FDH: A Novel Determinant of Tumor Suppression

描述（由申请方提供）：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与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（嘌呤从头生物合成的必需底物）转化为四氢叶酸。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，预测癌细胞中FDH的下调是促存活的，而通过稳定转染的人工升高将是有毒的。我们最近发现FDH在肿瘤组织和癌细胞系中普遍下调，并且FDH启动子超甲基化可能参与了这种下调。我们已经进一步证明，在FDH缺陷的癌细胞中适度的FDH表达诱导凋亡性细胞死亡。此外，最近获得的证据表明，需要p53介导FDH诱导的细胞毒性。相反，非癌细胞对FDH升高不敏感。因此，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的FDH下调是恶性肿瘤获得比正常细胞更好的生存优势的重要手段之一。将用于解决该假设的具体目的是：（1）确定转染的癌细胞中FDH诱导的细胞凋亡的分子机制，（2）阐明保护非恶性细胞免受FDH诱导的细胞凋亡的机制，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， 已经获得组成型表达FDH的能力的抗性癌细胞克隆和FDH不敏感的非恶性细胞将用于实现该项目的目标。有人提出，研究FDH在癌细胞生存中的关键作用将为恶性过程本身提供重要的见解，并将重要代谢途径的失调与细胞死亡联系起来。

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.