Cyclin A Function in Normal Proliferation and in Neoplasia

说明(申请人提供)：细胞周期蛋白是核心细胞周期机制的组成部分。它们的作用是激活相关的细胞周期蛋白依赖性蛋白激酶(CDK)。这项建议侧重于细胞周期蛋白A及其在正常发育和肿瘤形成中的作用。哺乳动物细胞表达两种A型细胞周期蛋白：普遍存在的细胞周期蛋白A2和睾丸特异的细胞周期蛋白A1。根据我们目前对细胞周期机制的理解，细胞周期蛋白A是一种必不可少的S期细胞周期蛋白，是哺乳动物所有胚胎和体细胞周期所必需的。细胞周期蛋白A2缺失的小鼠在胚胎第5.5天死亡的观察结果支持了这一观点。细胞周期蛋白A2的过度表达在许多人类肿瘤的发病机制中起着重要作用。因此，细胞周期蛋白A可能是治疗人类肿瘤的潜在靶点。然而，目前认为细胞周期蛋白A是核心细胞周期机制的重要组成部分的观点排除了探索这种可能性的可能性。我们通过产生条件性细胞周期蛋白A基因敲除小鼠(两种A细胞周期蛋白都缺失)对这一观点进行了实验测试。这种方法使我们绕过了早期的胚胎死亡，并获得了细胞周期蛋白A缺失的小鼠胚胎成纤维细胞。令人惊讶的是，我们发现，在体外培养的成纤维细胞中，A型周期蛋白的急性关闭对细胞增殖没有影响。因此，与目前的概念相反，至少在体外培养的成纤维细胞中，细胞周期蛋白A对于正常的、非致癌的增殖是必不可少的。这些意想不到的发现提出了几个重要的问题：(1)在没有细胞周期蛋白A的情况下，S进展的分子基础是什么？(2)在小鼠正常发育期间，A型细胞周期蛋白对体内细胞周期的进展也是必不可少的吗？(3)细胞周期蛋白A对于癌细胞的致癌增殖是必需的吗？在拟议的工作中，我们将解决这些问题。有待检验的总体假设是，对于大多数细胞类型的正常、非致癌增殖，细胞周期蛋白A是必不可少的，但对于至少一部分癌症的致癌增殖来说，它是至关重要的。如果我们的假设得到证实，它将改变我们对细胞周期机制的理解，并可能为癌症患者带来新的治疗方法。在目标1中，我们将检验我们的假设，即在哺乳动物细胞中，A型和E型细胞周期蛋白在驱动S阶段进展中扮演重叠的、多余的角色。因此，我们假设Cyclin A缺陷细胞由于Cyclin E的存在而正常增殖。我们进一步假设，在Cyclin A缺失的背景下消融E-型Cyclins将完全停止细胞周期进程。在目标2中，我们将使用组织特异性和可诱导的Cre菌株来绕过种植周围的致死性，并研究在发育的后期阶段对细胞周期蛋白A功能的需求。最后，在目标3中，我们将研究在Myc驱动的转化以及体内乳腺癌和肺癌中对细胞周期蛋白A功能的需求。目的：1.研究细胞周期蛋白A在细胞周期进程中作用的分子基础。目的2.确定正常发育过程中不同谱系对细胞周期蛋白A功能的需求。目的3.确定细胞周期蛋白A在肿瘤细胞增殖中的功能需求。公共卫生相关性：在许多人类癌症中，细胞周期蛋白A的过度表达已得到很好的证明，这表明细胞周期蛋白A可能是人类肿瘤的潜在治疗靶点。然而，目前的教科书知识是周期蛋白A对所有细胞的正常增殖是必不可少的，从而排除了对抗周期蛋白A治疗策略的探索。这项申请中提出的工作可能会改变这一概念，并将导致我们重新关注人类癌症中细胞周期蛋白A的抑制作用。因此，这项拟议的工作可能会为人类癌症患者带来新的治疗方法。

DESCRIPTION (provided by applicant): Cyclins are components of the core cell cycle machinery. They serve to activate the associated cyclin-dependent kinases (CDKs). This proposal focuses on cyclin A and its role in normal development and in neoplasia. Mammalian cells express two A-type cyclins: the ubiquitous cyclin A2 and testis-specific cyclin A1. According to our current understanding of the cell cycle machinery, cyclin A represents an essential S-phase cyclin which is required for all embryonic and somatic cell cycles in mammals. This notion is supported by the observation that cyclin A2-null mice die at embryonal day 5.5. Overexpression of cyclin A2 has been shown to play an important role in pathogenesis of many human cancers. Hence, cyclin A might represent a potential therapeutic target in human neoplasia. However, the current notion that cyclin A represents an essential component of the core cell cycle machinery has precluded exploring this possibility. We experimentally tested this notion by generating conditional cyclin A knockout mice (lacking both A-cyclins). This approach allowed us to bypass the early embryonic lethality and to derive cyclin A-null mouse embryonal fibroblasts. Surprisingly, we found that an acute shutdown of A-type cyclins in in vitro cultured fibroblasts had no impact on cell proliferation. Hence, contrary to the current notion, cyclin A is dispensable for normal, non-oncogenic proliferation at least in in vitro cultured fibroblasts. These unexpected findings raise several important issues: (1) What is the molecular basis of the S-progression in the absence of cyclin A? (2) Are A-type cyclins also dispensable for cell cycle progression in vivo, during normal mouse development? (3) Is cyclin A required for the oncogenic proliferation of cancer cells? In the proposed work we will address these issues. The overall hypothesis to be tested is that cyclin A is dispensable for normal, non-oncogenic proliferation of the majority of cell types, but it is critically required for oncogenic proliferation of at least a subset of cancers. If our hypothesis is confirmed, it will change our understanding of the cell cycle machinery, and it may lead to novel therapeutic approaches for cancer patients. In Aim 1 we will test our hypothesis that in mammalian cells A- type and E-type cyclins perform overlapping, redundant roles in driving S phase progression. Consequently, we hypothesize that cyclin A-deficient cells proliferate normally due to the presence of cyclin E. We further hypothesize that ablation of the E-type cyclins in cyclin A-null background would completely halt cell cycle progression. In Aim 2, we will use tissue-specific and inducible Cre strains to bypass the peri-implantational lethality, and to study the requirement for cyclin A function at later stages of development. Lastly in Aim 3, we will study the requirement for cyclin A function in Myc-driven transformation, and in breast and lung cancers in vivo. The Specific Aims are: Aim 1. To study the molecular basis of cyclin A function in cell cycle progression. Aim 2. To determine the requirement for cyclin A function in various lineages during normal development. Aim 3. To determine the requirement for cyclin A function in oncogenic cell proliferation. PUBLIC HEALTH RELEVANCE: The well-documented overexpression of cyclin A in a many human cancers suggests that cyclin A might represent a potential therapeutic target in human neoplasia. However, it is currently a textbook knowledge that cyclin A is essential for normal proliferation of all cells, thereby precluding exploration of anti-cyclin A therapeutic strategies. The work proposed in this application will likely change this notion, and will lead to re-focusing our efforts on cyclin A inhibition in human cancers. Hence, the proposed work will likely lead to novel therapeutic approaches for human cancer patients.