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

• 批准号: 9239553
• 负责人: Jieya Shao
• 金额: $ 34.88万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9239553
• 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; Recruitment Activity; Resistance; Role; Sampling; Staining method; Stains; 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; protein E; slug; stem; trafficking; transcriptome; tumor; tumor progression

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.

项目总结 改变的蛋白质核质运输越来越被认为是癌症的真正驱动因素 进步。许多肿瘤抑制蛋白(如P53、Rb)在细胞核中发挥作用，但经历了 核出口在许多类型的癌症中是由其核出口蛋白XPO1/CRM1过度表达引起的。 Profilin-1(PFN1)是一种肌动蛋白结合蛋白，具有广泛的抗肿瘤和抗转移活性。 各种类型的癌症。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 突变对细胞生长和生存至关重要，因为它促进了肌动蛋白聚合。 虽然普遍认为PFN1是一种细胞质蛋白，但它存在于许多哺乳动物细胞的细胞核中 对其功能知之甚少。我们先前已经发现，PFN1在体外对乳腺的抗肿瘤作用 癌细胞需要具备进入细胞核的能力。这提示Pfn1‘S抑瘤活性可能抑制了 由于它在细胞核中的“兼职”功能与其基本的细胞质肌动蛋白在空间上分开-- 监管职能。在与肌动蛋白的复合体中，PFN1经历了核出口国的积极核出口 Exportin-6(XPO6)。与XPO1/CRM1不同，XPO6具有高度的选择性，其唯一已知的载物是PFN1/肌动蛋白。 我们在TCGA数据集中发现，XPO6的mRNA水平在65%-100%的肿瘤中显著上调 12种不同类型的样本(包括乳房)。我们还发现XPO6蛋白水平是 在一组管腔和基底样乳腺癌细胞系中显著增加 未转化的乳腺上皮细胞。XPO6基因敲除选择性地抑制了几种 乳腺癌细胞株，而对未转化的对照细胞几乎没有。总而言之，这些数据表明 在癌症中，PFN1解除管制的主要方式可能是其增加的核出口，其原因是 其核出口国XPO6的过度表达。此外，我们还发现了一种新的相互作用 核PFN1和多蛋白超延长复合体(SEC)的共IP和质谱学分析。证交会 通过以下途径正向调节许多亲肿瘤和亲EMT/转移基因的转录延伸 磷酸化(通过其组分CDK9/Cyclin T1)和非停顿启动子近端RNA聚合酶 II以触发生产性延伸。我们证明，PFN1基因敲除增加了Pol II的磷酸化RNA水平 并使乳腺癌细胞对CDK9抑制反应敏感。总而言之，我们的数据支持这样的假设 PFN1通过抑制SEC介导的基因转录在细胞核内发挥肿瘤抑制作用 在癌症中，它的亚细胞定位经常通过其过度表达而被解除调控。 核出口国XPO6。在这笔赠款中，我们将使用包括体外在内的各种方法来检验这一假说。 体内肿瘤模型、转录组和表观基因组分析以及原发肿瘤样本分析。