MicroRNA-200 family regulation by the apical-basal polarity complexes in EMT and

描述（由申请人提供）：肺癌是癌症相关死亡的主要原因，主要是由于转移性疾病。开发改进的转移预防和治疗方案将需要更好地了解肿瘤进展和转移中涉及的特定事件。我们已经证明，表达在人肺癌中常见的突变（KrasG 12 D和p53 R172 H）的小鼠发生转移性肺腺癌。从这些小鼠的肿瘤中分离的细胞系在皮下注射到同基因小鼠中后以高或低的频率转移，这取决于microRNA-200（miR-200）家族成员的表达。由于miR-200成员抑制Zeb家族的转录抑制因子，因此miR-200表达的缺失导致上皮-间充质转化（EMT）基因的上调和转移。与EMT一致，易转移的肿瘤细胞失去上皮极性复合物的表达，并且miR-200的强制表达恢复上皮极性并消除转移。反过来，在无转移能力的细胞中，基底外侧极性复合物的关键组分scribble的敲低抑制miR-200，增加Zeb-1，并诱导转移。这些发现表明细胞极性、miR-200和转移之间存在明显的相互作用。我们的总体假设是，需要功能性顶部-基底极性复合物来维持miR-200水平，从而抑制癌细胞EMT和转移。我们将通过完成两个目标来检验这一假设：目标1将确定scribble缺陷型肿瘤的侵袭和转移是否完全是由于miR-200介导的Zeb 1抑制的丧失;目的2将确定在scribble缺陷型肿瘤中miR-200的抑制和转移是否是由于scribble本身的丢失，包括scribble（Scrib）的较大基底外侧复合体的功能障碍，盘大（Dlg 1），和致命的巨大幼虫（Lg 1）蛋白，或异常的相互作用的基底外侧复合体与顶端极性复合体，含有面包屑同源物3（Crb 3）或分区缺陷6（Pard 6）。将使用2D和3D体外模型以及同基因动物研究的组合来进行这项工作，目的是了解顶-底极性因子如何通过miR-200功能调节细胞表型。 公共卫生相关性：肺癌是美国癌症死亡的主要原因，主要是由于疾病转移到远处器官的影响。在此，我们提出了一种新的体外和体内模型的组合，以更好地了解转移的过程。具体来说，我们计划研究肿瘤的迁移和侵袭特征是如何由决定细胞结构和细胞间相互作用的蛋白质控制的。

DESCRIPTION (provided by applicant): Lung cancer is the leading cause of cancer-related mortality, primarily due to metastatic disease. Developing improved metastasis prevention and treatment options will require a better understanding of the specific events involved in tumor progression and metastasis. We have shown that mice expressing mutations commonly found in human lung cancer (KrasG12D and p53R172H) develop metastatic lung adenocarcinomas. Cell lines isolated from tumors in these mice metastasize with high or low frequency following subcutaneous injection into syngeneic mice, dependent upon the expression of the microRNA-200 (miR-200) family members. Since the miR-200 members repress the Zeb family of transcriptional repressors, loss of miR-200 expression results in upregulation of epithelial-to-mesenchymal transition (EMT) genes and metastasis. Coincident with EMT, the metastasis-prone tumor cells lose expression of epithelial polarity complexes, and forced expression of miR-200 restores epithelial polarity and abrogates metastasis. Reciprocally, knockdown of a key component of the basolateral polarity complex, scribble, in a metastasis-incompetent cell suppresses miR-200, increases Zeb-1, and induces metastasis. These findings demonstrate a clear interplay between cell polarity, miR-200, and metastasis. Our global hypothesis is that functional apical-basal polarity complexes are required to maintain miR-200 levels and thereby suppress cancer cell EMT & metastasis. We will test this hypothesis by completing two Aims: Aim 1 will determine whether invasion and metastasis in scribble-deficient tumors is due entirely to loss of miR-200-mediated suppression of Zeb1; Aim 2 will determine whether miR-200 suppression and metastasis in the scribble-deficient tumors is due to loss of scribble itself, dysfunction of the larger basolateral complex, which includes the scribble (Scrib), discs large (Dlg1), and lethal giant larvae (Lgl1) proteins, or aberrant interactions of the basolateral complex with the apical polarity complexes, containing the crumbs homolog 3 (Crb3) or partitioning defective 6 (Pard6). A combination of 2D and 3D in vitro models, and syngeneic animal studies will be used to carry out this work, with the aim of understanding how the apical- basal polarity factors regulate cell phenotype through miR-200 fucntion. PUBLIC HEALTH RELEVANCE: Lung cancer is the leading cause of cancer mortality in the U.S., due mainly to the impact of disease metastasis to distant organs. Herein we propose to work with a new combination of in vitro and in vivo models to better understand the process of metastasis. Specifically we plan to study how migratory and invasive characteristics of the tumor are controlled by proteins that determine cellular architecture and cell-cell interaction.