The role of phosphorylation of isocitrate dehydrogenase in breast cancer

项目概要-项目负责人范晨光 乳腺癌是对妇女健康的最大威胁之一。大约八分之一的美国女性（约12%）将发展为侵袭性 患乳腺癌的几率。以前的研究已经表明了一把钥匙的重要作用 三羧酸循环（TCA）酶异柠檬酸脱氢酶（IDH）在乳腺癌中的作用。各种IDH 已经观察到包括体细胞突变、异常表达和改变的活性在内的改变， 乳腺癌患者。最近，乳腺癌细胞的磷酸化蛋白质组学研究已经确定了几个独特的 在IDH同种型中存在磷酸化位点，并且这些位点对不同类型的乳腺癌是特异性的。等 异质性对有效的诊断和治疗提出了挑战，因此迫切需要发现 IDH磷酸化对乳腺癌的特异性机制。该项目的总体目标是确定 IDH磷酸化在乳腺癌中的作用。由于已知细菌中的IDH同系物受 磷酸化，假设是人细胞中IDH的磷酸化改变了IDH的功能和细胞增殖。 代谢，从而促进癌变。为了检验这一假设，提出了三个具体目标： 目的1是在分子水平上确定磷酸化对IDH酶功能的影响。目标二是 确定IDH磷酸化对细胞代谢和代谢水平上的氧化还原状态的影响。 目的3是确定IDH磷酸化在乳腺癌中发挥作用的机制或途径， 细胞水平。将对正常人乳腺上皮细胞进行致癌转化试验， 鉴定IDH磷酸化是否是乳腺癌引发剂或促进癌症的细胞适应 阵这一建议是创新的，因为：1）IDH磷酸化在人类中的功能主要是 未知的，这个建议将是第一个系统地研究所有三个IDH的磷酸化 2）为了解决经典的谷氨酸替代方法在人类细胞中的问题， 磷酸化研究并不总是有效的，遗传密码扩展技术将应用于这一领域。 在靶蛋白受控位点共选择性地掺入磷酸氨基酸（pAA）， 以产生已在乳腺癌中鉴定的位点特异性磷酸化IDH变体。这 一项提案将是第一个将pAA掺入系统引入人类细胞系以研究磷酸化的提案 在活细胞中。拟议的研究是重要的，因为它将提供关键证据的影响， IDH磷酸化在乳腺癌中的作用。鉴于蛋白质磷酸化通常参与信号传导 通过不同的激酶级联途径，IDH磷酸化在乳腺癌中的作用可能不同 其他IDH替代品。最后，对IDH磷酸化的拟议研究预计将确定 用于乳腺癌诊断和治疗的新生物标志物和靶标。

Project Summary – Project Leader Chenguang Fan Breast cancer is one of the top threats to women’s health. About 1 in 8 US women (~12%) will develop invasive breast cancer over the course of her lifetime. Previous studies have shown the important role of a key tricarboxylic acid cycle (TCA) enzyme isocitrate dehydrogenase (IDH) in breast cancer. A variety of IDH alternations including somatic mutations, aberrant expression, and altered activities have been observed in breast cancer patients. Recently, phosphoproteomic studies of breast cancer cells have identified several unique phosphorylation sites in IDH isoforms, and those sites are specific for different types of breast cancer. Such heterogeneity poses challenges for effective diagnosis and treatment, so there is a critical need to discover specific mechanisms of IDH phosphorylation for breast cancer. The overall goal of this project is to identify the role of IDH phosphorylation in breast cancer. As IDH homologues in bacteria are known to be regulated by phosphorylation, the hypothesis is that phosphorylation of IDH in human cells alters IDH functions and cellular metabolism, thus facilitating cancerous transformations. To test this hypothesis, three specific aims are proposed: Aim 1 is to identify the effects of phosphorylation on IDH enzyme functions at the molecular level. Aim 2 is to identify the influences of IDH phosphorylation on cellular metabolism and redox status at the metabolic level. Aim 3 is to identify mechanisms or pathways in which IDH phosphorylation plays roles in breast cancer at the cellular level. The oncogenic transformation test on normal human mammary epithelial cells will be performed to identify whether IDH phosphorylation is an initiator for breast cancer or a cellular adaption to facilitate cancer formation. This proposal is innovative because: 1) the functions of IDH phosphorylation in human remain largely unknown, and this proposal will be the first to systematically study the phosphorylation of all the three IDH isoforms in human cells; 2) To address the problem that the classic glutamate-substitution method for phosphorylation studies is not always effective, the genetic code expansion technique will be applied in this proposal to co-translationally incorporate phosphoamino acid (pAA) at a controlled site in target proteins in order to produce site-specifically phosphorylated IDH variants which have been identified in breast cancer. This proposal will be the first to introduce pAA-incorporation systems into human cell lines to study phosphorylation in living cells. The proposed research is significant because it will provide key evidence for the influences and roles of IDH phosphorylation in breast cancer. Given that protein phosphorylation is usually involved in signaling pathways through different kinase cascades, the role of IDH phosphorylation in breast cancer could be different from other IDH alternations. Ultimately, the proposed studies on IDH phosphorylation are expected to identify novel biomarker and targets for breast cancer diagnosis and treatment.