Understanding Spatially Regulated Tumor-Inhibitory Function of Profilin-1 and its Deregulation in Breast Cancer

了解 Profilin-1 的空间调节肿瘤抑制功能及其在乳腺癌中的失调

基本信息

批准号：
10062480
负责人：
Jieya Shao
金额：
$ 34.88万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-12-01 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10062480
关键词：
Actin-Binding Protein Actins Binding Binding Sites Bladder Breast Breast Cancer Cell Breast Cancer cell line Breast Epithelial Cells Cancer cell line Cell Line Cell Nucleus Cell Survival Cells Chromatin Co-Immunoprecipitations Complex Consensus Cytoplasm Cytoplasmic Protein DNA Polymerase II Data Data Set Development Down-Regulation Epithelial Eukaryotic Cell Exportins Foundations GTP-Binding Protein alpha Subunits, Gs Gene Expression Genes Genetic Transcription Goals Grant Growth In Vitro Investigation Liver Location MCF7 cell MDA MB 231 Malignant Neoplasms Mammalian Cell Mammary Neoplasms Mass Spectrum Analysis Mediating Mesenchymal Messenger RNA Modeling Mus Mutate Mutation Neoplasm Metastasis Normal tissue morphology Nuclear Nuclear Export Nuclear Protein Oncogenes Pancreas Phenotype Phosphorylation Phosphotransferases Play Primary Neoplasm Proline Protein Deregulation Protein Export Pathway Proteins RNA RNA Interference RNA Polymerase II Resistance Role Sampling Stains Structure TP53 gene Testing The Cancer Genome Atlas Tissues Transcription Elongation Tumor Suppressor Genes Tumor Suppressor Proteins Xenograft procedure antitumor effect cancer cell cancer type cell growth clinically relevant cyclin T1 epigenome experimental study gene repression in vivo knock-down malignant breast neoplasm novel novel therapeutics overexpression polymerization prevent profilin 1 promoter recruit slug stem trafficking transcriptome tumor tumor progression

项目摘要

PROJECT SUMMARY Altered protein nucleocytoplasmic trafficking is increasingly recognized as a bona fide driver of cancer progression. Many tumor suppressor proteins (e.g. p53, Rb) function in the nucleus, but undergo increased nuclear export in many types of cancer resulting from overexpression of their nuclear exporter XPO1/CRM1. Profilin-1 (Pfn1) is an actin-binding protein with well-documented anti-tumor and anti-metastatic activities in various types of cancer. However, its role as a tumor suppressor remains controversial because it is rarely mutated and paradoxically essential for cell growth and survival owing to its facilitation of actin polymerization. Though generally considered a cytoplasmic protein, Pfn1 is present in the nucleus of many mammalian cells with poorly understood function. We have previously found that the in vitro anti-tumor effect of Pfn1 in breast cancer cells requires its ability to enter nucleus. This suggests that Pfn1's tumor suppressor activity may stem from its “moonlighting” function in the nucleus that is spatially separated from its essential cytoplasmic actin- regulatory functions. Pfn1, in a complex with actin, undergoes active nuclear export by the nuclear exporter exportin-6 (XPO6). Unlike XPO1/CRM1, XPO6 is highly selective with Pfn1/actin being its only known cargo. We found in the TCGA datasets that the mRNA level of XPO6 is significantly upregulated in 65-100% of tumor samples across 12 different types (including breast). We also discovered that XPO6 protein level is significantly increased in a panel of luminal and basal-like breast cancer cell lines as compared to untransformed breast epithelial cells. XPO6 knockdown selectively inhibited the growth and survival of several breast cancer cell lines while having little on the untransformed control cells. Together, these data suggest that the primary mode of Pfn1 deregulation in cancer may be its increased nuclear export resulting from overexpression of its nuclear exporter XPO6. In addition, we have uncovered a novel interaction between nuclear Pfn1 and the multi-protein super elongation complex (SEC) by co-IP and mass spectrometry. SEC positively regulates the transcription elongation of many pro-tumor and pro-EMT/metastasis genes by phosphorylating (through its components CDK9/cyclin T1) and un-pausing promoter proximal RNA polymerase II to trigger productive elongation. We demonstrate that Pfn1 knockdown increases phospho-RNA Pol II level in breast cancer cells and sensitizes them to CDK9 inhibition. Together, our data support the hypothesis that Pfn1 functions in the nucleus as a tumor suppressor by inhibiting SEC-mediated gene transcription and its subcellular localization is frequently deregulated in cancer through overexpression of its nuclear exporter XPO6. In this grant, we will test this hypothesis using various approaches including in vitro and in vivo tumor models, transcriptome and epigenome analysis, and primary tumor sample analysis.

项目概要改变的蛋白质核细胞质运输越来越被认为是癌症的真正驱动因素许多肿瘤抑制蛋白（例如 p53、Rb）在细胞核中发挥作用，但会增加。许多类型的癌症中的核输出是由于其核输出蛋白 XPO1/CRM1 的过度表达而导致的。 Profilin-1 (Pfn1) 是一种肌动蛋白结合蛋白，具有充分记录的抗肿瘤和抗转移活性然而，它作为肿瘤抑制因子的作用仍然存在争议，因为它很少见。由于其促进肌动蛋白聚合，它发生了突变，并且对细胞生长和存活至关重要。虽然 Pfn1 通常被认为是细胞质蛋白，但它存在于许多哺乳动物细胞的细胞核中我们之前发现 Pfn1 在乳腺中的体外抗肿瘤作用。癌细胞需要其进入细胞核的能力，这表明 Pfn1 的肿瘤抑制活性可能源于此。其在细胞核中的“兼职”功能与其必需的细胞质肌动蛋白在空间上是分离的 Pfn1 与肌动蛋白形成复合物，通过核输出蛋白进行主动核输出。与 XPO1/CRM1 不同，XPO6 具有高度选择性，Pfn1/肌动蛋白是其唯一已知的货物。我们在TCGA数据集中发现XPO6的mRNA水平在65-100%的肿瘤中显着上调我们还发现 12 种不同类型（包括乳房）的样本中 XPO6 蛋白水平为与相比，在一组管腔和基底样乳腺癌细胞系中显着增加未转化的乳腺上皮细胞的 XPO6 敲低选择性地抑制了几种细胞的生长和存活。乳腺癌细胞系与未转化的对照细胞相比几乎没有，这些数据表明：癌症中 Pfn1 失调的主要模式可能是其核输出增加此外，我们还发现了其核输出蛋白 XPO6 之间的一种新的相互作用。通过 co-IP 和 SEC 分析核 Pfn1 和多蛋白超延伸复合物 (SEC)。通过以下方式正向调节许多促肿瘤和促 EMT/转移基因的转录延伸磷酸化（通过其组件 CDK9/细胞周期蛋白 T1）和非暂停启动子近端 RNA 聚合酶我们证明 Pfn1 敲低会增加磷酸化 RNA Pol II 的水平。乳腺癌细胞并使它们对 CDK9 抑制敏感，我们的数据支持这样的假设： Pfn1 在细胞核中通过抑制 SEC 介导的基因转录发挥肿瘤抑制因子的作用并且其亚细胞定位在癌症中经常通过其过度表达而失调在这笔资助中，我们将使用包括体外在内的各种方法来检验这一假设。以及体内肿瘤模型、转录组和表观基因组分析以及原发性肿瘤样本分析。