Blocking NFATc3 Ameliorates Colitis-Associated Colorectal Cancer In Mice

Ulcerative colitis(UC)is an idiopathic chronic and non-specific inflammatory bowel disease characterized by ulcer and erosion of the rectum and colon.UC is one of the three highest risk factors for developing colorectal cancer(CRC).It is considered as one of the most serious complications of UC.Ulcerative colitis-associated colorectal cancer(UCCRC),a type of colitis-associated CRC(CAC),may have distinct different pathogenesis to sporadic CRC.Nuclear factor of activated T cells(NFAT)is a transcription factor,which expressed in a variety of cell and tissue types.NFATc3 is closely related to the development of inflammation and cancer.It has been proven that NFATc3 can promote proliferation and invasion ability in CRC cell lines.These suggest that NFATc3 could promote inflammation in some diseases,and promote CRC.However,whether NFATc3 is involved in the development of UC-CRC remains unknown.Objective: Administration of Azoxymethane(AOM)/ Dextran sulfate sodium(DSS)to mice can cause inflammation and ulceration of colon,which can simulate the development of UC inhuman patients.In our study,we investigated whether NFATc3 affected the development of UC-CRC in AOM/DSS-induced C57BL/6 mouse model,and elucidated the related mechanisms.Methods: UC-CRC mouse model was induced using azoxymethane and dextran sulfate sodium.Six-to eight-week-old male C57BL/6 mice,weighting 15–20 g,were used for the experiments.Mice were fed with a regular basal diet and tap water and kept in a regulated environment.A total of 120 mice were acclimated for one week and then randomly divided into four groups(Control,UC-CRC,Ad-sh-control,and Ad-sh-NFATc3).In UC-CRC group,the mice were intraperitoneally injected with 10 mg/kg body weight of AOM.Seven days after AOM administration,3% DSS was administered through the drinking water for7 consecutive days.Then,untreated water was given for 14 days.DSS induction procedure was repeated twice.In Ad-sh-control group,and Ad-sh-NFATc3 group,the above steps were performed to induce UC-CRC model.During induction,two weeks after AOM administration,mice were injected with control interfering adenovirus(Ad-sh-control)and NFATc3 interfering adenovirus(Ad-sh-NFATc3)by tail intravenous injection respectively,twice a week until the end.Mice of control group were treated with physiological saline instead of AOM and DSS,and drank untreated water(Control).During the course of the experiment,the mice were weighed once a week,and the survival of these mice was recorded weekly.Six survival mice in every group were selected randomly and sacrificed at week 14.The colon tissues were removed and collected.Detected the expression of Ecadherin and PCNA by immunohistochemistry.The RNA levels of NFATc3 were determined by real-time PCR.Detected the expression of NFATc3,PCNA,E-cadherin,Ncadherin,Vimentin,P38,p-P38,JNK1/2,p-JNK1/2,and GAPDH by Western blot analysis.Results: We found that NFATc3 expression was significantly up-regulated in AOM/DSS treated mice compared with control.Mice lacking NFATc3 showed decreased tumor number and size,decreased mucosal damage,and increased survival rate.Histologically,normal crypt structure and no infiltration of inflammatory cells were observed in colon tissues of control group.AOM/DSS treatment resulted in crypt branching,crypt distortion and infiltration of inflammatory cells.Ad-sh-NFATc3 treatment could partially restore the structure of the crypt and reduce infiltration of inflammatory cells.These indicated that silencing NFATc3 could inhibit the development of UC-CRC in vivo.We detected expression levels of PCNA and E-cadherin in colon tissues using immunohistochemistry.The expression of E-cadherin was dramatically down-regulated,and the expression of PCNA was markedly up-regulated in UC-CRC and Ad-sh-control rats.In contrast,E-cadherin expression was increased by down-regulation of NFATc3,and PCNA expression was decreased by down-regulation of NFATc3 in colon tissues.Besides,using Western blot analysis,decreased levels of E-cadherin and increased levels of Ncadherin and vimentin were observed in colon tissues of UC-CRC and Ad-sh-control group.Inhibition of NFATc3 expression resulted in increased levels of E-cadherin,and decreased levels of N-cadherin and vimentin.Accordingly,we examined the phosphorylation levels of P38 and JNK.AOM/DSS treatment promoted the activation of P38 and JNK,while knockdown of NFATc3 attenuated this activation.Conclusions: Silencing NFATc3 could inhibit the development of UC-CRC,and prevent UC-CRC progression by inhibiting P38/JNK signaling pathway.