The Role of miRNAs in Epithelial-Mesenchymal Transition and Metastasis

描述(申请人提供)：microRNAs(MiRNAs)已被越来越多地认识到在正常生理和病理过程中发挥重要作用，包括癌症。我们最近发现miR-200家族和miR-194/192簇miRNAs是上皮-间充质转化(EMT)的负调控因子，而EMT被认为是癌症转移的关键起始步骤。这些miRNAs通过抑制E-钙粘附素的转录抑制物，包括ZEB1/ZEB2和MeCP2的表达，维持上皮细胞的表型并抑制EMT。这些miRNAs的过表达抑制了转化生长因子β诱导的正常乳腺上皮细胞的EMT。此外，EMT相关miRNAs在浸润性乳腺肿瘤细胞中的异位表达可诱导间充质-上皮转化(MET)，这是EMT的反向过程，显著减少了迁移和侵袭。令人惊讶的是，我们还发现miR-200的高表达与肿瘤细胞在继发转移部位产生宏观病变的能力增强相关。总体而言，这些数据暗示这些miRNAs在转移中具有阶段特异性的作用。在原发肿瘤中miRNA表达降低可能会刺激EMT和随后的肿瘤细胞扩散，而miRNA表达增加会导致MET和远处器官的定植。作为潜在的抗转移治疗的新药物，这些miRNAs在肿瘤不同发展阶段的功能作用和分子机制需要进一步的研究。我们推测这些miRNAs是肿瘤细胞上皮和间充质状态之间转换的关键调控因子，在原发肿瘤的初始侵袭和次级靶器官的转移性定植中发挥重要作用。我们将利用体外和体内模型以及实验室现有的基因组和蛋白质组研究平台，采用综合方法研究miRNA介导的EMT和肿瘤转移的分子机制。为了研究这些miRNAs在转移中的潜在双相作用，我们将采用原位和实验转移分析。我们将在不同的小鼠和人乳腺癌细胞系中过表达miRNAs并调节其下游感兴趣的基因，并利用体内动物模型来研究乳腺癌转移的阶段特异性影响(目标2)。我们将使用基因表达谱和质谱学的组合分析来确定与EMT相关的miRNAs的新的靶基因，并使用成熟的体外和体内分析来测试这些基因在EMT和转移中的功能重要性(目标2)。最后，我们将使用转基因小鼠模型评估EMT相关miRNAs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，并测试这些miRNAs在乳腺组织中过表达癌基因Neu或PyMT的易患肿瘤动物的乳腺癌进展中的作用(目标3)。通过这些实验，我们将揭示EMT相关miRNAs在肿瘤转移中的潜在阶段特异性作用和作用机制，并为改进转移癌的预防和治疗提供重要的新见解。 公共卫生相关性：超过90%的癌症相关死亡是由于原发肿瘤细胞转移到远处的重要器官造成的。上皮-间充质转化(EMT)及其逆转过程(MET)被认为是肿瘤细胞获得迁移和侵袭周围组织的能力以及在到达靶器官后重新获得上皮表型以促进定植的重要过程。这项研究将探讨一类新的遗传调节因子microRNAs如何调控EMT，并将提供一些迄今最好的证据，证明EMT是否可以促进肿瘤细胞的早期扩散，而MET可能是有效定植远处靶器官所必需的。

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.