Heavy metal-stimulated signal transduction: new metal-regulatory and -responsive mechanisms

重金属刺激的信号转导：新的金属调节和响应机制

基本信息

批准号：
10592144
负责人：
Matthew Oliver Ross
金额：
$ 8.76万
依托单位：
UNIVERSITY OF CHICAGO
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：
2022-12-01 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10592144
关键词：
A549 Airborne Particulate Matter Antineoplastic Agents Binding Binding Sites Biochemical Biological Biological Markers Biophysical Process Biophysics Cancer Prognosis Categories Cell Line Cell Surface Receptors Cells ChIP-seq Chelating Agents Cisplatin Complement Complex Development Disease EGF gene Elements Environment Environmental Exposure Environmental Pollutants Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Event Exhibits Exposure to Extracellular Domain Fingers Foundations Functional disorder Gene Expression Profile Genetic Transcription Genomics Geometry Goals Growth Health Heavy Metals Heme Human Human Cell Line In Vitro Link Location Lung MAP Kinase Gene Malignant Neoplasms Malignant neoplasm of lung Metal exposure Metals Modification Molecular Molecular Chaperones Mus Nerve Growth Factors Non-Small-Cell Lung Carcinoma Nutrient Pathway interactions Phenotype Phosphorylation Physiological Platinum Post-Transcriptional Regulation Prognostic Marker Proteins Receptor Protein-Tyrosine Kinases Regulation Regulator Genes Regulatory Pathway Repression Research Role SP1 gene Signal Transduction Signal Transduction Pathway Supplementation Techniques Tissue Sample Tissue-Specific Gene Expression Tissues Toxicology Transcript WT1 gene Work Xenograft Model anti-cancer biomarker identification carcinogenesis career cellular imaging colon cancer cell line crosslink experimental study exposure pathway extracellular gene repression genetic corepressor human disease human tissue in vivo inhibitor insight mutant next generation sequencing novel programs promoter receptor binding response sensor stable cell line stoichiometry synergism toxic metal trafficking transcription factor transcriptome sequencing transcriptomics tumor progression

项目摘要

PROJECT SUMMARY/ABSTRACT From a human physiological perspective, heavy metals (HM) are divided into two groups: essential (eHM) and toxic (tHM). eHM and tHM overexposure are both linked to numerous pathophysiological conditions; however, the signal transduction pathways and genomic activities stimulated by eHM dyshomeostasis and tHM exposure have yet to be adequately established. For example, the mammalian Cu-sensor protein responsible for relaying changes in extracellular Cu levels into signaling responses has not been identified. The goals of this application are to define (i) eHM- and tHM-stimulated and -regulatory signaling networks (including resultant genomic activities), and (ii) the underlying molecular biological, biochemical, and biophysical mechanisms. Through the work in this application, I have characterized the signaling and genomic responses induced by Cu exposure and identified a mechanism of Cu-stimulated epidermal growth factor receptor (EGFR, a cell surface receptor tyrosine kinase (RTK)) signal transduction along the MAPK/ERK pathway, thereby identifying EGFR as a candidate Cu- sensor protein. Biochemical experiments are underway investigating the EGFR-Cu binding site and geometry. Relatedly, through application of newly-developed next generation sequencing (NGS), molecular biological, and transcriptomic (in vitro and in vivo) techniques, I discovered a novel mechanism by which intracellular Cu levels are regulated. Elevated extracellular Cu levels stimulate EGFR/MAPK/ERK activation, which induces expression of the transcription factor (TF) EGR1 and EGR1-corepressor NAB1/2; the EGR1-NAB1/2 complex subsequently represses transcription of the mammalian Cu-importer, CTR1. EGR1 ChIP-seq experiments are underway for further corroboration. This work will pair with experiments assessing synergism of an inhibitor of Cu-chaperones and MAPK/ERK signaling (to increase CTR1 levels) with a platinum anticancer drug (cisplatin, imported via CTR1), as well as genomic insights into the TF activities of EGR1 in EGFR-driven lung cancer (a role that is currently unknown). My research establishing the Cu-regulatory network lays the foundation for my planned independent career (i) evaluating eHM- and tHM-regulatory and -responsive mechanisms and (ii) characterizing disruption of native signaling by tHM. In my independent career, I will apply phosphorylation arrays and NGS techniques to cells stimulated with non-Cu eHM and high-priority environmental pollutant tHM, with emphasis on the effects of tHM exposure in disrupting the eHM-regulatory and -responsive signal transduction networks. In summary, my identification of EGFR as a conduit for relaying elevated extracellular Cu levels into genomic activities via the MAPK/ERK pathway has linked a major signal transduction network (implicated in cancer pathophysiology, for example) to eHM-regulation, and thereby opens new avenues of understanding HM- regulatory networks in general. More broadly, the comprehensive research program established in this proposal stands to identify biomarkers of harm preceding development and progression of cancer and other human diseases, establish novel HM-regulatory mechanisms, and enrich tHM-toxicological mechanistic understanding.

