The Role of miRNAs in Epithelial-Mesenchymal Transition and Metastasis

描述（由申请人提供）：微RNA（miRNAs）已被越来越多地认识到在正常生理学和病理过程（包括癌症）中发挥重要作用。 我们最近发现miR-200家族和miR-194/192簇miRNA作为上皮间质转化（EMT）的负调节因子，EMT被认为是癌症转移的关键起始步骤。 这些miRNA维持上皮细胞表型并通过抑制E-钙粘蛋白的转录抑制剂（包括ZEB 1/ZEB 2和MeCP 2）的表达来抑制EMT。 这些miRNAs的过表达抑制TGF β诱导的正常乳腺上皮细胞EMT。 此外，在侵袭性乳腺肿瘤细胞中EMT相关miRNA的异位表达诱导间充质-上皮转化（MET），这是EMT的逆转过程，具有显著减少的迁移和侵袭。 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转移部位产生肉眼可见病变的能力增加相关。 总的来说，这些数据意味着这些miRNA在转移中具有阶段特异性作用。 原发性肿瘤中miRNA表达降低可能刺激EMT和随后的肿瘤细胞播散，而miRNA表达增加将导致MET和远处器官的定植。 作为潜在的抗转移治疗新药物，这些miRNA在癌症进展的不同阶段的功能作用和分子机制需要进一步表征。 我们假设这些miRNAs是肿瘤细胞上皮和间充质状态之间转变的关键主调节因子，并且在原发性肿瘤的初始侵袭以及继发性靶器官的转移性定植中起重要作用。 我们将使用一种综合的方法来研究miRNA介导的EMT和癌症转移的分子机制，利用体外和体内模型以及我们实验室提供的基因组和蛋白质组学研究平台。 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转移测定。 我们将在各种小鼠和人乳腺癌细胞系中过表达miRNA并调节其下游相关基因，并利用体内动物模型研究阶段特异性对乳腺癌转移的影响（目的2）。 我们将使用基因表达谱和质谱的组合分析来鉴定EMT相关miRNA的新靶基因，并使用成熟的体外和体内测定来测试这些基因在EMT和转移中的功能重要性（Aim 2）。 最后，我们将使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miRNAs产生MMTV转基因小鼠，并在乳腺组织中过表达癌基因Neu或PyMT的肿瘤易感动物中测试这些miRNAs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在癌症转移中的阶段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.