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

总结/摘要 肿瘤抑制基因TP53被称为“基因组的守护者”，是人类中突变频率最高的基因。 人癌症，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错义突变谱，并且许多TP53错义突变产生了 具有致癌性功能获得（GOF）活性的突变的p53蛋白。许多GOF致癌表型是 然而，目前还不清楚p53突变形式是否以及如何参与替代性的前 mRNA加工。特别是，替代性多聚腺苷酸化加工（APA）是有据可查的发挥作用 在许多癌症中。我们试图研究p53突变体在肺癌中的这种潜力。我们的试点研究使用 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 等基因人p53缺失型非小细胞肺癌（NSCLC）H1299稳定转染株的实验 表达p53-R273 H、R175 H、H179 Q、C238 Y、C242 F或对照载体。我们的高通量分析 多聚腺苷酸化位点（PAS）鉴定突变型p53全面调节内含子多聚腺苷酸化（IPA）事件， 从而能够生产多种RNA和蛋白质产物。重要的是，许多DNA修复基因携带IPA 位点，我们确定这些位点对p53突变体的表达特别敏感。这些 这些变化在TCGA队列中得到验证。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位点上游区域最显著富集的基序 与p53突变体相关;而hnRNPK表达在NSCLC中被p53突变体上调。进一步的实验 显示hnRNPK敲低导致DNA修复基因IPA同种型的表达降低， 恢复DNA修复活性。因此，我们推断在NSCLC中常见的p53错义突变体 通过以hnRNPK作为关键调节因子促进DNA修复基因中IPA加工而损害DNA修复。在这 项目，我们将通过两个具体目标验证这一假设。在具体目标1中，我们将测试另一组 NSCLC常见的p53突变体，并包括更多的人细胞系，以获得p53突变体的综合特征。 相关PAS。这将决定前mRNA加工的调节是否是一种常见的 这是在NSCLC中发现的大多数p53突变体的特征，也描述了各种p53突变体之间的差异。 变种人在具体目标2中，我们将描述p53突变体调节关键蛋白的机制。 剪接因子hnRNPK在IPA加工中的作用，以及抑制hnRNPK是否会抑制IPA加工， DNA修复基因，影响DNA修复效率和细胞凋亡.成功完成这项研究 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 处理.这一主题具有很强的潜力，在未来进行详细的长期研究，也许会导致新的 了解p53突变体在肿瘤发生过程中的作用，并为新的癌症提供机会 p53突变型癌症患者的治疗方法。

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.