FDH: A Novel Determinant of Tumor Suppression

摘要 这项提议的总体目标是表征一种关键代谢产物的新的肿瘤抑制活性。 酶，并确定将代谢效应转化为增殖调节的机制。外佣 (10-甲酰基四氢叶酸脱氢酶)不可逆地转化10-甲酰基四氢叶酸，这是一种必不可少的 从头合成嘌呤的底物，合成四氢叶酸。通过消耗这种底物，外佣可以 限制嘌呤的生物合成。反过来，这又干扰了重要的下游细胞过程，包括 DNA/RNA生物合成与DNA修复。由于这一关键的代谢功能，外佣在 癌细胞被预测是支持生存的。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。我们有 进一步证明在FDH缺陷的癌细胞中适度表达FDH可诱导细胞凋亡 死亡。相比之下，非癌细胞对高水平的这种酶不敏感。因此，现建议 癌细胞沉默FDH基因以逃避细胞毒性。表型效应的研究 FDH缺陷肿瘤细胞中正常FDH表达的重新激活进一步探讨了JNK1/2和P53作为 FDH诱导细胞凋亡信号的关键成分，并确定DHFR和叶酸补充为 核扩散救援因素。重要的是，一种新的途径通过细胞内叶酸调节将外佣连接到 发现了对细胞运动的控制。目前的提案延长了之前对抗增殖剂的研究 FDH和相关叶酸直接与P53相互作用的机制及其在体内的作用 老鼠模型。我们的中心假设是，外佣通过启动子超甲基化下调表达是 恶性肿瘤获得比正常细胞更有利的生存优势的重要手段。我们进一步建议 外佣通过多种机制发挥其监管作用。具体目的是探讨这些 机制和验证我们的假设是：(1)研究FDH与P53肿瘤的功能相互作用 胞质中的抑制蛋白。(2)确定外佣沉默对肿瘤发生/发展的影响 脱氢表雄酮缺乏小鼠模型。FDH在癌细胞存活/叶酸诱导中的关键作用研究 疾病开始时的压力，将提供对恶性过程本身和联系的重要洞察 放松对导致癌症的关键代谢途径的管制，以及建立新的诊断目标 恶变。

ABSTRACT 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 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.