The Role of ZEB1 in promoting therapeutic resistance through its interaction with 53BP1

ZEB1 通过与 53BP1 相互作用促进治疗耐药的作用

基本信息

批准号：
10551845
负责人：
James M Larner
金额：
$ 36.2万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10551845
关键词：
Area BRCA deficient BRCA1 gene BRCA2 gene Base Excision Repairs Binding Binding Sites Biological Assay Biological Markers Breast Breast Cancer Cell Breast Cancer Model Breast Cancer Patient Breast Cancer cell line CHEK1 gene Cell Cycle Cell Hypoxia Cells Cessation of life Chemotherapy and/or radiation Chromatin Chromatin Remodeling Factor Chromatin Structure Chromosome abnormality DNA Damage DNA Double Strand Break DNA Repair DNA-PKcs DNA-dependent protein kinase Development Double Strand Break Repair Environment Euchromatin Excision G2 Phase Gene Silencing Genetic Recombination Genetic Transcription Genome Genome Stability Genomic Segment Goals Hypoxia Ionizing radiation Lesion Link Malignant Neoplasms Mammalian Cell Mammary Neoplasms Maps Mediating Mutation Nonhomologous DNA End Joining Pathway interactions Phase Phosphorylation Play Poly(ADP-ribose) Polymerase Inhibitor Polymerase Population Proteins Radiation Radiation therapy Radiobiology Reactive Oxygen Species Regulation Resistance Role S phase Site Solid Neoplasm Source Testing Therapeutic Transcription Repressor Treatment Efficacy Xenograft procedure cancer cell cancer therapy chromatin modification cytotoxic epithelial to mesenchymal transition gene repression homologous recombination malignant breast neoplasm mouse model mutant normoxia novel novel strategies ovarian neoplasm p53-binding protein 1 patient response permissiveness predictive marker public database radiation resistance recombinational repair recruit repaired response restoration therapy resistant treatment response tumor tumor hypoxia tumor progression

项目摘要

ABSTRACT DNA double strand breaks (DSBs) are the most mutagenic and cytotoxic insults to the genome. DSBs are repaired through homology directed recombination (HDR), which is predominant in S and G2 phases, and also by error prone non-homologous end joining (NHEJ), which is active in all cell cycle phases. In cancer cells, heightened activity of these repair pathways has been linked to radioresistance. The transcriptional repressor ZEB1 is a well-established driver of the epithelial-to-mesenchymal transition (EMT) in both normal development and tumor progression. Recent studies suggest that ZEB1 plays new roles in the regulation of the DNA damage response (DDR), via enhancing the stability of the checkpoint kinase CHK1, and in regulating the repair of DSBs through the transcriptional silencing of polymerase theta, a major driver of microhomology- mediated alternative NHEJ (alt-NHEJ). We have discovered that ZEB1 is rapidly recruited to DSBs induced selectively in euchromatic genomic regions in a DNA-PK-dependent manner, and is essential for the recruitment 53BP1 to these sites. The recruitment of ZEB1 to these break sites was associated with local chromatin modifications permissive for NHEJ repair. Consequently, depletion or deletion of ZEB1 suppressed canonical NHEJ in cell-based DSB repair assays, and significantly increased both DSB-associated hyper- resection and HDR, This correlated with ATM-independent increases in chromosomal aberrations and enhanced sensitivity to IR. On the other hand, loss of ZEB1 was associated with decreased sensitivity of BRCA-deficient cells to PARP inhibitors (PARPi), indicative of restoration of HDR in these cell population, presumably through inhibiting 53BP1 recruitment to DSBs and stimulation of hyper-resection. Importantly, we found that ZEB1-dependent recruitment of 53BP1 to DSB is significantly amplified in hypoxia. Based on these novel observations, we hypothesize that ZEB1 promotes DSB repair via both direct and indirect mechanisms and this is critical for promoting therapeutic resistance to IR and for conferring sensitivity of BRCA-deficient cells to PARPi; on one hand, ZEB1 directly promotes c-NHEJ through the recruitment of the anti-resection and c-NHEJ repair protein 53BP1 to euchromatin-bound DSBs. On the other hand, ZEB1 facilitates the repair of DSBs through augmenting the DDR and through indirectly enhancing c-NHEJ repair through its chromatin- modifying as well as transcriptional silencing activity. Aim 1 of this proposal focuses on understanding the mechanism(s) by which ZEB1 regulates DSB repair. In Aim 2, we will determine the impact of ZEB1 on the therapeutic response of breast cancer to IR and PARP inhibitors in a panel of breast cancer cell lines and in xenograft mouse models of breast cancer. In Aim 3, we will determine how ZEB1 modulates the therapeutic response of hypoxic breast tumors to IR and PARPi. Understanding how the ZEB1/53BP1 axis regulates DSB repair in normoxia and hypoxia will allow us to exploit this axis as a biomarker to predict IR/PARPi sensitivity and well as to target the axis to decrease therapeutic resistance.

