Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jan;20(1):28-35.
doi: 10.1038/s41556-017-0009-8. Epub 2017 Dec 18.

Mechanical cues control mutant p53 stability through a mevalonate-RhoA axis

Eleonora Ingallina  1 , Giovanni Sorrentino  1   2 , Rebecca Bertolio  1   3 , Kamil Lisek  1   4 , Alessandro Zannini  1   3 , Luca Azzolin  5 , Luisa Ulloa Severino  3   6 , Denis Scaini  3   6 , Miguel Mano  7   8 , Fiamma Mantovani  1   3 , Antonio Rosato  9 , Silvio Bicciato  10 , Stefano Piccolo  5 , Giannino Del Sal  11   12
Affiliations

Mechanical cues control mutant p53 stability through a mevalonate-RhoA axis

Eleonora Ingallina et al. Nat Cell Biol. 2018 Jan.

Abstract

Tumour-associated p53 missense mutants act as driver oncogenes affecting cancer progression, metastatic potential and drug resistance (gain-of-function) 1 . Mutant p53 protein stabilization is a prerequisite for gain-of-function manifestation; however, it does not represent an intrinsic property of p53 mutants, but rather requires secondary events 2 . Moreover, mutant p53 protein levels are often heterogeneous even within the same tumour, raising questions on the mechanisms that control local mutant p53 accumulation in some tumour cells but not in their neighbours 2,3 . By investigating the cellular pathways that induce protection of mutant p53 from ubiquitin-mediated proteolysis, we found that HDAC6/Hsp90-dependent mutant p53 accumulation is sustained by RhoA geranylgeranylation downstream of the mevalonate pathway, as well as by RhoA- and actin-dependent transduction of mechanical inputs, such as the stiffness of the extracellular environment. Our results provide evidence for an unpredicted layer of mutant p53 regulation that relies on metabolic and mechanical cues.

