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

• 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
• 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; 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

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）是对基因组的诱变和细胞毒性侮辱。 DSB是 通过同源性重组（HDR）修复，该重组（HDR）在S和G2相中是主要的，也是 通过易于非同源末端连接的误差（NHEJ），在所有细胞周期阶段都活跃。在癌细胞中， 这些修复途径的活动增强与放射线相关。转录阻遏物 Zeb1是正常的上皮到间质转变（EMT）的良好驱动力 发育和肿瘤进展。最近的研究表明，Zeb1在调节中起新作用 DNA损伤响应（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，这是微观学的主要驱动力 介导的替代NHEJ（Alt-Nhej）。我们发现Zeb1迅速招募到DSB诱导 以DNA-PK依赖性方式选择性地在圣体基因组区域中，对于 招聘53BP1到这些站点。将Zeb1招募到这些休息地点与本地有关 NHEJ修复允许的染色质修饰。因此，抑制Zeb1的耗尽或删除 基于细胞的DSB修复测定法中的典型NHEJ，并显着增加了与DSB相关的高度增加 切除和HDR，这与非ATM无关的染色体畸变和 增强对IR的敏感性。另一方面，Zeb1的损失与降低的灵敏度有关 缺乏BRCA的细胞对PARP抑制剂（PARPI），指示这些细胞种群中HDR的恢复， 大概是通过抑制53BP1募集到DSB的刺激并刺激了高分辨率。重要的是，我们 发现在缺氧中依赖于53BP1对DSB的Zeb1依赖性募集显着放大。基于这些 新的观察结果，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的治疗性抗性和赋予BRCA缺陷敏感性至关重要 细胞到parpi；一方面，Zeb1直接通过招募反切除和 C-NHEJ修复蛋白53BP1至共染色质结合的DSB。另一方面，Zeb1促进了修复 DSB通过增强DDR并通过间接增强C-NHEJ修复的染色质 - 修改以及转录沉默活性。该提案的目标1着重理解 Zeb1调节DSB修复的机制。在AIM 2中，我们将确定Zeb1对 乳腺癌对IR和PARP抑制剂的治疗反应 异种移植小鼠乳腺癌模型。在AIM 3中，我们将确定Zeb1如何调节治疗性 低氧乳腺肿瘤对IR和PARPI的反应。了解ZEB1/53BP1轴如何调节DSB 正常氧和缺氧的维修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 并靶向轴以降低治疗性抗性。