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型肿瘤抑制活性，并确定将代谢作用转化为增殖调节的机制。FDH（10-甲酰四氢叶酸脱氢酶）不可逆地将10-甲酰四氢叶酸转化为四氢叶酸，而10-甲酰四氢叶酸是嘌呤生物合成的基本底物。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。由于这种关键的代谢功能，FDH在癌细胞中的下调被预测为有利于生存。事实上，我们最初已经做出了重要的观察，即FDH在肿瘤中通过启动子超甲基化被强烈而普遍地下调。我们进一步证明，FDH缺陷癌细胞中适度表达FDH可诱导凋亡细胞死亡。相反，非癌细胞对高水平的酶不敏感。因此，有人提出癌细胞沉默FDH基因以逃避细胞毒性。通过对FDH缺陷肿瘤细胞中正常FDH表达再激活的表型效应研究，进一步探索了JNK1/2和p53作为FDH诱导的凋亡信号的关键组分，并确定了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与细胞运动控制联系起来的新途径。目前的建议扩展了先前对FDH和相关叶酸的抗增殖机制的研究，以直接与p53相互作用，其在运动和可能的转移中的作用，并在小鼠模型中评估其在体内的作用。我们的中心假设是，通过启动子超甲基化的FDH下调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。我们进一步认为外佣通过多种机制发挥其调节作用。探讨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肿瘤抑制蛋白的功能相互作用。(2)阐明叶酸胁迫抑制细胞运动的分子机制。(3)利用FDH缺陷小鼠模型确定FDH沉默对肿瘤发生/发展的影响。研究FDH在癌症细胞存活/发病时叶酸胁迫诱导中的关键作用，将为恶性过程本身提供重要的见解，并将关键代谢途径的失调与癌症疾病联系起来，并为恶性转化的诊断建立新的靶点。

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.