PubMed Disclaimer

Conflict of interest statement

Competing financial interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Statins reduce missense mutant p53 protein levels in cancer cells.
(A) Results of high-content screening. Mutant p53 positive cells were detected by immunofluorescence and quantified through automated image analysis. (B) Representative images of p53 immunofluorescence from the screening. MDA-MB-231 cells were treated with dimethylsulphoxide (DMSO) or with Cerivastatin 10 μM for 24h. Scale bar 15 μm. (C) MDA-MB-231 cells were treated with dimethylsulphoxide (DMSO) or with Cerivastatin (+) 10 μM for 48h. Representative western blots are shown. (D) MDA-MB-231 cells treated with Cerivastatin 10 μM for the indicated time points. Representative western blots are shown. (E) MDA-MB-231 cells expressing pcDNA3-HA-p53R280K vector treated with dimethylsulphoxide (DMSO) (-) or treated (+) with Cerivastatin 10 μM for 48h. Representative blots are shown. (F) The indicated cell lines were treated with dimethylsulphoxide (DMSO)(-) or with Cerivastatin 10 μM for 48h. Representative blots are shown. (G) Quantification of BrdU-positive cells. The indicated cell lines were treated with Cerivastatin (CER) 0.1 μM for 48h. Error bars represent mean ± s.d. from n=3 biologically independent experiments. **P < 0.01 *P < 0.05; P values (from left to right): 0.008; 0.016; 0.007; 0.008; 0.039; 0.017; 0.005; 0.266; 0.801; 0.221, 0.341. Two-tailed Student’s t-test is used. All experiments were repeated three times, apart from 1A, which was performed twice. Source data for panel 1G is available in Supplementary Table 6. Unprocessed scans of blots are shown in Supplementary Figure 6.
Figure 2
Figure 2. Statins unleash MDM2-mediated degradation of mutant p53 by disrupting its interaction with Hsp90.
(A) Evaluation of mutant p53 half-life in MDA-MB-231 cells. Cells were pre-treated with Cerivastatin (1 μM), alone or with nutlin (10 μM) and after 24h cells were treated with cycloheximide (CHX) (50 μM) for the indicated times. MG132 (50 μM) was added to inhibit the proteasome. Representative western blots with the indicated antibodies are shown. The graph indicates normalized quantification of mutant p53 protein amounts. Each replicate from n=3 biologically independent experiments is shown. *P = 0.012. Two-tailed Student’s t-test. (B) Left: MDA-MB-231 cells were transfected with constructs expressing HA-ubiquitin and pcDNA3-p53R280K and then treated with Cerivastatin 1 μM for 48h. Right: mutant p53 was immunoprecipitated from lysates and anti-HA blot was performed to detect ubiquitinated forms of mutant p53. (C) MDA-MB-231 cells were treated with Nutlin-3 10 μM for 12h and then Cerivastatin 10 μM was added to the medium for additional 48 h. Representative blots are shown. (D) MDA-MB-231 cells were treated with Cerivastatin 10 μM for 48h after transfection with indicated siRNA for 24h. Representative blots are shown. (E) Schematic representation of the mechanism of mutant p53 stabilization by Hsp90 in cancer cells. (F) Mutant p53 was immunoprecipitated from lysates of MDA-MB-231 cells, untreated (-) or treated with Cerivastatin 1 or 10 μM for 24h. Co-immunoprecipitated Hsp90 and MDM2 were detected by Western blot. All experiments were repeated three independent times with similar results. Source data for panel 2A is avaibable in Supplementary Table 6. Unprocessed scans of blots are shown in Supplementary Figure 6.
Figure 3
Figure 3. SREBP/mevalonate pathway controls mutant p53 levels via GGPP.
(A) Schematic overview of the mevalonate pathway. Enzymes are shown in red and inhibitors in blue. (B) MDA-MB-231 cells transfected with siRNAs targeting either SREBP1 (BP1) or SREBP2 (BP2) or SREBP1/2 together (BP1/2) for 48h. Representative western blots with the indicated antibodies are shown. SCD-1, a SREBP1/2 target involved in lipid metabolism, was used as positive control. (C) MDA-MB-231 cells were grown in medium supplemented with 10% FBS or 2% Lipoprotein-depleted serum (LDS) with or without Cerivastatin (CER) 10 μM for 48h. Representative blots are shown. (D) MDA-MB-231 cells were treated with Cerivastatin 10 μM (CER) alone or with mevalonic acid (MVA) 0.5 mM for 48h. Representative blots are shown. (E) MDA-MB-231 cells were treated with different inhibitors: Cerivastatin (CER) 10 μM, Zoledronic Acid (ZA) 50 μM, geranylgeranyl transferase I inhibitor (GGTI-298) 10 μM, squalene synthase inhibitor (YM-53601) 20 μM, farnesyl transferase inhibitor (FTI-277) 20 μM, for 48h. Representative blots are shown. (F) MDA-MB-231 cells were treated with GGTI-298 (10 μM) for the indicated times. Representative blots are shown. (G) MDA-MB-231 cells were treated with Cerivastatin 10 μM (CER) either alone or in combination with geranygeranyl pyrophosphate 20 μM (CER+GGPP) for 48h. Representative blots are shown. (H) Colony formation assay. The indicated cell lines were treated with Cerivastatin (CER) 0.1 μM either alone or in combination with geranygeranyl pyrophosphate 20 μM (CER + GGPP) for 6 days. (I) Mutant p53 was immunoprecipitated from lysates of MDA-MB-231 cells either untreated (-) or treated (+) with GGTI-298 10 μM for 24h. Co-immunoprecipitated Hsp90 was detected by western blot. All experiments were repeated three independent times with similar results. Unprocessed scans of blots are shown in Supplementary Figure 6.
