Mechanism Of CEBPD/EAF2 Axis Inhibiting Colorectal Cancer Progression By Regulating STAT3/TGF-β1 Signaling Pathway

Objective:The Global Cancer Statistics 2020 showed that CRC was estimated to be the third most diagnosed malignant tumor(10%)and the second leading cause of cancer death(9.4%)worldwide.As an important process of CRC occurrence and development,tumor metastasis is a key factor affecting the survival rate of CRC.Therefore,it is urgent to identify the potential mechanism of CRC invasion and metastasis and explore new therapeutic targets.ELL-associated factor 2(EAF2)is an androgen-responsive gene expressed by luminal epithelial cells in benign prostatic tissue.More and more researches have shown that EAF2 plays an important role in tumor suppression in a variety of malignant tumors.Furthermore,there are scholars identified intratumor heterogeneity(ITH)of EAF2frameshift mutation in CRC and the inactivation of EAF2 in microsatellite instability(MSI)-H CRC.It suggests that EAF2 inactivation may occur during tumor progression rather than tumorigenesis.Besides,EAF2 gene silencing could modulate the cytotoxic response of colon cancer cell line HCT116 to Statins.However,studies on the expression and role of EAF2 protein in CRC are still lacking.The molecular mechanism of EAF2’s involvement in CRC invasion and metastasis remains unclear.EAF2 has been shown to attenuate transforming growth factor(TGF)-β1-induced G1 cell cycle arrest and cell migration in a variety of tumor cells,such as renal carcinoma cells,human hepatocellular carcinoma cells,and breast cancer cells.It is worth noting that blocking the TGF-β1 signaling pathway may be a therapeutic strategy for attenuating tumor metastasis in CRC metastatic models.In addition,signal transducer and activator of transcription 3(STAT3)pathway is involved in the process of TGF-β1-induced epithelial-mesenchymal transition(EMT),cell migration and invasion in malignant tumor.As well,EAF2 knockout induced STAT3 phosphorylation(Tyr705)in prostate cancer cells,which drove tumor progression in prostate cancer.Therefore,in this study,we investigated the effect of EAF2 on the STAT3/TGF-β1 pathway in CRC cells to explore the possible mechanism of EAF2 acting in CRC.CCAAT enhancer binding proteinδ(CEBPD)is a multifunctional transcription factor that plays a regulatory role in cell differentiation,proliferation,motility,growth arrest,and cell death.In the past few years,accumulated research has identified the dual function of CEBPD,which displays oncogenes or tumor suppressor genes based on tumor type and microenvironment.However,the expression of CEBPD in CRC and its role in inhibiting or promoting cancer are still unclear.The website predicts that CEBPD expression decreases in the colon cancer and binds to the EAF2 promoter.Therefore,we speculate that CEBPD targets the regulation of EAF2 expression and regulates the progression of CRC by STAT3/TGF-β1 signaling pathway.In this study,we first used bioinformatics analysis to predict the expression of related molecules,and collected samples from clinical CRC cases for further testing to investigate the expression of EAF2 protein in advanced CRC tissues.Secondly,in vitro and in vivo experiments were conducted to investigate the expression of EAF2 protein in CRC cells and its effect on the biological function of tumor cells,and to preliminarily explore the mutual regulatory role of EAF2 protein in CRC cells and its role and mechanism in mediating TGF-β1 related pathways.Finally,we utilized the HT29 cell line assay to investigate the interaction between CEBPD and EAF2,and evaluated the biological effects of CEBPD on CRC cells;Simultaneously,we verifed the regulation of CRC malignant phenotype by the CEBPD/EAF2 axis.This study provided important theoretical basis for elucidating the development mechanism of CRC and exploring new molecular targeted therapies.Methods:Part