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允许系统性鉴定与癌症进展相关的磷酸化事件。然而，验证和翻译这些大型数据集从体外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磷酸化事件如何促进乳腺癌的发展。本探索性研究的目的是开发一种简化的模型系统，用于快速体内分析ABL介导的蛋白磷酸化在乳腺癌生长和转移中的功能。我们最近开发了一种策略，通过使用基于短发夹RNA的技术，有条件地用其突变体对应物的表达替换内源基因表达。我们假设，通过使用这种策略与重组介导的盒式交换（RMCE）技术相结合，可以开发一种简化的系统，以提供一种快速，可逆和具有成本效益的方法来分析给定的磷酸化缺陷或模拟突变体在乳腺癌发展中的功能。我们将通过定义ABL相互作用因子1（Abi 1）酪氨酸磷酸化在乳腺癌发展中的作用来验证这一假设，ABL相互作用因子1是ABL底物，也是肌动蛋白细胞骨架重塑的关键调节因子。这项研究背后的基本原理是，一旦该系统被开发出来，我们将能够快速分析从癌症蛋白质组学研究中获得的大量ABL介导的磷酸化事件的功能，并确定那些对乳腺癌发展至关重要的事件，用于预后和治疗目的。在具体目标1中，将开发一组条件性Abi 1基因沉默/再表达盒，以分析ABL介导的Abi 1酪氨酸磷酸化如何影响乳腺癌细胞功能和信号传导。在目标2中，ABL介导的Abi 1磷酸化如何在体内促进乳腺癌进展将通过使用条件基因替换策略结合我们实验室最近开发的脑转移小鼠模型来确定。该项目具有创新性，因为它提出了一种不同于传统基因敲入的体内蛋白质突变分析新策略。如果成功的话，这一系列的研究将能够对乳腺癌发展中的蛋白质突变进行高通量功能分析。确定ABL介导的磷酸化事件对乳腺癌的发展至关重要，这不仅有助于我们了解ABL激酶如何促进乳腺癌的发展，而且还为选择可能受益于靶向分子治疗的乳腺癌患者提供了预后标志物。

 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.