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

摘要/摘要 肿瘤抑制基因tp53，被称为基因组的守护者，是人类基因组中最常发生突变的基因。 并导致其野生型P53蛋白保护功能减弱或丧失。 此外，在癌症中观察到广泛的TP53错义突变谱，并产生许多 具有致癌功能增益(GOF)活性的突变的P53蛋白。许多GOF致癌表型是 然而，在很大程度上还不清楚突变型p53是否以及如何参与替代的Pre-Pre。 M RNA的加工。特别是，交替多聚腺苷处理(APA)被很好地证明起到了作用 在许多癌症中。我们试图研究p53突变在肺癌中的这种可能性。我们的初步研究使用 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 人非小细胞肺癌(NSCLC)H1299稳定转染系的建立 表达P53-R273H、R175H、H179Q、C238Y、C242F或对照载体。我们的高通量分析 多聚腺苷酸化位点(PASS)发现突变体P53全局调控内含子多聚腺苷酸化(IPA)事件， 使各种RNA和蛋白质产品的生产成为可能。重要的是，许多DNA修复基因含有IPA 我们发现这些位点对突变型p53的表达特别敏感。这些 这些变化在TCGA队列中得到了验证。我们还发现剪接因子(SF)的序列基序 随着使用量的增加，hnRNPK是IPA站点上游区域最显著富含的基序 在非小细胞肺癌中，hnRNPK的表达被突变型p53上调。进一步的实验 研究表明，hnRNPK基因敲除导致DNA修复基因IPA亚型表达减少，并 DNA修复活性的恢复。因此，我们推测在非小细胞肺癌中常见的P53错义突变 通过促进DNA修复基因中的IPA处理，以hnRNPK作为关键调节因子来损害DNA修复。在这 项目中，我们将通过两个具体目标来验证这一假设。在特定的目标1中，我们将测试另一组 在非小细胞肺癌常见的p53突变中，包括更多的人类细胞株，以全面了解p53突变- 肺癌相关的PASS。这将决定前mRNA加工的调制是否是常见的 NSCLC中发现的大多数P53突变的特征，并描述了不同P53之间的差异 变种人。在特定的目标2中，我们将描述p53突变调控关键基因的机制。 剪接因子hnRNPK在IPA加工中的作用，以及抑制hnRNPK是否抑制IPA加工 DNA修复基因，并影响细胞的DNA修复效率和细胞凋亡。顺利完成这项研究 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 正在处理。这一主题很有可能在未来进行详细的长期研究，也许会导致新的 关于p53突变在肿瘤发生中的作用和为新的癌症提供机会的见解 P53基因突变癌症患者的治疗。

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.