Molecular Mechanism of Mutant p53 Stabilization: The Role of HSP70 and MDM2

<div><p>Numerous p53 missense mutations possess gain-of-function activities. Studies in mouse models have demonstrated that the stabilization of p53 R172H (R175H in human) mutant protein, by currently unknown factors, is a prerequisite for its oncogenic gain-of-function phenotype such as tumour progression and metastasis. Here we show that MDM2-dependent ubiquitination and degradation of p53 R175H mutant protein in mouse embryonic fibroblasts is partially inhibited by increasing concentration of heat shock protein 70 (HSP70/HSPA1-A). These phenomena correlate well with the appearance of HSP70-dependent folding intermediates in the form of dynamic cytoplasmic spots containing aggregate-prone p53 R175H and several molecular chaperones. We propose that a transient but recurrent interaction with HSP70 may lead to an increase in mutant p53 protein half-life. In the presence of MDM2 these pseudoaggregates can form stable amyloid-like structures, which occasionally merge into an aggresome. Interestingly, formation of folding intermediates is not observed in the presence of HSC70/HSPA8, the dominant-negative K71S variant of HSP70 or HSP70 inhibitor. In cancer cells, where endogenous HSP70 levels are already elevated, mutant p53 protein forms nuclear aggregates without the addition of exogenous HSP70. Aggregates containing p53 are also visible under conditions where p53 is partially unfolded: 37°C for temperature-sensitive variant p53 V143A and 42°C for wild-type p53. Refolding kinetics of p53 indicate that HSP70 causes transient exposure of p53 aggregate-prone domain(s). We propose that formation of HSP70- and MDM2-dependent protein coaggregates in tumours with high levels of these two proteins could be one of the mechanisms by which mutant p53 is stabilized. Moreover, sequestration of p73 tumour suppressor protein by these nuclear aggregates may lead to gain-of-function phenotypes.</p> </div

HSP70 regulates CHIP- and MDM2-dependent degradation of p53 R175H in opposite ways.

<p>Analysis of mutant p53 degradation was performed in double knockout MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>). (<b>A</b>) CHIP-mediated degradation is accelerated by HSP70. (<b>B</b>) HSP70 WT partially inhibits MDM2-dependent p53 R175H degradation. ANOVA statistical test was carried out, where P values were counted for 3 independent experiments. (<b>C)</b> The steady-state level of p53 R175H is increased or decreased upon co-expression of WT or K71S HSP70, respectively. MEF cells were transfected with plasmids encoding p53 R175H and MDM2 together with increasing amounts of WT HSP70. Additionally, for the highest concentration of WT HSP70 titration of HSP70 K71S was applied. 24 hours later the cells were harvested and analysed by immunoblotting with p53 specific antibody (DO-1). The graph depicts the densitometric analysis performed using Image Quant software. Mean and standard deviation (s.d.) of two independent experiments are shown. (<b>D</b>) Overexpression of HSP70 downregulates the level of MDM2-dependent ubiquitination of p53 R175H<b>.</b> Cell based ubiquitination was carried out in MEF cells treated with MG132 (10 µM) for 4 hours. Mutant p53 protein was immunoprecipitated from the lysate using DO-1 antibody and analysed by western blotting using HA tag-specific antibody.</p

MDM2 and HSP70/HSPA family members change the localization and aggregation state of EYFP-p53 R175H in MEFs.

<p>(<b>A</b>) MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>) were transfected with various combinations of plasmids encoding EYFP-p53 R175H, MDM2, HSC70, WT HSP70 and HSP70 K71S, as labelled above. Cells shown in 9–16 were treated with MG132 (2,5 µM) for 16 hours before imaging. Detailed description in text. Scale bar 10 µm. (<b>B</b>) MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>) were transfected with plasmid encoding EYFP-p53 R175H together with HSP70 and optionally MDM2 (lower panel). 24 hours post-transfection cells were treated with MG132 and scanned using Olympus ScanR Station widefield fluorescent microscope (time-lapse imaging) for additional 16 hours. 0 h represents 8 hours post-transfection, subsequent frames were acquired every 1 hour. The obtained images were analysed by ScanR Analysis software.</p

MDM2 and HSP70 colocalize with p53R175H in an aggressome.

