loading of protein. (B) Same as in (A) except supernatants of overnight incubated colons (n=4) were collected. Detection of inflammatory cytokines was measured by multiplex mouse inflammatory flow cytometric analysis. Values are presented as mean ± SE taken from individual mice. This experiment was performed twice, 4 mice/group. *, P < 0.05. (C) Sections with adenomatous tissue of representative colons from wild type (WT, left panel) and STAT2^−/− mice (right panel) were stained for phospho-STAT3 Y705. Immunostaining was scored on a 0–3 scale (3 being the highest intensity). WT adenomas were scored 2–3 whereas STAT2^−/− were scored 1–2.

Figure 5

STAT2 deletion attenuates DMBA/TPA induced skin carcinogenesis. Age matched wild type (n=10) and STAT2 ^−/− mice (n=10) were initiated with a single dose of DMBA (week 1) followed by tumor promoter TPA treatment for 20 weeks, twice a week. X-axis indicates number of weeks after the initial DMBA application. (A) Tumor incidence is the number of mice with palpable papillomas. (B) Number of papillomas per mouse scored weekly. Significant statistical differences were detected at week 11 in tumor incidence and from 23 and forward for papilloma multiplicity (P <0.05). Results represent mean ± SE.

Figure 6

STAT2 deficient skin carcinoma cells show reduced STAT3 activation and secrete less IL-6. (A) STAT2 expression in cell lines from STAT2^−/− and WT mice (left panel) and from STAT2^−/− cells transfected with empty vector or STAT2 expression vector (right panel) were assessed by Western blot analysis. (B) Cells were examined for phospho-STAT3-Y705. (C) Cells were treated with IL-6 (100ng/ml) and analyzed for phospho-STAT3-Y705. Actin was used to monitor for equal loading of protein. (D) IL-6 concentrations were measured in supernatants collected from skin carcinoma cell lines (WT = LD751, STAT2^−/− = AG1279 and STAT2 ^/− reconstituted with STAT2).