期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：11
页码：E1220-E1229
DOI：10.1073/pnas.1416318112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceMutant p53 (mtp53) is a driver oncogene of breast cancer. Here, for the first time, to our knowledge, using an inducible endogenous knockdown system, we explore the mtp53 driven proteome. We report this key data set that highlights mtp53-driven proteome diversity at the level of protein localization, as well as changes in protein levels without corresponding changes in transcription. We validated two protein pathways that include increased chromatin association of poly(ADP ribose) polymerase 1, and the increase of nuclear replication proteins minichromosome maintenance 4 and proliferating cell nuclear antigen. The addition of mtp53 proteomic targets to the previously identified transcriptional targets suggests that effective treatment of mtp53-driven breast cancers may be facilitated by new combination protocols blocking proteins of the metabolic pathways of cholesterol biosynthesis, DNA replication, and DNA repair. The gain-of-function mutant p53 (mtp53) transcriptome has been studied, but, to date, no detailed analysis of the mtp53-associated proteome has been described. We coupled cell fractionation with stable isotope labeling with amino acids in cell culture (SILAC) and inducible knockdown of endogenous mtp53 to determine the mtp53-driven proteome. Our fractionation data highlight the underappreciated biology that missense mtp53 proteins R273H, R280K, and L194F are tightly associated with chromatin. Using SILAC coupled to tandem MS, we identified that R273H mtp53 expression in MDA-MB-468 breast cancer cells up- and down-regulated multiple proteins and metabolic pathways. Here we provide the data set obtained from sequencing 73,154 peptide pairs that then corresponded to 3,010 proteins detected under reciprocal labeling conditions. Importantly, the high impact regulated targets included the previously identified transcriptionally regulated mevalonate pathway proteins but also identified two new levels of mtp53 protein regulation for nontranscriptional targets. Interestingly, mtp53 depletion profoundly influenced poly(ADP ribose) polymerase 1 (PARP1) localization, with increased cytoplasmic and decreased chromatin-associated protein. An enzymatic PARP shift occurred with high mtp53 expression, resulting in increased poly-ADP-ribosylated proteins in the nucleus. Mtp53 increased the level of proliferating cell nuclear antigen (PCNA) and minichromosome maintenance 4 (MCM4) proteins without changing the amount of pcna and mcm4 transcripts. Pathway enrichment analysis ranked the DNA replication pathway above the cholesterol biosynthesis pathway as a R273H mtp53 activated proteomic target. Knowledge of the proteome diversity driven by mtp53 suggests that DNA replication and repair pathways are major targets of mtp53 and highlights consideration of combination chemotherapeutic strategies targeting cholesterol biosynthesis and PARP inhibition.
