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

描述(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。开发更好的转移预防和治疗方案将需要更好地了解与肿瘤进展和转移有关的具体事件。我们已经证明，表达在人类肺癌中常见的突变(KrasG12D和p53R172H)的小鼠会发生转移性肺腺癌。从这些小鼠的肿瘤中分离出来的细胞系在皮下注射到同基因小鼠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，miR-200的表达缺失会导致上皮间充质转化(EMT)基因上调和转移。与EMT一致的是，易转移的肿瘤细胞失去了上皮极性复合体的表达，强制表达miR-200可以恢复上皮极性，消除转移。反过来，在没有转移能力的细胞中敲除基底侧极性复合体的关键成分Scrible会抑制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。我们的全球假设是，功能性顶端-基底极性复合体需要维持miR-200水平，从而抑制癌细胞的EMT和转移。我们将通过完成两个目标来验证这一假说：目标1将确定Scrible缺陷肿瘤中的侵袭和转移是否是完全由于失去了miR-200介导的ZEB1抑制；目标2将确定Scrible缺陷肿瘤中miR-200的抑制和转移是否是由于Scribble本身的丢失，更大的基侧复合体(包括Scribble(Scrib)、Diss Large(Dlg1)和致死性巨型幼虫(Lgl1)蛋白)的功能障碍，或者基侧复合体与包含碎屑同源3(Crb3)或分割缺陷6(Pard6)的顶端极性复合体的异常相互作用。这项工作将使用2D和3D体外模型以及同基因动物研究相结合的方法来进行，目的是了解心尖-基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。在这里，我们建议使用一种新的体外和体内模型的组合，以更好地了解转移过程。具体地说，我们计划研究决定细胞结构和细胞间相互作用的蛋白质如何控制肿瘤的迁移和侵袭特性。

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.