Roles for Rho Proteins During Specific Stages of Breast Cancer Metastasis.

描述（由申请人提供）：尽管RhoA和RhoC与侵袭和转移有关，但Rho介导的转移背后的分子机制尚不清楚，并且不清楚哪些转移过程涉及Rho蛋白。我们这个项目的长期目标是了解这两种蛋白在转移级联的关键阶段的重要性，并确定哪些Rho效应途径参与其中。我们假设RhoA和RhoC都可以通过促进多阶段转移来促进乳腺癌的扩散。我们进一步预测Rho介导的转移将需要特定的Rho效应物，并且不同的Rho效应物可能在转移过程中介导每个Rho介导的过程。具体目的：(1)确定乳腺癌转移是否需要RhoA或RhoC，并确定转移级联的哪些阶段依赖于这些蛋白。(2)确定RhoA或RhoC表达增加促进乳腺癌转移的阶段。(3)确定Rho介导的转移所需的Rho效应物。(4)检测RhoA、RhoC和Rho效应物在乳腺癌发生、进展和转移过程中的作用。研究设计：我们将使用逆转录病毒表达构建体和基于mir30的shrna来改变一系列具有不同转移潜力的乳腺癌（MC）细胞系中RhoA或RhoC的表达。然后将细胞植入同基因小鼠的乳腺脂肪垫，以测定几种转移过程（侵袭、内渗、外渗和转移性肿瘤形成）的差异。我们还将筛选参与转移的Rho效应物，方法是通过原位植入表达基于mir30的条形码shrna的MC细胞池，每个细胞靶向一种已知的Rho效应物，然后筛选在肺部形成的转移病变中每种条形码的富集或急剧减少。作为一个额外的模型系统，我们还将从携带肿瘤的MMTV多瘤中间T小鼠中分离原代MC细胞，操纵Rho蛋白和效应物的表达，然后将细胞原位植入供体小鼠并检测对转移过程的影响。公共卫生相关性：乳腺癌转移每年导致40,000名美国妇女死亡，在我们完全了解特定蛋白质如何影响转移过程之前，我们将无法设计出对抗这种疾病的治疗方法。我们的研究旨在确定哪些转移过程涉及Rho蛋白，以及确定Rho蛋白下游的哪些分子途径是这些过程所必需的。

DESCRIPTION (provided by applicant): Although RhoA and RhoC have been implicated in invasion and metastasis, the molecular mechanisms behind Rho-mediated metastasis are unclear, and it is unknown which metastatic processes involve Rho proteins. Our long-term goals for this project are to understand the importance of these two proteins during key stages of the metastatic cascade, and to identify which Rho effector pathways are involved. We hypothesize that both RhoA and RhoC will promote the spread of breast cancer by enhancing multiple stages of metastasis. We further predict that Rho-mediated metastasis will require specific Rho effectors, and that different Rho effectors may mediate each Rho-mediated process during metastasis. Specific Aims: (1) Determine if breast cancer metastasis requires RhoA or RhoC, and identify which stages of the metastatic cascade are dependent upon these proteins. (2) Identify which stages of breast cancer metastasis are promoted by increased expression of RhoA or RhoC. (3) Identify Rho effectors that are required for Rho-mediated metastasis. (4) Test the involvement of RhoA, RhoC, and Rho effectors during mammary carcinoma development, progression, and metastasis. Study design: We will use retroviral expression constructs and miR30-based shRNAs to alter the expression of RhoA or RhoC in a series of mammary carcinoma (MC) cell lines with varying metastatic potentials. Cells will then be implanted into the mammary fat pads of syngeneic mice to assay differences in several metastatic processes (invasion, intravasation, extravasation and metastatic tumor formation). We will also screen for Rho effectors involved in metastasis by orthotopically implanting pools of MC cells expressing barcoded miR30-based shRNAs that each target a known Rho effector, and then screening for enrichment or dramatic reduction of each barcode in metastatic lesions that form in the lungs. As an additional model system, we will also isolate primary MC cells from tumor-bearing MMTV Polyoma middle T mice, manipulate Rho protein and effector expression, and then orthotopically implant the cells into donor mice and assay the effects on metastatic processes. PUBLIC HEALTH RELEVANCE: Breast cancer metastasis kills 40,000 American women each year, and until we fully understand how specific proteins influence the metastatic processes, we will be unable to design therapies that combat this disease. Our studies aim to identify which metastatic processes involve Rho proteins, as well as determine which molecular pathways downstream of Rho proteins are required for each of these processes.