Figure 4
Figure 4. RhoA geranylgeranylation controls mutant p53 levels downstream of mevalonate pathway.
(A) The indicated cell lines were transfected with two independent siRNAs targeting RhoA for 72h. Representative blots are shown. (B) MDA-MB-231 cells were treated with C3 toxin (100 ng/ml) for 48 h. Representative blots are shown. (C) MDA-MB-231 cells stably expressing control vector (CTL) or the active form of RhoA (RhoA-G14V) were treated with increasing amount of GGTI-298 (0; 10μM; 20μM) for 48h. Representative blots are shown. (D) Fluorescence microscopy analysis of MDA-MB-231 cells stably expressing the construct coding for a mutant RhoA-G14V bearing a farnesylation consensus sequence (GFP-RhoA-G14V-F) either left untreated (-) or treated with Cerivastatin 1 μM or with GGTI-298 10μM for 48h. Scale bar 15 μm. (E) MDA-MB-231 cells stably expressing control vector (CTL) or GFP-RhoA-G14V-F were left untreated (-) or treated with GGTI-298 10 μM or ZA 50 μM for 48 h. Representative blots are shown. (F) Mutant p53 was immunoprecipitated from lysates of MDA-MB-231 cells transfected with control (CTL) or two independent RhoA siRNAs (siR#1 and siR#2) for 72h. Co-immunoprecipitated Hsp90 was detected by western blot. All experiments were repeated three independent times with similar results. Unprocessed scans of blots are shown in Supplementary Figure 6.
Figure 5
Figure 5. Mechanical cues control mutant p53 levels and activity via RhoA/actin cytoskeleton.
(A) MDA-MB-231 cells treated with Latrunculin A 0.5 μM (LAT.A) or Cerivastatin 10μM for 48h. Scale bar 15 μm. (B) Cell stiffness 48h after treatment with indicated compounds. Data for NT come from n=291 cells (pooled across 5 independent experiments), for ZOL, CER and CER-GGPP from n=171, n=156 and n=163 cells, respectively (pooled across 3 independent experiments each), while for BLEB and Y from n=114 and n=110 cells, respectively (pooled across 2 independent experiments each). Box plots range from 25th to 75th percentiles, bold lines inside the box represent the median. P values obtained by two-sample Kolmogorov-Smirnov test based on probability distribution. **P<0.01. (C) MDA-MB-231 cells treated with Latrunculin A 0.5 μM (LAT.A) for 48h. Scale bar 15 μm. (D) Lysates of MDA-MB-231-derived xenograft tumours from saline- or zoledronic acid (ZA)-treated mice. (E) Upper: schematic overview of the experiment. Lower: mammary epithelial cells were plated on soft or stiff fibronectin-coated hydrogels for 3 days with or without Cerivastatin 10μM. (F) The indicated cell lines were either grown on plastic, or plated on soft fibronectin-coated hydrogels for 3 days. Cells grown on plastic were also treated with Latrunculin A 0.5 μM (LAT.A) or Cerivastatin 10μM for the last 48h. (G) SUM149 cells stably expressing control vector (CTL) or the active form of RhoA (G14V) were plated on plastic or on fibronectin-coated soft hydrogels for 3 days. (H) Mutant p53 was immunoprecipitated from lysates of MDA-MB-231 cells grown on plastic, or plated on soft hydrogels for 3 days. (I) MDA-MB-231 cells were plated on soft or stiff hydrogels for 3 days with or without Sulforaphane (SFN) 20μM. (J) Average gene expression values of “mutant p53 signature” genes in missense mutant p53 breast cancer samples, classified according to the “stiffness” signature. (K) Average gene expression values of “YAP/TAZ signature” genes in missense mutant p53 breast cancer samples, classified according to the “stiffness” signature. Data are shown as individual samples (n = 117 independent breast cancer patients; dots) and the mean ± s.e.m. (standard error of the mean; back lines). P-value <0.0001 in a two-tailed unpaired t-test. Experiments in 1A, 1E, 1F, 1G, 1H and 1I were repeated three independent times with similar results. The experiment in 5D was performed once. Unprocessed scans of blots are shown in Supplementary Figure 6.
See this image and copyright information in PMC

References

    1. Mantovani F, Walerych D, Sal G Del. Targeting mutant p53 in cancer: a long road to precision therapy. FEBS J. 2016 doi: 10.1111/febs.13948. - DOI - PubMed
    1. Terzian T, et al. The inherent instability of mutant p53 is alleviated by Mdm2 or p16 INK4a loss. Genes Dev. 2008;22:1337–1344. - PMC - PubMed
    1. Koga T, et al. Heterogeneous distribution of P53 immunoreactivity in human lung adenocarcinoma correlates with MDM2 protein expression, rather than with P53 gene mutation. Int J Cancer. 2001;95:232–239. - PubMed
    1. Freed-Pastor WA, Prives C. Mutant p53: One name, many proteins. Genes Dev. 2012;26:1268–1286. - PMC - PubMed
    1. Girardini JE, et al. A Pin1/Mutant p53 Axis Promotes Aggressiveness in Breast Cancer. Cancer Cell. 2011;20:79–91. - PubMed

Publication types

MeSH terms