Ⅰ Clinical and prognostic significance of EAF2 in advanced CRCWe collected histological sections of advanced CRC from November 2012 to June2015 in The First Affiliated Hospital of China Medical University.This study included 70pairs of colorectal adenocarcinomas and corresponding paracancerous tissue that had been surgically removed.In addition,another 8 pairs of fresh cancer and adjacent tissue were selected for Western blot assay.The following clinicopathological variables were considered:age,sex,tumor site,tumor size,histological type,degree of differentiation,angiolymphatic and/or perineural invasion,tumor stage,tumor invasion depth,lymph node status,distant metastasis,and follow-up.Included samples were also examined for protein expression(including CEA,CA19-9,P53,and CDX2).Immunohistochemistry and Western blot were used to detect the expression of EAF2.The basic characteristics of patients with advanced colorectal cancer and their correlation with EAF2 immunohistochemical expression was analyzed.Correlation analysis between EAF2 protein and clinical pathological parameters.Survival analysis of EAF2 expression levels in patients with advanced colorectal cancer,including EAF2expression and survival analysis,as well as Cox proportional risk model assessment of influencing factors for OS.Part Ⅱ The biological effects of EAF2 on CRC and its effects on STAT3/TGF-β1pathway(1)Colon cancer cell lines(SW480,RKO,HCT116,HT29,Lo Vo)and human normal colon cell lines(NCM460)were cultured in RPMI-1640 medium containing 10%fetal bovine serum.Western blot was used to detect the expression of EAF2.(2)RKO cells with low expression of EAF2 were subjected to overexpression treatment(stable transformation)and divided into Parental group,NC-OE-1 group and EAF2-OE group.HT29 cells with high expression of EAF2 were subjected to knockdown treatment(stable transformation)and divided into Parental group,NC-sh RNA group,EAF2-sh RNA-1 group and EAF2-sh RNA-2 group.Western blot detection of EAF2 expression to verify transfection efficiency.After constructing an EAF2 stable transfection cell line,the effect of EAF2 on the biological behavior of CRC cells was investigated.CCK8 was used to detect cell proliferation at 0,24,48,and 72hours.Clone formation assay was used to detect cell proliferation ability.Immunofluorescence examination for Ki67 expression.Wound healing assay was used to examine the cell migration(0,24 hours).Transwell was used to detect the cell invasion(0,24 hours).Western blot was used to detect the expression of N-cadherin and E-cadherin.Flow cytometry was used to detect cell cycle.Flow cytometry was used to detect cell apoptosis.Western blot was used to detect the expression of p-STAT3,STAT3and p-Smad.(3)The effect of EAF2 on tumor formation in CRC cells in vivo.Twenty-four BALB/c male nude mice were subcutaneously injected with the above-mentioned stable transfected cells(4×10~6/mouse),with 6 mice in each group.Mice were divided into NC-sh RNA group and EAF2 sh RNA group.Mice were injected the aforementioned stably transformed RKO cells and divideed into NC-OE-1 group and EAF2-OE group.The tumor volume was measured every 3 days and collected around 30 days after injection.The tumor growth curve was recorded.Western blot was used to detect the expression of EAF2.Immunohistochemical was used to detect EAF2 and Ki67expression.Part Ⅲ CEBPD/EAF2 axis regulation of CRC malignant phenotype(1)GEPIA database predicted the expression of CEBPD in CRC.(2)Target Scan website predicted the combination of EAF2 and CEBPD.Double luciferase was used to validaten whether CEBPD bound to the EAF2 promoter(in HT29).Oligonucleotide pull down experiment was used to detect whether the EAF2promoter bound to CEBPD,and then Western blot was used to detect the expression of CEBPD(in HT29).