Regulation of the Selenocysteine Stress Response in Cancer Metastasis

癌症转移中硒代半胱氨酸应激反应的调节

• 批准号: 10445241
• 负责人: Leona Nease
• 金额: $ 4.68万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-14 至 2023-07-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10445241
• 关键词: Address; Amino Acids; Anabolism; Anticodon; Binding Proteins; Biochemical; Biological Assay; Biology; Biotin; Cancer Etiology; Cause of Death; Cell Line; Cell Survival; Cells; Cellular Stress; Cellular Stress Response; Cessation of life; Codon Nucleotides; Cysteine; Development; Diet; Disease; Endoplasmic Reticulum; Enzymes; Equilibrium; Event; Family; Genetic; Genetic Transcription; Goals; Human; Immunoprecipitation; In Vitro; Investigation; Laboratories; Link; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Medicine; Melanoma Cell; Metabolic; Metastatic Melanoma; Methods; Methylation; Methyltransferase; Modeling; Modification; Molecular; Molecular Chaperones; Molecular Conformation; Mus; Mutate; Neoplasm Metastasis; Nodule; Nucleosides; Oxidation-Reduction; Oxidative Stress; Patient-Focused Outcomes; Patients; Pharmacology; Primary Neoplasm; Protein Family; Proteins; Reactive Oxygen Species; Regulation; Reporter; Research Personnel; Ribose; Role; Science; Scientist; Selenium; Selenocysteine; Site-Directed Mutagenesis; Solid Neoplasm; Speed; Stress; Sulfur; Techniques; Terminator Codon; Testing; Therapeutic; Trans-Activators; Transfer RNA; Translational Regulation; Translations; Tumor-Derived; Uridine; Work; X-linked mental retardation 9; Yeasts; antioxidant enzyme; base; biological adaptation to stress; cancer cell; career; crosslink; effective therapy; glutathione peroxidase; in vivo; insight; liquid chromatography mass spectrometry; malignant breast neoplasm; melanoma; novel; patient derived xenograft model; protein complex; protein folding; rat Secp43 protein; selenocysteine-tRNA; selenoprotein; small hairpin RNA; tRNA Methyltransferases; thioredoxin reductase; tool

PROJECT SUMMARY The adaptations that metastasizing cancer cells undergo are not well understood, contributing to a lack of effective therapies, making metastasis the leading cause of cancer-related deaths. Genetic drivers of metastatic progression have not been identified, thus it is imperative to study the disease at the transcriptional and translational level where cells are able to make reversible adaptations that allow them to survive throughout the metastatic cascade. Our long-term goal is to find novel and targetable vulnerabilities in cancer metastasis by identifying these molecular and metabolic adaptations. Early work from our lab shows that metastasizing cells undergo high levels of oxidative stress. Selenocysteine, the 21st amino acid, is incorporated into a family of proteins that are involved in detoxifying reactive oxygen species and maintaining redox balance in the cell. Translation of selenoproteins under cellular stress is regulated by a single 2’-O-ribose methylation on the wobble uridine (Um34) of the selenocysteine tRNA (tRNASec), completed by an unknown methyltransferase. Using our lab’s patient-derived tumor model of melanoma metastasis, I will test the hypothesis that Um34 methylation is increased in metastasizing cells and that this modification increases cell survival under oxidative stress by regulating a subset of stress response selenoproteins. To address this question, I will utilize an established patient-derived melanoma model where metastasis is predictive of patient outcome in parallel with melanoma cell lines in vitro. In Aim 1, I will determine the functional role of Um34 in metastasis and the oxidative stress response. I will develop tools to measure Um34 levels and I will perturb the modification event by targeting related enzymes, tRNASec and selenium availability. In Aim 2, I will identify the unknown Um34 methyltransferase by testing a candidate protein and through targeted immunoprecipitation approaches. I will use mass spectrometry to quantify Um34 levels and identify related protein complexes. I will then characterize the selenocysteine stress response and through depletion of the identified methyltransferase, I will determine its functional role in metastasis and the oxidative stress response. This work will be completed at Weill Cornell Medicine where I will develop scientifically and professionally into an independent researcher. I will receive expert guidance throughout my project from my committee of world-renowned scientists whose expertise span the many fields covered in my proposal. Through this work I will master biochemical analytical techniques, modeling a complicated disease in vivo and clearly communicating my ideas and results – all of which will prepare me for a career as an independent investigator in academic science. Identifying novel targets in metastatic disease is an urgent therapeutic need and I believe that this work will not only find one, but expand the current understanding of how metastasizing cancer cells adapt and survive.

项目概要 转移癌细胞所经历的适应尚不清楚，导致缺乏 有效的治疗方法使转移成为癌症相关死亡的主要原因。转移的遗传驱动因素 进展尚未确定，因此有必要在转录和转录方面研究该疾病 翻译水平，细胞能够进行可逆的适应，使它们能够在整个生命周期中生存 转移级联。我们的长期目标是通过以下方式找到癌症转移中新颖且可针对的脆弱性： 识别这些分子和代谢适应。我们实验室的早期工作表明，转移细胞 承受高水平的氧化应激。硒代半胱氨酸是第 21 个氨基酸，被纳入一个家族中 参与活性氧解毒和维持细胞内氧化还原平衡的蛋白质。 细胞应激下硒蛋白的翻译由摆动上的单个 2'-O-核糖甲基化调节 硒代半胱氨酸 tRNA (tRNASec) 的尿苷 (Um34)，由未知的甲基转移酶完成。使用我们的 实验室的患者来源的黑色素瘤转移肿瘤模型，我将检验 Um34 甲基化的假设 在转移细胞中增加，并且这种修饰增加了细胞在氧化条件下的存活率 通过调节应激反应硒蛋白的子集来调节应激。为了解决这个问题，我将利用 建立了源自患者的黑色素瘤模型，其中转移可同时预测患者的结果 体外黑色素瘤细胞系。在目标 1 中，我将确定 Um34 在转移和氧化中的功能作用 应激反应。我将开发工具来测量 Um34 水平，并且我将通过瞄准来干扰修改事件 相关酶、tRNASec 和硒的可用性。在目标 2 中，我将识别未知的 Um34 甲基转移酶 通过测试候选蛋白质和通过靶向免疫沉淀方法。我将使用质量 光谱法定量 Um34 水平并鉴定相关蛋白质复合物。然后我将描述 硒代半胱氨酸应激反应，并通过耗尽已识别的甲基转移酶，我将确定其 在转移和氧化应激反应中的功能作用。这项工作将在威尔康奈尔大学完成 医学方面我将科学、专业地发展成为一名独立的研究者。我会收到 我的世界知名科学家委员会在整个项目中提供专家指导，他们的专业知识涵盖 我的提案涵盖了许多领域。通过这项工作我将掌握生化分析技术， 体内模拟复杂的疾病并清楚地传达我的想法和结果 - 所有这些都将 为我作为学术科学领域的独立研究者的职业生涯做好准备。识别新目标 转移性疾病是一种紧迫的治疗需求，我相信这项工作不仅会找到一种转移性疾病，而且会扩展 目前对转移癌细胞如何适应和生存的理解。