FDH: A Novel Determinant of Tumor Suppression

这项建议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。这会干扰DNA/RNA的生物合成和DNA修复。由于这一关键的代谢功能，下调癌细胞中FDH的表达将有利于生存。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，并在小鼠模型中评估其作用。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)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。

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. This interferes with DNA/RNA biosynthesis and DNA repair. Because of this critical metabolic function, down-regulation of FDH in cancer cells would 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 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) 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.