(3)CEBPD stable cells were constructed in HT29,and knockdown-CEBPD cells were divided into Parental group,NC sh RNA group,CEBPD sh RNA 1 group,and CEBPD sh RNA 2 group.Overexpression-CEBPD cells were divided into Parental group,NC-OE-2 group,and CEBPD-OE group.Western blot was used to detect the expression of CEBPD and EAF2.The effect of CEBPD on the malignant phenotype of CRC:CCK8was used to detect cell proliferation(0,24,48,72 hours).Clone formation assay was used to detect cell proliferation ability.Immunofluorescence examination for Ki67expression.Wound healing assay was used to examine the cell migration(0,24 hours).Transwell was used to detect the cell invasion(0,24 hours).Western blot was used to detect the expression of N-cadherin and E-cadherin.Annexin V-FITC/PI flow cytometry was used to detect cell apoptosis.Hoechst staining was used to observe the morphology of apoptosis.Western blot was used to detect the expression of Bcl-2,Bax,cleaved caspase-3,and cleaved caspase-9.(4)The effect of CEBPD on tumor formation in CRC cells in vivo.Twenty-four BALB/c male nude mice were injected with the aforementioned stable HT29 cells and divided into NC sh RNA group,CEBPD sh RNA group(the one with high efficiency of CEBPD sh RNA-1 and 2),NC OE-2 group,and CEBPD OE group,with 6 in each group.The tumor volume was measured every 3 days and collected around 30 days after injection.The tumor growth curve was recorded.Western blot was used to detect the expression of CEBPD and EAF2.Immunohistochemical was used to detect CEBPD and Ki67 expression.(5)Verification of CEBPD/EAF2 axis regulation of CRC malignant phenotype.HT29 with CEBPD-OE and EAF2-sh RNA were divided into NC-OE-2+NC-sh RNA group,CEBPD-OE+NC-sh RNA group,and CEBPD-OE+EAF2-sh RNA group.After 24hours,CCK8 was used to detect cell proliferation.Wound healing assay was used to detect the cell migration(0,24 hours).Transwell was used to detect the cell invasion(0,24 hours).Hoechst staining was used to observe the morphology of apoptosis.Western blot was used to detect the expression of p-STAT3,STAT3,and p-Smad.Results:Part ⅠClinical and prognostic significance of EAF2 in advanced CRC(1)Immunohistochemical and Western blot results indicated that the expression level of EAF2 protein in colorectal adenocarcinoma tissue was significantly lower than that in adjacent normal tissues.(2)Kaplan Meier survival analysis showed that among patients with advanced CRC,those with high expression of EAF2 protein in adenocarcinoma tissue had a higher survival rate,and univariate analysis suggested that low expression of EAF2 protein was a factor affecting the survival prognosis of patients with advanced CRC.Part Ⅱ The biological effects of EAF2 on CRC and its effects on STAT3/TGF-β1pathway(1)Compared with normal colorectal epithelial cells(NCM460),the expression levels of EAF2 protein in human colorectal cancer cell lines(SW480,RKO,HCT116,HT29,and Lo Vo)significantly reduced(P(27)0.01).(2)Compared with the Parental group,the EAF2 sh RNA-1 and EAF2 sh RNA-2groups showed a significant decrease in EAF2 expression,while the EAF2 OE group showed a significant increase in EAF2 expression(P(27)0.01).Compared with the Parental group,the CCK8 OD value in EAF2-sh RNA-1 group increased,clone formation rate,Ki67 expression,migration and invasion rate increased(P(27)0.01),the protein expression of E-cadherin increased(P(27)0.01),the protein expression of N-cadherin decreased(P(27)0.01),the proportion of G1 phase cells increased,cell apoptosis rate decreased(P(27)0.01),Bcl-2 expression increased(P(27)0.01),P21,P27,Bax,cleaved caspase-3,and cleaved caspase-9 protein expression decreased(P(27)0.01),STAT3phosphorylation level and p-Smad protein expression increased(P(27)0.01).Compared with the Parental group,the CCK-8 OD value of EAF2-OE group cells decreased,clone formation rate,Ki67 expression,migration and invasion rate decreased(P(27)0.01),E-cadherin protein expression decreased(P(27)0.01),N-cadherin protein expression increased(P(27)0.01),the proportion of G1 phase cells increased(P(27)0.01),cell apoptosis rate increased,Bcl-2 expression decreased(P(27)0.01),P21,P27,Bax,cleaved caspase-3,and cleaved caspase-9 protein expression increased(P(27)0.01),STAT3 phosphorylation level and p-Smad protein expression decreased(P(27)0.01).