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.

研究摘要了解这一点的目的是了解Ptpen在DNA代谢和复制中的作用。在第十（PTEN）上删除的Tensin同源物是最常见或柔和的肿瘤之一癌症中的抑制剂，通常在子宫内膜癌，胶质母细胞瘤，乳腺癌和前列腺癌中丧失。 PTEN是一种双特异性磷酸酶华磷酸酶，其主要底物是脂质的第二使者磷脂酰肌醇3、4,5-三磷酸（PIP3），PTEN活性对磷酸肌醇3-- 激酶（PI3K）/Akt生长信号通路在我们的实验室和其他人的工作中都表明，两者俩均两者俩都均两者都删除。 PTEN的等位基因会导致多种正常组织的生长速率和繁殖率提高，而在原发性中无关紧要小鼠胚胎成纤维细胞（MEF）。将谷氨酰胺通量增加到从头嘧啶的合成中，并对药理学的增益抑制初级PTEN-/ - / - MEF中的嘧啶合成。核苷酸代谢，细胞周期和DNA复制，因为潜在的可能会导致我们认为使用Ptenflox/Flox MEF探索生命途径模型系统。在AIM 1中，我们将研究PTEN表达的丧失如何使细胞敏感到吸入嘧啶通过改变细胞周期动力学的合成。检查DNA损伤和复制应力，细胞死亡和细胞周期循环周期循环周期检查点的激活后激活药理后激活抑制幼胺的合成 - 鉴于潜在的使用左旋胺。患有PTEN缺乏肿瘤的患者，对细胞后果的全面了解在PTEN损失的背景下，Leflunomide治疗将有助于更有效的使用。在AIM 2中，我们将表征PTEN损失对S相和DNA复制效率持续时间的影响。为了确定主要PTEN - / - MEF中s相的长度，我们将使用两种允许的经验方法为了确定S相长：用胸腺类似物和活细胞进行双脉冲DNA复制标记与荧光标记的周期特异性蛋白结合成像。效率，我们将进行纤维梳理，直接可视化复制DNA。在PTEN损失的背景下改变复制动态的背后，有助于基本的理解逃避癌细胞生长调节的机制。