The role of PTEN in DNA metabolism and replication

PTEN 在 DNA 代谢和复制中的作用

基本信息

批准号：
10797177
负责人：
Abigail Rose Lubin
金额：
$ 2.3万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-01 至 2023-08-11
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10797177
关键词：
Alleles Biological Models Cancer Biology Cell Cycle Cell Cycle Checkpoint Cell Death Cells Chromosomes DNA Damage DNA biosynthesis DNA metabolism Dependence Dihydroorotate Dehydrogenase Inhibitor Embryo Endometrial Carcinoma Exhibits FDA approved Fiber Fibroblasts Glioblastoma Glutamine Goals Growth Knowledge Label Leflunomide Length Lipids LoxP-flanked allele Malignant Neoplasms Malignant neoplasm of prostate Methods Molecular Mus Mutate Normal tissue morphology Nucleotide Metabolism Pathway PTEN gene Pathway interactions Patients Pharmaceutical Preparations Phosphatidylinositols Phosphoric Monoester Hydrolases Phosphotransferases Physiologic pulse Proliferating 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 Visualization Work analog cancer cell design inorganic phosphate live cell imaging malignant breast neoplasm nucleotide metabolism pharmacologic replication stress response targeted treatment tumor

项目摘要

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.

研究概要该项目的目标是了解 PTEN 在 DNA 代谢和复制中的作用。十号染色体上缺失的张力蛋白同源物 (PTEN) 是最常见丢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 (PIP3) 和 PTEN 活性负向调节磷酸肌醇 3- 我们实验室和其他实验室的工作表明，激酶 (PI3K)/AKT 生长信号通路的缺失。 PTEN 等位基因会导致多种正常组织（包括原发性组织）的生长速度和增殖增加此外，我们还证明了小鼠胚胎成纤维细胞（MEF）的谷氨酰胺依赖性。增加谷氨酰胺进入嘧啶从头合成的通量，并增加对药理学的敏感性初级 Pten-/- MEF 中嘧啶从头合成的抑制我们认为这些结果意味着核苷酸代谢、细胞周期和 DNA 复制是 PTEN 丢失的潜在途径我们假设使用初级 Ptenflox/flox MEF 作为探索这些途径。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变得敏感为了研究这种敏感性的分子基础，我们将通过改变细胞周期动力学来合成。检查药物后 DNA 损伤和复制应激、细胞死亡和细胞周期检查点激活鉴于来氟米特的潜在用途，抑制初级 Pten-/- MEF 中的嘧啶合成。 PTEN 缺陷肿瘤患者，全面了解 PTEN 缺陷的细胞后果在 PTEN 缺失的情况下进行来氟米特治疗将能够更有效地使用。在目标 2 中，我们将描述 PTEN 丢失对 S 期持续时间和 DNA 复制效率的影响。为了确定初级 Pten–/– MEF 中 S 期的长度，我们将使用两种实验方法，允许用于确定 S 期长度：用胸苷类似物和活细胞进行双脉冲 DNA 复制标记结合荧光标记的细胞周期特异性蛋白进行成像以研究 DNA 复制。效率，我们将进行纤维梳理以直接可视化复制 DNA 的机制知识。 PTEN 丢失背景下复制动力学改变背后的原因将有助于基本理解癌细胞逃避生长调节的机制。