CDC25 PHOSPHATASES IN CELL CYCLE CONTROL AND CANCER

描述（由申请人提供）：Cdc25磷酸酶通过激活Cdks积极调节细胞周期。在哺乳动物中，存在三个家族成员，分别为Cdc25A、Cdc25B和Cdc25C。目前尚不清楚为什么高等真核生物编码三个Cdc25s，而酵母细胞只有一个Cdc25s存活。生成了Cdc25A基因可被删除的小鼠。此外，还产生了缺乏Cdc25B、Cdc25C或两者都缺乏的小鼠，这些小鼠也可以因Cdc25A而被有条件地删除。这些小鼠将被用来确定Cdc25家族成员对成年细胞周期的贡献。本提案的另一个目标是利用高通量和分子成像策略来鉴定Cdc25A的新调节因子。Cdc25A在整个细胞周期中发挥作用，调节细胞周期转变，在几种癌症中Cdc25A过量产生。Cdc25A的活性、稳定性以及与其他蛋白的相互作用受可逆磷酸化调控。因此，确定调节Cdc25A的蛋白激酶将有助于深入了解Cdc25A在正常和应激条件下是如何被调节的，以及在人类癌症中导致Cdc25A过量产生的途径。最后，我们提出了实验来研究ATR、Chk1和PP2A的新调控途径，并研究小鼠中Chk1和Cdc25A之间的相互作用。除了促进我们对基本细胞周期原理的理解外，这些研究也具有临床影响。例如，Cdc25A和Cdc25B在许多人类癌症中过量产生，人们正在努力开发可用于治疗人类癌症的Cdc25抑制剂。在Cdc25抑制剂进入临床之前，需要评估Cdc25抑制剂的细胞毒性。这项资助中提出的基因敲除研究将评估Cdc25家族成员缺失对成年小鼠的影响，并可能预测患者对Cdc25整体抑制和个体抑制的反应。目前用于治疗癌症患者的另一种策略是将DNA损伤剂与诱导Cdc25A积累的药物结合使用。在临床前模型中，该策略诱导检查点绕过和优先杀死p53缺陷细胞。Chk1抑制剂UCN-01正在与DNA损伤剂一起进行I期和II期临床试验。因此，在我们的研究过程中发现的PP2A/Chk1通路的新激酶和/或调节因子是潜在的可药物靶点。靶向这些通路成分的抑制剂可以在上述联合治疗中替代Chk1抑制剂。希望这些抑制剂能比Chk1抑制剂诱导更少的基因组不稳定性。公共卫生相关性：提出了实验，以充分剖析PP2A/Chk1/Cdc25A调控途径的分子基础，并在小鼠中功能表征Cdc25磷酸酶。从拟议的研究中获得的信息不仅有望增强我们对哺乳动物细胞周期控制基本原理的理解，而且有望直接影响未来癌症治疗的治疗策略。

DESCRIPTION (provided by applicant): The Cdc25 phosphatases positively regulate the cell cycle by activating Cdks. In mammals, three family members, denoted Cdc25A, Cdc25B, and Cdc25C, exist. It is unclear why higher eukaryotic organisms encode three Cdc25s while yeast cells survive with one. Mice that can be deleted for Cdc25A were generated. In addition, mice lacking Cdc25B, Cdc25C or both and that can also be conditionally deleted for Cdc25A were generated. These mice will be used to determine the contributions made by Cdc25 family members to adult cell cycles. Another goal of this proposal is to utilize high throughput and molecular imaging strategies to identify novel regulators of Cdc25A. Cdc25A functions throughout the cell cycle to regulate cell cycle transitions and Cdc25A is overproduced in several cancers. Cdc25A activity, stability and interactions with other proteins are regulated by reversible phosphorylation. Thus, identifying the protein kinases that regulate Cdc25A will provide insight into how Cdc25A is regulated under normal and stressed conditions and what pathways leading to Cdc25A overproduction are derailed in human cancers. Finally, experiments are proposed to study a novel regulatory pathway involving ATR, Chk1 and PP2A and to investigate interactions between Chk1 and Cdc25A in mice. In addition to advancing our understanding of basic cell cycle principles, these studies have clinical impact as well. For example, Cdc25A and Cdc25B are overproduced in many human cancers and efforts are underway to develop Cdc25 inhibitors that can be used to treat human cancers. The cytotoxicity of Cdc25 inhibition needs to be assessed before Cdc25 inhibitors can proceed to the clinic. The gene knockout studies proposed in this grant will assess effects of loss of Cdc25 family members in adult mice and may predict how patients will respond to global versus individual Cdc25 inhibition. Another strategy that is currently being used to treat cancer patients is to combine DNA damaging agents with drugs that induce Cdc25A accumulation. In preclinical models, this strategy induces checkpoint bypass and preferential killing of p53-deficient cells. The Chk1 inhibitor UCN-01 is being tested with DNA damaging agents in Phase I and II clinical trials. Thus, the novel kinases and/or regulators of the PP2A/Chk1 pathway identified in the course of our studies are potential druggable targets. Inhibitors that target components of these pathways could substitute for Chk1 inhibitors in the combination therapy described above. The hope is that these inhibitors may induce less genome instability than Chk1 inhibitors. PUBLIC HEALTH RELEVANCE: Experiments are proposed to fully dissect the molecular underpinnings of the PP2A/Chk1/Cdc25A regulatory pathway and to functionally characterize the Cdc25 phosphatases in mice. The information gained from the proposed studies is expected not only to enhance our understanding of basic principles of cell cycle control in mammals but is also expected to directly impact future therapeutic strategies for cancer treatment.