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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：
10358369
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 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10358369
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 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 Light 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 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 Translations Tumor Suppressor Proteins Untranslated RNA Up-Regulation base 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/文摘