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.

尽管活性氧 (ROS) 在 Cr(VI) 诱导的恶性细胞中被认为很重要 的转化，其背后的机制还有待研究。最近的研究表明，ROS 在诱导自噬和随后 p62 的上调中很重要。我们的初步结果显示 人支气管上皮 (BEAS-2B) 细胞长期暴露于 Cr(VI) 会上调 p62，并且长期暴露于 Cr(VI) 会上调 p62 动物暴露于 Cr(VI) 颗粒后，肺部的这种蛋白质上调。 BEAS 中 p62 的强制表达 2B细胞引起这些细胞的恶性转化并在异种移植模型中产生肿瘤。因此，它是 在 BEAS-2B 细胞中，ROS 可能上调 p62 及其下游，从而导致细胞转化。我们的 初步研究表明，在 Cr(VI) 转化细胞中，p62 上调。其中六大主要 p62 的结构域/区域，我们将研究 (a) TRAF6 结合结构域，它与 TRAF6 结合并导致其 磷酸化，导致 NF-κB 激活，以及 (b) Keap 相互作用区域，与 Keap1 结合 （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 Cr(VI) 激活NADPH氧化酶，产生ROS，然后上调p62，导致细胞恶性转化 在 Cr(VI) 转化细胞中，p62 通过 TRAF6 结合域激活 NF-κB，并导致 通过 Keap 相互作用区域组成型 Nrf2 激活，并随后上调其下游抗 炎症、抗氧化和抗凋亡蛋白，导致细胞存活率和肿瘤发生增加 这些转化的细胞。目标 1 将检验正常细胞中 Cr(VI) 激活 NADPH 氧化酶的假设， 产生 ROS，并上调 p62，导致恶性细胞转化。我们将进行以下工作 研究。 (a) Cr(VI) 产生的 ROS 上调 p62。我们将抑制ROS来显示p62的减少。 (b) 我们 将抑制 ROS 或 p62，以证明对 Cr(VI) 诱导的恶性细胞转化的抑制。 (c) 我们将 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 恶性转化。目标 2 将检验 p62 通过其 TRAF6- 上调 NF-κB 的假设 p62 通过其 Keap 相互作用区引起 Nrf2 组成型激活，从而导致 产生有利于 Cr(VI) 转化细胞生存的微环境以及随后的 肿瘤发生。我们将展示 (a) TRAF6 结合域在 NF-κB 激活中的作用； (b) 凯普的角色- Nrf2 组成型激活及其下游炎症、抗氧化、 和抗凋亡蛋白； (c) p62 及其下游 NF-κB 和 Nrf2 通路在 Cr(VI) 转化细胞的肿瘤发生。目标 3 将研究 p62 在 Cr(VI) 机制中的作用 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 长期接触 Cr(VI) 的动物和职业接触 Cr(VI) 的工人的肺部问题 加强细胞和动物 Cr(VI) 暴露研究。