The Role And Mechanism Of IL-17A In Colitis Associated Colorectal Cancer

Human colorectal cancer is an important contributor to cancer mortality and morbidity in developed countries, which remains the third most common malignancy and third leading cause of cancer-related deaths in the United States. The American Cancer Society estimates that there were approximately 136 830 new cases and 50 310 deaths due to colorectal cancer in American men and women in 2014. In China, colorectal cancer incidence and mortality ranked the fourth and sixth in urban and rural areas, respectively. In recent years, the colorectal cancer incidence in China has increased. There are several factors that have been associated with the increased risk of developing colorectal cancer, such as old age, polyps in the colorectum, ulcerative colitis and Crohn’s disease, a family history of colorectal cancer, ethnicity, type 2 diabetes, and certain family syndromes, such as familial adenomatous polyposis(FAP) or hereditary nonpolyposis colon cancer(HNPCC). Some lifestyle-related factors have also been linked to a higher risk of colorectal cancer, such as diet with red meats(beef, lamb, or liver) and processed meats(like hot dogs and bologna), cooking meats at very high heat(frying, broiling, or grilling), lack of exercise, overweight and obesity, smoking, and heavy alcohol use. Inflammatory bowel diseases, including ulcerative colitis and Crohn’s disease, are well-known for their risk to develop colorectal cancer. IBD is formed due to various inflammatory cells and cytokines in tumor microenvironment, chronic intestinal infection, defects of mucosa antigen barrier and abnormal immune reaction caused by antigens in inner lumen. It is generally accepted that CRC is linked to chronic inflammation. However, the interaction between immune cells, inflammatory cytokines, and cancer evolution is still largely unknown.Interleukin-17(IL-17 or IL-17A) is a proinflammatory cytokine, which is the founding member of IL-17 cytokine family consisting of six homologous proteins(from IL-17 A to IL-17F). It binds to a heterodimer of IL-17 receptor A(IL-17RA) and IL-17 receptor C(IL-17RC) complex, and subsequently activates nuclear factor-κB(NF-κB) activator 1(Act1) through a similar expression to fibroblast growth factor genes, IL-17 receptors, and the Toll-IL-1R(SEFIR) domain. Act1 is an E3 ubiquitin ligase, which activates tumor necrosis factor(TNF) receptor-associated factor 6(TRAF6) through lysine-63-linked ubiquitination. The polyubiquitinated TRAF6 activates transforming growth factor-β-activated kinase(TAK1) and then IκB kinase(IKK) complex, which subsequently activates the NF-κB pathway to initiate transcription of a variety of chemokines, cytokines, and growth factor, such as C-X-C motif ligand 1(CXCL1), IL-6, and vascular endothelial growth factor(VEGF). Some previous studies have demonstrated that IL-17 can stabilize downstream CXCL1 messenger RNA through an inducible kinase IKKi-dependent Act1–TRAF2–TRAF5 complex, which binds to splicing factor 2(SF2) and prevents SF2/ASF-mediated mRNA degradation. IL-17 also activates mitogen-activated protein kinases(MAPK), including the extracellular signal-regulated kinase(ERK) 1/2, p38, and c-Jun N-terminal kinase(JNK1/2/3) pathways. ERK1/2 and p38 may act through increasing target mRNA stability. The JNK pathway acts through activator protein-1(AP-1) transcription factor. However, the role of IL-17 A in tumorigenesis remains debated. Several studies have shown that IL-17 A has either a protumor or antitumor role in different cancer models. But in CRC, the majority of studies consider that IL-17 A acts as a promoter in tumor initiation and progression.In the present study, we firstly assessed IL-17A-driven inflammatory responses in human colon carcinogenesis, then investigated the effects and mechanisms of IL-17 A on the expression of MMPS and VEGF in colitis-associated cancer model mouse in vivo, lastly detected the effects of IL-17 A on HCT116 cell line in vitro to assess IL-17 A and IL-17 RA levels and to assess the biological changes that IL-17 A signaling may have caused in colorectum adenocarcinoma. Part one Human colon carcinogenesis is associated with increased interleukin-17-driven inflammatory responsesObjective: To explore the effects of interleukin-17-driven inflammatory responses on human colon carcinogenesis.Methods: IL-17-driven inflammatory responses in 17 cases of human colon adenocarcinomas, 16 cases of human normal colon tissues adjacent to the resected colon adenocarcinomas, ten cases of human ulcerative colitis tissues from biopsies, and eight cases of human colon polyps diagnosed as benign adenomas were assessed. The IL-17 