Mutant p53 disrupts mammary tissue architecture via the mevalonate pathway

Abstract

p53 is a frequent target for mutation in human tumors, and mutant p53 proteins can actively contribute to tumorigenesis. We employed a three-dimensional culture model in which nonmalignant breast epithelial cells form spheroids reminiscent of acinar structures found in vivo, whereas breast cancer cells display highly disorganized morphology. We found that mutant p53 depletion is sufficient to phenotypically revert breast cancer cells to a more acinar-like morphology. Genome-wide expression analysis identified the mevalonate pathway as significantly upregulated by mutant p53. Statins and sterol biosynthesis intermediates reveal that this pathway is both necessary and sufficient for the phenotypic effects of mutant p53 on breast tissue architecture. Mutant p53 associates with sterol gene promoters at least partly via SREBP transcription factors. Finally, p53 mutation correlates with highly expressed sterol biosynthesis genes in human breast tumors. These findings implicate the mevalonate pathway as a therapeutic target for tumors bearing mutations in p53.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

Figure 1. Depletion of mutant p53 from breast cancer cells induces a phenotypic reversion in 3D culture

(A) MDA-231.shp53 cells were grown under 3D conditions for 8 days in the absence or presence of DOX to induce an shRNA targeting p53. Representative differential interference contrast (DIC) images are shown. Scale Bar, 200 μm. (B) MDA-231.shp53 cells grown as in (A) prior to immunoblotting analysis. p53 was detected using anti-p53 antibody (PAb1801). (C) MDA-468.shp53 cells were grown in 3D cultures for 8 days and confocal microscopic structures were grouped into the three indicated categories. Actin (Green) and nuclear (Red) staining. Scale bar, 50 μm. (D) MDA-468.shp53 cells were grown in 3D cultures for 8 days in the presence of DOX to deplete mutant p53. Left panel: GFP (Green) serves as a marker for shRNA induction. Right panel: Nuclei (Red). The larger structure is representative of intermediate colony morphologies, while the smaller structure is representative of acinus-like structures with hollow lumen morphology. White arrow indicates apoptotic cell debris within the luminal space. Scale bar, 50 μm. (E) MDA-468.shp53 cells were grown and processed as in (B). (F) Morphometry: A stable pool of MDA-468.shp53 cells was grown in 3D cultures for 8 days in the presence or absence of DOX and structures were analyzed by confocal microscopy and categorized as in (C). Left panel: population distribution. Right panel: percent structures with hollow lumens. Data presented as mean ± SD. *denotes p

Figure 2. Mutant p53 requires functional transactivation sub-domains to disrupt morphology of mammary cells in 3D culture

(A) MDA-468.shp53 cells expressing a control vector were grown in 3D cultures for 5 days in the absence (left panel) or presence (right panel) of DOX. Representative DIC images are shown. Scale bar, 200 μm. (B) MDA-468.shp53 cells expressing an shRNA-resistant Flag-tagged p53-R273H were grown in 3D cultures as in (A). Scale bar, 200 μm. (C) MDA-468.shp53 cells expressing an shRNA-resistant Flag-tagged p53-R273H-mTAD (mutant p53 with non-functional transactivation region, p53-R273H-L22Q/W23S/W53Q/F54S) were grown in 3D cultures as in (A). Scale bar, 200 μm. (D) Cell lines in (A–C) were grown in 3D cultures as in (A) and processed for immunoblotting for p53 (PAb240) or for actin. Flag-tagged mutant p53 variants migrate more slowly than endogenously expressed mutant p53.

Figure 3. Knockdown of mutant p53 from breast cancer cells in 3D culture significantly downregulates the mevalonate pathway

(A) Data were analyzed through the use of Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com). Blue bars that cross the threshold line (p<0.05) represent pathways that are significantly changed following mutant p53 depletion from MDA-468 cells. (B) Gene Ontology (GO) analysis. 1, 2, 3 represent three independent experiments. GO terms were sorted based on their significance and redundant terms were discarded. (C) MDA-468.shp53 cells were grown in 3D cultures for 8 days in the presence or absence of DOX as indicated. qRT-PCR for the 7 sterol biosynthesis genes identified by IPA. Data represent mean ± SD of three independent experiments. **indicates p

Figure 4. The mevalonate pathway is both necessary and sufficient to maintain the malignant state of breast cancer cells in 3D culture

(A) MDA-468.shp53 cells were grown in 3D cultures for 8 days in the presence or absence of DOX to deplete mutant p53. Parallel cultures grown in the presence of DOX were supplemented with mevalonate pathway metabolites: mevalonic acid/mevalonic acid-phosphate (MVA/MVAP) beginning on Day 1. Morphological categories as indicated were determined using confocal microscopy and plotted as a percentage of the population. A representative experiment is shown here and a second representative experiment is shown in Figure S4. (B) MDA-468 cells grown in 3D cultures for 13 days untreated or treated with DMSO, Simvastatin (100 nM) or (1 μM) as indicated. Representative DIC images are shown. Drugs were added on Day 4. Scale Bar, 200 μm. (C) MDA-231 cells grown in 3D cultures and treated as in (B). Scale Bar, 200 μm. (D) MDA-468 cells (top panel) or MDA-231 cells (bottom panel) were grown in 3D cultures for 13 days with Simvastatin (1 μM) as in (B) and (C), respectively, but were supplemented with MVA/MVAP. Scale Bar, 200 μm. (E) MDA-468 cells were grown in 3D cultures for 8 days with DMSO or 6-Fluoromevalonate (200 μM), added on Day 1 of the experiment. Scale Bar, 200 μm. (F) MDA-231 cells were grown as in (E). Scale Bar, 200 μm.

