Proteome-Wide Analysis Of Gain-Of-Function Mutant P53 Targets In Breast Cancer Implicates Parp, Pcna And Mcm4 As Oncogenic Drivers

Gain-of-function mutant p53 (mtp53) changes the cancer cell transcriptome. However no analysis of mtp53-associated proteome diversity has been carried out. 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. Using SILAC followed by tandem mass spectrometry (LC-MS/MS) we identified that R273H mtp53 expression in MDA-MB-468 breast cancer cells both up and down-regulated multiple proteins and metabolic pathways. We sequenced 73,154 peptide pairs that corresponded to 3010 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 non-transcriptional targets. Interestingly, mtp53 depletion profoundly influenced poly (ADP-ribose) polymerase 1 (PARP1) localization, with increased cytoplasmic and decreased chromatin-associated PARP1. An enzymatic PARP shift occurred with high mtp53 expression resulting in increased poly-ADP-ribosylated proteins in the nucleus. Mtp53 increased the level of PCNA and MCM4 protein without changing the amount of pcna or mcm4 transcript. Pathway enrichment analysis ranked the DNA replication pathway above the cholesterol biosynthesis pathway as an R273H mtp53 activated proteomic target. Knowledge of the proteome diversity driven by mtp53 suggests DNA replication and repair pathways are major targets of mtp53 and highlights consideration of combination chemotherapeutic strategies targeting cholesterol biosynthesis and PARP inhibition. Acknowledgements. This work was supported by a grant to JB from the Breast Cancer Research Foundation. Citation Format: Alla Polotskaia, Gu Xiao, Katherine Reynoso, Che Martin, Wei-Gang Qiu, Ronald Hendrickson, Jill Bargonetti. Proteome-wide analysis of gain-of-function mutant p53 targets in breast cancer implicates PARP, PCNA and MCM4 as oncogenic drivers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1225. doi:10.1158/1538-7445.AM2015-1225