How do Tumor Cells Gain Anchorage Independency?

描述(申请人提供)：DNA肿瘤病毒的转化蛋白与内源性细胞因子相互作用，可在调节细胞生长方面发挥广泛的作用。内源性细胞周期调节蛋白与DNA病毒转化蛋白相互作用的最典型的例子是pRb。然而，还有其他例子。一种非常高分子量(600 KDa)的细胞蛋白(以下简称P600)与人和牛乳头瘤病毒(HPV)的E7转化因子形成紧密的蛋白质：蛋白质复合体。在初步研究中，我和我的合作者已经证明，P600在许多肿瘤细胞类型的锚定非依赖性生长和恶性转化中起着普遍的作用。这里提出的工作的广泛目标是通过E7可置换的P600结合蛋白的纯化和酶特性来了解锚定依赖性和恶性转化。我有两个明确的目标：目标1是识别以E7依赖的方式与P600结合的细胞因子。P600蛋白太大，不能用传统方法进行遗传操作或异位表达。在初步研究中，我使用了一种“敲入”策略来创建表达串联亲和表位标记的P600的ES细胞。在这些细胞的抗体下拉实验中，我有几个与P600结合的细胞蛋白被HPVE7取代。Aim 1的研究计划描述了我将如何使用生化方案和质谱学来提纯和鉴定这些E7可置换因子。目的2是了解E7和E7可置换的细胞蛋白如何调节P600的转化功能。在初步研究中，我已经证明P600具有泛素连接酶活性。我推测P600的泛素连接酶活性受E7和E7置换细胞因子相互作用的调节。为了验证这一假设，我将描述P600的泛素连接酶结构域，并鉴定E7特定的泛素连接酶底物。在针对这两个特定目的的概念验证研究中，我已经确定了一种与P600结合、E7可置换的蛋白质为PKM2--一种最近被证明在肿瘤的合成代谢生长中发挥广泛作用的蛋白激酶。我相信，这些实验将为恶性表型的分子机制提供新的见解，包括失巢凋亡和锚定依赖生长。随着时间的推移，这项工作可能会为抗癌药物的开发带来新的靶点。 公共卫生相关性：P600是一种多功能蛋白质，调节细胞运动、锚定依赖性、蛋白质降解和细胞凋亡，以响应钙信号。我们认为，P600复合体的生化特征将揭示锚定依赖性的分子功能，这是理解转移机制的主要问题。这一应用不仅对基础科学具有重要意义，而且对药物发现也具有重要意义。如果特定的蛋白质与癌细胞中的P600结合以支持非锚定生长，这些相互作用可能成为抗癌药物的潜在靶点。

DESCRIPTION (provided by applicant): The transforming proteins of DNA tumor viruses interact with endogenous cellular factors that can play broad roles in the regulation of cell growth. The best-characterized example of an endogenous cell cycle regulator protein that interacts with a DNA virus transforming protein is pRB. However, there are other examples. A very high molecular weight (600 kDa) cellular protein (hereafter termed "p600") forms a tight protein:protein complex with the E7 transforming factor of human and bovine papillomaviruses (HPV). In preliminary studies, my collaborators and I have shown that p600 plays a generalized role in anchorage independent growth and malignant transformation of many tumor cell types. The broad objective of work proposed here is to understand anchorage dependency and malignant transformation through purification and enzymatic characterization of E7-displacible, p600 binding proteins. I have two specific aims: Aim 1 is to identify cellular factors that bind to p600 in an E7-dependent fashion. The p600 protein is too large for genetic manipulation or ectopic expression using conventional approaches. In preliminary studies, I have used a "knock in" strategy to create ES cells that express tandem affinity epitope-tagged p600. In antibody pull down experiments with these cells, I have several p600- binding cellular proteins that are displaced by HPV E7. The study plan for aim 1 describes how I will purify and identify these E7-displacible factors using biochemical protocols and mass spectroscopy. Aim 2 is to understand how E7 and E7-displacible cellular proteins might regulate p600 transforming functions. In preliminary studies, I have shown that p600 has ubiquitin ligase activity. I hypothesize that the ubiquitin ligase activity of p600 is regulated by interactions with E7 and E7-displacibile cellular factors. To test this hypothesis, I will characterize the ubiquitin ligase domain of p600 and identify E7-specific ubiquitin ligase substrates. In proof-of-concept studies for these two specific aims, I have already identified one p600-binding, E7-displacible protein as PKM2 - a protein kinase that has recently been shown to play a broad role in anabolic growth of tumors. I believe that these experiments will provide new insights into the molecular mechanisms of the malignant phenotype including anoikis and anchorage-dependent growth. In the fullness of time, the work could lead to novel new targets for anti cancer drug development. PUBLIC HEALTH RELEVANCE: p600 is a multifunctional protein that regulates cell motility, anchorage dependency, protein degradation, and apoptosis in response to Ca2+ signals. We believe that biochemical characterization of the p600 complexes would reveal molecular functions of anchorage-dependency, which is a major problem in understanding mechanisms of metastasis. This application would be significant not only for basic sciences, but also for drug discovery. If specific proteins bind to p600 in carcinoma cells to support anchorage-independent growth, these interactions could be potential targets for anti-cancer drugs.