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 ?1 (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.
Project description:Colorectal cancers (CRCs) often show a dense infiltrate of cytokine-producing immune/inflammatory cells. The exact contribution of each immune cell subset and cytokine in the activation of the intracellular pathways sustaining CRC cell growth is not understood. Herein, we isolate tumor-infiltrating leukocytes (TILs) and lamina propria mononuclear cells (LPMCs) from the tumor area and the macroscopically unaffected, adjacent, colonic mucosa of patients who underwent resection for sporadic CRC and show that the culture supernatants of TILs, but not of LPMCs, potently enhance the growth of human CRC cell lines through the activation of the oncogenic transcription factors signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-kB). Characterization of immune cell complexity of TILs and LPMCs reveals no differences in the percentages of T cells, natural killer T cells, natural killer (NK) cells, macrophages and B cells. However, T cells from TILs show a functional switch compared with those from LPMCs to produce large amounts of T helper type 17 (Th17)-related cytokines (that is, interleukin-17A (IL-17A), IL-17F, IL-21 and IL-22), tumor necrosis factor-? (TNF-?) and IL-6. Individual neutralization of IL-17A, IL-17F, IL-21, IL-22, TNF-? or IL-6 does not change TIL-derived supernatant-driven STAT3 and NF-kB activation, as well as their proproliferative effect in CRC cells. In contrast, simultaneous neutralization of both IL-17A and TNF-?, which abrogates NF-kB signaling, and IL-22 and IL-6, which abrogates STAT3 signaling, reduces the mitogenic effect of supernatants in CRC cells. IL-17A, IL-21, IL-22, TNF-? and IL-6 are also produced in excess in the early colonic lesions in a mouse model of sporadic CRC, associated with enhanced STAT3/NF-kB activation. Mice therapeutically given BP-1-102, an orally bioavailable compound targeting STAT3/NF-kB activation and cross-talk, exhibit reduced colon tumorigenesis and diminished expression of STAT3/NF-kB-activating cytokines in the neoplastic areas. These data suggest that strategies aimed at the cotargeting of STAT3/NF-kB activation and interaction between them might represent an attractive and novel approach to combat CRC.
Project description:The tumor-derived factors involved in the expansion and accumulation of myeloid-derived suppressor cells (MDSCs) in metastatic dissemination of colorectal cancer (CRC) to the liver has not been studied. Immunohistochemistry was used to detect sphingosine-1-phosphate receptor 1 (S1PR1) and signal transducer and activator of transcription-3 (STAT3) in human colorectal tumors. IL-6 and interferon-γ were detected by enzyme-linked immunosorbent assay (ELISA). Tumor growth, invasion, and migration were evaluated by MTT, transwell, and wound healing assays, respectively. Subcutaneous tumor-bearing and CRC liver metastasis (CRLM) nude mouse models were constructed. The percentage of MDSCs was measured using multicolor flow cytometry. Western blot assay was used to evaluate S1PR1 and p-STAT3 expression in MDSCs after separation from the liver and tumor by magnetic antibody. T-cell suppression assay was detected by carboxyfluorescein succinimidyl ester (CFSE). Aberrant co-expressed S1PR1 and p-STAT3 was correlated with metachronous liver metastasis and poor prognosis in CRC. A mutual activation loop between S1PR1 and STAT3 can enhance CRC cell proliferation, migration, and invasion in vitro and in vivo. The expression of p-STAT3 and its downstream proteins can be regulated by S1PR1. p-STAT3 was the dependent signaling pathway of S1PR1 in the promotion of cell growth and liver metastasis in CRC. The level of IL-6 and the associated MDSCs stimulated by the S1PR1-STAT3 correlated with the number of liver metastatic nodes in the CRLM mouse models and patients. Increased CD14+HLA-DR-/low MDSCs from CRLM patients inhibited autologous T-cell proliferation and predict poor prognosis. The S1PR1-STAT3-IL-6-MDSCs axis operates in both tumor cells and MDSCs involved in the promotion of growth and liver metastasis in CRC. MDSCs induced by S1PR1-STAT3 in CRC cells formed the premetastatic niche in the liver can promote organ-specific metastasis.
Project description:Expression of IL-22 is induced in several human inflammatory conditions, including inflammatory bowel disease (IBD). Expression of the IL-22 receptor is restricted to innate immune cells; however, the role of IL-22 in colitis has not yet been defined. We developed what we believe to be a novel microinjection-based local gene-delivery system that is capable of targeting the inflamed intestine. Using this approach, we demonstrated a therapeutic potency for IL-22-mediated activation of the innate immune pathway in a mouse model of Th2-mediated colitis that induces disease with characteristics similar to that of IBD ulcerative colitis (UC). IL-22 gene delivery enhanced STAT3 activation specifically within colonic epithelial cells and induced both STAT3-dependent expression of mucus-associated molecules and restitution of mucus-producing goblet cells. Importantly, IL-22 gene delivery led to rapid amelioration of local intestinal inflammation. The amelioration of disease by IL-22 was mediated by enhanced mucus production. In addition, local gene delivery was used to inhibit IL-22 activity through overexpression of IL-22-binding protein. Treatment with IL-22-binding protein suppressed goblet cell restitution during the recovery phase of a dextran sulfate sodium-induced model of acute colitis. These data demonstrate what we believe to be a novel function for IL-22 in the intestine and suggest the potency of a local IL-22 gene-delivery system for treating UC.
