Proteomic identification of heat shock protein 90 as a candidate target for p53 mutation reactivation by PRIMA-1 in breast cancer cells
© Rehman et al.; licensee BioMed Central Ltd. 2005
Received: 27 July 2004
Accepted: 29 June 2005
Published: 27 July 2005
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.
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.
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 α isoform of Hsp90 (Hsp90α). Co-immunoprecipitation with anti-Hsp90α antibody followed by immunoblotting with DO-1 confirmed that p53 and Hsp90α were interacting proteins. PRIMA-1 treatment also resulted in the translocation of Hsp90α 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.
The studies demonstrate that PRIMA-1 restores the p53-Hsp90α interaction, enhances the translocation of the p53-Hsp90α 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.
Many clinical studies have shown that mutations in p53 are a strong predictor of relapse and are associated with resistance to several therapeutic regimens [1, 2]. Studies in our laboratories and others, for example, showed that mutations in p53 in human tumor cells were correlated with decreased sensitivity to DNA-damaging agents [3–6]. An improved understanding of the relationship between p53 and chemosensitivity might therefore lay the groundwork for new cancer therapies. To understand this relationship better, we recently used a pharmacogenomic approach with complementary DNA microarrays to characterize gene expression profiles of cells containing wild-type p53 (p53+/+) and those containing an isogenic p53 knockout counterpart (p53-/-) after treatment with topotecan, a specific topoisomerase I inhibitor and a DNA-targeted agent . About 10% of the transcripts detected were differentially expressed in the p53+/+ cells in response to topotecan, whereas only 1% of the transcripts changed in the p53-/- cells . These data clearly showed the broad effect of p53 on the transcriptional response to DNA damage, which can lead to growth arrest or apoptosis.
Given that p53 is the most commonly mutated gene in human cancers and that more than 50% of breast tumors are defective in p53 [8–10], extensive research efforts are centered on restoring normal function to mutant p53 to promote tumor suppression. This effort includes the use of modifying peptides [11, 12], antisense oligonucleotides  and small molecules [14, 15]. Unfortunately, the problem of in vivo delivery and lack of selectivity to tumor cells has limited the practical application of most of these efforts. Recently, PRIMA-1 has emerged from an in vitro screen of small molecules that reactivate the transcriptional activity of mutant p53 . PRIMA-1 has the capability of restoring the transcriptional transactivation function to mutant p53 in vitro and in vivo with subsequent tumor regression. PRIMA-1 also has the ability to trigger apoptosis in tumor cells as a function of its mutant p53 reactivation response . We reported recently  that PRIMA-1 (in a effect dependent on both dose and time) restored the transcriptional activity of p53 target genes such as p21Waf1/cip1in breast cancer cells possessing a p53 mutation. However, the exact molecular mechanisms for mutant p53 reactivation by PRIMA-1 are not yet determined. A direct interaction between PRIMA-1 and p53 has not yet been demonstrated. It is possible that PRIMA-1 affects cellular chaperones, resulting in the refolding of mutant p53. Alternatively, PRIMA-1 may block complex formation between mutant p53 and p73, leading to the release of active p73, which triggers proapoptotic target genes . To identify the possible molecular candidates of mutant p53 reactivation by PRIMA-1 in breast tumor cells, in this study we used tools available for a functional proteomics approach.
Our study indicates that the restoration of the transcriptional transactivation of p53 target genes such as p21Waf1/cip1is dependent on p53 and that the α isoform of heat shock protein 90 (Hsp90α) is associated with mutant p53 reactivation by PRIMA-1. As a result of refolding of mutant p53 by Hsp90, we show that both p53 and Hsp90α are translocated to the nucleus of the tumor cells for the activation of p53 target genes. The use of PRIMA-1 to reactivate mutant p53 may therefore be considered further as an approach for adjuvant chemotherapy in the treatment of breast tumors, especially in cancers with aberrant p53 function.
Materials and methods
Drugs and materials
PRIMA-1 (NSC-281668) was obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute (Bethesda, MD); pifithrin-α (PFTα) and doxorubicin (adriamycin hydrochloride) were purchased from Biomol Research Inc. (Plymouth Meeting, PA). Primary antibodies against p53 and p21 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); Hsp 90 primary antibodies were from Stressgen (Victoria, BC, Canada). The goat anti-mouse, anti-rat and anti-rabbit secondary antibodies labeled with IRDye™ 38 were purchased from LI-COR, Inc. Biosciences (Lincoln, NE) or with Alex680 from Molecular Probes, Inc. (Eugene, OR). All other chemicals were of reagent grade.