(3)Compared with the NC sh RNA group,the tumor volume of nude mice in the EAF2 sh RNA group increased(P(27)0.01),EAF2 expression decreased(P(27)0.01),and Ki67 expression increased(P(27)0.01).The tumor volume growth of nude mice in the EAF2-OE group slowed down,EAF2 expression increased(P(27)0.01),and Ki67expression decreased(P(27)0.01).Part Ⅲ CEBPD/EAF2 axis regulation of CRC malignant phenotype(1)GEPIA showed a significant decrease in the expression level of CEBPD in colon cancer compared to normal tissues.(2)Target Scan website predicted that the EAF2 promoter can bind to CEBPD.The dual luciferase activity assay and Oligonucleiotide pull down experiment confirmed the binding between EAF2 and CEBPD.(3)Compared with the Parental group,the protein expression levels of CEBPD and EAF2 in the CEBPD-sh RNA-1 and CEBPD-sh RNA-2 groups were significantly reduced(P(27)0.01),the CCK-8 OD value increased(P(27)0.01),Ki67 expression,cell migration and invasion rate,E-cadherin protein expression increased,N-cadherin protein expression decreased(P(27)0.01),apoptosis rate decreased(P(27)0.01),Bcl-2 protein expression increased(P(27)0.01),Bax,cleaved caspase-3,and cleaved caspase-9 protein expression decreased(P(27)0.01).The protein expression levels of CEBPD and EAF2 in the CEBPD-OE group cells increased,the CCK-8 OD value decreased(P(27)0.01),the expression of Ki67 decreased,cell migration and invasion rate decreased(P(27)0.01),E-cadherin protein expression decreased(P(27)0.01),N-cadherin protein expression increased(P(27)0.01),apoptosis rate increased(P(27)0.01),Bcl-2 protein expression decreased(P(27)0.01),Bax,cleaved caspase-3,and cleaved caspase-9 protein expression increased(P(27)0.01).(4)Compared with the NC sh RNA group,the tumor volume of nude mice in the CEBPD sh RNA group increased,while the expression of CEBPD and EAF2 decreased,and the number of Ki67 positive cells increased.The tumor growth of nude mice in the CEBPD-OE group was slow,with the increased expression of CEBPD and EAF2,and the decrease in the number of Ki67 positive cells(P(27)0.01).(5)Compared with the CEBPD-OE+NC sh RNA group,the CEBPD-OE+EAF2sh RNA group showed the increase in CCK8 OD value,migration and invasion rates,and the phosphorylation levels of STAT3 and p-Smad(P(27)0.01).Conclusion:1.In patients with advanced CRC,the expression level of EAF2 protein in colorectal adenocarcinoma tissue was significantly lower than that in normal adjacent tissues,and its low expression level might be associated with poor prognosis in patients with advanced CRC.Moreover,univariate analysis suggested that low expression of EAF2 protein was an important factor for the survival and prognosis of patients with advanced CRC,suggesting that EAF2 protein might become a new prognostic indicator for CRC.2.Overexpression of EAF2 protein in CRC cells inhibited cell cycle,invasion,and migration,promotes cell apoptosis,and reduced tumor volume in nude mice.In addition,overexpression of EAF2 downregulated the activity of STAT3/TGF-β1 pathway,further inhibited the biological processes of CRC cells.3.CEBPD positively regulates the expression of EAF2 by binding to the promoter.Overexpression of CEBPD inhibited the proliferation,migration,and invasion of CRC cells,inhibit the EMT process of cells,promote cancer cell apoptosis,and inhibit tumor growth in nude mice.Knocking down of EAF2 reversed the inhibition of overexpression of CEBPD on CRC cell proliferation,migration,and invasion,reversed the induction of cancer cell apoptosis,and enhanced the activity of STAT3/TGF-βsignaling pathway.