Functional Role of Metadherin Subcellular Localization in Breast Cancer

Metadherin 亚细胞定位在乳腺癌中的功能作用

• 批准号: 7546001
• 负责人: Mario Andres Blanco
• 金额: $ 4.1万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2010-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7546001
• 关键词: 8q22; Accounting; Adhesions; Affect; Biochemistry; Biological Assay; Biomedical Research; Breast Cancer Cell; Cancer Biology; Cancer Patient; Cancer cell line; Cell Adhesion; Cell Line; Cells; Cessation of life; Clinical; Co-Immunoprecipitations; Coculture Techniques; Complement; Drug Delivery Systems; Endothelial Cells; Endothelium; Exclusion; Fatty acid glycerol esters; Fluorescence Microscopy; Genes; Genomics; Goals; Green Fluorescent Proteins; Human; Image; Immunoblotting; Immunofluorescence Immunologic; In Vitro; Injection of therapeutic agent; Laboratories; Life; Localized; Location; Lung; Malignant Neoplasms; Mammary Neoplasms; Mammary gland; Maps; Mediating; Modeling; Molecular; Monitor; Morbidity - disease rate; Mus; Neoplasm Metastasis; Nuclear; Nuclear Localization Signal; Pharmaceutical Preparations; Phenotype; Play; Process; Protein Overexpression; Proteins; Public Health; Range; Recurrence; Research; Risk; Role; Sampling; Series; Site-Directed Mutagenesis; Source; Specimen; Staging; Tail; Testing; Therapeutic; Tissue Microarray; Tissue Sample; Transfection; Translations; Variant; Vascular Endothelium; Veins; Western Blotting; Work; Yeasts; cancer cell; cancer therapy; chemotherapeutic agent; in vitro Assay; in vivo; in vivo Model; interest; knock-down; malignant breast neoplasm; migration; monolayer; mutant; neoplastic cell; outcome forecast; research study; small hairpin RNA; tumor progression; tumorigenic; yeast two hybrid system

DESCRIPTION (provided by applicant): The proposed research aims to increase the molecular understanding of metastasis - a process that accounts for most of cancer-related death and morbidity. Recent work in our laboratory identified the gene Metadherin (MTDH) as the target of recurrent genomic amplification in poor prognosis breast cancer. We determined MTDH to be a dual-functional gene that enhances metastasis by mediating cancer cell adhesion to the lung endothelium while simultaneously promoting increased chemoresistance. As such, MTDH is potentially an excellent drug target of great interest to clinicians; however the molecular mechanisms underlying MTDH-mediated metastasis and chemoresistance must first be uncovered. The proposed study will initiate a rigorous mechanistic analysis of MTDH functionality using a combined approach that will utilize in vitro biochemistry, in vivo models of cancer progression, and extensive clinical correlation analyses with human breast cancer tissue samples. The overarching goal is to determine in what cellular compartments and with which interacting partners MTDH promotes its multiple phenotypes. As a first step, I will use immunofluorescence and immunoblotting analyses to investigate endogenous MTDH localization in early and late stage breast cancer cell lines and in a large-scale set of clinical breast cancer samples. I will also investigate the functional role of MTDH localization by assessing its subcellular localization before and after chemotherapeutic drug treatment and with or without attachment to lung endothelial cells. Secondly, I will determine the role of nuclear-localized MTDH by investigating the phenotypic consequences of expressing a variant of MTDH with blocked nuclear access in relevant cell lines. Functionality of nuclear-blocked MTDH will be assessed in a set in vitro and in vivo chemoresistance, adhesion, and metastasis assays. Finally, I will perform GST-pulldown and co-immunoprecipitation experiments using fractionated cancer cell samples to determine the proteins with which MTDH interacts in various cellular compartments. I will then test the functional significance of these interacting partners by knocking them down with shRNA and testing subsequent effects via the aforementioned functional assays. PUBLIC HEALTH RELEVANCE: Metastasis is the most deadly yet least understood aspect of cancer progression. Improvements in clinical cancer treatment rely on advances in the molecular understanding of metastasis. By studying the molecular mechanisms through which a gene called Metadherin promotes chemoresistant, metastatic breast cancer, I aim to provide support for the translation of basic biomedical research into clinical therapeutics that saves lives.

描述（由申请人提供）：拟议的研究旨在增加对转移的分子理解，这是一个占与癌症相关的死亡和发病率大多数的过程。我们实验室的最新工作确定基因元隐蛋白（MTDH）是不良预后乳腺癌基因组扩增的靶标。我们确定MTDH是一种双功能基因，通过介导癌细胞粘附到肺内皮，同时促进化学抗性增加，从而增强转移。因此，MTDH可能是临床医生引起极大兴趣的优秀药物靶标。但是，必须首先发现MTDH介导的转移和化学抗性的分子机制。拟议的研究将使用一种合并的方法对MTDH功能进行严格的机械分析，该方法将利用体外生物化学，在癌症进展的体内模型以及与人类乳腺癌组织样品进行广泛的临床相关分析。总体目标是确定哪些细胞隔室以及与之相互作用的伴侣MTDH促进其多种表型。作为第一步，我将使用免疫荧光和免疫印迹分析来研究早期和晚期乳腺癌细胞系和大规模临床乳腺癌样品中的内源性MTDH定位。我还将通过评估化学治疗药物治疗前后的亚细胞定位以及在肺内皮细胞附着或不附着的情况下，通过评估其亚细胞的定位来研究MTDH定位的功能作用。其次，我将通过研究表达MTDH变体并在相关细胞系中核能阻塞的表达变体的表型后果来确定核能定位的MTDH的作用。将在体外和体内化学耐药，粘附和转移测定中评估核封锁MTDH的功能。最后，我将使用分离的癌细胞样品进行GST-Pulldown和共免疫沉淀实验，以确定MTDH在各种细胞室中相互作用的蛋白质。然后，我将通过上述功能测定法对这些相互作用伙伴的功能意义来测试这些相互作用的伙伴的功能意义。公共卫生相关性：转移是癌症发展中最鲜为人知的方面。临床癌症治疗的改善取决于转移的分子理解的进步。通过研究一种称为元杜德蛋白的基因促进化学抗性的转移性乳腺癌的分子机制，我旨在为将基本生物医学研究转化为挽救生命的临床治疗剂的基本生物医学研究提供支持。