Regulation of the Selenocysteine Stress Response in Cancer Metastasis

项目总结 转移的癌细胞所经历的适应还不是很清楚，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。转移瘤的遗传驱动因素 进展尚未确定，因此必须从转录和转录水平研究该病。 翻译水平，即细胞能够进行可逆的适应，使其能够在整个 转移性级联。我们的长期目标是通过以下方式发现癌症转移的新的和有针对性的弱点 确定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。硒半胱氨酸，第21个氨基酸，被合并到一个家族 参与解毒活性氧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受摆动上单个2‘-O-核糖甲基化的调节 硒半胱氨酸tRNA(TRNASec)的尿苷(Um34)，由一种未知的甲基转移酶完成。使用我们的 Lab的黑色素瘤转移患者来源的肿瘤模型，我将测试Um34甲基化的假设 在转移细胞中增加，这种修饰提高了细胞在氧化作用下的存活率 通过调节应激反应硒蛋白的一个子集来调节应激。为了解决这个问题，我将利用 建立了患者来源的黑色素瘤模型，其中转移与患者预后平行预测 体外培养黑色素瘤细胞株。在目标1中，我将确定Um34在转移和氧化中的功能作用。 压力反应。我会开发工具来测量Um34水平，我会通过瞄准目标来扰乱修改事件 相关酶、tRNASec和硒的有效性。在目标2中，我将确定未知的Um34甲基转移酶 通过测试候选蛋白质和通过有针对性的免疫沉淀方法。我会用弥撒 光谱分析以量化Um34水平并识别相关蛋白质复合体。然后，我将描述 硒半胱氨酸的应激反应和通过耗尽已鉴定的甲基转移酶，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完成 在医学方面，我将以科学和专业的方式发展成为一名独立的研究人员。我会收到 在我的项目过程中，由世界知名科学家组成的委员会提供了专家指导，他们的专业知识涵盖了 我的提案所涵盖的许多领域。通过这项工作，我将掌握生化分析技术， 在体内模拟一种复杂的疾病，并清楚地传达我的想法和结果-所有这些都将 让我做好准备，成为一名学术科学领域的独立研究员。确定新的目标 转移性疾病是一种迫切的治疗需求，我相信这项工作不仅会找到一个，而且会扩大 目前对转移性癌细胞如何适应和生存的理解。

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.