Unraveling the MALAT1 lncRNA-protein interaction networks that drive lung cancer metastasis

项目摘要/摘要 长的非编码RNA(LncRNAs)通过与RNA结合蛋白的相互作用，在 发育和细胞动态平衡。许多lncRNA与癌症和疾病有关，代表着一种 基本未被触及的潜在治疗靶点。转移相关的肺腺癌转录本 1(MALAT1)是一种高度保守的lncRNA，其表达与预后不良和药物相关。 耐药及其活性可加速细胞增殖并增加多发性肿瘤的转移潜能 类型。MALAT1在许多正常细胞系中普遍高水平表达，但MALAT1基因缺失 转录本对小鼠的发育或存活没有负面影响。MALAT1在两者中的作用机制 正常细胞和癌细胞尚不清楚，更好地了解这些机制将释放潜在的 将MALAT1作为治疗癌症的靶点。我的初步数据显示，虽然 肺腺癌细胞系中MALAT1转录本低于正常人肺上皮细胞 细胞中，腺癌的转移活性依赖于MALAT1：因此MALAT1高表达 不足以促进转移。我观察到不同细胞系之间的MALAT1 RNA结构没有差异， 但许多MALAT1结合蛋白的表达水平显著增加。这些数据有力地表明 肺癌细胞中存在的蛋白质正在赋予MALAT1促转移活性。我的目标是 将尖端测序和定量蛋白质组学技术与基于细胞的功能 了解MALAT1的RNA-蛋白质相互作用网络如何促进肿瘤转移活性的方法 人类肺癌。在目标1中，我将从正常肺细胞和 肺癌细胞，并从机械上详细说明转移特异性RNA的RNA和蛋白质组分- 蛋白质相互作用网络。我将在肺癌细胞系中表达MALAT1片段并鉴定序列 足以组装RNP复合体并重新编程细胞培养中的转移行为。在目标2中，我将聘用 全基因组总蛋白质和磷酸蛋白质，核酸相互作用组捕获，以及荧光显微镜观察 了解组装功能失调的MALAT1 RNPs基因组和转录组的信号通路 很宽。总之，这些实验将确定MALAT1-蛋白质相互作用网络的机制 促进肺癌转移的每一步。我希望这项工作将改善我们的基础 了解MALAT1，提供治疗靶向MALAT1的策略，并作为 转录组中促进转移的lncRNA-蛋白质复合体的特征。发展中的 这些提出的目标将为作为癌症RBPome新领域领导者的成功职业生涯奠定基础。

PROJECT SUMMARY/ABSTRACT Long non-coding RNAs (lncRNAs), through interactions with RNA-binding proteins, play critical roles in development and cellular homeostasis. Many lncRNAs are associated with cancer and disease, representing a largely untouched pool of potential therapeutic targets. Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) is a highly conserved lncRNA whose expression is associated with poor prognosis and drug- resistance and whose activity accelerates cell proliferation and increases metastatic potential in multiple cancer types. MALAT1 is ubiquitously expressed at high levels in many normal cell lines, and yet genetic deletion of the transcript has no negative effects on development or viability in mice. The mechanisms of MALAT1 in both normal and cancer cells are unclear, and a better understanding of these mechanisms would unlock the potential of MALAT1 as a therapeutic target in the treatment of cancer. My preliminary data show that while levels of MALAT1 transcript are lower in a lung adenocarcinoma cell line than in normal human lung airway epithelial cells, metastatic activity of the adenocarcinoma is dependent on MALAT1: therefore high expression of MALAT1 is not sufficient to promote metastasis. I observe no differences in MALAT1 RNA structure between cell lines, but levels of expression of many MALAT1-binding proteins increase significantly. These data strongly indicate that proteins present in lung cancer cells are imparting pro-metastatic activity onto MALAT1. My goal is to integrate cutting-edge sequencing and quantitative proteomics technologies with cell-based functional approaches to understand how RNA-protein interaction networks of MALAT1 promote metastatic activity in human lung cancers. In Aim 1, I will purify MALAT1 RNA-protein complexes (RNPs) from normal lung cells and lung cancer cells and mechanistically detail the RNA and protein components of metastases-specific RNA- protein interaction networks. I will express fragments of MALAT1 in lung cancer cell lines and identify sequences sufficient to assemble RNP complexes and reprogram metastatic behavior in cell culture. In Aim 2, I will employ RBPome-wide total and phospho-protemics, nucleic acid interactome capture, and fluorescent microscopy to understand the signaling pathways that assemble dysfunctional MALAT1 RNPs genome- and transcriptome- wide. Together, these experiments will identify the mechanisms by which MALAT1-protein interaction networks promote each step of metastasis in lung cancer. I expect that this work will improve our fundamental understanding of MALAT1, inform strategies to target MALAT1 therapeutically, and act as a framework for the characterization of pro-metastatic lncRNA-protein complexes throughout the transcriptome. Development of these proposed aims will lay the foundation for a successful career as a leader in a new field of cancer RBPomes.