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，并确定其对恶性细胞和非恶性细胞的选择性毒性机制。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，这干扰了重要的下游细胞内过程，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，而通过稳定转染人工升高FDH则是有毒的。我们最近发现，FDH在肿瘤组织和癌细胞系中普遍下调，FDH启动子超甲基化可能参与了这种下调。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。此外，最近获得的证据表明，p53是介导fdh诱导的细胞毒性所必需的。相反，非癌细胞对FDH升高不敏感。因此，有人提出癌细胞沉默FDH基因以逃避细胞毒性。我们的中心假设是通过启动子超甲基化来下调FDH是恶性肿瘤比正常细胞获得促生存优势的重要手段之一。具体目标将用于解决这一假设：(1)确定FDH诱导转染癌细胞凋亡的分子机制，(2)阐明保护非恶性细胞免受FDH诱导的凋亡的机制，以及(3)确定启动子超甲基化在癌细胞中FDH下调中的作用。诱导表达FDH的恶性细胞的稳定克隆，

DESCRIPTION (provided by applicant): The overall goal of this proposal is to characterize the novel tumor suppressor activity of a key metabolic enzyme and to determine the mechanism(s) of its selective toxicity to malignant vs. non-malignant cells. 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 intracellular 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 prosurvival, while artificial elevation through stable transfection, would be toxic. We have recently made the important observation that FDH is strongly and ubiquitously down-regulated in tumor tissues and cancer cell lines and that FDH promoter hypermethylation is likely involved in this down-regulation. We have further demonstrated that moderate FDH expression in FDH-deficient cancer cells induces apoptotic cell death. Furthermore, evidence was recently obtained that p53 is required to mediate FDH-induced cytotoxicity. In contrast, non-cancer cells were insensitive to FDH elevation. Therefore, it is proposed that cancer cells silence the FDH gene in order to escape cytotoxicity. 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. The Specific Aims that will be used to address this hypothesis are: (1) Determine the molecular mechanisms of FDH-induced apoptosis in transfected cancer cells, (2) Elucidate the mechanism(s) that protects non-malignant cells from FDH-induced apoptosis, and (3) Determine the role of promoter hypermethylation in down-regulation of FDH in cancer cells. Stable clones of malignant cells that inducibly express FDH, resistant cancer cell clones that have acquired the ability to constitutively express FDH, and FDH-insensitive non-malignant cells, will be used to pursue the goals of this project. It is proposed that investigation of the critical role of FDH in cancer cell survival will provide important insight into the malignant process itself and link disregulation of important metabolic pathways to cell death.