Friday, May 20, 2022
5/20/2022
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.
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