Redox and Protein Homeostasis in Arsenic Tumorigenicity

砷致瘤性中的氧化还原和蛋白质稳态

• 批准号: 10613495
• 负责人: Eli Chapman
• 金额: $ 29.9万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-09 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10613495
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Agar; Air; Antioxidants; Arsenic; Autophagosome; Binding; CRISPR/Cas technology; CUL3 gene; Cancer Model; Carcinogenicity Tests; Carcinogens; Cell Line; Cells; Cellular Stress; Chronic; Classification; Clinical; Clinical Trials; Complex; Cytoprotection; Data; Developing Countries; Disabled Persons; Dose; Ensure; Environment; Environmental Carcinogens; Epithelial Cells; Event; Exposure to; Feedback; Genes; Genetic Transcription; Growth; Health; Homeostasis; Human; In Vitro; Infection; Isogenic transplantation; Laboratories; Letters; Lung; Lung Neoplasms; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of urinary bladder; Mediating; Modeling; Molecular; Molecular Chaperones; Mus; Mutagenesis; Mutate; Mutation; Nude Mice; Oxidation-Reduction; Oxidative Stress; Persons; Play; Procedures; Proteins; RBX1 gene; Reporting; Response Elements; Risk; Role; Skin Cancer; Soil; Stress; Testing; Therapeutic; Transformed Cell Line; Tumor Tissue; Tumorigenicity; Up-Regulation; Xenograft Model; biological adaptation to stress; carcinogenesis; contaminated water; in vivo; inhibitor; metaplastic cell transformation; mouse model; multicatalytic endopeptidase complex; novel; pre-clinical; prevent; promoter; proteostasis; proteotoxicity; response; segregation; subcutaneous; tumor; ubiquitin-protein ligase

PROJECT SUMMARY Arsenic is classified as a human carcinogen that has been associated with increased risk of developing cancers of the lung, skin, and bladder. However, the mechanism of arsenic tumorgenicity remains incompletely described. Arsenic has been shown to upregulate NRF2, the master regulator of the cellular stress response in vitro and in vivo. Under non-stress conditions, the basal level of NRF2 is kept low by KEAP1-CUL3-RBX1- mediated ubiquitylation and subsequent 26S proteasomal degradation. During canonical NRF2 activation KEAP1-Cys151 is modified, preventing NRF2 ubiquitylation and increasing the level of NRF2 and its downstream genes to protect cells from environmental insults. Once cellular homeostasis is restored, NRF2 returns to low basal level, ensuring transient NRF2 upregulation. However, we discovered arsenic activates NRF2 by a non- canonical mechanism. Environmentally relevant doses of arsenic block autophagosome maturation, which leads to p62-mediated sequestration of KEAP1 into autophagosomes, blocking NRF2 ubiquitylation and degradation. This leads to sustained hyperexpression of NRF2 and its antioxidant response element (ARE) containing target genes, conferring a cellular survival advantage. Interestingly, our novel unpublished results demonstrate that the ATPase associated with various cellular activities (AAA+) chaperone, p97, contains a functional ARE in its promoter. Functionally, p97 uses the binding and hydrolysis of ATP to generate a force to segregate ubiquitylated proteins from other biomolecules, often for subsequent degradation by the proteasome. We have demonstrated that p97 negatively regulates NRF2 by extracting ubiquitinated NRF2 from the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex that facilitates NRF2 degradation by proteasome. This segregase activity of p97 plays a prominent role in protein homeostasis – proteostasis. Our finding of p97 as a novel NRF2 target gene indicates that NRF2 controls not only oxidative stress but also proteotoxic stress. Like NRF2, hyperexpression of p97 in human tumors has been reported and p97 is considered a target for treating cancer. We also observed that the protein levels of both NRF2 and p97 were increased in arsenic transformed cell lines and in human lung tumor tissues (preliminary data). Based on these findings, we propose the following model: Under basal conditions, low level expression of NRF2 and p97 is maintained through the NRF2-p97-NRF2 negative feedback loop. However, during chronic arsenic exposure, this NRF2-p97-NRF2 negative feed bask loop is disabled since NRF2 is no longer ubiquitylated, resulting in sustained upregulation of both NRF2 and p97. Thus, arsenic increases both the oxidative stress response and the proteotoxic stress response. We will test our hypothesis that sustained NRF2 and NRF2-mediated p97 upregulation in response to environmental arsenic exposure provides a survival advantage that enables arsenic-exposed cells to survive both oxidative and proteotoxic stress and to accumulate sufficient molecular events that drive malignant transformation.

项目摘要 砷被列为人类致癌物质，与癌症风险增加有关 肺皮肤和膀胱然而，砷致瘤的机制仍不完全 介绍了砷已被证明上调NRF 2，NRF 2是细胞应激反应的主要调节因子， 体外和体内。在非胁迫条件下，NRF 2的基础水平被KEAP 1-CUL 3-RBX 1-BX 1-B 介导的泛素化和随后的26 S蛋白酶体降解。在典型NRF 2激活期间 KEAP 1-Cys 151被修饰，阻止NRF 2泛素化并增加NRF 2及其下游的水平。 保护细胞免受环境侵害的基因。一旦细胞内稳态恢复，NRF 2恢复到低水平， 基础水平，确保瞬时NRF 2上调。然而，我们发现砷激活NRF 2的非- 规范机制环境相关剂量的砷阻断自噬体成熟， p62介导的KEAP 1进入自噬体的隔离，阻断NRF 2泛素化和降解。 这导致NRF 2及其含有靶点的抗氧化反应元件（ARE）的持续高表达。 基因，赋予细胞生存优势。有趣的是，我们新的未发表的结果表明， 与各种细胞活性相关的ATP酶（AAA+）伴侣蛋白，p97，在其分子中含有功能性ARE。 启动子在功能上，p97利用ATP的结合和水解来产生分离泛素化的力。 蛋白质从其他生物分子中分离出来，通常随后被蛋白酶体降解。我们已经证明 p97通过从KEAP 1-CUL 3-RBX 1 E3泛素中提取泛素化的NRF 2来负调控NRF 2 连接酶复合物，促进蛋白酶体降解NRF 2。p97的这种分离酶活性起着重要作用。 在蛋白质稳态中的重要作用。我们发现p97是一个新的NRF 2靶基因，这表明 NRF 2不仅控制氧化应激，还控制蛋白毒性应激。与NRF 2一样，p97在 已经报道了人肿瘤，并且p97被认为是治疗癌症的靶点。我们还观察到， 在砷转化细胞系和人肺肿瘤中，NRF 2和p97蛋白水平均升高 组织（初步数据）。基于这些发现，我们提出了以下模型：在基础条件下， 通过NRF 2-p97-NRF 2负反馈环维持NRF 2和p97的低水平表达。 然而，在慢性砷暴露期间，由于NRF 2-p97-NRF 2负反馈晒环被禁用， 不再被泛素化，导致NRF 2和p97的持续上调。因此，砷增加 氧化应激反应和蛋白毒性应激反应。我们将测试我们的假设， 环境砷暴露后NRF 2和NRF 2介导的p97持续上调 提供了一种生存优势，使砷暴露的细胞能够在氧化和 蛋白毒性应激和积累足够的分子事件，驱动恶性转化。