The Role of miRNAs in Epithelial-Mesenchymal Transition and Metastasis

描述（由申请人提供）：MicroRNAs （miRNAs）在包括癌症在内的正常生理和病理过程中发挥着越来越重要的作用。我们最近发现miR-200家族和miR-194/192 microrna簇是上皮-间质转化（EMT）的负调控因子，而上皮-间质转化被认为是癌症转移的关键启动步骤。这些mirna维持上皮细胞表型，并通过抑制E-cadherin转录抑制剂（包括ZEB1/ZEB2和MeCP2）的表达来抑制EMT。这些mirna的过表达可抑制正常乳腺上皮细胞中TGF -诱导的EMT。此外，侵袭性乳腺肿瘤细胞中EMT相关mirna的异位表达诱导了EMT的反向过程间充质-上皮转化（MET），显著减少了EMT的迁移和侵袭。令人惊讶的是，我们还发现miR-200表达升高与肿瘤细胞在继发性转移部位产生宏观病变的能力增加相关。这些数据表明，这些mirna在转移中具有分期特异性作用。原发肿瘤中miRNA表达的减少可能会刺激EMT和随后的肿瘤细胞播散，而miRNA表达的增加将导致MET和远端器官的定植。作为潜在的抗转移治疗新药物，这些mirna在不同癌症进展阶段的功能作用和分子机制有待进一步研究。我们假设这些mirna是肿瘤细胞在上皮和间质状态之间转换的关键主调控因子，在原发性肿瘤的初始侵袭和次要靶器官的转移定殖中发挥重要作用。我们将利用体外和体内模型以及实验室现有的基因组学和蛋白质组学研究平台，采用综合方法研究mirna介导的EMT和癌症转移的分子机制。为了研究这些mirna在转移中的潜在双相作用，我们将采用原位和实验性转移分析。我们将在各种小鼠和人类乳腺癌细胞系中过表达mirna并调节其下游基因，并利用体内动物模型研究乳腺癌转移的分期特异性作用（目的2）。我们将使用基因表达谱和质谱的联合分析来鉴定EMT相关mirna的新靶基因，并使用成熟的体外和体内试验来测试这些基因在EMT和转移中的功能重要性（目的2）。最后，我们将利用转基因小鼠模型评估emt相关mirna在乳腺功能和乳腺肿瘤进展中的重要性。我们将为这些mirna生成MMTV转基因小鼠，并在乳腺组织中过度表达癌基因Neu或PyMT的易发肿瘤动物中测试这些mirna在乳腺癌进展中的作用（目的3）。通过这些实验，我们将揭示emt相关mirna在癌症转移中的潜在分期特异性作用和功能机制，并为改善转移性癌症的预防和治疗提供重要的新见解。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) have been increasingly recognized to play important roles in normal physiology and in pathological processes, including cancer. We recently identified the miR-200 family and the miR-194/192 cluster of miRNAs as negative regulators of epithelial-mesenchymal transition (EMT), which is thought to be the critical initiating step of cancer metastasis. These miRNAs maintain the epithelial cell phenotype and inhibit EMT by repressing the expression of transcriptional inhibitors of E-cadherin, including ZEB1/ZEB2 and MeCP2. Overexpression of these miRNAs inhibits TGF¿-induced EMT in normal mammary epithelial cells. Furthermore, ectopic expression of the EMT-related miRNAs in invasive breast tumor cells induces mesenchymal-epithelial transition (MET), the reverse process of EMT, with significant reduction of migration and invasion. Surprisingly, we also found that elevated miR-200 expression is correlated with increased ability of tumor cells to generate macroscopic lesions at secondary metastasis sites. These data in aggregate imply that there is a stage-specific role of these miRNAs in metastasis. Decreased miRNA expression in the primary tumor may stimulate EMT and subsequent dissemination of tumor cells, whereas increased miRNA expression will lead to MET and colonization of distant organs. As potentially new agents for anti-metastasis therapeutics, the functional role and molecular mechanism of these miRNAs in different stages of cancer progression require further characterization. We hypothesize that these miRNAs are critical master regulators of the transition between the epithelial and mesenchymal states of tumor cells and play important roles in both the initial invasion of primary tumors as well as metastatic colonization of secondary target organs. We will use a comprehensive approach to study the molecular mechanism of miRNA-mediated EMT and cancer metastasis, taking advantage of in vitro and in vivo models as well as genomic and proteomic research platforms available in our laboratory. To study the potential biphasic role of these miRNAs in metastasis, we will employ orthotopic and experimental metastasis assays. We will overexpress the miRNAs and modulate their downstream genes of interest in various mouse and human breast cancer cell lines and utilize in vivo animal models to investigate stage-specific effects on breast cancer metastasis (Aim 2). We will use combined analysis of gene expression profiling and mass spectrometry to identify novel target genes of EMT-related miRNAs and test the functional importance of these genes in EMT and metastasis using well-established in vitro and in vivo assays (Aim 2). Finally, we will evaluate the importance of EMT-related miRNAs in mammary gland function and mammary tumor progression using transgenic mouse models. We will generate MMTV transgenic mice for these miRNAs and test the role of these miRNAs in breast cancer progression in tumor-prone animals that overexpress the oncogene Neu or PyMT in their mammary tissue (Aim 3). Through these experiments, we will reveal the potential stage-specific roles and functional mechanisms for EMT-related miRNAs in cancer metastasis and provide important novel insights for improving the prevention and treatment of metastatic cancer. PUBLIC HEALTH RELEVANCE: Over 90% of cancer related deaths are due to the metastatic spread of primary tumor cells to distant vital organs. Epithelial-mesenchymal transition (EMT) and the reverse process, MET, have been postulated to be important processes for tumor cells to gain the ability to migrate and invade into their surrounding tissue and to regain their epithelial phenotype to facilitate colonization once they reach the target organs. This study will investigate how microRNAs, a novel class of genetic regulators, regulate EMT and will provide some of the best evidence to date as to whether EMT can promote early dissemination of tumor cells while MET may be essential for efficient colonization of distant target organs.