How do Tumor Cells Gain Anchorage Independency?

描述（由申请人提供）：DNA肿瘤病毒的转化蛋白与内源性细胞因子相互作用，在细胞生长调节中发挥广泛作用。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具特征的例子是pRB。然而，还有其他的例子。一种非常高分子量（600 kDa）的细胞蛋白（以下称为“p600”）与人乳头瘤病毒和牛乳头瘤病毒（HPV）的E7转化因子形成紧密蛋白复合物。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独立生长和恶性转化中起着广泛的作用。本文提出的工作的主要目的是通过纯化和酶学表征e7 -可置换p600结合蛋白来了解锚定依赖性和恶性转化。我有两个具体目标：目标1是确定以e7依赖性方式结合p600的细胞因子。p600蛋白太大，无法使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“敲入”策略来创建表达串联亲和表位标记p600的胚胎干细胞。在这些细胞的抗体下拉实验中，我有几个p600结合的细胞蛋白被HPV E7取代。目标1的研究计划描述了我将如何使用生化协议和质谱法纯化和鉴定这些e7可置换因子。目的2是了解E7和E7可置换细胞蛋白如何调节p600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。我假设p600的泛素连接酶活性是通过与E7和E7置换细胞因子的相互作用来调节的。为了验证这一假设，我将表征p600的泛素连接酶结构域，并鉴定e7特异性泛素连接酶底物。在这两个特定目标的概念验证研究中，我已经确定了一种p600结合，e7置换蛋白PKM2 -一种最近被证明在肿瘤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，包括缺失和锚定依赖性生长。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。

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.