Functional Role of Metadherin Subcellular Localization in Breast Cancer

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-这是一个导致大多数癌症相关死亡和发病的过程。我们实验室最近的工作确定了Metadherin（MTDH）基因作为预后不良乳腺癌复发性基因组扩增的靶点。我们确定MTDH是一个双功能基因，它通过介导癌细胞粘附到肺内皮而增强转移，同时促进化疗耐药性的增加。因此，MTDH可能是临床医生非常感兴趣的一个极好的药物靶标;然而，必须首先揭示MTDH介导的转移和耐药性的分子机制。拟议的研究将使用一种组合方法对MTDH功能进行严格的机制分析，该方法将利用体外生物化学、癌症进展的体内模型以及与人类乳腺癌组织样本的广泛临床相关性分析。首要目标是确定MTDH在哪些细胞隔室中以及与哪些相互作用伙伴促进其多种表型。作为第一步，我将使用免疫荧光和免疫印迹分析，以调查内源性MTDH定位在早期和晚期乳腺癌细胞系和大规模的一组临床乳腺癌样本。我还将研究MTDH定位的功能作用，通过评估其亚细胞定位之前和之后的化疗药物治疗和有或没有附着到肺内皮细胞。其次，我将确定核定位MTDH的作用，通过研究在相关细胞系中表达MTDH变体与阻断核通路的表型后果。将在一组体外和体内化学抗性、粘附和转移测定中评估核阻断的MTDH的功能。最后，我将使用分级分离的癌细胞样品进行GST-下拉和免疫共沉淀实验，以确定MTDH在各种细胞区室中相互作用的蛋白质。然后，我将通过用shRNA敲除这些相互作用的伴侣，并通过上述功能测定测试随后的影响，来测试这些相互作用的伴侣的功能意义。公共卫生相关性：转移是癌症进展中最致命但最不了解的方面。临床癌症治疗的改进依赖于对转移的分子理解的进展。通过研究一种名为Metadherin的基因促进化疗耐药的转移性乳腺癌的分子机制，我的目标是为基础生物医学研究转化为拯救生命的临床疗法提供支持。

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.