项目概要/摘要从人体生理角度来看，重金属（HM）分为两类：必需金属（eHM）和有毒（tHM）。 eHM 和 tHM 过度暴露均与多种病理生理状况有关；然而， eHM 稳态失调和 tHM 暴露刺激的信号转导途径和基因组活性尚未得到充分确立。例如，哺乳动物的铜传感器蛋白负责传递细胞外铜水平变化对信号反应的影响尚未确定。此应用程序的目标定义 (i) eHM 和 tHM 刺激和调节信号网络（包括由此产生的基因组活动），以及（ii）潜在的分子生物学、生化和生物物理机制。通过在这个应用程序的工作中，我描述了铜暴露和诱导的信号传导和基因组反应确定了铜刺激表皮生长因子受体（EGFR，一种细胞表面受体酪氨酸）的机制激酶（RTK））沿 MAPK/ERK 途径进行信号转导，从而将 EGFR 鉴定为候选 Cu- 传感器蛋白。生化实验正在进行中，研究 EGFR-Cu 结合位点和几何结构。相关地，通过应用新开发的下一代测序（NGS）、分子生物学和转录组（体外和体内）技术，我发现了一种新机制，通过该机制细胞内铜水平受到监管。细胞外铜水平升高刺激 EGFR/MAPK/ERK 激活，从而诱导表达转录因子 (TF) EGR1 和 EGR1 辅阻遏物 NAB1/2；随后EGR1-NAB1/2复合物抑制哺乳动物铜输入蛋白 CTR1 的转录。 EGR1 ChIP-seq 实验正在进行中进一步佐证。这项工作将与评估铜伴侣抑制剂协同作用的实验相结合和 MAPK/ERK 信号传导（以增加 CTR1 水平）与铂类抗癌药物（顺铂，通过进口） CTR1），以及对 EGR1 在 EGFR 驱动的肺癌中的 TF 活性的基因组见解（这一作用是目前未知）。我建立铜监管网络的研究为我的计划奠定了基础独立职业 (i) 评估 eHM 和 tHM 监管和响应机制以及 (ii) 表征 tHM 破坏天然信号传导。在我的独立职业生涯中，我将应用磷酸化芯片和NGS 使用非 Cu eHM 和高优先级环境污染物 tHM 刺激细胞的技术，重点是 tHM 暴露对破坏 eHM 调节和响应信号转导网络的影响。在总结一下，我认为 EGFR 作为将升高的细胞外铜水平传递到基因组的渠道通过 MAPK/ERK 通路的活动连接了一个主要的信号转导网络（与癌症有关）例如，病理生理学）到 eHM 调节，从而开辟了理解 HM 的新途径总体监管网络。更广泛地说，本提案中建立的综合研究计划旨在识别癌症和其他人类疾病发生和进展之前的危害生物标志物疾病，建立新的 HM 调节机制，并丰富 tHM 毒理学机制的理解。