Proteome-wide analysis of mutant p53 targets in breast cancer identifies new levels of gain-of-function that influence PARP, PCNA, and MCM4.
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.
Department of Biological Sciences, Hunter College, City University of New York, New York, NY 10065; and.
Alla
A
Polotskaia
Department of Biological Sciences, Hunter College, City University of New York, New York, NY 10065; and.
Gu
G
Xiao
Department of Biological Sciences, Hunter College, City University of New York, New York, NY 10065; and.
Katherine
K
Reynoso
Department of Biological Sciences, Hunter College, City University of New York, New York, NY 10065; and.
Che
C
Martin
Department of Biological Sciences, Hunter College, City University of New York, New York, NY 10065; and.
Wei-Gang
WG
Qiu
Proteomics Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY 10065.
Ronald C
RC
Hendrickson
Department of Biological Sciences, Hunter College, City University of New York, New York, NY 10065; and bargonetti@genectr.hunter.cuny.edu.
Jill
J
Bargonetti
D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D007553:Isotope Labeling; D008798:Mevalonic Acid / Q000378:metabolism; D064148:Minichromosome Maintenance Complex Component 4 / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D055550:Protein Stability / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D015398:Signal Transduction / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D002843:Chromatin; D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D018809:Proliferating Cell Nuclear Antigen; D020543:Proteome; D016159:Tumor Suppressor Protein p53; D011065:Poly(ADP-ribose) Polymerases; C099243:MCM4 protein, human; D064148:Minichromosome Maintenance Complex Component 4; D008798:Mevalonic Acid
MCM4; PARP; chromatin; mutant p53; proteome