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

揭示驱动肺癌转移的 MALAT1 lncRNA-蛋白质相互作用网络

• 批准号: 10283482
• 负责人: Chase A Weidmann
• 金额: $ 16.2万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-22 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10283482
• 关键词: Adenocarcinoma; Automobile Driving; Behavior; Binding Proteins; Bioinformatics; Biological Process; Cancer Biology; Cancer cell line; Cell Culture Techniques; Cell Line; Cell Proliferation; Cells; Chemicals; Chromatin; Complex; Cytoplasmic Granules; DNA; Data; Development; Disease; Drug resistance; Epithelial Cells; Event; Fluorescent in Situ Hybridization; Foundations; Functional disorder; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Goals; High-Throughput Nucleotide Sequencing; Homeostasis; Human; Immunofluorescence Microscopy; Laboratories; Link; Lung; Lung Adenocarcinoma; MALAT1 gene; Malignant Neoplasms; Malignant neoplasm of lung; Maps; Mass Spectrum Analysis; Metastatic Neoplasm to the Lung; Metastatic to; Microscopy; Molecular; Molecular Conformation; Monitor; Mus; Neoplasm Metastasis; Normal Cell; Nuclear; Nucleic Acids; Organelles; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Positioning Attribute; Prognosis; Prognostic Marker; Proteins; Proteomics; RNA; RNA Splicing; RNA-Binding Proteins; RNA-Protein Interaction; Regulator Genes; Regulatory Pathway; Research; Resolution; Ribonucleoproteins; Role; Route; Signal Pathway; Structure; Technology; Testing; Therapeutic; Transcript; Translations; Untranslated RNA; Validation; Vision; Work; airway epithelium; base; cancer cell; cancer risk; cancer therapy; cancer type; career; cell motility; experience; experimental study; follow-up; improved; insight; lung cancer cell; lung metastatic; metaplastic cell transformation; migration; new therapeutic target; novel; novel therapeutic intervention; novel therapeutics; programs; protein complex; protein expression; stoichiometry; therapeutic target; transcriptome; transcriptome sequencing

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.

项目总结/摘要 长链非编码RNA（lncRNA）通过与RNA结合蛋白相互作用，在 发育和细胞内稳态。许多lncRNA与癌症和疾病相关，代表了一种新的癌症。 基本上未被触及的潜在治疗靶点。转移相关肺腺癌转录本 1（MALAT 1）是一种高度保守的lncRNA，其表达与不良预后和药物相关。 其活性加速细胞增殖并增加多种癌症的转移潜力 类型MALAT 1在许多正常细胞系中以高水平普遍表达，但MALAT 1的基因缺失导致MALAT 1基因的缺失。 转录物对小鼠的发育或生存能力没有负面影响。MALAT 1在这两种疾病中的作用机制 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 MALAT 1作为癌症治疗中的治疗靶点。我的初步数据显示， 肺腺癌细胞系中MALAT 1转录低于正常人肺气道上皮细胞 细胞，腺癌的转移活性依赖于MALAT 1：因此MALAT 1的高表达 不足以促进转移。我没有观察到细胞系之间MALAT 1 RNA结构的差异， 但许多MALAT 1结合蛋白的表达水平显著增加。这些数据有力地表明 存在于肺癌细胞中的蛋白质赋予MALAT 1促转移活性。我的目标是 将尖端的测序和定量蛋白质组学技术与基于细胞的功能 了解MALAT 1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，我将从正常肺细胞中纯化MALAT 1 RNA-蛋白复合物（RNP）， 肺癌细胞和机械细节的RNA和蛋白质成分的端粒酶特异性RNA- 蛋白质相互作用网络我将在肺癌细胞系中表达MALAT 1的片段，并鉴定序列 足以在细胞培养物中组装RNP复合物和重编程转移行为。在目标2中，我将使用 全RBPome总蛋白质组学和磷酸蛋白质组学、核酸相互作用组捕获和荧光显微镜检查， 了解组装功能失调的MALAT 1 RNP基因组和转录组的信号通路， 宽总之，这些实验将确定MALAT 1-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本 了解MALAT 1，为治疗靶向MALAT 1的策略提供信息，并作为一个框架， 在整个转录组中的促转移性lncRNA-蛋白质复合物的表征。发展 这些提出的目标将奠定一个成功的职业生涯作为一个领导者在一个新的领域的癌症RBPomes的基础。