A novel role of mutant p53 in intronic polyadenylation and impairment of DNA repair

突变体 p53 在内含子多聚腺苷酸化和 DNA 修复损伤中的新作用

基本信息

批准号：
10535488
负责人：
Liang Liu
金额：
$ 7.75万
依托单位：
WAKE FOREST UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-12-08 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10535488
关键词：
3&apos Untranslated Regions Address Affect Amino Acid Substitution Apoptosis Binding Bioinformatics Cancer Patient Cancer cell line Cell Line Cells Characteristics DNA Damage DNA Repair DNA Repair Disorder DNA Repair Gene Data Development Event Foundations Frequencies Future Gene Expression Gene Expression Regulation Genes Genetic Transcription Genome H1299 Heterogeneity Heterogeneous-Nuclear Ribonucleoprotein K Human Human Cell Line Impairment Knowledge Longitudinal Studies Lung Adenocarcinoma Malignant Neoplasms Malignant neoplasm of lung Mediating Messenger RNA Missense Mutation Molecular Mutate Mutation Non-Small-Cell Lung Carcinoma Oncogenic Patients Phenotype Physiology Pilot Projects Play Poly A Polyadenylation Production Protein Isoforms Proteins Publications RNA RNA Processing RNA Splicing RNA-Binding Proteins Recovery Regulation Reporting Research Role Sentinel Site Solid TP53 gene Techniques Testing The Cancer Genome Atlas Transfection Translations Tumor Suppressor Proteins Untranslated RNA Up-Regulation cancer cell cancer therapy cell growth regulation cohort experimental study gain of function insight knock-down lung cancer cell mRNA Precursor mRNA Stability mRNA sequencing mutant next generation sequencing novel novel therapeutics targeted cancer therapy tumor tumorigenesis tumorigenic vector vector control

项目摘要

SUMMARY/ABSTRACT The tumor suppressor TP53, known as “the guardian of the genome”, is the most frequently mutated gene in human cancers and results in the diminished or abolished protective function of its wildtype p53 protein. Additionally, an extensive spectrum of TP53 missense mutations is observed in cancer and many produce mutated p53 protein with oncogenic gain-of-function (GOF) activities. Many GOF oncogenic phenotypes are described, however, it is largely unknown whether and how p53 mutant forms are involved in alternative pre- mRNA processing. In particular, alternative polyadenylation processing (APA) is well-documented to play roles in many cancers. We sought to investigate this potential of p53 mutants in lung cancer. Our pilot studies use patient tumor data in the Cancer Genome Atlas (TCGA) cohort and Next Generation Sequencing (NGS) experiments on isogenic human p53-null non-small cell lung cancer (NSCLC) H1299 stable transfectant lines expressing p53-R273H, R175H, H179Q, C238Y, C242F or control vector. Our high-throughput profiling of polyadenylation sites (PASs) identified that mutant p53 globally regulates intronic polyadenylation (IPA) events, enabling the production of diverse RNA and protein products. Importantly, many DNA repair genes harbor IPA sites and we determined that these sites are particularly sensitive to the expression of p53 mutants. These changes were validated in the TCGA cohort. We also found that the sequence motif for splicing factor (SF) hnRNPK was the top significantly enriched motif in the upstream regions of IPA sites with increased usage related to p53 mutants; while hnRNPK expression is upregulated by p53 mutants in NSCLC. Further experiments show that knock-down of hnRNPK leads to the reduced expression of IPA isoforms of DNA repair genes and recovery of DNA repair activity. Thus we deduce that commonly occurring p53 missense mutants in NSCLC impair DNA repair by promoting IPA processing in DNA repair genes with hnRNPK as a key regulator. In this project, we will validate this hypothesis through two specific aims. In Specific Aim 1, we will test an additional set of p53 mutants common to NSCLC and include more human cell lines for a comprehensive profile of p53 mutant- associated PASs in lung cancer. This will determine whether modulation of pre-mRNA processing is a common characteristic of the majority of p53 mutants found in NSCLC, and also depict the differences among various p53 mutants. In Specific Aim 2, we will characterize the mechanism through which p53 mutants regulate the key splicing factor, hnRNPK, in IPA processing, and whether inhibition of hnRNPK suppresses IPA processing of DNA repair genes and affects DNA repair efficiency and apoptosis of cells. Successful completion of this research will provide a solid foundation in our understanding of the regulatory role of p53 mutant proteins in RNA processing. This topic has strong potential for detailed long-term studies in the future, perhaps leading to new insights on how p53 mutants function during tumorigenesis and providing opportunities for novel cancer therapies for p53-mutant cancer patients.

摘要/摘要被称为“基因组卫士”的抑癌基因TP53是人类中突变频率最高的基因。人类癌症，并导致其野生型 p53 蛋白的保护功能减弱或消失。此外，在癌症中观察到广泛的 TP53 错义突变，并且许多产生具有致癌功能获得 (GOF) 活性的突变 p53 蛋白许多 GOF 致癌表型是然而，目前尚不清楚 p53 突变体形式是否以及如何参与替代预- 特别是，替代性聚腺苷酸化加工 (APA) 的作用已得到充分证明。我们试图研究 p53 突变体在肺癌中的这种潜力。癌症基因组图谱 (TCGA) 队列和下一代测序 (NGS) 中的患者肿瘤数据同基因人 p53 缺失非小细胞肺癌 (NSCLC) H1299 稳定转染系的实验表达 p53-R273H、R175H、H179Q、C238Y、C242F 或我们的高通量分析。多腺苷酸化位点 (PAS) 发现突变体 p53 全局调节内含子多腺苷酸化 (IPA) 事件，重要的是，许多 DNA 修复基因都含有 IPA。位点，我们确定这些位点对 p53 突变体的表达特别敏感。我们还发现了剪接因子 (SF) 的序列基序。 hnRNPK 是 IPA 位点上游区域中最显着富集的基序，且使用量增加与 p53 突变体相关；而 hnRNPK 表达在 NSCLC 中被 p53 突变体上调。表明 hnRNPK 的敲低会导致 DNA 修复基因的 IPA 亚型表达减少， DNA修复活性的恢复因此我们推断出NSCLC中常见的p53错义突变体。以 hnRNPK 作为关键调节因子，通过促进 DNA 修复基因中的 IPA 加工来损害 DNA 修复。在项目中，我们将通过两个具体目标验证这一假设。在具体目标 1 中，我们将测试另外一组目标。 NSCLC 常见的 p53 突变体，包括更多人类细胞系，以获得 p53 突变体的全面概况 - 这将决定前 mRNA 加工的调节是否常见。 NSCLC 中发现的大多数 p53 突变体的特征，并且还描述了各种 p53 之间的差异在具体目标 2 中，我们将描述 p53 突变体调节关键的机制。 IPA 加工中的剪接因子 hnRNPK，以及抑制 hnRNPK 是否会抑制 IPA 加工 DNA修复基因并影响DNA修复效率和细胞凋亡。将为我们理解 p53 突变蛋白在 RNA 中的调节作用提供坚实的基础这个主题在未来进行详细的长期研究具有很大的潜力，也许会带来新的结果。关于 p53 突变体在肿瘤发生过程中如何发挥作用并为新型癌症提供机会的见解 p53 突变癌症患者的疗法。