Proteome-wide analysis of mutant p53 targets in breast cancer identifies new levels of gain-of-function that influence PARP, PCNA, and MCM4

Abstract

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.

Keywords: MCM4; PARP; chromatin; mutant p53; proteome.

Fig. 1.

Inducible depletion of mtp53 proteins R273H, R280K, and L194F in breast cancer cell lines shows they are efficiently depleted in the cytoplasm and strongly associated with chromatin. (A) The mtp53 expression in whole-cell extract from vector controls and clones MDA-468.shp53 1F5 and 2F3, MDA-231.shp53 2C9 and 1D10, and T47D.shp53 3F2 and 4F6 is shown. Cells were grown in the presence or absence of doxycycline (4 μg/mL for T47D and 8 μg/mL for other cell lines) for 6 d. Whole-cell lysates were prepared as described in Materials and Methods, and 50 μg of protein was separated by gradient 4–12% SDS/PAGE and analyzed by Western blot using antibody to p53. Actin was used as a loading control. Relative depletion of mtp53 was quantified by ImageJ analysis. (B) Biochemical fractionation of MCF-7, MDA-MB-231, T47D, and MDA-MB-468 breast cancer cells was carried out according to the well-established method of Méndez and Stillman (23). The cytoplasmic, nuclear-soluble, and chromatin-enriched protein samples were resolved in a gradient 4–12% SDS/PAGE in amounts proportional to the subcellular fraction of 100 μg of protein derived from a cell pellet. The p53 subcellular localization was determined by immunoblotting. ImageJ was used to quantify the percent distribution. (C) Biochemical fractionation of MDA-468.shp53 2F3 clone was carried out with or without p53 depletion. Fractions were run with 50 μg of protein loaded per lane and normalized for actin in the cytoplasmic and nuclear-soluble fractions and fibrillarin in the chromatin-enriched fraction. ImageJ was used to quantify the values. The Western blot is a representative image for doxycycline knockdown for 12 d, and the histogram is a mean (SD) of two independent experiments.

Fig. 2.

Proteomics screen of stable isotope-labeled cells in culture (SILAC) determines multiple mtp53 targets and regulated pathways. (A) Overview of the proteomics strategy to determine mtp53 targets in MDA-MB-468 cells. (B) mtp53 depleted and untreated cytoplasmic fraction from MDA-468.shp53 2F3 were mixed from cells grown in heavy (labeled “H”) vs. light (labeled “L”) media. Normalized LC-MS/MS H/L ratios of 3,010 proteins identified from forward-labeled (y axis for p53 depletion in cells grown in heavy media) and reverse-labeled (x axis for p53 depletion in cells grown in light media) experiments were plotted. An H/L ratio > 1 or < 1 indicates an mtp53-dependent change in the amount of a protein in the cytoplasmic fraction. The diagonal line indicates a lack of change in the H/L ratio between the two experiments, which corresponds to nontarget proteins (those with H/L ratios close to 1 in both experiments) or proteins inconsistently expressed between the two experiments (those with H/L > 2 or H/L < 1 in both experiments). Targets with reciprocal H/L ratios greater than 1.5 and less than 0.5 (blue and yellow dots) have changed strongly and consistently between the depleted and untreated cells. The change in p53 (purple dot) is labeled TP53 as the positive control. The cholesterol biosynthesis enzymes are shown as green dots. The DNA replication proteins are shown as pink dots (PCNA and MCM4 are labeled), and PARP1 is one of the labeled blue dots. (C) Reverse labeling of R273H depletion shows high-quality LC-MS/MS peptides for PARP in the chromatin fraction. Representative MS spectra from PARP in the chromatin fraction is shown with forward and reverse labeling in a and b, respectively. The selected ion current chromatograms for the (M+2H)²⁺ ions from tryptic peptide IAPPEAPVTGYMFGK at the selected retention time (109–113 min) for the forward and reverse labeling are shown in c and d, respectively. The light isoform with m/z 789.4 is shown in blue, and heavy isoform with m/z 792.4 is shown in red. The integrated area under the curve was used to calculate the change in abundance. (D) Representative MS spectra from PCNA in the chromatin fraction is shown with forward and reverse labeling in a and b, respectively. The selected ion current chromatograms for the (M+2H)²⁺ ions from tryptic peptide YLNFFTK at the selected retention time (107–112 min) for the forward and reverse labeling are shown in c and d, respectively. The light isoform with m/z 466.7 is shown in blue, and heavy isoform with m/z 469.7 is shown in red. The integrated area under the curve was used to calculate the change in abundance.

Fig. 3.

Depletion of mtp53 modulates PARP localization and PARP enzymatic activity. (A) Cells MDA-468 vector and MDA-468.shp53 were grown in the presence or absence of 8 μg of doxycycline for 6 d, and fractionation was carried out as described. Samples were resolved on a gradient 4–12% SDS/PAGE. Protein levels of p53, PARP1, Actin, and Fibrillarin in the fractions were determined by Western blot analysis. Actin was used to normalize the cytoplasmic and nuclear-soluble fractions, and fibrillarin was used to normalize the chromatin fraction. A total of 50 μg of protein from the cytoplasmic fraction and nuclear-soluble fractions per lane and 5 μg of the chromatin fraction per lane were resolved. ImageJ was used to quantify p53 and PARP1 change in each fraction compared with the corresponding control. (B) Confocal microscopy images of PAR proteins were obtained by using anti-PAR antibody. DAPI staining was used to determine the nucleus, and GFP was an indicator of doxycycline-mediated induction. Images are representative of three independent experiments.