抽象的 DNA 双链断裂 (DSB) 是对基因组最具致突变性和细胞毒性的损伤。 DSB 是通过同源定向重组 (HDR) 进行修复，该重组在 S 期和 G2 期占主导地位，并且通过容易出错的非同源末端连接（NHEJ），它在所有细胞周期阶段都很活跃。在癌细胞中，这些修复途径的活性增强与放射抗性有关。转录抑制子 ZEB1 是正常细胞中上皮间质转化 (EMT) 的公认驱动因素。发育和肿瘤进展。最近的研究表明ZEB1在调节中发挥新的作用 DNA 损伤反应 (DDR)，通过增强检查点激酶 CHK1 的稳定性，并调节通过聚合酶 theta 的转录沉默来修复 DSB，聚合酶 theta 是微同源性的主要驱动因素介导的替代 NHEJ (alt-NHEJ)。我们发现ZEB1被快速招募到诱导的DSB中以 DNA-PK 依赖性方式选择性地作用于常染色质基因组区域，并且对于将 53BP1 招募到这些位点。 ZEB1 招募到这些断裂位点与局部相关染色质修饰允许 NHEJ 修复。因此，ZEB1 的耗尽或缺失抑制了典型的 NHEJ 在基于细胞的 DSB 修复测定中，并显着增加了 DSB 相关的超切除和 HDR，这与染色体畸变的 ATM 独立增加相关增强对红外的敏感性。另一方面，ZEB1 的缺失与 BRCA 缺陷细胞对 PARP 抑制剂 (PARPi) 的依赖，表明这些细胞群中 HDR 的恢复，推测是通过抑制 53BP1 募集到 DSB 并刺激超切除。重要的是，我们发现在缺氧条件下，ZEB1 依赖性 53BP1 向 DSB 的募集显着放大。基于这些新颖的观察结果，我们假设 ZEB1 通过直接和间接机制促进 DSB 修复这对于促进对 IR 的治疗抵抗和赋予 BRCA 缺陷的敏感性至关重要细胞至 PARPi；一方面，ZEB1通过招募抗切除和 c-NHEJ 将蛋白 53BP1 修复为常染色质结合的 DSB。另一方面，ZEB1 有助于修复 DSB 通过增强 DDR 并通过其染色质间接增强 c-NHEJ 修复修饰以及转录沉默活性。该提案的目标 1 侧重于理解 ZEB1 调节 DSB 修复的机制。在目标 2 中，我们将确定 ZEB1 对一组乳腺癌细胞系中乳腺癌对 IR 和 PARP 抑制剂的治疗反应乳腺癌异种移植小鼠模型。在目标 3 中，我们将确定 ZEB1 如何调节治疗缺氧乳腺肿瘤对 IR 和 PARPi 的反应。了解 ZEB1/53BP1 轴如何调节 DSB 常氧和缺氧状态下的修复将使我们能够利用该轴作为生物标志物来预测 IR/PARPi 敏感性以及瞄准轴以降低治疗阻力。