Project description:Mesenchymal stem cells (MSCs) have been reported to localize in colorectal carcinomas, and participate in the formation of the tumor microenvironment. They have recently been isolated from colorectal cancer tissues, and are implicated in the growth, invasion, and metastasis of cancer cells. However, the roles and detailed mechanisms associated with human colorectal cancer-derived MSCs (CC-MSCs) have not been fully addressed. In this study, we found that CC-MSCs increased the migration and invasion of colorectal cancer cells and promoted the tumorigenesis of colorectal cancer through epithelial-to-mesenchymal transition (EMT) in vitro. We also found that CC-MSCs enhanced the growth and metastasis of colorectal cancer in vivo. Mechanistically, we determined that interleukin-6 (IL-6) was the most highly expressed cytokine in the CC-MSC conditioned medium, and promoted the progression of colorectal cancer cells through IL-6/JAK2/STAT3 signaling, which activated PI3K/AKT signaling. We used anti-IL-6 antibody to target IL-6. Collectively, these results reveal that the IL-6 secreted by CC-MSCs enhances the progression of colorectal cancer cells through IL-6/JAK2/STAT3 signaling, and could provide a novel therapeutic or preventive target.
Project description:Tumor-infiltrating cytotoxic T lymphocytes (TILs), including CD8 TILs, have been associated with favorable clinical outcomes in multiple tumor types. Tumor-infiltrating CD8 T cells and major histocompatibility complex (MHC) class I expression in urothelial carcinoma (UC) have not been previously reported. Most immune responses are mediated by local cytotoxic lymphocytes (CD8 T cells), which can eradicate tumor cells by recognizing tumor-associated antigens presented by MHC class I molecules. Here we analyzed the presence of intratumoral CD8 T cells, the expression of MHC class I antigen, and the expression of the NY-ESO-1 tumor antigen in UC samples and correlated our findings with clinical outcome. Immunohistochemical staining for intratumoral CD8 T cells in tissue samples from 69 patients with UC showed that patients with advanced UC (pT2, pT3, or pT4) and higher numbers of CD8 TILs within the tumor (> or =8) had better disease-free survival (P < 0.001) and overall survival (P = 0.018) than did patients with similar-staged UC and fewer intratumoral CD8 TILs. We conclude that the extent of intratumoral CD8 TILs is an important prognostic indicator in advanced UC.
Project description:Inflammatory conditions elicited by extrinsic environmental factors promote malignant cell transformation, tumor growth, and metastasis. Although most attention has been focused on innate immune mechanisms of inflammatory carcinogenesis, more recently the role of T cells in cancer promotion has been examined. Although IFN-dependent Th1 responses that promote Stat1 signaling inhibit tumor growth, the role of T helper type 17 responses, and interleukin-17 (IL-17) in particular, has been controversial. Indeed, IL-17 has been reported to either enhance or inhibit the growth of transplantable tumors, depending on the system. Little is known about the role of IL-17 in de novo carcinogenesis. Using IL-17 knockout mice, we examined the role of IL-17 in the classic DMBA/TPA-induced skin carcinogenesis model. Disruption of IL-17 dramatically reduced tumorigenesis in this model in a manner correlated with diminished Stat3 activation in the tumor microenvironment. IL-17 loss reduced Stat3-associated proliferative and antiapoptotic gene expression along with epidermal cell proliferation and hyperplasia. In addition, IL-17 loss was associated with reduced expression of Stat3-regulated chemokines that attract myeloid cells and a decreased infiltration of myeloid cells into the local tumor microenvironment. Together, our findings point to a critical role of the IL-17-Stat3 pathway in supporting cancer-associated inflammation in the tumor microenvironment. Therapeutic approaches that target this pathway may therefore be effective to inhibit carcinogenesis.
Project description:Ulcerative colitis (UC) is a chronic condition in which the overreacting immune system may play an important role. It has been confirmed that the interleukin (IL) 9 (IL-9) participates in the pathogenesis of UC but the molecular mechanism is not fully illustrated. Here, we show that levels of peripheral blood cytokines IL-9, IL-8, IL-10, IL-6, IL-1β, IL-12, and tumor necrosis factor (TNF) were higher in patients with UC than normal control, and serum and local IL-9 levels were positively correlated with the disease activity grade. Moreover, IL-9 stimulation inhibited suppressor of cytokine signaling 3 (SOCS3) expression and wound healing ability in colonic epithelial cells and promoted the phosphorylation level of signal transducers and activators of transcription 3 (STAT3). And IL-9 stimulation promoted claudin-2 expression while inhibited claudin-3 and occludin expression. Furthermore, SOCS3 overexpression rescued the IL-9-induced effects. Altogether, IL-9 participates in the pathogenesis of UC through STAT3/SOCS3 signaling pathway and has the potential to serve as a possible therapeutic candidate in patients with UC.
