The role of PTEN in DNA metabolism and replication

PTEN 在 DNA 代谢和复制中的作用

• 批准号: 10343770
• 负责人: Abigail Rose Lubin
• 金额: $ 4.61万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10343770
• 关键词: Alleles; Biological Models; Cancer Biology; Cell Cycle; Cell Cycle Checkpoint; Cell Death; Cells; Chromosomes; Comb animal structure; DHODH gene; DNA; DNA Damage; DNA biosynthesis; DNA metabolism; Dependence; Dihydroorotate Dehydrogenase Inhibitor; Embryo; Endometrial Carcinoma; Exhibits; FDA approved; Fiber; Fibroblasts; Glioblastoma; Glutamine; Goals; Growth; Knowledge; Label; Lead; Leflunomide; Length; Lipids; Malignant Neoplasms; Malignant neoplasm of prostate; Methods; Molecular; Mus; Mutate; Normal tissue morphology; Nucleotide Metabolism Pathway; PTEN gene; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacology; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphotransferases; Physiologic pulse; Proteins; Proto-Oncogene Proteins c-akt; Pyrimidine; Regulation; Research; Role; S phase; Second Messenger Systems; Signal Pathway; Signal Transduction; Solid Neoplasm; Specificity; Therapeutic; Thymidine; Tumor Suppressor Proteins; Work; analog; cancer cell; design; live cell imaging; malignant breast neoplasm; nucleotide metabolism; replication stress; response; targeted treatment; tumor

RESEARCH SUMMARY The goal of this project is to understand the role of PTEN in DNA metabolism and replication. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is one of the most frequently lost or mutated tumor suppressors in cancer, commonly lost in endometrial cancer, glioblastoma, breast cancer, and prostate cancer. PTEN is a dual-specificity phosphatase whose main substrate is the lipid second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3), and PTEN activity negatively regulates the phosphoinositide 3- kinase (PI3K)/AKT growth signaling pathway. Work in our lab and others has shown that the deletion of both alleles of PTEN causes increased growth rate and proliferation in a variety of normal tissues, including in primary mouse embryonic fibroblasts (MEFs). Additionally, we have demonstrated a glutamine dependency, an increased glutamine flux into de novo pyrimidine synthesis, and an increased sensitivity to pharmacologic inhibition of de novo pyrimidine synthesis in primary Pten–/– MEFs. We propose that these results implicate the nucleotide metabolism, the cell cycle, and DNA replication as potential avenues through which the loss of PTEN could contribute to deregulated growth. We posit to explore these pathways using primary Ptenflox/flox MEFs as a model system. In Aim 1, we will investigate how the loss of PTEN expression sensitizes cells to inhibition of de novo pyrimidine synthesis through altered cell cycle dynamics. To investigate the molecular basis of this sensitivity, we will examine DNA damage and replication stress, cell death, and cell cycle checkpoint activation after pharmacologic inhibition of de novo pyrimidine synthesis in primary Pten–/– MEFs. Given the potential use of leflunomide for patients with PTEN-deficient tumors, a comprehensive understanding of the cellular consequences of leflunomide treatment in the context of PTEN loss will enable more effective use. In Aim 2, we will characterize the impact of PTEN loss on the duration of S phase and DNA replication efficiency. To determine the length of S phase in primary Pten–/– MEFs, we will use two experimental methods that allow for the determination of S phase length: dual-pulse DNA replication labeling with thymidine analogs and live cell imaging in combination with fluorescent-labeled cell cycle-specific proteins. To investigate DNA replication efficiency, we will perform fiber combing to directly visualize replicating DNA. Knowledge of the mechanism behind altered replication dynamics in the context of PTEN loss would contribute to the basic understanding of the mechanisms of evading growth regulation in cancer cells.

研究综述 该项目的目标是了解PTEN在DNA代谢和复制中的作用。磷酸酶和 第10号染色体上的张力蛋白同源物缺失（tensin homolog deleted on chromosome ten，PTEN）是最常见的缺失或突变的肿瘤之一 癌症中的抑制因子，通常在子宫内膜癌、胶质母细胞瘤、乳腺癌和前列腺癌中丢失。 PTEN是一种双特异性磷酸酶，其主要底物是脂质第二信使 磷脂酰肌醇-3，4，5-三磷酸（PIP 3），和PTEN活性负调节磷酸肌醇3-磷酸 激酶（PI 3 K）/AKT生长信号通路。我们实验室和其他实验室的工作表明， PTEN的等位基因导致多种正常组织中生长速率和增殖增加，包括原发性肝癌中的生长速率和增殖。 小鼠胚胎成纤维细胞（MEFs）。此外，我们已经证明了谷氨酰胺依赖性， 增加谷氨酰胺流入从头嘧啶合成，并增加对药理学的敏感性， 抑制原代Pten-/-MEF中从头嘧啶合成。我们认为这些结果暗示了 核苷酸代谢，细胞周期和DNA复制作为潜在的途径，通过该损失的PTEN 可能有助于放松管制的增长。我们尝试使用初级Ptenflox/flox MEFs作为 模型系统 在目标1中，我们将研究PTEN表达的缺失如何使细胞对从头嘧啶的抑制敏感 通过改变细胞周期动力学合成。为了研究这种敏感性的分子基础，我们将 检查DNA损伤和复制应激，细胞死亡和细胞周期检查点激活后，药理学 抑制原代Pten-/-MEF中从头嘧啶合成。鉴于来氟米特的潜在用途， 与PTEN缺陷型肿瘤患者，细胞后果的全面了解， 来氟米特治疗在PTEN丢失的情况下将能够更有效地使用。 在目标2中，我们将描述PTEN缺失对S期持续时间和DNA复制效率的影响。 为了确定原代Pten-/- MEFs中S相的长度，我们将使用两种实验方法， 用于测定S期长度：用胸苷类似物和活细胞进行双脉冲DNA复制标记 与荧光标记的细胞周期特异性蛋白质组合成像。为了研究DNA复制 为了提高效率，我们将进行纤维梳理以直接可视化复制DNA。机制知识 在PTEN缺失的背景下改变复制动力学的背后，将有助于对 逃避癌细胞生长调节的机制。