Novel Animal Models for Functional Analysis of Protein Phosphorylation in Breast Cancer Progression.

 描述(由申请人提供)：癌症蛋白质组学的最新进展使系统地识别与癌症进展相关的磷酸化事件成为可能。然而，验证并将这些大型数据集从体外系统转换到体内系统仍然是一个重大挑战。我们研究的长期目标是了解Abelson(ABL)酪氨酸激酶诱导的蛋白质磷酸化事件如何促进乳腺癌的发展。这项探索性研究的目的是建立一个简化的模型系统，用于在体内快速分析ABL介导的蛋白磷酸化在乳腺癌生长和转移中的作用。我们最近开发了一种策略，通过使用基于短发夹RNA的技术，有条件地用突变基因的表达替换内源基因的表达。我们假设，通过使用该策略和重组介导盒交换(RMCE)技术相结合，可以开发一个简化的系统来提供快速、可逆和经济有效的方法来分析给定的磷酸化缺陷或模拟突变在乳腺癌发生中的功能。我们将通过确定ABL相互作用因子1(ABL底物和肌动蛋白细胞骨架重塑的关键调节因子)的酪氨酸磷酸化在乳腺癌发生中的作用来检验这一假说。这项研究背后的理论基础是，一旦这个系统被开发出来，我们将能够快速分析从癌症蛋白质组研究中获得的大量ABL介导的磷酸化事件的功能，并识别那些对于乳腺癌的发展至关重要的预后和治疗目的。在具体目标1中，将开发一套条件Abi1基因沉默/再表达盒，以分析ABL介导的Abi1酪氨酸磷酸化如何影响乳腺癌细胞的功能和信号转导。在目标2中，ABL介导的Abi1磷酸化如何在体内促进乳腺癌的进展将通过使用条件基因替换策略结合我们实验室最近建立的脑转移小鼠模型来确定。这个项目具有创新性，因为它为体内蛋白质突变分析提出了一种不同于传统基因敲击的新策略。如果成功，这一系列研究将能够对乳腺癌发生过程中的蛋白质突变进行高通量功能分析。识别ABL介导的磷酸化事件对乳腺癌的发生发展至关重要，这不仅有助于我们了解ABL激酶在乳腺癌发生中的作用，还将为选择可能受益于靶向分子治疗的乳腺癌患者提供预后标志物。

 DESCRIPTION (provided by applicant): Recent advances in cancer proteomics have allowed for systemic identification of the phosphorylation events associated with cancer progression. However, validating and translating these large datasets from in vitro to in vivo systems remains a major challenge. The long-term goal of our research is to understand how the protein phosphorylation events induced by Abelson (ABL) tyrosine kinases contribute to breast cancer development. The objective of this exploratory study is to develop a streamlined model system for rapid in vivo analysis of the function of ABL-mediated protein phosphorylation in breast cancer growth and metastasis. We have recently developed a strategy to conditionally replace an endogenous gene expression with the expression of its mutant counterpart by using short hairpin RNA-based technology. We hypothesize that, by using this strategy combined with the recombination-mediated cassette exchange (RMCE) technology, a streamlined system can be developed to provide a fast, reversible, and cost-effective way to analyze the function of a given phosphorylation-deficient or -mimetic mutant in breast cancer development. We will test this hypothesis by defining the role of tyrosine phosphorylation of ABL interactor 1 (Abi1), an ABL substrate and key regulator of actin cytoskeleton remodeling, in breast cancer development. The rationale behind this research is that once this system is developed, we will be able to quickly analyze the function of a large set of ABL-mediated phosphorylation events obtained from cancer proteomic studies and identify those that are essential for breast cancer development for prognostic and therapeutic purposes. In specific aim 1, a set of conditional Abi1 gene silencing/re-expressing cassettes will be developed to analyze how ABL-mediated Abi1 tyrosine phosphorylation affects breast cancer cell function and signaling. In aim 2, how the ABL-mediated Abi1 phosphorylation contributes to breast cancer progression in vivo will be determined by using conditional gene replacement strategy combined with a brain metastasis mouse model recently developed in our lab. This project is innovative because it proposes a new strategy different from conventional gene knockin for in vivo protein mutation analysis. If successful, this line of research will enable a high-throughput function analysis of protein mutation in breast cancer development. Identifying ABL-mediated phosphorylation events essential for breast cancer development will not only help us to understand how ABL kinases contribute to breast cancer development, but also provide prognostic markers for selection of breast cancer patients who may benefit from targeted molecular therapy.