High Expression Of Collagen Triple Helix Repeat Containing 1 (CTHRC1) Facilitates Progression Of Esophageal Squamous Cell Carcinoma By Activating FRA-1 Through ERK Pathway

Esophageal cancer is one of the most common malignancies and remains a serious threat to human health.China is a country with high incidence of esophageal cancer,where more than 90%of esophageal cancer is squamous cell carcinoma.It is warrant to disclose the underlying molecular mechanisms that drive the development of ESCC,helping to identify new therapeutic target and markers for diagnosis or predicting prognosis,providing new ideas for the treatment of ESCC.CTHRC1(College Triple Helix Repeat Containing 1)is a secreted glycoprotein that can reduce collagen matrix deposition and promote mobility of fibroblasts and smooth muscle cells.Germline mutation in CTHRC1 was identified to be associated with Barrett esophagus and esophageal adenocarcinoma,while overexpression of CTHRC1 was reported in ESCC by expression profiling studies in cohorts with small number of cases.Yet its role in tumorigenesis and progression of ESCC remains unclear.Here we examined the clinical significance of CTHRC1 in ESCC and explored the effect of CTHRC1 on the phenotype of ESCC as well as the underlying molecular mechanism in vitro and in vivo.Through transcriptome-wide microarray profiling,we identified that CTHRC1 was significantly overexpressed in ESCC tumor tissues(n = 119,Fold change = 20.5,P = 2.12E-66,Wilcoxon test;FDR = 2.93E-64,Benjamini-Hochberg procedure).On the other side,we confirmed marked elevated expression of CTHRC1 protein in tumor tissues by detecting the expression of CTHRC1 protein in 204 cases of ESCC by immunohistochemistry(IHC).High expression of CTHRC1 was positively correlated with T stage(P = 0.043,Chi square test),lymph node metastasis(P = 0.023,Chi square test)and TNM staging(P = 0.024,Chi square test).Kaplan-Meier survival analysis showed that ESCC patients with high expression of CTHRC1 had shorter overall survival than those with low expression of CTHRC1(P = 0.020,Log-Rank test).To investigate the mechanism contributing to the elevated expression of CTHRC1 expression in ESCC,we tested the methylation of the promoter region of CTHRC1 in 50 pairs of ESCC and non-tumor tissues by pyrosequencing.Methylation of cg07757887 in the promoter region of CTHRC1 was significantly lower in ESCC tumor tissues compared with the corresponding non-tumor tissues(n = 50,P<0.0001,t-test).92%(46/50)of the cases showed hypomethylation of CTHRC1 in tumor tissues.The expression level of CTHRC1 was dramatically increased in the cells with reduced methylation degree after treated with DNA methyltransferase inhibitor,5-aza-2'-deoxycytidine(5-Aza-dc).These data indicated that expression of CTHRC1 is closely regulated by promoter methylation in ESCC.Overexpression of CTHRC1 enhanced the proliferation,invasion and metastasis,whereas depletion of CTHRC1 suppressed these biological functions in ESCC cells and xenografts.We next explored the downstream signaling pathways responsible for CTHRC1-mediated aggressiveness of ESCC cells by RNA sequencing on KYSE510-ShCTHRC1 and KYSE510-vector cells.Differential expression genes were selected for KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis,enriching PI3K-Akt and MAPK pathways.Western blot verified that the expression of p-c-Raf,p-MEK1/2 and p-ERK1/2 sharply decreased in KYSE510-ShCTHRC1 and KYSE30-ShCTHRC1 cells,increased in KYSE450-CTHRC1 cells compared with their control cells.Treatment with MEK1/2 inhibitor,U0126,reversed CTHRC1-induced proliferation,migration and invasion of KYSE450 cells.Furthermore,CTHRC1 activated MAPK/MEK/ERK signaling pathway to activate FRA-1(Fos-related antigen 1),an important transcription factor mediating upregulating the expression of cyclin D1 and snail 1.Snail 1 further induced expression of MMP14(Matrix metallopeptidase 14).Compatible with the above in vitro studies,CTHRC1 mRNA level was significantly positively correlated with CCND1 and SNAH1 mRNA level in ESCC tumour tissues(n =119,r=0.227,P = 0.013;r = 0.550,P<0.0001,Pearson's correlation coefficient).Significant positive correlation between SNAI1 and MMP14 mRNA levels was also found(r =0.318,P ? 0.0004,Pearson's correlation coefficient).Moreover,expression of CTHRC 1 was positively associated with expression of cyclin D1,as well as MMP14,at protein level(n = 204,P = 0.018,Chi square test;P = 0.022,Chi square test).Kaplan-Meier analysis revealed the significant association between shorter overall survival of ESCC patients and high expression of cyclin D1(n = 204,P = 0.049,Log-Rank test),or MMP14(n = 196,P = 0.0062,Log-Rank test).In summary,CTHRC 1 may act as an oncogenic driver in progression and metastasis of ESCC,and may serve as a potential biomarker for prognosis and personalized therapy.