The role of ATF2 in 5-Fluorouracil resistance of colorectal cancer cells

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Doctoral Thesis
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Yang, Hao

Background: The Activating transcription factor-2 (ATF2) is a member of the ATF/CREB bZIP family of transcription factors, it regulates transcription of various genes, which are involved in cell proliferation, apoptosis, inflammation, and DNA damage response. ATF2 behaves as a double-edged sword and plays a carcinogenic or anticarcinogenic role in different types of cancer. Colorectal cancer (CRC) is the third most common disease in the world in terms of new cases and second in terms of death. 5-Fluorouracil (5-FU) is an essential chemotherapeutic drug for CRC. The development of tumor resistance can impede tumor cell death and affect the clinical outcome of patients. So far, the role of ATF2 in 5-FU resistance mechanisms in CRC is not known. Therefore, the aim of this study is to investigate the potential role and mechanism of ATF2 in CRC. Methods: CRISPR-Cas9 mediated knockout of ATF2 in HCT116 (wildtype p53) and HT29 (mutant p53) cell lines were performed to evaluate the sensitivity to 5-FU drug. Drug effects were investigated by Annexin-PI staining, Western Blotting for apoptosis and autophagy markers, tumor-forming ability assay in vitro and the chicken chorioallantoic membrane (CAM) model in vivo. The effects of ATF2 on 5-FU resistance and apoptosis after blockade of the ATR/Chk1 pathway by the Chk1 inhibitor PF-477736 were examined. Co-immunoprecipitation, proximity ligation assay and in silico modeling were performed to discover the molecular mechanisms. RNA interference (RNAi) was used to knock down ATF2 in HCT116 p53-/- cells. The expression of ATR/Chk1 pathway molecules and apoptosis-related markers were compared in control and RNAi-treated groups under 5-FU by Western Blotting. Results: HCT116 cells lacking ATF2 exhibited increased resistance to 5-FU with significantly reduced DNA damage, apoptosis, and enhanced cell proliferation in vitro and in vivo. These cells had a higher colony formation ability. Western Blotting showed that the deletion of ATF2 induced an activation of the ATR/Chk1 signaling pathway. The Chk1 inhibitor PF-477736 blocked the activation of the ATR/Chk1 signaling cascade and caused more DNA damage and DNA-induced apoptosis in ATF2-deficient cells. Co-immunoprecipitation and proximity ligation assay revealed that ATF2 built a complex with p-ATR, which phosphorylated Chk1. In silico modeling indicated an unstable triple complex consisting of ATR/Chk1/ATF2 and loss of ATF2 induced a stronger binding between ATR and Chk1. In HT29 cells, ATF2 hindered the inhibitory effect of mutant p53 on the ATR/Chk1 axis. When cells lost p53, the ATR/Chk1 pathway no longer played a key role in regulating 5-FU-induced apoptosis and DNA damage. Conclusion: For the first time, we showed that ATF2 mediated 5-FU resistance by affecting the DDR in a p53-dependent manner. As a scaffold protein, ATF2 seemed to affect the efficacy of phosphorylation of the repair protein Chk1 by the damage sensor ATR.

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