Cyclin A Function in Normal Proliferation and in Neoplasia

描述（由申请人提供）：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周期蛋白依赖性激酶（CDKs）。本提案的重点是细胞周期蛋白A及其在正常发育和肿瘤中的作用。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和睾丸特异性细胞周期蛋白A1。根据我们目前对细胞周期机制的理解，周期蛋白A代表了哺乳动物所有胚胎和体细胞周期所必需的s期周期蛋白。这一观点得到了细胞周期蛋白a2缺失小鼠在胚胎期第5.5天死亡的观察结果的支持。细胞周期蛋白A2的过度表达已被证明在许多人类癌症的发病机制中发挥重要作用。因此，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。我们通过产生条件细胞周期蛋白A敲除小鼠（缺乏A-细胞周期蛋白）实验验证了这一概念。这种方法使我们能够绕过早期胚胎致死性，并获得细胞周期蛋白a缺失的小鼠胚胎成纤维细胞。令人惊讶的是，我们发现体外培养成纤维细胞中a型细胞周期蛋白的急性关闭对细胞增殖没有影响。因此，与目前的观点相反，至少在体外培养的成纤维细胞中，细胞周期蛋白A对于正常的非致癌性增殖是必不可少的。这些意想不到的发现提出了几个重要的问题：(1)在缺乏细胞周期蛋白A的情况下，s -进展的分子基础是什么？(2)在正常小鼠的体内发育过程中，a型细胞周期蛋白对细胞周期进程是否也是不可或缺的？(3)癌细胞的癌性增殖是否需要细胞周期蛋白A ？在拟议的工作中，我们将解决这些问题。需要验证的总体假设是，对于大多数细胞类型的正常、非致癌性增殖，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性增殖，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Aim 1中，我们将验证我们的假设，即在哺乳动物细胞中，A型和e型细胞周期蛋白在驱动S期进展中发挥重叠、冗余的作用。因此，我们假设周期蛋白a缺陷的细胞由于周期蛋白e的存在而正常增殖。我们进一步假设，在周期蛋白a缺失的背景下，e型周期蛋白的消融将完全停止细胞周期进程。在Aim 2中，我们将使用组织特异性和可诱导的Cre菌株来绕过植入期的致死率，并研究细胞周期蛋白A在发育后期的功能需求。最后，在Aim 3中，我们将在体内研究myc驱动转化以及乳腺癌和肺癌中对细胞周期蛋白A功能的需求。具体目标是：目标1。研究细胞周期蛋白A在细胞周期进程中的分子基础。目标2。确定不同谱系在正常发育过程中对细胞周期蛋白A功能的需求。目标3。目的：探讨肿瘤细胞增殖对细胞周期蛋白A功能的要求。公共卫生相关性：细胞周期蛋白A在许多人类癌症中的过度表达表明，细胞周期蛋白A可能是人类肿瘤的潜在治疗靶点。然而，目前的教科书知识表明，细胞周期蛋白a对所有细胞的正常增殖至关重要，因此阻碍了对抗细胞周期蛋白a治疗策略的探索。本应用程序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细胞周期蛋白A在人类癌症中的抑制作用。因此，这项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.