Cell and culture conditions
The human MCF-7 breast carcinoma cells (p53+/+), MDA-MB-231 and GI-101A (p53 mutant) were routinely maintained in monolayer cultures in RPMI-1640 medium (Invitrogen, Inc., Carlsbad, CA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) as reported previously . As we reported previously , p53 protein in GI-101A cells contains mutations at Y236C, A278P and R72P, whereas p53 protein in MDA-231 cells contains mutations at A278P, R280K and M385T. Exponentially growing cultures at 80% confluence were used in all experiments. For cytotoxicity assays, cells from exponentially growing cultures were plated in 24-well tissue culture plates in RPMI-1640 medium plus 10% fetal calf serum at about 104 cells per well, and the IC50 doses of PRIMA-1 in MDA-231 and GI-101A cells were determined as reported previously . For drug combination studies, we used 100 μM PRIMA-1 and 0.2 μM adriamycin. Both cell lines were treated with the following drug sequence: A3, cells were treated with adriamycin for 3 hours; A24, cells were treated with adriamycin for 24 hours; P24, cells were treated with PRIMA-1 for 24 hours; AP24, cells were treated with both adriamycin and PRIMA-1; and A3P24, cells were treated with adriamycin for 3 hours followed by the removal of adriamycin-containing medium, washing with fresh medium and then incubation with PRIMA-1 for 24 hours. Each treatment was performed in quadruplicate wells in two independent experiments. The wells were washed twice with prewarmed medium, followed by the addition of 2 ml of drug-free medium. The plates were then incubated for a further 3 days. The surviving fraction of cells was determined with the crystal violet assay, as reported previously . The precision of this method with quadruplicate determinations is 10% (SD). The data were analyzed with Student's two-tailed t-test; P < 0.01 was considered statistically significant.
Immunoblotting and immunoprecipitation
Cells were incubated with 0 or 100 μM PRIMA-1 for 0, 2, 4 or 8 hours, then washed twice with PBS (pH 7.4) and harvested. Harvested cells were either used to prepare whole cell lysates or for subcellular fractionation studies. Cell lysates were prepared as described previously . For immunoblotting, 20 μg protein samples were separated by SDS-PAGE (4 to 20% polyacrylamide gradient gel) and transferred on nitrocellulose (Millipore, Bedford, MA). The membrane was developed in accordance with a protocol provided by LI-COR, Inc. Biosciences using anti-β-actin, anti-p53 and anti-p21 primary antibodies (Santa Cruz Biotechnology) or anti-Hsp90α and anti-Hsp90β primary antibodies (Stressgen). The goat anti-mouse, anti-rat and anti-rabbit secondary antibodies labeled with IRDye™ 38 (ex/em: 774/800 nm; LI-COR) or with Alexafluor® 680 (ex/em: 680/707 nm; Molecular Probes) were used. The reactive bands were revealed and detected with the Odyssey™ Infrared Imaging System (LI-COR, Inc.).
For immunoprecipitation studies, 107 MDA-MB-231 or GI-101A cell cultures were grown in T-150 tissue culture flasks. Cultures were treated with 100 μM PRIMA-1 for 4 hours. Cleared cell lysates (total 1 mg of protein) were immunoprecipitated either with 5 μg of DO-1 anti-p53 monoclonal antibody or 5 μg of anti-Hsp90α monoclonal antibody in a co-immunoprecipitation assay in accordance with the protocol provided by eBiosciences, Inc. (San Diego, CA) using Protein A or G-plus agarose beads (Santa Cruz Biotechnology). After SDS-PAGE (4 to 20% polyacrylamide), gels were either stained with Coomassie blue (Bio-Rad, Inc., Hercules, CA) or subjected to immunoblotting.
In-gel enzymatic digestion and mass spectrometry
An OmniFlex MALDI-TOF mass spectrometer (Bruker Daltonics, Billerica, MA) was used for peptide mass fingerprinting. Desalted peptide solution (1 μl) was mixed with 1 μl of matrix solution (α-cyano-4-hydroxycinnamic acid in 0.1% trifluoroacetic acid and 50% acetonitrile) and was then spotted directly on the MALDI target. All data used for protein identification and calibration were acquired in reflectron mode and averaged for 100 shots. These studies employed external mass calibration and used angiotensin I, corticotropin (ACTH) clip (1 to 17) and ACTH clip (18 to 39) peptides as calibrants. After spectral acquisition, the peak m/z values were extracted and used to search the Swiss Protein Database with the ProFound program  search engine. The database search was performed with an implementation of the following search parameters: taxonomy (Homo sapiens), enzyme (trypsin), missing cleavage, mass tolerance (0.24 Da), modification (carbamidomethyl for cysteine and oxidation for methionine) and database (NCBRnr).
