Deciphering the Role of Reductive Stress in Non Small Cell Lung Cancer

解读还原应激在非小细胞肺癌中的作用

基本信息

批准号：
10540372
负责人：
Liron Bar-Peled
金额：
$ 37.66万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-12-13 至 2026-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10540372
关键词：
Antioxidants Applications Grants Aspartate Biochemical Biologic Characteristic Biological Assay Biological Markers Biology CRISPR screen Cancer Model Cancer Patient Cancer cell line Cell Proliferation Cells Chemicals Companions Complex Cysteine Dependence Drug Metabolic Detoxication Electron Transport Environment Enzymes Equilibrium Foundations Functional disorder Gene Deletion Generations Genes Genetic Genetic Transcription Genomics Goals Growth Homeostasis Impairment In Vitro Lead Maintenance Malignant Neoplasms Malignant neoplasm of lung Measures Mediating Metabolic Metabolic Pathway Metabolism Methods Mitochondria Modeling Modification Mutation Non-Small-Cell Lung Carcinoma Oncogenic Oxidation-Reduction Oxidative Stress Pathway interactions Patients Pharmacology Play Point Mutation Production Proliferating Proteins Proteomics Reactive Oxygen Species Regulation Research Respiration Role Series Signal Transduction Signal Transduction Pathway Signaling Protein Stress Supplementation Technology Testing Therapeutic Tumor Suppressor Proteins Work anti-cancer therapeutic biomarker identification cancer cell cancer therapy cancer type cell growth cohort efficacy evaluation efficacy testing experimental study functional genomics genome-wide in vivo insight lung cancer cell metabolomics mitochondrial metabolism mutant next generation novel novel therapeutic intervention pharmacologic potential biomarker protein function reconstitution response small molecule inhibitor synergism therapeutic development therapeutic evaluation transcription factor transcriptomics translational therapeutics tumor growth

项目摘要

Project Summary Control of the redox homeostasis is essential to cancer cell proliferation and requires the delicate maintenance of oxidative and reductive metabolic pathways. This equilibrium is controlled by signal transduction pathways and imbalances lead to redox stress that potently blocks cancer growth. Much work has focused on the role of oxidative stress in cancer proliferation, however, the converse– reductive stress and its impact on malignant cells is poorly understood. We have studied the role of redox control in cancer in the context of modification of proteinaceous cysteines by reactive oxygen species and the NRF2 transcription factor pathway. NRF2 functions as the master regulator of the cellular antioxidant response and promotes the expression of key metabolic and detoxification genes to generate a reductive environment and negate oxidative stress. NRF2 is activated in many cancers including ~30% of non small cell lung cancers (NSCLC) through mutation of its negative regulator KEAP1. While NRF2 has been extensively studied in KEAP1-mutant NSCLCs, we wondered what role this pathway plays in the proliferation of NSCLC cell lines which are wildtype (WT) for KEAP1. To this end, we pharmacologically activated NRF2 in 50+ NSCLC cell lines (WT for KEAP1) and measured their proliferation. Unexpectedly, we find that in ~16% of NSCLC cell lines, NRF2 activation results in a severe block in proliferation. A genome wide CRISPR screen identifies that genes involved in mitochondrial metabolism, mitochondrial fusion and the electron transport chain (ETC) are major sensitizers to NRF2 activation when lost and can function as companion biomarkers for NRF2-sensitivity. In line with the generation of reductive stress following NRF2 activation, key cysteines on enzymes involved in mitochondrial metabolism and mitochondrial fusion are reduced as determined by chemical proteomic platforms. To explain these surprising biological characteristics we propose the following hypothesis: NRF2 activation in a subset of NSCLC cell lines promotes an overly reductive environment that decreases the activity of key enzymes in mitochondrial metabolism and mitochondrial respiration and fusion. The inactivation of these pathways synergize to block cell growth. In this grant application, we build on our research surrounding NRF2 sensitization and mechanistically characterize the role of reductive stress in NSCLC proliferation. In this grant application, we will comprehensively define KEAP1-dependence by identifying NRF2 regulation of mitochondrial metabolism/fusion at the protein, cellular and organismal levels. The research proposed herein, takes full advantage of a series of recently conceived methods: chemical proteomics, genome-wide CRISPR screens and untargeted metabolomics, which have previously been deployed in isolation, to be used in an integrated manner to effectively dissect how protein reduction underlies protein malfunction and KEAP1-dependence. These studies will not only provide a comprehensive understanding of NRF2/KEAP1 biology but may also lay the foundation for developing translational therapeutics to benefit lung cancer patients with deregulated NRF2 signaling.

项目摘要控制氧化还原稳态对于癌细胞增殖至关重要，需要精致的维护氧化和还原的代谢途径。该等效物受信号转导途径的控制不平衡导致氧化还原压力可能阻止癌症的生长。很多工作都集中在然而，癌症增殖中的氧化应激，相反的应激及其对恶性肿瘤的影响细胞知之甚少。我们已经研究了氧化还原控制在癌症中的作用活性氧和NRF2转录因子途径通过蛋白质半胱氨酸。 NRF2功能作为细胞抗氧化剂反应的主要调节剂，并促进了关键代谢和解毒基因产生降低的环境并消除氧化应激。 NRF2在许多中被激活癌症包括约30％的非小细胞肺癌（NSCLC）通过其阴性调节剂的突变 Keap1。虽然NRF2在KEAP1突变的NSCLC中已广泛研究，但我们想知道这是什么作用途径在KEAP1的NSCLC细胞系的增殖中发挥作用。为此，我们在50+ NSCLC细胞系（KEAP1的WT）中，药理学激活的NRF2并测量了它们的增殖。出乎意料的是，我们发现在约16％的NSCLC细胞系中，NRF2激活会导致严重的增殖阻滞。基因组宽CRISPR屏幕鉴定出涉及线粒体代谢，线粒体融合的基因当丢失时，电子传输链（ETC）是NRF2激活的主要传感器，并且可以用作 NRF2敏感性的伴侣生物标志物。与NRF2之后的减轻应力的产生一致激活，涉及线粒体代谢和线粒体融合的酶的关键半胱氨酸被降低由化学蛋白质组学平台确定。为了解释这些令人惊讶的生物学特征我们提案以下假设：NSCLC细胞系的子集中的NRF2激活促进了过度降低的降低线粒体代谢和线粒体中关键酶活性的环境呼吸和融合。这些途径的失活协同以阻断细胞生长。在此赠款申请中，我们以围绕NRF2灵敏度的研究为基础，并在机械上表征了减少的作用 NSCLC增殖中的压力。在此赠款应用中，我们将通过鉴定NRF2调节线粒体代谢/融合在蛋白质，细胞和有机水平上的调节。本文提出的研究充分利用了一系列最近构思的方法：化学蛋白质组学，全基因组CRISPR屏幕和非靶向代谢组学，以前曾经是孤立地部署，以整合的方式使用，以有效剖析蛋白质还原的基础蛋白质故障和KEAP1依赖性。这些研究不仅将提供全面的了解NRF2/KEAP1生物学，但也可能为发展转化疗法奠定基础受益于肺癌患者的NRF2信号传导。