Effects Of Corticotropin-Releasing Hormone In The Development Of Colon Cancer And The Molecular Mechanisms

Colon cancer is one of the most common malignancies worldwide,with high incidence and lethality.Inflammatory diseases such as inflammatory bowel disease(IBD)and ulcerative colitis(UC)are associated with high risk for colon cancer.corticotropin-releasing hormone receptor 1(CRHR1)and its ligands,corticotropin-releasing hormone(CRH)and urocortin(Ucnl),are well-characterized with an established role in the regulation of intestinal inflammation.However,their roles in colon cancer development remain unclear.CRH is a 41-aminoacid peptide.It belongs to the CRH family peptides,which include CRH,Ucnl,Ucn2,and Ucn3.They act through 2 kinds of G-protein-coupled receptors,CRHR1 and CRHR2.CRH preferentially binds to CRHR1,the affinity of CRH to CRHR1 is about 20-fold of that to CRHR2,whereas Ucnl has equal affinity for both receptors,Ucn2 and Ucn3 are specific ligands of CRHR2.Although CRH family members are best known as neuroendocrine regulators of the stress response in the central nervous system,they are widely expressed in peripheral tissues and act as inflammation regulators in several diseases.Recently,they have also been found to be involved in tumorigenesis.The reported roles of CRH and CRHR1 in cancer development are diverse.It has been reported that CRH and CRHR1 were observed in UC patients and acted as pro-inflammatory factors.CRHR1 deficiency relieved the colitis induced by dextran sodium sulfate(DSS)in mice.These studies suggest CRH/CRHR1 system has a pro-inflammatory action in intestinal inflammation.CRH/CRHR1 signaling also plays a role in cardiovascular system,regulating vascular permeability,inflammation and angiogenesis.Given the pro-inflammatory action of CRH/CRHR1 system in colitis,we hypothesized that CRH/CRHR1 system is involved in colon cancer development.Part I The role of CRHR1 in the development of colitis-associated cancer in mouse modelAim:We determined the role of CRHR1 in the development of colitis-associated cancer(CAC)in miceMethods:CRHRl-/-mice were generated by gene knockout technology.Azoxymethane(AOM)and DSS were used to induce chronic CAC in CRHRl-/-and CRHR1+/+ mice.The expressions of CRHR1,Ucnl and CRH were detected by immunohistochemistry(IHC),qRT-PCR and Western blot.Mortality and body weight changes were monitored throughout the study duration and colonic tumor burden was determined at day 80.Besides,the ulceration,inflammation,and hyperplasia of the mucosa in colon tissues were analyzed by histological examination.At last,TUNEL and BRDU staining were used to determine the colonic epithelial cells apoptosis and proliferation in CAC mice.In addition,western blot analysis was used to detect the expressions and phosphorylation of nuclear factor kappa B(NF-κB),signal transducers and activators of transcription(STAT3)and apoptosis-associated signaling proteins.Results:CAC model in mouse was successfully induced by AOM and DSS.The expressions of CRHRl,Ucnl and CRH were obviously higher in the colon of CAC mice than the controls.Compared with CRHR1-/-CAC mice,CRHR1+/+ CAC mice displayed increased mortality,weight loss and tumorigenesis.The colon samples in 70%of CRHR1+/+ mice developed high-grade dysplasia,of which 30%were identified as adenocarcinoma with invasive growth.Whereas only 40%of CRHR1-/-mice displayed high-grade dysplasia and no adenocarcinoma was observed.Moreover,immunohistochemistry results showed that samples from CRHR1+/+ mice colon exhibited more TUNEL-staining cells and less BRDU-positive cells than CRHR1-/-mice.Western blot analysis showed that significantly higher phosphorylation of p65 and STAT3 was observed in the colon tissues of CRHR1+/+mice than of CRHRl-/－ mice.In addition,the colon tissues of CRHRl+/+ mice had a higher expression of anti-apoptosis protein Bcl-2 and a lower expression of the pro-apoptosis protein Bax than CRHR1-/-mice.Conclusion:CRHR1 mediated the development of CAC by promoting cell growth and inhibiting cell apoptosis,and the action of CRHR1 in CAC development may be associated with NF-κB and STAT3 signaling.Part Ⅱ Effect and mechanisms of CRH/CRHR1 signaling in colon cancer cell proliferationAim:We determined the regulatory action and mechanism of CRH/CRHR1 signaling in colon cancer cell proliferation.Methods:IHC,qRT-PCR and Western blot were used to detect the expressions of CRH,Ucnl and CRHR1 in colon tissues of colorectal patients,colon cancer cell lines and colonic epithelial cell line.DLD1 and HCT116 cells were used as in vitro model of colon cancer cells.Cell proliferation and colony formation assays were performed to determine the effect of CRH/CRHR1 signaling on colon cancer cell proliferation.The effects of CRH on cell cycle progression and apoptosis of colon cancer cells were detected by flow cytometry(FCM).Western blot and qRT-PCR were performed to detect the effect of CRH on the expression of interleukin-6(IL-6).We further determined the effect of CRH on the phosphorylation of NF-κB,JAK2 and STAT3.The NF-κB inhibitor CAPE and STAT3 inhibitor Stattic were used to detect the roles of NF-κB and STAT3.Results:We found high expressions of CRH and CRHR1 in tumor tissues and colon cancer cell lines DLD1 and HCT116 cells.Cell proliferation and colony formation assays revealed that CRH promoted cell growth through CRHR1,Further investigation showed that CRH significantly up-regulated the expressions of IL-6 in a CRHR1-dependent manner.Mechanistically,CRH induced phosphorylation of NF-κB,JAK2 and STAT3.STAT3 inhibition by Stattic significantly attenuated the CRH-induced cell growth.Knockdown of IL-6 by RNA interference or neutralization by anti-IL-6 antibody displayed strong suppression of CRH-induced phosphorylation of JAK2 and STAT3.Conclusion:CRH/CRHR1 signaling promoted human colon cancer cell proliferation via IL-6/JAK2/STAT3 signaling pathway.Part Ⅲ Effect and mechanisms of CRH/CRHR1 signaling on angiogenesis of HUVECsAim:We determined the effect of CRH on tumor angiogenesis in vitro.Methods:ELISA and qRT-PCR were performed to detect the effect of CRH on expression of vascular endothelial growth factor(VEGF)in colon cancer cell line DLD1 and HCT116 cells.And CRHR1 antagonist Antalarmin and NF-κB inhibitor CAPE were used to determine the roles of CRHR1 and NF-κB.Tube formation of HUVECs on metrigel was used as the angiogenesis model in vitro.The conditioned medium of DLD1 cells was used to induce angiogenesis of HUVECs,which was detected by tube formation assay.Expression of VEGF was interrupted by siRNA.Results:ELISA and qRT-PCR showed that CRH significantly increased VEGF expression in DLD1 and HCT116 cells.And the CRH-induced VEGF was attenuated by Antalarmin and CAPE.Tube formation assay showed that CRH-treated DLD1 cells supernatant obviously promoted angiogenesis of HUVECs compared with control groups.Silence of VEGF by siRNA suppressed the CRH-induced angiogenesis of HUVECs.Conclusion:CRH enhanced the expression of VEGF through NF-κB signaling by CRHR1 in colon cancer cells,and the CRH-induced VEGF promoted angiogenesis of HUVECs.