The Role Of FOXP3 On Self-renewal Of Colorectal Cancer Stem Cell And Its Mechanism

Background Colorectal cancer is one of the most common cancers in the world.Despite the conventional treatment,most of patients with colorectal cancer will show tumor metastasis and recurrence.Although the reason for this is not yet been fully elucidated,the presence of a small number of cancer stem cells(CSCs)with self-renewal,infinite differentiation,and chemotherapy resistance in the tumor is thought to play a major role in tumor recurrence and metastasis.In recent years,cancer stem cell theory explaining the recurrence and metastasis of the tumor has been widely recognized by scientists.Therefore,it is need for development of novel treatment strategies for recurrent colorectal cancer by targeting CSCs.Cyclooxygenase-2(COX2)is one of the cyclooxygenase(COX)isoenzymes,which is required for the metabolic conversion of arachidonic acid to prostaglandins,including PGE2.Studies have shown that COX2 and PGE2 are critical in promoting stem cell self-renewal.In recent years,COX2 was been identified in a wide variety of tumors,including colorectal cancer,COX2 specific inhibitors can reduce the formation efficiency of Colonosphere.However,the mechanism of COX2 abnormal expression in colorectal cancer stem cells is unclear.Therefore,discovery of molecules that can inhibit COX2 will better advance our understanding of colorectal cancer stem cells.FOXP3(Forkhead box protein 3)is one of the members of the forkhead transcription factor family,which is widely known for its role in the development and function of Tregs(Regulatory cells).However,in recent years,FOXP3 was been identified in many tumors,including colorectal cancer and a tumor suppressor role in breast and prostate cancer.In our previous study,we found that FOXP3 could inhibit the expression of COX2 by interacting with NF-κB and play an inhibitory effect on gastric cancer metastasis.This suggests that FOXP3 is a tumor suppressor gene that inhibits tumors by interacting with NF-κB.However,the role of FOXP3 in cancer stem cells formation and self-renewal is unclear.Aims This study focused on the effects of FOXP3 on colorectal cancer stem cells and the underlying molecular mechanism.Methods 1.The Colonospheres were cultivated with several different human colorectal cancer cell lines.In order to analyze the differences in the expression levels of FOXP3 and COX2 between colorectal cancer cells and colorectal cancer stem cells,we performed Real-Time PCR and Western Blot experiments.2.The expression of FOXP3 in colorectal cancer cells was overexpressed / knocked down by infecting adenovirus.Identify the effect of overexpressed / knockdown FOXP3 on Sphere formation efficiency by microscopic observation.Identify the effect of overexpressed / knockdown FOXP3 on the proportion of side population cells by Flow Cytometry.Identify the effect of overexpressed / knockdown FOXP3 on stem cell markers of Colonospheres by Real-Time PCR.Identify the effect of overexpressed / knockdown FOXP3 on the carcinogenicity of Colonospheres by tumor-bearing mice.3.Identify the interactions between FOXP3 and P65 by Co-immunoprecipitation.Identify the effect of knockdown FOXP3 as well as together knockdown FOXP3 and p65 on expression of COX2 by Real-Time PCR and Western Blot.Identify the effect of overexpressed FOXP3 as well as together overexpressed FOXP3 and p65 on the COX2 promoter activity by dual luciferase reporter gene.Explore whether FOXP3 binds to the NFκB binding site on the promoter of the COX2 gene by CHIP.4.Down-regulation of FOXP3 and COX2 expression in Colonospheres by infecting adenovirus.Identify the effect of knockdown FOXP3 as well as together knockdown FOXP3 and COX2 on Sphere formation efficiency by microscopic observation.Identify the effect of knockdown FOXP3 as well as together knockdown FOXP3 and COX2 on the proportion of side population cells by Flow Cytometry.Identify the effect of knockdown FOXP3 as well as together knockdown FOXP3 and COX2 on stem cell markers of Colonospheres by Real-Time PCR.Identify the effect of knockdown FOXP3 as well as together knockdown FOXP3 and COX2 on the carcinogenicity of Colonospheres by tumorbearing mice.Results 1.All the colorectal cancer cell lines produced sufficient numbers of Colonospheres.Real-time PCR and flow cytometry analysis showed that the expression level of surface molecular markers of colon cancer stem cells in Colonosphere was significantly higher than that of colon cancer cells.Colonospheres could be regarded as colorectal cancer stem cells for follow-up experiments.Compared with the parental cells,the Colonospheres displayed much lower expression of FOXP3 at both m RNA and protein levels.Consistent with the reduced expression of FOXP3 in the Colonospheres,there was much higher expression of COX2 observed in the Colonospheres.2.FOXP3 overexpression significantly decreased the Sphere formation efficiency.FOXP3 overexpression significantly decreased the proportion of SP Cells in the Colonospheres.FOXP3 overexpression significantly decreased the expression of stem cell markers in the Colonospheres.FOXP3 overexpression significantly decreased the tumorigenic ability of the Colonospheres.In contrast,knockdown FOXP3 significantly increased the Sphere formation efficiency,side population proportion,carcinogenicity,together with the increased expression of stem cell markers.3.FOXP3 interacted p65 in Colonospheres.FOXP3 overexpression repressed COX2 expression at both m RNA and protein levels.In contrast,knockdown FOXP3 increased COX2 expression at both m RNA and protein levels.Although inhibiting p65 only can decreased expression of COX2,the promoting effects of knockdown FOXP3 on COX2 expression was impaired when together knockdown FOXP3 and p65.FOXP3 overexpression inhibited the COX2 promoter activity,the inhibition effects of FOXP3 overexpression on the COX2 promoter activity was impaired when together overexpressed FOXP3 and p65.Chip assay revealed occupation of FOXP3 on the NFκB binding sites on the COX2 promoter.4.Knockdown FOXP3 significantly increased the Sphere formation efficiency.Knockdown FOXP3 significantly increased the proportion of SP Cells in the Colonosphere.Knockdown FOXP3 significantly increased the expression of stem cell markers in the Colonospheres.Knockdown FOXP3 significantly increased the tumorigenic ability of the Colonospheres.However,the promoting effects of knockdown FOXP3 on the Sphere formation efficiency,side population proportion,tumorigenic ability,together with the increased expression of stem cell markers was impaired when together knockdown FOXP3 and COX2.Conclusion In conclusion,FOXP3 was down-regulated in Colonospheres.FOXP3 overexpression significantly decreased the self-renewal ability of cancer stem cells.Mechanistically,FOXP3 interacts with p65 and in turn inhibits NF-κB mediated transcriptional activation of COX2.Our study here has revealed a negative regulatory role of FOXP3 in tuning the selfrenewal of cancer stem cells by inhibiting COX2 expression,which provides a new target and strategy for the eradication of colorectal cancer stem cells.