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

• 批准号: 10365388
• 负责人: Liron Bar-Peled
• 金额: $ 38.43万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 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/10365388
• 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; Oxides; Pathway interactions; Patients; Pharmacology; Play; Point Mutation; Production; 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; base; cancer cell; cancer therapy; cancer type; cell growth; cohort; efficacy testing; experimental study; functional genomics; genome-wide; in vivo; insight; lung cancer cell; metabolomics; mitochondrial metabolism; mutant; next generation; novel; novel therapeutic intervention; potential biomarker; protein function; reconstitution; response; small molecule inhibitor; stress reduction; therapeutic development; therapeutic evaluation; transcription factor; transcriptomics; translational therapeutics; tumor growth

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 控制氧化还原动态平衡对癌细胞的增殖至关重要，需要微妙的维护 氧化和还原代谢途径。这种平衡是由信号转导途径控制的 而失衡会导致氧化还原压力，从而有力地阻止癌症的生长。许多工作都集中在 然而，氧化应激在肿瘤增殖中的反向还原应激及其对恶性肿瘤的影响 人们对细胞知之甚少。我们研究了氧化还原控制在癌症中的作用。 蛋白质类半胱氨酸通过活性氧和NRF2转录因子途径。NRF2函数 作为细胞抗氧化反应的主要调节者，并促进关键代谢和 解毒基因，产生还原环境，消除氧化应激。NRF2在许多情况下被激活 包括约30%的非小细胞肺癌(NSCLC)在内的肿瘤通过其负调控因子的突变 Keap1。虽然NRF2在Keap1突变的NSCLC中已经得到了广泛的研究，但我们想知道这是什么作用 途径在野生型(WT)Keap1非小细胞肺癌细胞系的增殖中起作用。为此，我们 药物激活的NRF2在50+NSCLC细胞系(WT为Keap1)中，并检测其增殖情况。 出乎意料的是，我们发现在~16%的非小细胞肺癌细胞系中，NRF2的激活导致了严重的增殖抑制。 全基因组CRISPR筛查发现参与线粒体新陈代谢、线粒体融合的基因 而电子传输链(ETC)在丢失时是NRF2激活的主要敏感剂，可以作为 NRF2敏感性的伴随生物标志物。与NRF2之后的还原应力的产生相一致 参与线粒体新陈代谢和线粒体融合的酶的关键半胱氨酸的激活减少 由化学蛋白质组平台决定。为了解释这些令人惊讶的生物学特征，我们 提出以下假设：Nrf2在NSCLC细胞亚群中的激活促进过度还原 降低线粒体代谢和线粒体关键酶活性的环境 呼吸和融合。这些途径的失活协同作用，阻止了细胞的生长。在这项拨款申请中， 我们建立在我们围绕NRF2敏化的研究基础上，并从机械上表征了还原剂的作用 NSCLC增殖中的应激反应。在这项拨款申请中，我们将通过以下方式全面定义Keap1依赖 在蛋白质、细胞和生物水平上确定NRF2对线粒体代谢/融合的调控。 在这里提出的研究，充分利用了最近构思的一系列方法：化学 蛋白质组学、全基因组CRISPR筛查和非靶向代谢组学，这些都是以前 单独部署，以集成的方式使用，有效地剖析蛋白质减少的基础 蛋白质功能障碍和Keap1依赖。这些研究不仅将提供全面的 对NRF2/Keap1生物学的理解，但也可能为开发翻译治疗学奠定基础 通过去调控的NRF2信号使肺癌患者受益。