The role of p62 in the mechanism of Cr(VI) carcinogenesis

p62在Cr(VI)致癌机制中的作用

• 批准号: 9753486
• 负责人: Xianglin Shi
• 金额: $ 34.43万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-08 至 2019-08-22
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9753486
• 关键词: Animals; Anti-inflammatory; Antioxidants; Apoptosis; Apoptotic; Autophagocytosis; BCL2 gene; Binding; Cell Survival; Cells; Chronic; Development; Drug resistance; Epithelial; Epithelial Cells; Exhibits; Exposure to; Generations; Genes; Inflammation; Inflammatory; Investigation; Knockout Mice; Laboratories; Lung; Malignant - descriptor; Malignant Neoplasms; Measures; Mus; NADPH Oxidase; Normal Cell; Occupational Exposure; Oncogenic; Oxidative Stress; Pathway interactions; Phosphorylation; Play; Poison; Process; Property; Proteins; Reactive Oxygen Species; Response Elements; Role; Signal Pathway; Signal Transduction; Structure of parenchyma of lung; TNF gene; TRAF6 gene; Testing; Up-Regulation; Xenograft Model; animal tissue; cancer cell; carcinogenesis; carcinogenicity; catalase; cell transformation; chromium hexavalent ion; exposed human population; human tissue; inhibitor/antagonist; particle; promoter; transcription factor; transcriptome; transcriptome sequencing; tumor; tumorigenesis

Although reactive oxygen species (ROS) are considered important in Cr(VI)-induced malignant cell transformation, the underlying mechanism remains to be investigated. Recent studies show that ROS are important in the induction of autophagy and subsequently upregulation of p62. Our preliminary results show that chronic exposure of human bronchial epithelial (BEAS-2B) cells to Cr(VI) upregulated p62 and that chronic exposure of animals to Cr(VI) particles upregulated this protein in the lung. Forced expression of p62 in BEAS- 2B cells caused malignant transformation of these cells and generated tumor in xenograft model. Thus, it is likely that in BEAS-2B cells ROS upregulate p62 and its downstream, leading to cell transformation. Our preliminary studies show that in Cr(VI)-transformed cells p62 was upregulated. Among six main domains/regions of p62, we will study (a) the TRAF6-binding domain, which binds to TRAF6 and causes its phosphorylation, leading to activation of NF-κB, and (b) the Keap-interacting region, which binds to Keap1 (Nrf2 inhibitor) and causes constitutive activation of Nrf2. The central hypothesis is that in normal cells Cr(VI) activates NADPH oxidase, generates ROS, and then upregulates p62, leading to malignant cell transformation and that in Cr(VI)-transformed cells p62 activates NF-κB through TRAF6-binding domain and causes constitutive Nrf2 activation through Keap-interacting region and subsequently upregulates its downstream anti- inflammatory, antioxidant, and anti-apoptotic proteins, resulting in increased survival and tumorigenesis of these transformed cells. Aim 1 will test the hypothesis that in normal cells Cr(VI) activates NADPH oxidase, generates ROS, and upregulates p62, leading to malignant cell transformation. We will carry out the following studies. (a) ROS generated by Cr(VI) upregulate p62. We will inhibit ROS to show the decrease of p62. (b) We will inhibit ROS or p62 to demonstrate the inhibition of Cr(VI)-induced malignant cell transformation. (c) We will use whole transcriptome (RNA Seq) analysis to identify p62 downstream genes responsible for Cr(VI)-induced malignantly transformation. Aim 2 will test the hypothesis that p62 upregulates NF-κB through its TRAF6- binding domain and that p62 causes constitutive Nrf2 activation through its Keap-interacting region, resulting in the generation of microenvironment favorable for survival of Cr(VI)-transformed cells and subsequent tumorigenesis. We will show (a) the role of TRAF6-binding domain in NF-κB activation; (b) the role of Keap- interacting region in constitutive Nrf2 activation and upregulation of its downstream inflammatory, antioxidant, and anti-apoptotic proteins; and (c) the role of p62 and its downstream NF-κB and Nrf2 pathways in tumorigenesis of Cr(VI)-transformed cells. Aim 3 will investigate the role of p62 in the mechanism of Cr(VI) carcinogenesis using p62 wildtype and knockout mice. We will measure p62 and its downstream proteins in lung issues of animals chronically exposed to Cr(VI) and of workers occupationally exposed to Cr(VI) to strengthen the cellular and animal Cr(VI) exposure studies.

尽管反应性氧（ROS）在CR（VI）诱导的恶性细胞中被认为很重要 转化，基本机制仍有待研究。最近的研究表明ROS是 对于诱导自噬和随后上调p62重要。我们的初步结果显示 人支气管上皮（BEAS-2B）细胞的长期暴露于Cr（VI）更新P62和慢性 动物暴露于Cr（VI）颗粒中，在肺中更新了该蛋白质。 p62强迫表达在beas- 2b细胞在Xenographic模型中引起这些细胞的恶性转化并产生肿瘤。那是 在Beas-2b细胞中，ROS上调p62及其下游，导致细胞转化。我们的 初步研究表明，在CR（VI）转化的细胞中，P62更新了。六个主要 p62的域/区域，我们将研究（a）traf6结合域，该域与traf6结合并导致其 磷酸化，导致NF-κB的激活，以及（b）与Keap1结合的Keap相互作用区域 （NRF2抑制剂）并引起NRF2的组成型激活。中心假设是在正常细胞中（VI） 激活NADPH氧化酶，产生ROS，然后上调p62，导致恶性细胞转化 在CR（VI）转化的细胞中，P62通过TRAF6结合域激活NF-κB，并引起原因 构成NRF2通过KEAP相互作用区域激活，随后上调其下游抗 - 炎症，抗氧化剂和抗凋亡蛋白，导致生存率增加和肿瘤发生 这些转化的细胞。 AIM 1将检验以下假设：正常细胞中Cr（VI）激活NADPH氧化物， 产生ROS并上调p62，导致恶性细胞转化。我们将执行以下 研究。 （a）Cr（VI）产生的ROS上调p62。我们将抑制ROS显示p62的减少。 （b）我们 将抑制ROS或P62以证明CR（VI）诱导的恶性细胞转化的抑制。 （c）我们会的 使用全转录组（RNA SEQ）分析来识别负责CR（VI）诱导的p62下游基因 恶性转变。 AIM 2将检验p62通过其TRAF6-上调NF-κB的假设 结合域，p62通过其Keap交互区域引起构造NRF2激活，导致 有利于CR（VI）转化细胞生存的微环境的产生 肿瘤发生。我们将显示（a）TRAF6结合结构域在NF-κB激活中的作用； （b）Keap-的作用 本构nrf2激活中的相互作用区域及其下游炎症，抗氧化剂的上调， 和抗凋亡蛋白； （c）p62及其下游NF-κB和NRF2途径的作用 CR（VI）转化细胞的肿瘤发生。 AIM 3将研究p62在CR（VI）机理中的作用 使用p62野生型和基因敲除小鼠进行致癌作用。我们将测量P62及其下游蛋白 长期暴露于CR（VI）的动物的肺部问题和占用CR（VI）的工人的肺部问题 加强细胞和动物CR（VI）暴露研究。