<p>(<b>A</b>) Immunostaining of MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>) transfected with plasmids encoding p53 R175H, HSP70 and optionally MDM2 (lower panel) shows that only in the presence of MDM2, A11 antibodies (labelling amyloid-oligomers) recognize aggregates composed of mutant p53 and HSP70. (<b>B</b>) Immunostaining of MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>) transfected with plasmids encoding p53R175H, MDM2 and HSP70 revealed recruitment of both <b>(1)</b> the E3 ubiquitin ligase and <b>(2)</b> the chaperone to the large inclusion body. (<b>3)</b> The inclusion is formed at the centrosome marked by the presence of gamma-tubulin, suggesting that the large inclusion body containing p53 R175H is an aggresome. (<b>4</b>) Disruption of microtubules with nocodazole treatment impairs protein transport and results in the disappearance of the aggresome; smaller, scattered cytoplasmic p53-containing aggregates form instead. Scale bar 10 µm.</p

Aggregation propensity of p53 is conformation-dependent and promoted by HSP70.

<p>(<b>A</b>) Unfolding of p53 induces its nuclear aggregation<b>.</b> H1299 cells were transfected with plasmids encoding WT p53 (1, 3, 5) or p53 V143A (2, 4) and the next day incubated for 24 hours at the indicated temperature. In case of 42°C treatment, cells were incubated for 23 hours at 37°C and for 1 hour at 42°C. Small nuclear p53-containing aggregates can be seen in 4 and 5. Scale bar 10 µm. (<b>B</b>) HSP70 delays the disappearance of severely unfolded p53 temperature sensitive mutant V143A. Lysates from H1299 cells, transfected with plasmids encoding for indicated proteins, were subjected to p53 conformation-specific immunoprecipitation and analysed by western blotting. For WT conformation two exposures of the same blot are shown.</p

Nuclear diffusion of p53 R175H is limited.

<p>H1299 cells were transfected with plasmids encoding for indicated proteins and 24 hours post-transfection subjected to the FRAP analysis. Mobility of tandem EYFP protein and EYFP fused to indicated p53 mutant was measured in the nuclei. Recovery curves were normalized and each set of replicates was fitted to one or two phase exponential association equation with an R² of no less than 0.95. The differences between all curves shown are statistically significant (larger than 95% confidence intervals, not shown for chart clarity).</p

HSP70 inhibition prevents the formation of cytoplasmic p53 R175H speckles in MEFs.

<p>MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>) were transfected with plasmids encoding p53 R175H and HSP70 and treated (or not) with HSP70 inhibitor (VER155008, Tocris Bioscience) and subsequently labelled for p53 and HSP70. In non-treated cells cytoplasmic spots containing p53 and HSP70 were observed. Small folding intermediates were visible when HSP70 inhibitor was added 6h post-transfection and no spots were detected in the case of HSP70 inhibition induced in parallel with transfection. Scale bar 10 µm.</p

Endogenous HSP70 protein levels in H1299 cells are sufficient for nuclear aggregation of p53R175H.