Project description:Defects in T-cell function in patients with cancer might influence their capacity to mount efficient antitumor immune responses. Here, we identified highly reduced IL-4-, IL-10-, and IL-21-induced phosphorylation of STAT6 and STAT3 in tumor-infiltrating T cells (TILs) in follicular lymphoma (FL) tumors, contrasting other non-Hodgkin lymphoma TILs. By combining phospho-protein-specific flow cytometry with several T-cell markers, we identified that CD4(+)CD45RO(+)CD62L(-) FL TILs were largely nonresponsive to cytokines, in contrast to the corresponding autologous peripheral blood subset. We observed differential expression of the inhibitory receptor PD-1 in FL TILs and peripheral blood T cells. Furthermore, CD4(+)PD-1(hi) FL TILs, containing T(FH) and non-T(FH) cells, had lost their cytokine responsiveness, whereas PD-1 TILs had normal cytokine signaling. However, this phenomenon was not tumor specific, because tonsil T cells were similar to FL TILs. FL tumor cells were negative for PD-1 ligands, but PD-L1(+) histiocytes were found within the T cell-rich zone of the neoplastic follicles. Disruption of the microenvironment and in vitro culture of FL TILs could restore cytokine signaling in the PD-1(hi) subset. Because FL TILs in vivo probably receive suppressive signals through PD-1, this provides a rationale for testing PD-1 Ab in combination with immunotherapy in patients with FL.
Project description:Interleukin (IL)-22 is a member of the IL-10 family. Its main targets are epithelial cells, not immune cells. We examined IL-22 signal transduction in oral squamous cell carcinoma (OSCC) cells. Immunohistochemical staining revealed that IL-22R was expressed more highly in OSCC compared to normal regions. An IL-22R signal was also observed in metastatic OSCC cells in the lymph node. RT-PCR showed that the human OSCC cell lines MISK81-5, HSC-3, HSC-4, SAS and SQUU-B expressed IL-22 receptor chains. Immunoblotting showed that IL-22 induced a transient tyrosine phosphorylation of STAT3 (pY705-STAT3) in MISK81-5 cells. The change in the serine phosphorylation of STAT3 was subtle during the examination periods. Simultaneously, pY705-STAT3 activation in HSC-3 cells was undetectable after IL-22 stimulation. Immunocytochemistry demonstrated that IL-22 induced the translocation of phosphorylated STAT3 into the nucleus of MISK81-5 cells. IL-22 temporarily upregulated the expression of anti-apoptotic and mitogenic genes such as Bcl-x, survivin and c-Myc, as well as SOCS3. IL-22 transiently activated ERK1/2 and induced a delayed phosphorylation of p38 MAP kinase, but negligibly involved the activation of NF-?B in MISK81-5 cells. MISK81-5 and SQUU-B cells treated with IL-22 showed mild cellular proliferation. MISK81-5, HSC-4 and SAS cells treated with IL-22 downregulated the keratinocyte differentiation-related genes compared with unstimulated cells. Conversely, STAT3 suppression by STAT3 siRNA strongly disrupted the downregulation of these genes by IL-22, but it did not significantly affect the activation of ERK1/2 by IL-22. The OSCC cells used in this study upregulated the expression of SERPINB3/4 (SCCA1/2), well-known SCC markers, following treatment with IL-22. These results indicate that IL-22 differentially activates the STAT3 signaling system depending on the type of OSCC. IL-22 may therefore play a role in tumor growth, cell differentiation and progression through STAT3-dependent and -independent pathways.
Project description:The pathophysiology of inflammatory bowel disease (IBD) reflects a balance between mucosal injury and reparative mechanisms. Some regenerating gene (Reg) family members have been reported to be expressed in Crohn's disease (CD) and ulcerative colitis (UC) and to be involved as proliferative mucosal factors in IBD. However, expression of all REG family genes in IBD is still unclear. Here, we analyzed expression of all REG family genes (REG I?, REG I?, REG III, HIP/PAP, and REG IV) in biopsy specimens of UC and CD by real-time RT-PCR. REG I?, REG I?, and REG IV genes were overexpressed in CD samples. REG IV gene was also overexpressed in UC samples. We further analyzed the expression mechanisms of REG I?, REG I?, and REG IV genes in human colon cells. The expression of REG I? was significantly induced by IL-6 or IL-22, and REG I? was induced by IL-22. Deletion analyses revealed that three regions (- 220 to - 211, - 179 to - 156, and - 146 to - 130) in REG I? and the region (- 274 to- 260) in REG I? promoter were responsible for the activation by IL-22/IL-6. The promoters contain consensus transcription factor binding sequences for MZF1, RTEF1/TEAD4, and STAT3 in REG I?, and HLTF/FOXN2F in REG I?, respectively. The introduction of siRNAs for MZF1, RTEF1/TEAD4, STAT3, and HLTF/FOXN2F abolished the transcription of REG I? and REG I?. The gene activation mechanisms of REG I?/REG I? may play a role in colon mucosal regeneration in IBD.