Functional Analysis of metastasis-Associated Proteins

描述（由申请人提供）：该项目的总体目标是获得有关肿瘤转移分子机制的新信息，以有助于了解这一过程的发病机制并获得临床有用的数据。所提出的方法旨在从鉴定和验证转移相关蛋白和转录本，到匹配转移性和非转移性克隆细胞系中特定基因的遗传操作，并使用存档的人类肿瘤组织进行临床相关性研究。为了便于研究乳腺癌转移的分子机制，我们获得了一系列单克隆乳腺肿瘤细胞系，在这些细胞系中可以筛选和功能测试候选转移相关基因。从多克隆乳腺癌细胞系MDA-MB-435和MDA-MB-231中分离得到具有相反转移倾向的单克隆肿瘤细胞系。在本提案中，我们将使用新开发的二维液体分离/大规模定位蛋白质组学方法对一系列乳腺肿瘤细胞的蛋白质谱进行详细分析。该方法使用等电聚焦和无孔二氧化硅反相高效液相色谱来创建类似于二维凝胶电泳的pI与Mr图像。蛋白质将根据MALDI-TOF-MS肽图谱和完整Mr进行鉴定，并在细胞系裂解液样品之间进行定量比较。在转移表型方面被鉴定和验证为差异表达的蛋白质将被选择用于遗传操作，并通过体外和体内试验测试所产生的细胞系的行为。最后，候选蛋白/基因的表达将在存档的正常和患病人乳腺组织样本中进行研究。与临床病理信息的相关性将确定那些具有最有希望的诊断和预后标记效用的候选乳腺抗原。

DESCRIPTION (provided by applicant): The overall goal of this project is to obtain novel information on the molecular mechanisms of tumor metastasis, in order to contribute to understanding of the pathogenesis of this process and to obtain data that would be clinically useful. The proposed approach is designed to progress from identifying and verifying metastasis-associated proteins and transcripts, to genetic manipulation of specific genes in matched metastatic and non-metastatic clonal cell lines, and to lead into clinical correlation studies using archived human tumor tissues. In order to facilitate research investigations into the molecular mechanisms of breast tumor metastasis, we have derived a series of monoclonal breast tumor cell lines in which candidate metastasis-associated genes can be screened and functionally tested. Monoclonal tumor cell lines of opposite metastatic propensity were derived from the polyclonal breast carcinoma cell lines MDA-MB-435 and MDA-MB-231. Within this proposal, we will conduct a detailed analysis of the protein profile of the series of breast tumor cells using a newly developed 2D liquid separation/mass-mapping proteomic approach. This method uses isoelectric focusing and non-porous silica reversed-phase high-performance liquid chromatography to create an image of pI vs Mr analogous to 2D gel electrophoresis. Proteins will be identified based upon MALDI-TOF-MS peptide mapping and intact Mr, and quantitative comparisons will be performed between cell line lysate samples. Proteins identified and validated as being differentially expressed with regard to metastatic phenotype will be selected for genetic manipulation and resulting cell lines will be tested for behavior using in vitro and in vivo assays. Finally, the expression of candidate proteins/genes will be studied in archived normal and diseased human breast tissue samples. Correlation with clinicopathologic information will identify those candidate breast antigens with the most promising diagnostic and prognostic marker utility.