Proteomic identification of heat shock protein 90 as a candidate target for p53 mutation reactivation by PRIMA-1 in breast cancer cells

Abstract

Introduction: A loss of p53 function resulting from mutation is prevalent in human cancers. Thus, restoration of p53 function to mutant p53 using small compounds has been extensively studied for cancer therapy. We previously reported that PRIMA-1 (for 'p53 reactivation and induction of massive apoptosis') restored the transcriptional activity of p53 target genes in breast cancer cells with a p53 mutation. By using functional proteomics approach, we sought to identify molecular targets that are involved in the restoration of normal function to mutant p53.

Methods: PRIMA-1 treated cell lysates were subjected to immunoprecipitation with DO-1 primary antibody against p53 protein, and proteins bound to p53 were separated on a denaturing gel. Bands expressed differentially between control and PRIMA-1-treated cells were then identified by matrix-assisted laser desorption ionization-time-of-flight spectrometry. Protein expression in whole cell lysates and nuclear extracts were confirmed by Western blotting. The effect of combined treatment of PRIMA-1 and adriamycin in breast cancer cells was determined with a cytotoxicity assay in vitro.

Results: PRIMA-1 treated cells distinctly expressed a protein band of 90 kDa that was identified as heat shock protein 90 (Hsp90) by the analysis of the 90 kDa band tryptic digest. Immunoblotting with isoform-specific antibodies against Hsp90 identified this band as the alpha isoform of Hsp90 (Hsp90alpha). Co-immunoprecipitation with anti-Hsp90alpha antibody followed by immunoblotting with DO-1 confirmed that p53 and Hsp90alpha were interacting proteins. PRIMA-1 treatment also resulted in the translocation of Hsp90alpha to the nucleus by 8 hours. Treatment of cells with PRIMA-1 alone or in combination with adriamycin, a DNA-targeted agent, resulted in increased sensitivity of tumor cells.

Conclusion: The studies demonstrate that PRIMA-1 restores the p53-Hsp90alpha interaction, enhances the translocation of the p53-Hsp90alpha complex and reactivates p53 transcriptional activity. Our preliminary evidence also suggests that PRIMA-1 could be considered in combination therapy with DNA-targeted agents for the treatment of breast cancer, especially for tumors with aberrant p53 function.

Figure 1

Coomassie blue-stained gel of proteins co-immunoprecipitated with DO-1 primary antibody from MDA-MB-231 cells. Cleared cell lysates were immunoprecipitated with DO-1 primary antibody directed against p53, washed, and resolved by SDS-PAGE (4 to 20% polyacrylamide). Two independent co-immunoprecipitated samples from untreated control (-) and cells treated for 4 hours with 100 μM PRIMA-1 (+) were loaded. The gels were stained with Coomassie blue. Molecular masses of protein size markers are indicated (MW). The arrowhead indicates the band of stained proteins excised for enzymatic digestion by trypsin and subsequent mass fingerprinting with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

Figure 2

Inhibition of PRIMA-1 mediated transcriptional reactivation function of p53 with pifithrin-α (PFTα). MCF-7 (p53^+/+) and GI-101A (mut p53) cells were treated with 100 μM PRIMA-1 for 2, 4 and 8 hours (lanes 1, 2 and 3, respectively). Cells were treated with 20 μM PFTα for 6 hours (lane 4) or with 20 μM PFTα for 2 hours followed by PRIMA-1 for 4 hours (lane 5). 20 μg of protein samples of cell lysates were separated by SDS-PAGE (4 to 20% polyacrylamide) and subjected to Western blot analysis with p53 and p21 primary antibodies. The reactive bands were revealed and detected with the Odyssey™ Infrared Imaging System. β-Actin was used as a loading control for protein samples.

Figure 3

Peptide mass fingerprinting of in-gel tryptic digest of the 90 kDa band generated with a matrix-assisted laser desorption ionization-time-of-flight mass spectrometer. Protein bands indicated by the arrowhead in Fig. 1 were subjected to in-gel digestion and analyzed by mass spectrometry. The tryptic peptides from this band showed the presence of six peptides corresponding to heat shock protein 90 (Hsp90) as one of the proteins that were found in the altered protein-protein interaction pattern of p53 with and without PRIMA-1 treatment.

Figure 4

Protein-protein interaction analysis of p53 and the α isoform of heat shock protein 90 (Hsp90α). (a) MDA-231 cell lysates from untreated cells (lanes 1 and 3) and cells treated for 4 hours with 100 μM PRIMA-1 (lanes 2 and 4) were immunoprecipitated (IP) with anti-Hsp90α monoclonal antibody and subjected to Western blotting (WB) with anti-p53 (DO-1) monoclonal antibody (lanes 1 and 2) in addition to reciprocal immunoprecipitation with DO-1 and Western blotting with anti-Hsp90α (lanes 3 and 4). (b) GI-101A cell lysates from untreated cells (lanes 1 and 3) and cells treated for 4 hours with 100 μM PRIMA-1 (lanes 2 and 4) were immunoprecipitated with anti-Hsp90α monoclonal antibody and subjected to Western blotting with anti-p53 (DO-1) monoclonal antibody (lanes 1 and 2) in addition to reciprocal immunoprecipitation with DO-1 and Western blotting with anti-Hsp90α (lanes 3 and 4).

Figure 5

Western blots of heat shock protein 90 (Hsp90) proteins from control and PRIMA-1-treated cells. Cells were treated with 100 μM PRIMA-1 for 2, 4 or 8 hours. 20 μg of protein samples of cell lysates from the whole cell extracts (WCE) and nuclear extracts (NE) of the control (C) and treated samples were separated by SDS-PAGE (4 to 20% polyacrylamide) and Western blotted with antibodies directed against both the α and β isoforms of Hsp90 (a) and against p53 (b). β-Actin was used as a loading control. The reactive bands were detected with the Odyssey™ Infrared Imaging System.

Figure 6

The nuclear localization of the α isoform of heat shock protein 90 (Hsp90α) is enhanced by treatment of cells with PRIMA-1. MDA-231 cells treated with PRIMA-1 were subjected to nuclear isolation. A fraction of the isolated nuclear pellet was fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 and incubated overnight with anti-p53 and anti-Hsp90α monoclonal antibodies. Nuclear fractions were then immunostained with a secondary antibody conjugated with Oregon green to detect p53 (green) and a secondary antibody conjugated with Texas red to detect Hsp90α (red) before detection by fluorescence microscopy. Nuclei were stained with 4',6-diamidino-2-phenylindole (blue). Normal mouse immunoglobulin G was used as a negative control (data not shown). Arrows mark nuclear staining of Hsp90α. Scale bar, 5 μm.

Figure 7

Combination and sequential exposure of cells to PRIMA-1 and adriamycin. Exponentially growing MDA-MB-231 or GI-101A cells were seeded in 10% serum-supplemented RPMI-1640 medium at 10⁴ cells per well. After 24 hours, cells were treated with 100 μM PRIMA-1 for 24 hours (P24), 0.2 μM adriamycin for 3 hours (A3) or 24 hours (A24), and a combination of PRIMA-1 plus adriamycin for 24 hours (AP24) or adriamycin for 3 hours followed by PRIMA-1 for 24 hours (A3P24). After drug treatment, the cells were reincubated in drug-free medium for a further 3 days and cell survival was determined with the crystal violet assay. Results are means ± SD for quadruplicate determinations (SD<10%) representative of two to four independent experiments; P<0.01.