ZEB1 inhibition sensitizes cells to the ATR inhibitor VE-821 by abrogating epithelial–mesenchymal transition and enhancing DNA damage

The ataxia-telangiectasia-mutated (ATM) and rad3-related (ATR) checkpoint pathway plays an essential role in modulating cellular responses to replication stress and DNA damage to maintain genomic stability. In various tumors, cancer cells have increased dependence on ATR signaling for survival, making ATR a promising target for cancer therapy. ATR inhibitors sensitize multiple tumor cell types to radiation and DNA-damaging agents, but application of an ATR inhibitor alone shows limited efficacy. In the present study, we investigated the role of epithelial-to-mesenchymal transition (EMT) and the EMT transcription factor ZEB1 in regulating cell sensitivity to the ATR inhibitor VE-821. We found that VE-821 induced EMT with concomitant ZEB1 upregulation and promoted migration in cells in which the anti-proliferative effect of VE-821 was limited. Knocking down ZEB1 using siRNA partially reversed VE-821-induced EMT, and sensitized cells to VE-821 via effective attenuation of migration and AKT/ERK signaling. Moreover, ZEB1 inhibition promoted Chk1 phosphorylation and induced S-phase arrest by enhancing TopBP1 expression, which suggests a distinctive modulatory effect of ZEB1 on Chk1. Finally, combining VE-821 with ZEB1 inhibition enhanced DNA damage accumulation. These results demonstrate that EMT represents a novel mechanism for limiting the effectiveness of an ATR inhibitor, and thus suggest that ZEB1 inhibition might represent a new approach to increasing the efficiency of, or reversing resistance to, ATR inhibitors.共济失调毛细血管扩张症突变蛋白（ataxia-telangiectasia-mutated, ATM）与RAD3相关激酶（rad3-related, ATR）检查点通路在调控细胞应对复制应激与DNA损伤的应答、维持基因组稳定性过程中发挥关键作用。在多种肿瘤中，癌细胞的生存愈发依赖ATR信号通路，这使得ATR成为极具潜力的癌症治疗靶点。ATR抑制剂可使多种肿瘤细胞类型对放疗及DNA损伤剂增敏，但单独使用ATR抑制剂的疗效有限。本研究探讨了上皮间质转化（epithelial-to-mesenchymal transition, EMT）及其转录因子ZEB1在调控细胞对ATR抑制剂VE-821敏感性中的作用。研究发现，VE-821可诱导EMT并伴随ZEB1上调，同时在VE-821抗增殖作用受限的细胞中促进细胞迁移。通过小干扰RNA（small interfering RNA, siRNA）敲低ZEB1，可部分逆转VE-821诱导的EMT，并通过有效抑制细胞迁移及AKT/ERK信号通路，使细胞对VE-821重新敏感。此外，抑制ZEB1可促进Chk1磷酸化，并通过增强TopBP1的表达诱导S期阻滞，这表明ZEB1对Chk1具有独特的调控作用。最后，联合使用VE-821与ZEB1抑制剂可增加DNA损伤的积累。上述结果表明，EMT是限制ATR抑制剂疗效的全新机制，这提示抑制ZEB1或可成为提高ATR抑制剂疗效、逆转其耐药性的新策略。