Figure 5. Modulation of geranylgeranylation mediates many of the phenotypic effects of mutant p53 depletion and HMG-CoA reductase inhibition in MDA-231 cells

(A) MDA-231 cells were grown in 3D cultures for 8 days untreated or treated with DMSO, or inhibitors: YM-53601 (1 μM), FTI-277 (1 μM) or GGTI-2133 (1 μM) as indicated. Drugs were added on Day 1. Scale Bar, 200 μm. (B) MDA-231.shp53 cells were grown in 3D culture conditions for 9 days in the absence or presence of DOX as indicated. Parallel wells of cells which were grown in the presence of DOX were supplemented with geranylgeranyl pyrophosphate (GGPP) (25 μM) beginning on Day 1. Scale Bar, 200 μm. (C) MDA-231 cells were grown in 3D cultures for 13 days either treated with DMSO or Simvastatin (1 μM) as indicated. Parallel wells of cells which were grown in the presence of Simvastatin (1 μM) were supplemented with GGPP (25 μM) beginning on Day 1. Scale Bar, 200 μm.

Figure 6. Mutant p53 is recruited to mevalonate pathway gene promoters and this recruitment is dependent on SREBP proteins

(A) 293 cells were transfected with Flag-p53-R273H and either Myc-mSREBP-1a, -1c or -2. Cells were subjected to crosslinking with formaldehyde prior to lysis, sonication and immunoprecipitation as described in experimental procedures followed by SDS-PAGE and immunoblotting with anti-Myc (upper panel) and anti-Flag (lower panel). Input is 2.5% of IP sample. (B) Nuclear lysates from serum-starved MDA-468.shp53 cells were immunoprecipitated with an anti-SREBP-2 antibody (1D2) or Mouse IgG (Mock IP) and then immunoblotted with anti-SREBP-2 (1D2) and anti-p53 antibodies (DO-1). Input is 10% of IP sample. (C) MDA-468.shp53 cells were grown in 2D cultures for 8 days in the absence or presence of DOX and subjected to ChIP analysis as described in experimental procedures. Mock IP (C) serves as a negative control. Data is presented as mean ± SD of three independent experiments. Values were normalized to the highest immunoprecipitation signal. **indicates pHMGCR promoter. **indicates p<0.01 compared to all of the following: negative site, +DOX, Mock IP, upstream and downstream sites. Genomic locations of PCR primers are illustrated in the HMGCR promoter. SRE-1 denotes sterol regulatory element. (E) MDA-468 cells were treated with siRNA targeting SREBP-2 and subjected to ChIP analysis as in (C) for mutant p53 recruitment to the vicinity of the SRE-1 site in the HMGCR promoter (−150 bp). Data presented as mean ± SD of three independent experiments. *designates p<0.05. See Figure S5C for extent of SREBP-2 knockdown. (F) MDA-468.shp53 cells were treated with Fatostatin (20 μM) and subjected to ChIP analysis as in (E). Data is presented as mean ± SD of six independent experiments. **designates p

Figure 7. Mutant p53 is correlated with higher expression of a subset of mevalonate pathway genes in human breast cancer patient datasets

(A) Five human breast cancer patient datasets (see Supplemental Information for details) were analyzed to determine whether tumors bearing mutant p53 correlate with higher expression of sterol biosynthesis genes. Patients were stratified based on TP53 status (wild-type vs. mutant) and expression levels for sterol biosynthesis genes were analyzed. Isopentenyl Pyrophosphate Isomerase (IDI1), exhibited higher expression levels in mutant p53 tumors compared to wild-type p53 tumors (p<0.05) across all five datasets. p-value represents the result of a one-sided t-test. See Figure S7 and Table S1 for other genes. (B) Unsupervised hierarchical clustering with Euclidean distance and ward linkage of expression matrix from the 17 sterol biosynthesis genes on 812 samples (728 of which have TP53 mutational status). MVD was not present in the DBCG dataset and its missing expression values were grayed out on the heatmap. Rows indicate the identity of the genes and columns indicate the identity of the patients. The TP53 mutational status for each tumor is depicted directly above each column. Cluster I exhibits the lowest expression of the mevalonate pathway genes, cluster III exhibits an intermediate expression level and cluster II exhibits the highest expression level of the mevalonate pathway genes. (C) The Kaplan-Meier curves for the resulting clusters from the unsupervised hierarchical clustering in (B). (D) Estimated hazard ratios (HRs; the relative risk for 1 unit increasing in the gene expression) with 95% confidence interval for risk of breast cancer specific death. Expression levels of following genes were positively associated with the risk of breast cancer specific death at False Discovery Rate (FDR) (q) 5%: ACAT2 (HR = 1.23, q = 0.0069), HMGCS1 (HR = 1.21, q = 0.007), HMGCR (HR = 1.17, q = 0.032), IDI1 (HR = 1.26, q < 0.001), FDPS (HR = 1.17, q = 0.012), SQLE (HR = 1.35, q < 0.001), LSS (HR = 1.16, q = 0.032), NSDHL (HR = 1.17, q = 0.032), DHCR7 (HR = 1.26, q < 0.001). Blue indicates q<0.05, grey indicates q>0.05.