From the Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama 36604 and.
the Department of Dermatology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
J Biol Chem. 2014 Nov 7;289(45):31513-25. doi: 10.1074/jbc.M114.606483. Epub 2014 Sep 24.
Aberrant expression of hedgehog molecules, particularly Gli1, is common in cancers of many tissues and is responsible for their aggressive behavior and chemoresistance. Here we demonstrate a novel and tumor-specific role for aberrant Gli1 in the regulation of the S-phase checkpoint that suppresses replication stress and resistance to chemotherapy. Inhibition of Gli1 in tumor cells induced replication stress-mediated DNA damage response, attenuated their clonogenic potential, abrogated camptothecin (CPT)-induced Chk1 phosphorylation, and potentiated its cytotoxicity. However, in normal fibroblasts, Gli1 siRNAs showed no significant changes in CPT-induced Chk1 phosphorylation. Further analysis of ataxia telangiectasia and Rad3-related protein (ATR)/Chk1 signaling cascade genes in tumor cells revealed an unexpected mechanism whereby Gli1 regulates ATR-mediated Chk1 phosphorylation by transcriptional regulation of the BH3-only protein Bid. Consistent with its role in DNA damage response, Bid down-regulation in tumor cells abolished CPT-induced Chk1 phosphorylation and sensitized them to CPT. Correspondingly, Gli1 inhibition affected the expression of Bid and the association of replication protein A (RPA) with the ATR- interacting protein (ATRIP)-ATR complex, and this compromised the S-phase checkpoint. Conversely, complementation of Bid in Gli1-deficient cells restored CPT-induced Chk1 phosphorylation. An in silico analysis of the Bid promoter identified a putative Gli1 binding site, and further studies using luciferase reporter assays confirmed Gli1-dependent promoter activity. Collectively, our studies established a novel connection between aberrant Gli1 and Bid in the survival of tumor cells and their response to chemotherapy, at least in part, by regulating the S-phase checkpoint. Importantly, our data suggest a novel drug combination of Gli1 and Top1 inhibitors as an effective therapeutic strategy in treating tumors that expresses Gli1.
hedgehog 分子（尤其是 Gli1）的异常表达在许多组织的癌症中很常见，是其侵袭性行为和化疗耐药性的原因。在这里，我们证明了异常 Gli1 在调节 S 期检查点方面具有新颖的肿瘤特异性作用，该作用抑制复制应激并抵抗化疗。在肿瘤细胞中抑制 Gli1 会诱导复制应激介导的 DNA 损伤反应，减弱其集落形成能力，消除喜树碱（CPT）诱导的 Chk1 磷酸化，并增强其细胞毒性。然而，在正常成纤维细胞中，Gli1 siRNA 对 CPT 诱导的 Chk1 磷酸化没有明显变化。进一步分析肿瘤细胞中的共济失调毛细血管扩张症和 Rad3 相关蛋白（ATR）/Chk1 信号级联基因，揭示了一个意想不到的机制，即 Gli1 通过转录调节 BH3 仅蛋白 Bid 来调节 ATR 介导的 Chk1 磷酸化。与它在 DNA 损伤反应中的作用一致，Bid 在肿瘤细胞中的下调消除了 CPT 诱导的 Chk1 磷酸化，并使它们对 CPT 敏感。相应地，Gli1 抑制作用影响 Bid 的表达以及复制蛋白 A（RPA）与 ATR 相互作用蛋白（ATRIP）-ATR 复合物的结合，从而破坏 S 期检查点。相反，在 Gli1 缺陷细胞中补充 Bid 恢复了 CPT 诱导的 Chk1 磷酸化。Bid 启动子的计算机分析确定了一个推定的 Gli1 结合位点，进一步使用荧光素酶报告基因检测证实了 Gli1 依赖性启动子活性。总的来说，我们的研究在肿瘤细胞的存活及其对化疗的反应中建立了异常 Gli1 和 Bid 之间的新联系，至少部分是通过调节 S 期检查点实现的。重要的是，我们的数据表明，Gli1 和 Top1 抑制剂的新型药物联合可能是治疗表达 Gli1 的肿瘤的有效治疗策略。
