Regulation of Lung Cancer Metastasis by miR-200

描述(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，这可能会对公众健康产生巨大影响，因为在西方国家，肺癌是癌症相关死亡的主要原因，而转移是肺癌死亡的主要原因。根据我们以前的报道，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，并且是转移预防的靶点。在这里，我们表明，由于突变的K-ras和p53的表达而导致肺腺癌的小鼠的转移倾向肿瘤细胞同时表达Notch和Notch配体，并且Notch配体Jagged2促进肿瘤球体的形成、EMT、侵袭和转移。Jagged2可上调所有6个GATA转录因子家族成员的表达，而GATA3通过抑制miR-200水平促进肿瘤细胞的EMT和转移。我们假设Jagged2调控的每一个GATA因子都发挥着不同的转录作用，并共同调节Jagged2在该肿瘤模型中的不同生物效应。这一假说是创新的，因为它将Notch轴与对EMT和转移至关重要的microRNA联系起来，并开辟了一个新的研究领域，研究GATA因子作为转移的促进者而不是抑制者的作用。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紧密概括人类肺腺癌的生物学和转录特征的模型，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接口的前沿。我们提出了两个具体目标。第一个目的是确定在小鼠肺腺癌模型中Jagged2的失活是否可以消除转移。为此，我们将建立有条件地使Jagged2失活并在肺中表达致癌K-ras和P53的小鼠；我们将使用我们开发的细胞微图案技术来检测肿瘤细胞之间的Jagged2/Notch依赖的相互作用是否是极化球形成和EMT所必需的；我们将确定在人类肺腺癌活检样本中高Jagged2水平是否与低microRNA-200家族成员表达相关，以及这种表达模式是否与疾病复发和短生存期相关。第二个目的是检测Jagged2上调的GATA转录因子家族成员是否对肿瘤细胞极化、对TGF2诱导的EMT的敏感性和转移是必需的。为此，我们将使用遗传学方法从肺腺癌细胞中逐个去除GATA因子；我们将在培养中研究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测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有高复发风险的患者，并使用药理学方法来靶向这些患者中转移的关键介质。

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.