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

项目摘要/摘要： 尽管在治疗方面取得了进展，但某些类型的癌症的五年存活率仍然很低。为 例如，转移性乳腺癌患者中只有20%的人活了5年。不受控制的扩散是 这是癌症的一个标志，由包括细胞周期调节因子在内的多种基因突变引起。中心是 控制细胞增殖的是肿瘤抑制因子P53和Rb，以及调节Rb功能的CDK。 在设计和测试新型抗癌药物的过程中，我们发现P53、Rb和CDK2可以 戏剧性地调节细胞对铁下垂的敏感性，铁下垂是一种铁依赖的、ROS介导的细胞死亡形式 以灾难性的脂质过氧化为特征。我们观察到，升高的P53增强了铁性下垂，而 升高的CDK2阻断了这一反应。Rb蛋白缺失增强铁性下垂，而E2F1过表达 没有任何效果。Rb作为E2F1简单抑制剂的经典模型不能解释我们的观察。然而， 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 磷酸化形式的RB积极促进反铁上链状态的建立， 可能以独立于E2F的方式。在该模型的基础上，P53和Rb的缺失均增强 而CDK2通过产生磷酸化的Rb来阻止铁下垂。 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蛋白的细胞将与不能磷酸化的Rb突变体重组 被CDK。细胞将暴露在小分子中，以诱导铁下垂来确定敏感性。我们预测 与野生型蛋白不同，非磷酸化的Rb蛋白不能抑制铁下垂。其他内容 实验将绘制出调节铁性下垂的Rb磷酸化位点。目标2.下游目标 Rb在铁性下垂的调节中的作用。Rb蛋白调节铁性下垂的机制尚不清楚。 我们已经进行了微阵列转录图谱分析和公共数据库的审查，以确定 可能介导Rb对铁性下垂影响的基因。我们将通过过度表达或 用RNAi敲除来确定对铁性下垂的影响。目的3.P53、CDKs和Rb在卵巢癌中的协调作用 铁眼镜蛇反应。除了Rb家族外，P53和CDK都调节许多靶点，但这三个都是 各成分可调节铁性下垂。我们建议确定P53、CDK和Rb是否在单个 调节铁性下垂的途径，或者是否也涉及平行途径。这些实验将涉及 在一些缺乏这些蛋白质的突变细胞系中测试铁下垂的敏感性。P53是一个家庭的成员 这包括p63和p73蛋白，这两种蛋白的功能都与p53部分重叠。几乎什么都没有 已知p63和p73参与了铁性下垂的发生。我们将使用RNAi和过度表达来测试他们的 在这一过程中发挥作用。更好地了解铁性下垂是如何调节的，将填补知识中的一个重要空白 并将有助于设计更有效的癌症治疗方法。

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.