<p>(<b>A</b>) SK-BR-3, H1299 and MEF cells were seeded 24 hours prior to harvesting. Immunoblotting with HSP70-specific antibody revealed high levels of HSP70 in the cancer-derived SKBR3 and H1299 cell lines when compared to double knockout MEF (<i>Trp53^−/−, Mdm2^−/−</i>) cells. (<b>B</b>) H1299 cells were treated (or not) with a combination of two compounds –17-AAG (1 µM, HSP90 inhibitor) and VER155008 (10 µM, HSP70 inhibitor) – for 16 hours. Immunoblotting of cell lysates shows the accumulation of HSP70 protein upon treatment with 17-AAG and the decrease in p53 R175H protein level as a result of HSP70 inhibition by VER155008. (<b>C</b>) H1299 cells were transfected with plasmids encoding p53 R175H and optionally low or high amount of HSP70 and then treated with MG132 (2 µM) for 16 hours prior to fixation. Immunostaining with p53-specific antibody revealed that the endogenous HSP70 protein level is sufficient for induction of nuclear p53 R175H aggregate formation (left panel) and that an increase in HSP70 level results in appearance of cytoplasmic p53-containing speckles (middle and right panel). (<b>D</b>) Immunofluorescence of H1299 cells treated with 17-AAG further supports the notion that endogenous HSP70 shifts the equilibrium between various oligomeric states of mutated p53. The lower level of HSP70 (6 hours after 17-AAG treatment, middle panel) triggers p53 R175H nuclear aggregation, whereas the higher level of HSP70 (16 hours after 17-AAG treatment, right panel) causes the dissociation of the nuclear aggregates into smaller speckles and their relocalization to the cytoplasm. Scale bar 10 µm.</p

Mutant p53 sequesters p73 in cancer cells.

<p>(<b>A</b>) Overexpressed HSP70 restores transcriptional activity of TAp63α isoform inhibited by p53 R175H mutant in H1299 cells (left diagram), while TAp73α remains inhibited (right diagram). All data points were carried out in triplicate by means of Dual Luciferase Reporter Assay. (<b>B</b>) Overexpressed HSP70 molecular chaperone releases TAp63α isoform from the complex with p53 R175H mutant in H1299 cells, whereas maintains TAp73α in this interaction. 24h post-transfection cells were lysed and p53 protein was immunoprecipitated with anti-p53 antibody (DO-1). The immunoprecipitated protein complexes were subjected to Western blot analysis. (<b>C</b>) H1299 cells were transfected with plasmids encoding p53 R175H and p63 or p73 and treated with MG132 (2 µM) overnight. Labelling for specific antibodies showed that under such conditions p73 coaggregates with mutant p53 in the nucleus, while p63 remains diffused in the same compartment. Analysis performed in the ImageJ software with the JACoP plugin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426-Bolte1" target="_blank">[88]</a> confirmed that the colocalization is high for p73 and p53 R175H, whereas p63 overlaps with mutant p53 only partially (diagram). Two colocalization factors were determined – Pearson’s coefficient (r) and Mander’s coefficients (M1 and M2) - using threshold value of 110 for images in p63 analysis (upper panels) and 95 for p73 (lower panels). (<b>D</b>) p63 and p73 protein solubility assay performed in H1299 cells cotransfected with plasmids encoding one of those two proteins parallel with p53 R175H and HSP70 revealed that p63 is absent in the most detergent resistant fraction, while a part of p73 pool remains aggregated and is present in the Laemmli sample buffer together with mutant p53 and HSP70. Detailed description of the method available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s002" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s003" target="_blank">S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s004" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s005" target="_blank">S5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s006" target="_blank">S6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s007" target="_blank">S7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s008" target="_blank">S8</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s009" target="_blank">S9</a>.</p

Additional file 6: of Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

Figure S2. Predictive power of ID4 mRNA expression for overall survival (OS) was evaluated by Kaplan-Meier analysis on the TCGA cohort in BLBCs showing high or low CD68 (a and b) or macrophage signature (MacSig) (c and d) levels. Macrophage signature is composed of eight widely used markers for the mononuclear phagocyte system (CD14, CD105, CD11b, CD68, CD93, CD33, IL4R and CD163 [37]). e Evaluation of association between ID4 or CD68 and the pathological variables T, N, G and TP53 status in the BLBCs from the TCGA cohort. (PDF 4464 kb