Functional Role of Metadherin Subcellular Localization in Breast Cancer

描述(申请人提供)：这项拟议的研究旨在增加对转移的分子理解--这一过程是癌症相关死亡和发病率的主要原因。我们实验室最近的工作证实，在预后不良的乳腺癌中，Metherin(MTDH)基因是反复进行基因组扩增的靶基因。我们确定MTDH是一个双功能基因，通过介导癌细胞与肺内皮细胞的黏附而促进转移，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种极好的药物靶点；然而，必须首先揭示MTDH介导的转移和化疗耐药的分子机制。这项拟议的研究将采用一种综合方法，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，启动对MTDH功能的严格机制分析。首要目标是确定在哪些细胞隔间以及与哪些相互作用的合作伙伴中，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内源性MTDH在早期和晚期乳腺癌细胞系中的定位以及在大规模临床乳腺癌样本中的定位。我还将通过评估MTDH在化疗药物治疗前后的亚细胞定位以及是否附着于肺内皮细胞来研究其功能作用。其次，我将通过研究在相关细胞系中表达核通路受阻的MTDH变体的表型后果来确定核定位的MTDH的作用。在体外和体内的化疗耐药、黏附和转移试验中，将对核阻断MTDH的功能进行评估。最后，我将使用分离的癌细胞样本进行GST下拉和免疫共沉淀实验，以确定MTDH在各个细胞间隔中与之相互作用的蛋白质。然后，我将通过用shRNA击倒这些相互作用的伙伴来测试它们的功能意义，并通过前面提到的功能分析来测试后续的效果。公共卫生相关性：转移是癌症进展过程中最致命但最不为人所知的方面。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Metherin的基因促进化疗耐药、转移性乳腺癌的分子机制，我的目标是为将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.