The role of phosphorylation of isocitrate dehydrogenase in breast cancer

项目总结-项目负责人范晨光 乳腺癌是对女性健康的最大威胁之一。大约八分之一的美国女性(约12%)会患上侵袭性疾病 在她的一生中罹患乳腺癌。以往的研究已经表明，关键字的重要作用 乳腺癌中的三羧酸循环(TCA)酶异柠檬酸脱氢酶(IDH)各种IDH 已观察到包括体细胞突变、异常表达和活性改变在内的变化。 乳腺癌患者。最近，对乳腺癌细胞的磷蛋白质组学研究已经确定了几种独特的 IDH亚型中的磷酸化位点，这些位点是不同类型乳腺癌所特有的。是这样的 异质性给有效的诊断和治疗带来了挑战，因此迫切需要发现 IDH磷酸化在乳腺癌中的特殊作用机制。该项目的总体目标是确定 IDH磷酸化在乳腺癌中的作用。因为已知细菌中的IDH同系物受 磷酸化，假说是人类细胞中IDH的磷酸化改变了IDH的功能和细胞 新陈代谢，从而促进癌变。为了验证这一假设，我们提出了三个具体目标： 目的1从分子水平确定磷酸化对IDH酶功能的影响。目标2是 在代谢水平上确定IDH磷酸化对细胞代谢和氧化还原状态的影响。 目标3是确定idh磷酸化在乳腺癌中起作用的机制或途径。 细胞水平。对正常人乳腺上皮细胞进行致癌转化试验，以 确定IDH磷酸化是乳腺癌的始发者还是促进癌症的细胞适应 队形。这一建议的创新之处在于：1)人体内IDH的磷酸化功能基本保持不变 未知，这项提议将是第一次系统地研究所有三种idh的磷酸化。 人类细胞中的异构体；2)解决经典的谷氨酸取代法 磷酸化研究并不总是有效的，遗传密码扩展技术将应用于 建议在靶蛋白的受控位置共翻译掺入磷酸氨基酸(PAA) 以产生已在乳腺癌中发现的位点特异的IDH磷酸化变体。这 将首次将PAA-掺入系统引入人类细胞系以研究磷酸化 在活细胞中。这项拟议的研究具有重要意义，因为它将为这些影响和 IDH磷酸化在乳腺癌中的作用。鉴于蛋白质的磷酸化通常参与信号转导 IDH的磷酸化在乳腺癌中的作用可能是不同的。 与其他IDH替代方案不同。最终，拟议的idh磷酸化研究有望确定 乳腺癌诊断和治疗的新生物标志物和靶点。

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.