PPARβ/δ Inhibits The Malignant Phenotype Of Colorectal Cancer Stem Cells By The Regulation Of IRF1

Background and objective:Colorectal cancer(CRC)is one of the most common malignancies in the world,with the third highest morbidity and the second highest tumor-related mortality among all cancer types.Over past decades,with aging of population and changing of lifestyle,the incidence of colorectal cancer is increasing in China with a speed of 4.71%annually.Although the rapid development of treatments,including surgery,chemotherapy,radiotherapy,and targeted therapy,has doubled the overall survival(OS)of patients with advanced CRC,the high recurrence rate and poor prognosis remain troubling issues.Therefore,further exploration of CRC related genes and signaling pathways is of great significance for the in-depth understanding of the molecular mechanisms of tumorigenesis,as well as guiding the development of promising prognostic markers and optimal treatment strategies.Peroxisome proliferator-activated receptor beta/delta(PPARβ/δ),one of three members of the PPAR group in the nuclear receptor superfamily,is a ligand-activated transcription factor which has been suggested to be involved in lipid metabolism and multiple cellular functions.PPARβ/δ has been suggested as a therapeutic target for type 2 diabetes and metabolic syndrome.However,potential risks in the settings of cancer are less clear.A variety of studies have investigated PPARβ/δ expression or activation/inhibition in CRC,but the potential function of PPARβ/δ in CRC carcinogenesis remains an area of controversy.This is owing to conflicting reports with some studies suggesting that PPARβ/δ promotes CRC,whereas other studies indicate that PPARβ/δ inhibits CRC.Therefore,more studies are still needed to further explore the role of PPARβ/δ in CRC carcinogenesis,and the proposal and confirmation of the cancer stem cells(CSCs)provides a new perspective.Colorectal cancer stem cells(CCSCs)reside in the base of the colonic crypts and are essential for the initiation and maintenance of colorectal cancer,possess the capacity of self-renewal,differentiate into a heterogeneous lineage,and develop innate resistance to cytotoxic agents.CCSCs are reported to be responsible for the tumor initiation,growth,metastatic spread,relapse,and recurrence of colorectal cancer.However,very little is known about their biology and how they are regulated.Therefore,investigating the regulatory mechanisms that control the growth of these cancer stem cells is a promising area of investigation for possible therapeutic agents and preventive treatment.We therefore studied the effects of PPARβ/δ on CCSCs in order to unveil the role of PPARβ/δ in the occurrence and progression of CRC and its related mechanisms.Methods:The CCSCs model which has been successfully established in our laboratory was used for further study.Firstly,the CCSCs was activated by the PPARβ/δ selective agonist GW501516 followed by analyzing the stem cell frequency,cell cycle,proliferation ability,apoptosis,and xenograft tumorigenicity.Subsequently,the PPARβ/δ was knockdown to verify the effects of PPARβ/δ on CCSCs.Then,RNA-seq and Ch IP-seq were used to analyze the effect of PPARβ/δactivation on the transcription profile of CCSCs and to identify the target genes regulated by PPARβ/δ transcription.Finally,the Interferon Regulatory Factor 1(IRF1)was identified as the target genes of PPARβ/δ.Subsequently,we used The Cancer Genome Atlas(TCGA)and Cancer Cell Line Encyclopedia(CCLE)to analyze the co-expression relationship between PPARβ/δ and IRF1.Subsequently,the IRF1 gene expression was knockdown to verify the effects of PPARβ/δ on CCSCs.Then,we verified the transcriptional regulatory relationship between PPARβ/δ and IRF1 by analyzing the effect of knockdown of IRF1 gene on the tumor inhibition ability of PPARβ/δ.Subsequently,the expression of IRF1 in 141 cases of CRC was measured by immunohistochemical(IHC)followed by analyzing the relationship between IRF1 expression and tumor prognosis,and the results were verified by the TCGA database.Finally,we also analyzed the relationship between the co-expression level of PPARβ/δ and IRF1 and the prognosis of colorectal cancer.Results:1.The RT-PCR and Western Blot results demonstrated the GW501516 specifically activated the expression of PPARβ/δ in CCSCs.Subsequently,we found that the activation of PPARβ/δ can significantly inhibit the self-renewal ability of CCSCs by Sphere-forming assays.PPARβ/δ activation significantly inhibited the expression of stemness-related genes such as Lgr5,SMOC2 and MSI1(Musashi1)in