EphA3 Downregulation By Hypermethylation Associated With Lymph Node Metastasis And TNM Stage In Colorectal Cancer

Objective:EphA3 is a member of Eph receptors,which is involved in the development and tumorigenesis.The expression and clinical significance of EphA3 in colorectal cancer(CRC)have not been fully investigated.Methods:Four colon cancer and one normal intestinal cell lines and a set of CRC tissues were examined for EphA3 expression.The methylation status of a CpG island within the EphA3 promoter,the presence of four somatic EPHA3 mutations,and EPHA3 gene copy number variations(CNVs)were also analyzed in colon cancer cell lines.Furthermore,transfection of EphA3 plasmid into HT29 cancer cells with lost EphA3 expression was carried out.The function of EphA3 in colorectal cancer was analyzed by in vitro experiments including cell proliferation,apoptosis and invasiveness.Results:EphA3expression was lost in all colon cancer cell lines examined.EphA3 expression was lower in tumor tissues when compared with normal intestinal tissues(P<0.001).A comparison of EphA3 immunohistochemical scores for tumor and matched normal intestinal tissues revealed that the protein was downregulated in 82/164(50.0%), unchanged in 52/164(31.7%)and upregulated in 30/164(18.3%)cases of CRC. EphA3 expression was negatively associated with lymph node metastasis(P=0.014,r_s=-0.192)and TNM stage(P=0.001,r_s=-0.260).Downregulation of expression was more common in older patients(P=0.013,r_s=0.193).Methylated promoter DNA was detected in all four colon cancer cell lines.Somatic mutations or EphA3gene deletion were not detected.No EPHA3 gene deletions or amplifications were detected in the four cell lines tested.HT29 cells transfected with EphA3 plasmid show decreased proliferation rate,lower invasiveness,and higher apoptosis rate.Conclusions:EphA3 was downregulated in the majority of CRC.Hypermethylation of a CpG island within the EPHA3 promoter provides a possible mechanism.Loss of EphA3 expression was associated with lymph node metastasis and TNM stage. Experimental results in vitro suggest that EphA3 may play a tumor suppressor in CRC.EphA3 may be a useful predictor for tumor spread in CRC,and a potential target for CRC.