Regulation of Lung Cancer Metastasis by miR-200

描述（由申请人提供）： 我们的目标是更好地了解肺癌转移的生物学基础，这可能对公共卫生产生巨大影响，因为肺癌是西方国家癌症相关死亡的主要原因，而转移是肺癌死亡的主要原因。基于我们之前的报道，我们认为抑制肺癌细胞中microRNA-200（miR-200）水平的细胞外信号是转移的关键驱动因素，也是转移预防的靶点。在这里，我们表明，转移倾向的肿瘤细胞从小鼠发展肺腺癌，由于表达突变K-ras和p53表达Notch和Notch配体，和Notch配体锯齿状2促进肿瘤球的形成，EMT，侵袭和转移。Jagged 2增加所有六个加塔转录因子家族成员的表达，GATA 3通过抑制miR-200水平促进肿瘤细胞EMT和转移。我们证实，每个由锯齿状蛋白2调控的加塔因子都起着不同的转录作用，并共同介导了锯齿状蛋白2在该肿瘤模型中的多种生物学效应。这一假说是创新性的，因为它将Notch轴与EMT和转移的核心microRNA联系起来，并且它开辟了一个新的研究领域，即加塔因子作为转移的启动子而不是抑制子的作用。我们提出的实验方法是创新的;小鼠模型是基于我们创建的一个模型，该模型密切概括了人类肺腺癌的生物学和转录特征，我们开发的细胞微图案化技术处于生物工程和细胞生物学之间接口的最前沿。我们提出两个具体目标。第一个目的是确定Jagged 2的失活是否消除肺腺癌小鼠模型中的转移。为了这个目的，我们将建立小鼠，有条件地抑制锯齿状蛋白2和表达致癌K-ras和p53在肺中，我们将检查是否锯齿状蛋白2/缺口依赖性相互作用的肿瘤细胞所需的极化球形成和EMT使用细胞微图案技术，我们已经开发;我们将确定高jagged 2水平是否与低microRNA相关，200家族成员在人肺腺癌活检样品中的表达以及这种表达模式是否与疾病复发和短生存期相关。第二个目的是检查由jagged 2上调的加塔转录因子家族成员是否是肿瘤细胞极化、对TGF 2诱导的EMT的敏感性和转移所必需的。为此，我们将使用遗传学方法从肺腺癌细胞中单独去除加塔因子;我们将研究培养中这些细胞的生物学和转录特性;我们将在我们开发的原位肺肿瘤模型中检查它们的致瘤性和转移潜力。我们的长期目标是开发新的方法来识别那些在早期疾病治疗后复发风险高的患者，并使用药理学方法来靶向这些患者转移的关键介质。 公共卫生相关性： 肺癌是西方国家癌症相关死亡的主要原因，转移是肺癌死亡的主要原因，这表明迫切需要更好地了解转移的生物学基础。基于本文的研究结果，我们假设肿瘤细胞通过受体（Notch）与相邻肿瘤细胞上的配体（jagged 2）结合，以锁和钥匙的方式相互结合;然后Notch抑制称为miR-200的小RNA的水平，诱导细胞形状的变化和与相邻细胞的分离，这是转移的早期事件。如果这些发现被证明是真的，那么将有必要进行研究，以确定抑制Notch受体的药物（目前正在临床试验中）是否能预防肺癌转移，并且对具有高水平jagged 2和Notch的肿瘤最有效。

DESCRIPTION (provided by applicant): Our goal is to better understand the biologic basis for lung cancer metastasis, which could potentially have a tremendous public health impact because lung cancer is the primary cause of cancer-related death in Western countries, and metastasis is the primary cause of death from lung cancer. On the basis of our previous reports, we believe that the extracellular signals that repress microRNA-200 (miR-200) levels in lung cancer cells are key drivers of metastasis and are targets for metastasis prevention. Here we show that metastasis-prone tumor cells from mice that develop lung adenocarcinomas owing to expression of mutant K-ras and p53 express both Notch and Notch ligands, and the Notch ligand jagged2 promotes tumor sphere formation, EMT, invasion, and metastasis. Jagged2 increases the expression of all six GATA transcription factor family members, and GATA3 promotes tumor cell EMT and metastasis by suppressing miR-200 levels. We posit that each of the GATA factors regulated by jagged2 plays a distinct transcriptional role and collectively mediate the diverse biologic effects of jagged2 in this tumor model. This hypothesis is innovative because it links the Notch axis to a microRNA that is central to EMT and metastasis, and it opens a new area of investigation into the role of GATA factors as promoters, rather than repressors, of metastasis. The experimental approaches we have proposed are innovative; the mouse model is based on one we created that closely recapitulates biologic and transcriptional features of human lung adenocarcinoma, and the cell micropatterning techniques we have developed are at the forefront of the interface between bioengineering and cell biology. We propose two Specific Aims. The first Aim is to determine whether inactivation of Jagged2 abrogates metastasis in a mouse model of lung adenocarcinoma. For this Aim, we will create mice that conditionally inactivate jagged2 and express oncogenic K-ras and p53 in the lung; we will examine whether jagged2/Notch-dependent interactions between tumor cells are required for polarized sphere formation and EMT using cell micro-patterning techniques we have developed; and we will determine whether high jagged2 levels correlate with low microRNA-200 family member expression in human lung adenocarcinoma biopsy samples and whether this expression pattern correlates with disease recurrence and short duration of survival. The second Aim is to examine whether the GATA transcription factor family members up-regulated by jagged2 are required for tumor cell polarization, sensitivity to TGF2-induced EMT, and metastasis. For this Aim, we will use genetic approaches to deplete GATA factors individually from lung adenocarcinoma cells; we will study the biologic and transcriptional properties of those cells in culture; and we will examine their tumorigenicity and metastatic potential in an orthotopic lung tumor model we have developed. Our long-term goal is to develop novel approaches to identify those patients who are at high risk for recurrence following treatment of early-stage disease and to use pharmacologic approaches to target key mediators of metastasis in those patients. PUBLIC HEALTH RELEVANCE: Lung cancer is the primary cause of cancer-related death in western countries, and metastasis is the primary cause of death from lung cancer, which demonstrates a crucial need to better understand the biologic underpinnings of metastasis. On the basis of the findings presented here, we postulate that tumor cells bind to each other in a lock-and-key fashion through a receptor (Notch) that binds to its ligand (jagged2) on adjacent tumor cells; Notch then suppresses the levels of a small RNA called miR-200, inducing a change in cell shape and detachment from neighboring cells, an early event in metastasis. If proven true, these findings would warrant studies to determine whether drugs that inhibit Notch receptors, which are currently in clinical trials, prevent lung cancer metastasis and do so most effectively for tumors that have high levels of jagged2 and Notch.