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.

项目摘要 转移癌细胞的适应性尚不清楚，导致缺乏 有效的疗法，使转移成为与癌症相关死亡的主要原因。转移性的遗传驱动因素 尚未确定进展，因此必须在转录和 翻译水平，细胞能够进行可逆的适应性，使它们能够生存 转移性级联。我们的长期目标是通过通过 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 经历高水平的氧化应激。硒代半胱氨酸，第21氨基酸，将 参与排毒活性氧并维持细胞中的氧化还原平衡的蛋白质。 细胞应激下硒蛋白的翻译受到摇摆的单个2'-O-核糖甲基化调节 硒素半胱氨酸tRNA（TRNASEC）的尿苷（UM34），由未知的甲基转移酶完成。使用我们的 LAB的患者衍生的黑色素瘤转移肿瘤模型，我将测试UM34甲基化的假设 在转移细胞中增加，这种修饰会在氧化下增加细胞存活。 通过控制应力反应硒蛋白的子集的压力。为了解决这个问题，我将使用一个 建立的患者衍生的黑色素瘤模型，其中转移可以预测患者的结局与 体外黑色素瘤细胞系。在AIM 1中，我将确定UM34在转移和氧化中的功能作用 压力反应。我将开发用于测量UM34级别的工具，并通过定位来扰动修改事件 相关酶，TRNASEC和硒的可用性。在AIM 2中，我将确定未知的UM34甲基转移酶 通过测试候选蛋白质并通过靶向免疫沉淀方法。我会用弥撒 量化UM34水平并鉴定相关蛋白质复合物的光谱法。然后我将表征 硒代半胱氨酸应激反应和通过鉴定的甲基转移酶的部署，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Weill Cornell完成 我将科学和专业地发展成独立研究人员的医学。我会收到的 我的世界知名科学家委员会的整个项目的专家指导，其专业知识跨度 我的提议中涵盖的许多领域。通过这项工作，我将掌握生化分析技术， 在体内建模复杂的疾病，并清楚地传达我的想法和结果 - 所有这些都将 为我准备职业，担任学术科学独立研究者。确定新颖的目标 转移性疾病是一种紧急疗法，我相信这项工作不仅会找到一种，而且会扩大 当前对癌细胞如何适应和生存的理解。