Effect of overexpression of activating transcription factor 3 on biological behaviors of human colorectal cancer HCT116 cells

Colorectal cancer is the third most common cancer and the fourth leading cause of cancer-related mortality worldwide. Activating transcription factor 3 (ATF3) has been identified as both an oncogene and a tumor suppressor in colorectal cancer, which encourages further studies to validate the exact role of ATF3 in colorectal cancer development and progression. The aim of this study was to examine the effect of ATF3 overexpression on biological behaviors of human colorectal cancer HCT116 cells. A stable ATF3-overexpressed HCT116 cell line was established by transfecting HCT116 cells with lentivirus carrying a full-length of ATF3. Cell proliferation was measured with MTS and colony-formation assays, and cell migration and invasion was detected using Transwell migration/invasion assays. In addition, a sphere-forming assay was performed to assess the cancer-initiation capacity, while the gene and protein expression was quantified with qRT-PCR, Western blotting and flow cytometry. ATF3 overexpression was found to suppress proliferation, migration and invasion of HCT116 cells as compared with the control cells, while down-regulation of Survivin and PCNA expression was detected in HCT116 cells at both translational and transcriptional levels, and a reduction was measured in the CyclinD1 protein expression in HCT116 cells overexpressing ATF3 than in the control cells. In addition, MMP2 and MMP9 expression was found to be down-regulated in HCT116 cells overexpressing ATF3 at both translational and transcriptional levels, and Western blotting detected up-regulation of TIMP-2 and TIMP-1 expression in HCT116 cells overexpressing ATF3 relative to the control cells. Overexpression of ATF3 resulted in reversal of epithelial-mesenchymal transition (EMT), as revealed by changes of cell morphology and altered expression of E-cadherin, Snail, Slug and Twist. Most importantly, ATF3-overexpressed HCT117 cells produced less and smaller spheres as compared with the control cells. The expression of multiple markers associated with colorectal cancer-initiation including CD24, CD133, CD166, EpCAM, EZH2 and ID1 were down-regulated in HCT116 cells overexpressing ATF3 at both translational and transcriptional levels, while lower Β-catenin protein expression was detected in ATF3-overexpressed HCT116 cells relative to the control cells. The results of this study demonstrate that ATF3 overexpression inhibits colorectal cancer progression and suggest that ATF3 exhibits antitumor activity through reversing EMT and impairing cancer-initiation ability.