FDH: A Novel Determinant of Tumor Suppression

描述(由申请人提供)：本提案的总体目标是表征一种关键代谢酶的新的肿瘤抑制活性，并确定将代谢效应转化为增殖调节的机制。FDH(10-甲酰基四氢叶酸脱氢酶)不可逆地将10-甲酰基四氢叶酸转化为四氢叶酸，10-甲酰基四氢叶酸是从头合成嘌呤的必需底物。通过消耗这种底物，FDH可以限制嘌呤的生物合成。反过来，这会干扰重要的下游细胞过程，包括DNA/RNA生物合成和DNA修复。由于这一关键的代谢功能，癌细胞中FDH的下调被预测为有利于生存。事实上，我们最初的重要观察结果是，FDH通过启动子高甲基化在肿瘤中强烈且普遍下调。我们进一步证明，在FDH缺陷的癌细胞中适度表达FDH可以诱导细胞凋亡。相比之下，非癌细胞对高水平的这种酶不敏感。因此，有人认为癌细胞沉默FDH基因是为了逃避细胞毒作用。在FDH缺陷的肿瘤细胞中重新激活正常FDH表达的表型效应的研究进一步探索了JNK1/2和P53作为FDH诱导的凋亡信号的关键成分，并确定DHFR和叶酸补充作为增殖拯救因子。重要的是，发现了一种通过细胞内叶酸调节将FDH连接到细胞运动控制的新途径。目前的建议扩展了先前关于FDH和相关叶酸的抗增殖机制的研究，以直接与P53相互作用，它们在运动和可能的转移中的作用，以及在小鼠模型中对其作用的评估。我们的中心假设是，通过启动子超甲基化下调FDH是恶性肿瘤获得比正常细胞更有利的生存优势的重要手段之一。我们进一步建议，外佣通过多种机制发挥其调节作用。探索这些机制并验证我们的假设的具体目的是：(1)研究FDH与细胞质中P53抑癌蛋白的功能相互作用。(2)阐明叶酸应激抑制细胞运动的分子机制。(3)利用FDH基因缺陷小鼠模型，研究FDH沉默对肿瘤发生、发展的影响。研究FDH在癌细胞存活/疾病开始时诱导叶酸应激中的关键作用，将提供对恶性过程本身的重要洞察，并将关键代谢途径的放松与癌症疾病联系起来，以及为恶性转化的诊断建立新的靶点。 与公共健康相关：叶酸是人类饮食中重要和必要的一部分，调节许多细胞过程，包括核苷酸生物合成和甲基化，而叶酸缺乏会导致许多疾病。这一应用主要集中在FDH的一种新的肿瘤抑制功能上，FDH是一种丰富的人类酶，它是叶酸途径的重要调节因子。由于FDH可以作为过度增殖的限制因子，了解其在细胞新陈代谢中的作用将有助于更好地了解肿瘤的发生过程。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to characterize the novel tumor suppressor activity of a key metabolic enzyme and determine the mechanisms transforming metabolic effects into regulation of proliferation. FDH (10-formyltetrahydrofolate dehydrogenase) irreversibly converts 10-formyltetrahydrofolate, an essential substrate for de novo purine biosynthesis, to tetrahydrofolate. Through depletion of this substrate, FDH can restrict purine biosynthesis. In turn, this interferes with important downstream cellular processes, including DNA/RNA biosynthesis and DNA repair. Because of this critical metabolic function, down-regulation of FDH in cancer cells was predicted to be pro-survival. Indeed, we have initially made the important observation that FDH is strongly and ubiquitously down-regulated in tumors through the promoter hypermethylation. We have further demonstrated that moderate FDH expression in FDH-deficient cancer cells induces apoptotic cell death. In contrast, non-cancer cells are insensitive to high levels of the enzyme. Therefore, it is proposed that cancer cells silence the FDH gene in order to escape cytotoxicity. Studies of phenotypic effects upon reactivation of normal FDH expression in FDH-deficient tumor cells have further explored JNK1/2 and p53 as key components of FDH-induced apoptotic signaling, and determined DHFR and folate supplementation as proliferation rescue factors. Importantly, a novel pathway linking FDH, through intracellular folate regulation, to control of cell motility, was discovered. The current proposal extends previous studies of antiproliferative mechanisms of FDH, and related folates, to direct interaction with p53, their role in motility and possibly metastasis, and evaluation of its role in vivo in mouse model. Our central hypothesis is that FDH down- regulation through promoter hypermethylation is one of the important means by which malignancies gain pro- survival advantage over normal cells. We further suggest that FDH exerts its regulatory effects through multiple mechanisms. The Specific Aims to probe these mechanisms and test our hypothesis are: (1) Investigate the functional interaction of FDH with p53 tumor suppressor protein in cytoplasm. (2) Elucidate the molecular mechanisms leading to inhibition of cell motility by folate stress. (3) Determine the impact of FDH silencing on tumor initiation/progression using FDH deficient mouse model. Investigation of the critical role of FDH, in cancer cell survival/induction of folate stress at the onset of the disease, will provide important insight into the malignant process itself and link deregulation of key metabolic pathways to cancer disease, as well as establish new targets for diagnostics of the malignant transformation. PUBLIC HEALTH RELEVANCE: Folate, an important and essential part of the human diet, regulates many cellular processes including nucleotide biosynthesis and methylation, while folate deficiency promotes many diseases. This application is focused on a novel tumor suppressor function of FDH, an abundant human enzyme, which is an important regulator of folate pathways. Since FDH can function as a restrictor of excessive proliferation, understanding its role in cellular metabolism will provide better understanding of tumorigenic processes.