IL-22 is related to development of human colon cancer by activation of STAT3

Abstract

Background: It has been previously reported that IL-22, one of the cytokines secreted by Th17 cells, demonstrates both a protective and inflammatory promotion effect in inflammatory bowel disease (IBD) through STAT3 signaling activation. We sought to investigate the role of IL-22 expression in colon cancer (CC).

Methods: The expression of IL-22 and related molecules were detected in human CC, the detail function and mechanism of IL-22 were investigated by in vivo and in vitro model.

Results: Our results demonstrated significant upregulation of IL-22 in human CC tumor infiltrated leukocytes (TILs) compared to peripheral lymphocytes. Moreover, our findings demonstrated that IL-22 expression was significantly higher in ulcerative colitis (UC) tissues versus normal colon tissues. Both IL-22 receptor α1 (IL-22RA1) and IL-23 were highly expressed in CC and UC tissues compared to normal controls. TILs exhibiting various IL-22 expression levels isolated from CC patients were demonstrated to enhance tumor growth and metastasis co-transplanted with Hct-116 cells underwent subcutaneous transplantation in mice model. Tumor growth and metastasis was promoted by STAT3 phosphorylation and upregulation of its downstream genes such as Bcl-xl, CyclinD1, and VEGF. In vitro studies confirmed the anti-apoptotic and pro-proliferation effect of IL-22 according to the BrdU cooperation assay and peroxide induced apoptosis analysis with or without the presence of IL-22.

Conclusion: In this study we demonstrated that excessive IL-22 in the CC and UC microenvironment leads to tumor growth, inhibition of apoptosis, and promotion of metastasis depend on STAT3 activation.

Figure 1

Elevated expression of IL-22 was investigated in human colon cancer (CC). A: Expression of IL-22 in PBMCs (n = 40) and TILs (n = 82) from patients suffering from CC, as detected by real-time PCR (unpaired t-test). B: Average integrated optical density (IOD) was obtained by analyzing five fields for each slide evaluated by Image-Pro Plus software (version 5.0) for IHC staining of IL-22 in CC, UC and normal controls (unpaired t-test). C: Expression and distribution of IL-22 in normal colon tissues (n = 40), CC tumor tissues (indicated by arrows) (n = 82) and UC tissues (n = 40), as analyzed by IHC. D1: IL-22 expression in TILs (n = 32) and PBMCs (n = 32) by flow cytometry co-stained with antibody to CD3, D2. Comparison of IL-22 in TILs and corresponding PBMCs (paired t-test). ** represented P < 0.01 and * represented P < 0.05.

Figure 2

A representative case of IL-22 expression in TILs (n = 31) by flow cytometry co-stained with antibody to CD4, CD8, CD68, and CD3 + CD16 + CD56.

Figure 3

IL-22 related proteins are also over-expressed in human colon cancer and ulcerative colitis. A1-A3: Average integrated optical density (IOD) was obtained by analyzing five fields for each slide evaluated by Image-Pro Plus software (version 5.0) for IHC staining of IL-23, IL-22RA1, and p-STAT3 (S727) in human CC, UC, and normal colon tissues. B1-3, B4-6 and B7-B9: Expression and distribution of IL-23, IL-22RA1 and p-STAT3 (S727) in human CC (n = 84), UC (n = 40) and normal colon tissues (n = 40) respectively (Mann–Whitney U test). * represented P < 0.05 and ** represented P < 0.01.

Figure 4

IL-22 promotes tumor growth and metastasis in vivo. TILs were isolated from two CC patients and co-transplanted subcutaneously into nude mice with Hct-116 cells. Tumor growth and metastasis was investigated. A: Different IL-22 expression levels of TILs1 and TILs2 analyzed by FCMs. B1: Tumor tissues obtained from nude mice from each group. B2: Comparison of tumor volume of each group (unpaired t-test). C1: IL-22 positive cell distribution in tumor tissues of each group investigated by IHC. C2: Average integrated optical density (IOD) was obtained by analyzing five fields for each slide evaluated by Image-Pro Plus software (version 5.0) for IHC staining of IL-22. D: Western-blot detection of expression of p-STAT3 (S727), total STAT3, Bcl-xl, CyclinD1, and VEGF, all normalized to β-actin for every 3 samples in each group. E: IHC staining of CEA in lymph nodes around the tumor beneath the skin of nude mice. E1 (×40), E2, E3 (×100) and E4 (×200), ** represented P < 0.01.

Figure 5

IL-22 enhances tumor proliferation and anti-apoptosis ability by activating STAT3 signaling in vitro. A: Schematic diagram of experimental design. B1-B6: Immunofluorescence staining of BrdU in green and DAPI in blue, reflecting cell proliferation in each group detected by fluorescence microscopy (×200). B7: Immunofluorescense staining with non-specific antibody control (NC) (×200). B8: Comparison of cell proliferation in each group (the independent co-cultures experiments were repeated in triplicate) (unpaired t-test). C1: Analysis of apoptosis of Hct-116 cells induced by peroxide in each group by flow cytometry, C2: Comparison of apoptosis in each group (the independent co-cultures experiments were repeated in triplicate) (unpaired t-test). D. Western-blot detection of IL-22, p-STAT3 (S727), total STAT3, Bcl-XL, Bcl-2, and CyclinD1 expression, all normalized to the β-actin. *p <0.05; **p < 0.01.