CCSCs,and significantly enhance the expression of cell differentiation-related genes such as CK20,TFF3,Villin1,CHGA(Chromogranin A)and LYSZ(Lysozyme).Moreover,we also found that activation of PPARβ/δ can significantly inhibit cell cycle and proliferation of CCSCs,and induce cell differentiation and apoptosis(all P<0.05).In addition,we also found that knockdown the expression of PPARβ/δ in CCSCs significantly reduced the inhibitory effect of GW501516 on CCSCs cells(all P<0.05).Subsequently,through the nude mouse xenograft model,we found the activation of PPARβ/δ significantly reduced the growth rate and tumor volume of CCSCs xenograft tumors,and knockdown of PPARβ/δ expression partially reversed this inhibition.And then,we also found that knockdown of PPARβ/δ expression significantly reduced the expression of stem cell-related gene Lgr5 in xenograft tumor cells and enhanced the expression of cell differentiation-related gene CK20.The results showed that PPARβ/δ could significantly inhibit the malignant phenotype of CCSCs.2.RNA-seq results showed that the activation of PPARβ/δ resulted in upregulation of 486 genes and down-regulation of 298 genes in CCSCs(all P<0.05).Through bioinformatics analysis,we found the PPARβ/δ activation will significantly promote the biological processes of lipid catabolism,cell apoptosis and organic anion transport,and significantly inhibit DNA replication initiation,chromosome segregation,cell cycle G1/S phase transition and nuclear DNA replication.In addition,we also found the activation of PPARβ/δ significantly enhanced the PPAR signaling pathway,metabolic pathways,PI3K-Akt signaling pathway,MAPK signaling pathway,and fatty acid metabolism signaling pathways,and significantly inhibited the cell cycle signaling pathways,DNA replication signaling pathways,nucleotide excision repair signaling pathways as well as mismatch repair signaling pathways.Subsequently,we used Ch IP-Seq to detect the transcriptional binding sites of PPARβ/δ genes,and found that the activation of PPARβ/δ enhanced the transcriptional binding of 883 genes and decreased the transcriptional binding of 1747genes(all P<0.05).Through bioinformatics analysis,we found that PPARβ/δtranscriptional combination gene is mainly involved in the regulation of metabolic level in vivo,induction of cell apoptosis and cell differentiation,inhibition of tumor progression.Through RNA-seq and Ch IP-seq integration analysis,we found that PPARβ/δ may play a role in tumor inhibition by directly regulating the expression of CDKN1 A,FGFR2,IRF1,ME1,STRA6 and ZFP36 genes.Finally,IRF1 was identified as the target gene of PPARβ/δ.Then,through RT-PCR and Western Blot experiments,we found that the activation of PPARβ/δ specifically enhanced the expression of IRF1 in CCSCs.Subsequently,we found a significant co-expression relationship between PPARβ/δ and IRF1 through TCGA and CCLE databases.Subsequently,we also found that knockdown of IRF1 expression in CCSCs significantly reduced the inhibitory effect of PPARβ/δ on CCSCs.These results suggest that PPARβ/δ can significantly inhibit the malignant phenotype of CCSCs by regulating the expression of IRF1.3.In our previous work,we have found that the expression of PPARβ/δ was increased in colorectal cancer,and the high expression of PPARβ/δ was significantly associated with better prognosis of colorectal cancer.In this study,we found that the expression of IRF1 in tumor tissues was lower than that in normal mucosa,and the higher expression of IRF1 in tumor was associated with earlier tumor stage and better prognosis(all P <0.01).In addition,we verify these results with TCGA database.Finally,we analyzed the relationship between the co-expression levels of PPARβ/δ/IRF1 and the clinicopathological characteristics and prognosis of colorectal cancer.The results demonstrated that the co-high expression of PPARβ/δ and IRF1 is significantly associated with the earlier tumor stage,and is an independent prognostic factor for good prognosis of colorectal cancer.Conclusion:1.PPARβ/δ can inhibit the malignant phenotype of CCSCs by inhibiting cell cycle and proliferation,inducing cell differentiation and promoting apoptosis.2.PPARβ/δ inhibits the tumorigenicity of CCSCs by inhibiting nuclear DNA replication,chromosome segregation,cell cycle G1/S phase transition,and enhancing cell differentiation and inducing cell apoptosis biological processes by the regulation of IRF1.3.The co-high expression of PPARβ/δ and IRF1 is significantly associated with the earlier tumor stage,and is an independent prognostic factor for good prognosis of colorectal cancer.