Functional Outcomes of Cdk6 and Eya2 Interaction

描述(申请人提供)：本提案旨在继续原研究CDK6分化功能的基金的工作。该领域的最新证据表明，CDK6是多能性的早期靶点，表明它既促进自我更新，又抑制分化，这是多能性细胞和癌症干细胞的特征。我们的实验室发现了CDK6和发育重要的转录因子Eya2之间的一种新的相互作用。我们有证据表明，CDK6抑制了Eya2诱导的转录激活，我们开发了一种创新的方法，以卵巢癌组织培养细胞作为模型系统来研究CDK6。这项工作的目标是研究CDK6在分化和肿瘤发生过程中的新功能。这一提议的重要之处在于，CDK6癌基因正在成为多能性和自我更新的主要调节者的早期靶标之一。它已被证明是SOX2、NANOG和OCT4的三个主要多能性调节因子的直接靶子(Bonini等人。1998年；Zhang et al.2009年)。因此，了解CDK6和Eya2之间的相互作用可能会影响我们对癌症干细胞和发育生物学的理解。这一建议的相关模型系统是卵巢，在那里Eya2在果蝇突变体中具有展示的效果(Bonini等人。1998)和卵巢癌细胞株，其中Eya2升高(Zhang等人。2009年)。Eya2升高与卵巢癌的不良预后相关(Zhang等人。2009年)。我们设想了一个模型，在该模型中，CDK6和Eya2相互作用，影响细胞周期和分化的时间，从而影响发育和转化。这里提出的AIMS组成了一个试验性研究项目，研究这种新的相互作用的生物学相关性。这些目的包括：1)证实CDK6和Eya2在卵巢癌细胞系中的相互作用。确认CDK6影响Eya2的转录激活功能，并确定CDK6的哪些功能是这一影响所必需的。进行带移、CHIP和CHIP-SEQ研究，以检测Eya2/Six4异源二聚体的DNA结合和启动子占有率。目的2)通过酵母双杂交筛选获得破坏CDK6与Eya2等蛋白结合的CDK6突变体。这一遗传筛选将利用CDK6突变体池来寻找破坏或增强与Eya2结合的突变体。目的3)应用免疫荧光和激酶/磷酸酶检测等多种方法研究CDK6Eya2相互作用的功能后果。目的：探讨Eya2、CDK6及其突变型蛋白过表达对卵巢癌细胞增殖、迁移和肿瘤发生的影响。这里提出的目的是通过检验我们实验室确定的CDK6和Eya2新的相互作用的生物学相关性来解决CDK6和Eya2相互作用影响细胞增殖和分化-在发育和癌症中至关重要的过程-的假说。 公共卫生相关性：该提案中概述的工作的目标是研究细胞周期调节蛋白CDK6和发育重要的转录因子Eya2的相互作用。了解这些蛋白质是如何影响分化和细胞增殖的，对于癌症干细胞的生物学和发育至关重要，并可能促进我们对卵巢癌的理解。

DESCRIPTION (provided by applicant): This proposal aims to continue the work of the original grant that investigated differentiation functions of cdk6. Recent evidence in the field suggests that cdk6 is an early target of pluripotency, suggesting it both promotes self-renewal and blocks differentiation, characteristics of pluripotent cells and cancer stem cells. Our lab has identified a novel interaction between cdk6 and the developmentally important transcription factor, Eya2. We have evidence that cdk6 squelches Eya2-driven transcriptional activation and we have developed an innovative approach to studying cdk6 using ovarian cancer tissue culture cells as a model system. The goal of the work outlined in this proposal is to investigate novel functions of cdk6 that contribute to the regulation of differentiation and oncogenesis. Important to this proposal is the fact that the cdk6 oncogene is emerging as one of the early targets of the major regulators of pluripotency and self-renewal. It has been shown to be a direct target of the three major regulators of pluripotency SOX2, NANOG and OCT4 (Bonini et al. 1998; Zhang et al. 2009). Thus, an understanding of the interaction between cdk6 and Eya2 could impact our understanding of the biology of both cancer stem cells and development. The relevant model system for this proposal is the ovary, where Eya2 has a demonstrated effect in Drosophila mutants (Bonini et al. 1998) and ovarian cancer cell lines, where Eya2 is elevated (Zhang et al. 2009). Elevated Eya2 has been associated with poor outcomes in ovarian cancer (Zhang et al. 2009). We envision a model in which cdk6 and Eya2 interact to affect the timing of cell cycle and differentiation that impacts development and transformation. Aims proposed here makeup a pilot research project that examines the biological relevance of this novel interaction. These Aims include 1) To confirm the interaction of cdk6 and Eya2 in an ovarian cancer cell line. To confirm that cdk6 influences the transcriptional activation function of Eya2 and determine what functions of cdk6 are required for this affect. To perform bandshift, ChIP and ChIP-seq studies to examine the DNA-binding and promoter occupancy of the Eya2/Six4 heterodimer. Aim 2) To isolate cdk6 mutants that disrupt binding of cdk6 to Eya2 and other proteins identified in a yeast two-hybrid screen. This genetic screen will utilize a pool of cdk6 mutants to search for mutants that disrupt or enhance binding to Eya2. Aim 3) To determine the functional consequences of the cdk6 Eya2 interaction using a variety of assays including immunofluorescence and kinase/phosphatase assays. To determine the effect of over-expression of Eya2, cdk6, and mutant versions of these proteins, on proliferation, migration, and tumorigenesis of ovarian cancer cells. The Aims presented here address the hypothesis that cdk6 and Eya2 interact to influence cell proliferation and differentiation-processes important in development and cancer-by examining the biological relevance of the novel interaction of cdk6 and Eya2, which was identified in our laboratory. PUBLIC HEALTH RELEVANCE: The goal of the work outlined in this proposal is to investigate the interaction of the cell cycle regulatory protein, cdk6 and the developmentally important transcription factor, Eya2. An understanding of how these proteins influence differentiation and cell proliferation is crucial to the biology of cancer stem cells and development and may advance our understanding ovarian cancer.