Functional Analysis of metastasis-Associated Proteins

描述(申请人提供)：这个项目的总体目标是获得关于肿瘤转移分子机制的新信息，以便有助于理解这一过程的发病机制，并获得临床有用的数据。该方法旨在从识别和验证转移相关蛋白和转录本，到对匹配的转移和非转移克隆细胞系中特定基因的遗传操作，并利用存档的人类肿瘤组织进行临床相关性研究。为了便于研究乳腺肿瘤转移的分子机制，我们建立了一系列的乳腺肿瘤细胞株，在这些细胞系中可以筛选转移相关的候选基因并进行功能测试。从多克隆乳腺癌细胞系MDA-MB-435和MDA-MB-231中分离出转移倾向相反的单抗肿瘤细胞系。在这项建议中，我们将使用新开发的2D液体分离/质量映射蛋白质组学方法对一系列乳腺肿瘤细胞的蛋白质图谱进行详细分析。该方法使用等电聚焦和无孔硅胶反相高效液相色谱来产生类似于2D凝胶电泳的PI和MR的图像。蛋白质将根据MALDI-TOF-MS多肽图谱和完整的MR进行鉴定，并将在细胞系裂解液样本之间进行定量比较。被确认和验证为在转移表型方面差异表达的蛋白质将被选择进行基因操作，并将使用体外和体内测试来测试所产生的细胞系的行为。最后，将研究候选蛋白质/基因在存档的正常和疾病人类乳房组织样本中的表达。与临床病理信息的相关性将确定那些具有最有前景的诊断和预后标志物用途的候选乳腺抗原。

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.