Regulation of Ferroptosis by the p53/CDK/Rb Axis.

p53/CDK/Rb 轴对铁死亡的调节。

• 批准号: 10203213
• 负责人: William R. Taylor
• 金额: $ 45.15万
• 依托单位: UNIVERSITY OF TOLEDO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10203213
• 关键词: ATP Synthesis Pathway; Affect; Alanine; Antineoplastic Agents; Back; CDK2 gene; CRISPR/Cas technology; Candidate Disease Gene; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Death; Cell Death Process; Cell Line; Cell Proliferation; Cell division; Cells; Cellular biology; Cessation of life; Cytolysis; DNA Damage; Data; Databases; E2F transcription factors; Ensure; Exposure to; Family; Family member; Gene Expression Profiling; Gene Expression Regulation; Generations; Genes; Genetic Transcription; Growth; Human; Iron; Knowledge; Lead; Light; Lipid Peroxidation; MDA-MB-468; Malignant Neoplasms; Maps; Mediating; Membrane Lipids; Metabolic; Metabolism; Metastatic breast cancer; Modeling; Molecular Target; Movement; Mus; Mutation; Names; Null Lymphocytes; Organelles; Oxygen; Pathway interactions; Patients; Phase; Phosphorylation; Process; Protein Family; Proteins; RNA Interference; Reaction; Reactive Oxygen Species; Regulation; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; Role; Serine; Site; Stress; Survival Rate; TP53 gene; Testing; Threonine; Time; Viral Antigens; Work; base; cancer therapy; cancer type; cell type; cellular targeting; design; differential expression; effective therapy; experimental study; genome integrity; inhibitor/antagonist; interest; knock-down; macromolecule; member; mutant; novel; oncoprotein p21; overexpression; oxidation; protein p73; reconstitution; response; small molecule; tissue/cell culture; transcription factor; virtual

Project Summary/Abstract: Despite advances in treatment, the five-year survival rates for certain types of cancer are still low. For example, only ~20% of patients with metastatic breast cancer are alive at 5 years. Uncontrolled proliferation is a hallmark of cancer and is caused by multiple mutations in genes including cell cycle regulators. Central to the control of cell proliferation are the tumor suppressors p53 and RB along with CDKs that modulate RB function. In the course of designing and testing a new class of anticancer drugs, we found that p53, RB and CDK2 can dramatically modulate cell sensitivity to ferroptosis, an iron-dependent, ROS-mediated form of cell death characterized by catastrophic lipid peroxidation. We observed that elevated p53 enhances ferroptosis, while elevated CDK2 blocks the response. Deletion of RB proteins enhances ferroptosis while overexpression of E2F1 has no effect. The classical model of RB as a simple inhibitor of E2F1 cannot explain our observations. However, our results can be reconciled by recently described activity of phosphorylated RB. Thus, we hypothesize that phosphorylated forms of RB actively contribute to the establishment of an anti-ferroptotic state, potentially in an E2F-independent manner. Based on this model, both elevated p53 and RB deletion enhance ferroptosis by removing phosphorylated RB, while CDK2 blocks ferroptosis by generating phosphorylated RB. To test our hypothesis, we propose the following specific aims: AIM 1. The role of RB phosphorylation in ferroptosis. Cells lacking RB proteins will be reconstituted with mutants of RB that cannot be phosphorylated by CDKs. Cells will be exposed to small molecules to induce ferroptosis to determine sensitivity. We predict that non-phosphorylatable RB protein will fail to suppress ferroptosis unlike the wild-type protein. Additional experiments will map the sites of RB phosphorylation that modulate ferroptosis. AIM 2. Downstream targets of RB in the regulation of ferroptosis. The mechanism by which RB proteins regulate ferroptosis is unknown. We have carried out microarray transcriptional profiling and review of public databases to identify a panel of genes that may mediate the effects of RB on ferroptosis. We will test candidate genes by overexpression or knock-down using RNAi to determine effects on ferroptosis. AIM 3. Coordination of p53, CDKs and RB in the ferroptotic response. Both p53 and CDKs regulate a number of targets in addition to the RB family, yet all three components modulate ferroptosis. We propose to determine whether p53, CDKs and RB work in a single pathway to regulate ferroptosis or whether parallel pathways are also involved. These experiments will involve testing ferroptosis sensitivity in a number of mutant cell lines lacking these proteins. p53 is a member of a family that includes p63 and p73 proteins both of which have partially overlapping functions with p53. Virtually nothing is known about the involvement of p63 and p73 in ferroptosis. We will use RNAi and overexpression to test their role in this process. A better understanding of how ferroptosis is regulated will fill an important gap in knowledge in the biology of cell division and will help in design of more effective cancer treatments.

项目概要/摘要： 尽管在治疗方面取得了进展，但某些类型癌症的五年生存率仍然很低。为 例如，只有约20%的转移性乳腺癌患者在5年内存活。不受控制的扩散是 是癌症的标志，由包括细胞周期调节因子在内的基因的多个突变引起。的核心 控制细胞增殖的是肿瘤抑制因子p53和RB沿着调节RB功能的CDK。 在设计和测试一类新的抗癌药物的过程中，我们发现p53、RB和CDK 2可以 显著调节细胞对铁凋亡（一种铁依赖性ROS介导的细胞死亡形式）的敏感性 以灾难性的脂质过氧化为特征。我们观察到p53升高会增强铁凋亡， 升高的CDK 2阻断应答。RB蛋白的缺失增强了铁凋亡，而E2 F1的过表达 没有任何效果。RB作为E2 F1的简单抑制剂的经典模型不能解释我们的观察结果。然而，在这方面， 我们的结果可以与最近描述的磷酸化RB的活性相一致。因此，我们假设， RB的磷酸化形式积极地有助于建立抗铁凋亡状态， 可能以E2 F独立的方式。基于该模型，升高的p53和RB缺失都增强了 CDK 2通过去除磷酸化RB来阻断铁凋亡，而CDK 2通过产生磷酸化RB来阻断铁凋亡。 为了验证我们的假设，我们提出了以下具体目标：目的1。RB磷酸化在 铁性下垂缺乏RB蛋白的细胞将用不能磷酸化的RB突变体重建 的CDK。将细胞暴露于小分子以诱导铁凋亡，从而确定敏感性。我们预测 与野生型蛋白不同，不可磷酸化的RB蛋白将不能抑制铁凋亡。额外 实验将绘制调节铁凋亡的RB磷酸化位点。AIM 2.的下游靶标 RB在铁下垂调节中的作用。RB蛋白调节铁凋亡的机制尚不清楚。 我们已经进行了微阵列转录谱分析和公共数据库的审查，以确定一个小组， 可能介导RB对铁凋亡作用的基因。我们将通过过度表达或 使用RNAi敲低以确定对铁凋亡的影响。AIM 3. p53、CDK和RB在肿瘤中的协调作用 铁中毒反应p53和CDK都调节RB家族以外的许多靶点，但这三种靶点都不受调节。 组分调节铁下垂。我们建议确定p53，CDKs和RB是否在一个单一的工作机制中起作用。 调节铁凋亡的途径或是否也涉及平行途径。这些实验将涉及 在缺乏这些蛋白质的许多突变细胞系中测试铁凋亡敏感性。p53是一个家族的成员 包括p63和p73蛋白，两者都具有与p53部分重叠的功能。几乎没有 已知p63和p73参与了亚铁下垂。我们将使用RNAi和过表达来测试它们的 在这个过程中的作用。更好地了解铁凋亡是如何调节的将填补知识上的一个重要空白 在细胞分裂生物学中，将有助于设计更有效的癌症治疗方法。