Fig. 4.

Mutant p53 stabilizes chromatin-associated MCM4 and PCNA. (A) MDA-468 vector and MDA-468.shp53 cells were grown in the presence or absence of 8 μg of doxycycline for 12 d, and fractionation was carried out as described. A total of 50 μg of protein from the chromatin fraction was resolved in a 10% SDS/PAGE. Protein levels of mtp53, MCM4, PCNA, and Fibrillarin were determined by Western blot analysis. Fibrillarin was used to normalize the chromatin fraction. The Western blot is a representative image, and the histogram (B) represents the average (SD) of three independent experiments. (C) Knockdown of the mtp53 and decrease in MCM4 and PCNA protein levels did not change the cell cycle profile, which was analyzed by flow cytometry. Results are the mean (SD) of two independent experiments. (D) Quantitative PCR analysis of MCM4 and PCNA RNA levels in the MDA-468 vector and MDA-468.shp53 cells grown with or without 8 μg/mL of doxycycline were not influenced by mtp53 depletion. Results are the mean (SD) of two independent experiments. (E) MDA-231 vector and MDA-231.shp53 1D10 clone cells were grown in the presence or absence of 8 μg of doxycycline for 6 d, and fractionation was carried out as described. A total of 50 μg of protein from the chromatin fraction was resolved in a 10% SDS/PAGE. Protein levels of mtp53, MCM4, PCNA, and Fibrillarin were determined by Western blot analysis. (F) HT-29 colon cancer cells were transfected with p53-siRNA (p53) or control siRNA (contr). Biochemical fractionation was carried out after 72 h of transfection. A total of 50 μg of protein from cytoplasmic (Cyto), nuclear-soluble (N-Sol), and chromatin fractions were run per lane, and protein levels of mtp53, MCM4, and Fibrillarin in the fractions were determined by Western blot analysis.

Fig. 5.

Reduced cell viability of mtp53 R273H-expressing cells when PARP is pharmacologically inhibited. MDA-468.shp53 or control vector-expressing cells were treated with 8 μg/mL doxycycline (DOX) for 7 d to induce shRNA expression or left without doxycycline treatment. (A) Whole-cell extracts were analyzed by Western blotting for p53 depletion. Actin was used as a loading control. (B) MDA-MB-468 cell lines with p53.shRNA or control vector were treated with 8 μg/mL doxycycline (DOX) for 5 d to induce shRNA expression or left without doxycycline treatment, followed by treatment with 0, 10, 20, or 40 μM rucaparib for 48 h. A total of 100,000 cells per well were plated on the 12-well plate for cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay based on mitochondrial dehydrogenase activity (as in ref. 31). (C) Rucaparib reduced cell viability of MDA-468.shp53 cells in a dose-dependent fashion. Error bars indicate the SDs of two independent experiments.

Fig. 6.

8AA-induced cell death in MDA-468 cells does not have the same proteome change as p53 depletion. (A) MDA-468 vector cells were left untreated (control) or treated with 15 μM of 8AA for 16 h. Phase-contrast microscopy image of cells untreated or treated with 8AA at a magnification of 20×. MDA-468 vector cells were left untreated or treated with 15 μM of 8AA for 16 h and were fractionated into cytoplasmic (cyto), nuclear-soluble (N-Sol), and chromatin fractions. (B) Fractions were run with 50 μg of protein loaded per lane, and protein levels of PARP1, MCM4, Actin, and Fibrillarin in the fractions were determined by Western blot analysis. (C) Fractions were run with 50 μg of protein from the cytoplasmic and nuclear-soluble fractions and 5 μg of the chromatin fraction, and protein levels of mtp53, PCNA, Actin, and Fibrillarin in the fractions were determined by Western blot analysis.

Fig. 7.

Mutant p53 drives proteome changes with and without directly activating the transcription of the target. (A) Mevalonate pathway enzymes (green) were detected at high levels in the cytoplasm when R273H mtp53 protein levels (purple) were high, and, upon mtp53 depletion, these mevalonate protein enzymes were reduced. (B) PARP1 protein level (blue) was high in the nucleus when R273H mtp53 protein levels (purple) were high, and, upon mtp53 depletion, PARP1 nuclear protein levels were reduced and the cytoplasmic PARP1 was increased. The depletion of R273H mtp53 also coordinated a decrease in PAR proteins in the nucleus and an increase of PAR proteins in the cytoplasm. (C) PCNA and MCM4 protein levels (pink and yellow) were high when R273H mtp53 protein levels (purple) were high, and, upon mtp53 depletion, PCNA and MCM4 protein levels were reduced.