A protein levels in human colon tissues were measured using an ELISA kit, the IL-17, IL-17 RA, VEGF, MMP2, MMP9, ERK, p-ERK, JNK, p-JNK, CyclinD1, Bax and Bcl-2 proteins were measured using western blot. Immunohistochemistry was performed using antibodies against MMP9, VEGF, and CD34(to identify microvessels).Result: Human colon adenocarcinomas contained the highest levels of IL-17 A cytokine, which was significantly higher than the IL-17 A levels in the adenomas, ulcerative colitis, and normal colon tissues(P<0.01). The levels of IL-17 receptor A(IL-17RA) were also the highest in human colon adenocarcinomas, followed by adenomas and ulcerative colitis. The increased levels of IL-17 A and IL-17 RA were accompanied with increased IL-17-driven inflammatory responses, including activation of extracellular signal-regulated kinase(ERK)1/2 and c-Jun N-terminal kinase(JNK) pathways, increase in expression of matrix metalloproteinase9(MMP9), MMP7, MMP2, B-cell lymphoma(Bcl-2), and cyclin D1, decrease in Bcl-2-associated X protein(Bax) expression, and increase in vascular endothelial growth factor(VEGF) and VEGF receptor(VEGFR) expression that were associated with increased angiogenesis. Part two Effects of IL-17 A on the capacity of metastasis and invasion of HCT116 cellObjective: To explore the effects of IL-17 A on the capacity of invasion and metastasis of colorectal cancer cells.Methods: HCT116 cells in vitro culture were treated differently(blank control group, IL-17 A treatment group, PD98059+IL-17 A group and SP600125+IL-17 A group) and their cell metastasis and invasion capacity were detected by scratch and Transwell experiments respectively. IL-17 RA, ERK, p-ERK, JNK, p-JNK, MMP2 and MMP7, MMP9, CyclinD1 and Bcl-2, Bax, VEGF and VEGFR proteins were tested by Western blot.Results: Trypan blue staining and cell blue TM test showed that HCT116 cell proliferation activity in IL-17 A treatment group was higher than that of the control group, while HCT116 cell proliferation activity in PD98059 and SP600125 pretreatment groups significantly decreased. Transwell membranepermeating cell number in the control group, IL-17 A treatment group, PD98059 and SP600125 pretreatment groups were 9.5±1.06, 15.4±1.11, 3.7±0.53 and 4.3±0.55 respectively. There was no statistically significant difference between the last two groups, while there was statistically significant difference between the rest groups. In IL-17 A treatment group, HCT116 cell had the strongest metastasis capacity and narrower scratches, while in PD98059 and SP600125 pretreatment groups, HCT116 cells decreased its metastasis ability and no obvious scratch distance change. IL-17 A up-regulated the proteins expression of IL-17 RA, MMP2, MMP7, MMP9, CyclinD1, Bcl-2, VEGF and VEGFR, and increased the phosphorylation of ERK1/2, JNK in HCT116 cells, while PD98059 and SP600125 could inhibit its effects. Part three Therapeutical Effect of IL-17 A siRNA on colitis-associated colorectal cancer model mouseObjective: To explore the therapeutical effect of IL-17 A siRNA on colorectal cancer model mouse.Methods: The ulcerative colitis-associated colorectal cancer model using 1, 2-dimethylhydrazine and dextran sulfate sodium, was treated by IL-17 A siRNA interference after successful modeling. As to the control group, model group and IL-17 A siRNA treatment group, Real-time PCR was performed to detect their colon tissue Il-17 a and Il-17 ra mRNA level and western blot was carried to detect the proteins expression of IL-17 A, IL-17 RA, ERK, p-ERK, JNK, p-JNK, MMP2 and MMP7, MMP9, CyclinD1 and Bcl-2, Bax, VEGF and VEGFR.Results: Mouse weight in IL-17 A siRNA treatment group was higher than that in model group, while the mucosal epithelial damage and glandular structure in IL-17 A siRNA treatment group was lower than that in model group; Il-17 a and Il-17 ra gene and protein were the highest in the model group, followed by IL-17 A siRNA treatment group and the control group lowest(P<0.05); p-ERK1/2, p-JNK proteins in IL-17 A siRNA treatment group was lower than that in model group; The proteins expression of MMP2 and MMP7, MMP9, Bcl-2, CyclinD1, VEGF and VEGFR in colon tissue decreased in sequence in model group, IL-17 A siRNA treatment group and control group, while Bax was the highest in control group, lowest in model group.Conclusions:1 IL-17 A is increased in colorectal cancer tissues.2 IL-17 A might regulate downstream signal protein expression by activating ERK1/2 and JNK signaling pathways and play a role in the initiation and development of colorectal cancer.3 IL-17 A siRNA therapy can inhibit colorectal cancer progress, and may become the new target for prevention and treatment of colorectal cancer.