Abstract 3684: Proteome-wide triple negative breast cancer mutant p53 association index identifies chromatin unwinding for precision therapeutics

Over 80% of TNBCs express mutant p53 (mtp53) proteins. 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 in the cytoplasm and chromatin of triple negative breast cancer (TNBC) cells. Using SILAC coupled to 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 in the cytoplasm and 48,825 peptide pairs that corresponded to 5195 proteins in the chromatin fraction. Pathway enrichment analysis ranked the DNA unwinding pathway as the highest chromatin associated pathway regulated by mtp53. Moreover, to summarize and quantify the degree of under- or over-expression of a protein from two reciprocal experiments, a mutant p53 association index (mPAI) was defined as the log (of base 2) ratio of two readings from the two reciprocal experiments. Values of mPAI were normally distributed with a mean close to zero, consistent with the fact that levels of the majority of proteins were not affected by mtp53 knockdown. Standard deviation of mPAI is close to one and mtp53 itself shows mPAI values of greater than 2.0 (z-score > 2.0) in both cytosol and chromatin fractions, consistent with the expectation that its levels are significantly reduced by knockdown experiments. We thereby identified proteins and pathways significantly affected by mtp53 knockdowns as those with mPAI > 1.0 (indicating positive association) or mPAI < -1.0 (indicating negative association). A pathway that was positively associated in both the cytoplasm and the chromatin was the DNA unwinding pathway, which was represented by associated changes for all the minichromosome maintenance protein complex proteins 2 through 7 (MCM2-7). This hexomeric protein ring structure complex is the eukaryotic DNA helicase complex required for DNA replication and elongation of the replication fork. We validated that the MCM4 protein level is positively associated with mtp53 R273H in the TNBC cell line MDA-MB-468 and in the colon adenocarcinoma cell line HT-29. This suggests that targeting inhibition of the MCM hexomeric complex may be a good precision medicine approach for TNBCs. Further studies are in progress to determine the efficacy of targeting inhibi