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“基因组守护者”，是肿瘤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- mRNA 处理。特别是，替代多腺苷酸化加工 (APA) 已被充分证明可以发挥作用 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 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 或对照载体。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， 能够生产多种 RNA 和蛋白质产品。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。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 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。进一步的实验 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 NSCLC 中常见的 p53 错义突变体 以 hnRNPK 作为关键调节因子，促进 DNA 修复基因中的 IPA 加工，从而损害 DNA 修复。在这个 项目中，我们将通过两个具体目标来验证这一假设。在具体目标 1 中，我们将测试另外一组 NSCLC 常见的 p53 突变体，包括更多人类细胞系，以全面了解 p53 突变体 - 肺癌中相关的 PAS。这将确定前 mRNA 加工的调节是否是常见的 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 突变体。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。顺利完成本次研究 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。该主题具有在未来进行详细长期研究的巨大潜力，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.