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，并且不知道哪些转移过程涉及Rho蛋白。 我们对这个项目的长期目标是了解这两种蛋白在转移级联反应的关键阶段的重要性，并确定哪些Rho效应途径参与。 我们假设RhoA和RhoC都将通过增强多个阶段的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效应子，并且不同的Rho效应子可能在转移期间介导每个Rho介导的过程。 具体目标：（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质。 (2)确定哪些阶段的乳腺癌转移是由RhoA或RhoC的表达增加促进的。 (3)鉴定Rho介导的转移所需的Rho效应子。 (4)检测RhoA、RhoC和Rho效应子在乳腺癌发生、进展和转移过程中的参与。 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来改变具有不同转移潜能的一系列乳腺癌（MC）细胞系中RhoA或RhoC的表达。 然后将细胞植入同系小鼠的乳腺脂肪垫中，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。 我们还将通过原位植入表达条形码化的基于miR30的shRNA的MC细胞池来筛选参与转移的Rho效应子，每个shRNA靶向已知的Rho效应子，然后筛选在肺中形成的转移性病变中每个条形码的富集或显著减少。 作为额外的模型系统，我们还将从荷瘤MMTV多瘤中T小鼠分离原代MC细胞，操纵Rho蛋白和效应子表达，然后将细胞原位植入供体小鼠并测定对转移过程的影响。 公共卫生相关性：乳腺癌转移每年导致4万名美国妇女死亡，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疗法。 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每个这些过程所需的。

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.