Subcellular fractionation and immunocytochemistry
To determine the nuclear translocation of p53 and Hsp90α, cells treated with PRIMA-1 were subjected to nuclear isolation with the FOCUS cytoplasmic and nuclear protein extraction kit (Geno Technology, Inc., St. Louis, MO) in accordance with the manufacturer's instructions. A fraction of the isolated nuclear pellet was fixed in 4% paraformaldehyde in 1 × PBS to determine the intactness of nuclei by staining with 4',6-diamidino-2-phenylindole (DAPI). Fixed nuclei were mounted on SuperFrost-plus slides (Fisher Scientific, Pittsburgh, PA) with Prolong Gold antifade reagent with DAPI (Molecular Probes, Invitrogen Detection Technologies, Carlsbad, CA). Our nuclear preparation contained 99% intact nuclei. To determine the expression of p53, and Hsp90s in intact nuclear fractions, the nuclear pellet was lysed into 1 × SDS sample buffer and subjected to denaturing gel electrophoresis followed by immunoblotting as described above.
To confirm translocation of proteins into the nucleus, nuclear fractions were subjected to immunostaining for p53 and Hsp90α. Immunocytochemistry was performed in accordance with a previously described protocol, with modifications . In brief, fixed nuclear fractions were suspended in antibody buffer (0.1 M PBS, pH 7.4, containing 0.1% Triton X-100) and incubated overnight at 4°C with mouse monoclonal anti-p53 (DO-1) and rat monoclonal anti-Hsp90α antibody at 50:1 dilution. In addition, all appropriate negative controls without the primary antibodies were run in parallel. Unbound antibodies were removed by centrifuging nuclear preparations at 800 g for 3 min at room temperature (22–24°C) followed by three subsequent 5 min washes with shaking (300 r.p.m.) in antibody incubating buffer at room temperature. Next, the bound primary antibody was detected by using species-specific secondary antibody conjugated with different fluorescent dyes at 100:1 dilution in antibody incubation buffer at room temperature for 1 hour. The mouse IgG secondary antibody conjugated with Oregon green was used to detect mouse monoclonal antibody against p53 (DO-1) and the rat IgG secondary antibody conjugated with Texas red was used to detect rat monoclonal antibody against Hsp90α (Molecular Probes, Invitrogen Detection Technologies). The unbound secondary antibody was removed by using three washes as described earlier for primary antibody. After three washes, nuclear fractions were diluted in antibody incubating buffer without Triton X-100 and spread onto a SuperFrost-plus slides. The air-dried slides were then coverslipped with medium containing the nuclear stain DAPI, as before. The immunofluorescence for DAPI, Texas Red and Oregon green was detected with an Axioplan 2 epifluorescent microscope (Zeiss, Thornwood, NY) equipped with appropriate filters. Individual nuclei were visualized at × 20 magnification.
Results and discussion
Inhibition of transcriptional reactivation function of p53 with PFTα
Identification of Hsp90 as a candidate target for p53 mutation reactivation
Nuclear translocation of Hsp90α after treatment with PRIMA-1
Many studies showed that the Hsp90 protein has a major role in the stability of p53 protein and in its translocation to the nucleus. King and colleagues  reported that the wild-type p53 protein forms a complex with Hsp90 in the presence of Hop and that Hsp90 may assist p53 import into the nucleus. However, the type of Hsp90 isoform that is actually involved in the nuclear translocation of p53 was not mentioned in that study. Chen and Wang  recently reported that the stabilization of p53 conformation after heat shock is associated with its binding to Hsp90 protein and that the phosphorylation of p53 is dependent on the Ataxia-telangiectasia-mutated protein kinase (ATM)-mediated activation of human checkpoint 2 (ChK2) kinase. Although these studies investigated the role of Hsp90 in regulating p53 stability under stressful conditions (heat shock), the present studies confirmed the association between Hsp90 and p53 under non-stressful conditions, because there was no increase in Hsp90 expression in WCE in PRIMA-1-treated cells (Fig. 5b), although a small decrease in WCE of PRIMA-1-treated samples at 8 hours was concomitant with an increase in nuclear translocation of Hsp90α in both cell lines.
Recently, Walerych and colleagues  reported that Hsp90α interacts with wild-type p53 protein and that this interaction facilitates the binding of Hsp90α to the p21 promoter in ATP-dependent manner. Murphy and colleagues  showed that PFTα inhibits the p53 signaling after interaction with Hsp90α. Thus, both reports [37, 38] suggest that the interaction of p53 and Hsp90α enhances the transcriptional transactivation of genes containing p53-binding sites in their promoter region. Our findings can be interpreted as the restoration of the p53-Hsp90α interaction by PRIMA-1, enhancing the nuclear translocation of p53-Hsp90α and reactivating the transcriptional activity of p53.
Sensitization of breast cancer cells to DNA targeted agents with PRIMA-1
This study illustrates the use of a functional proteomics approach to identify target molecules that are associated with the reactivation of a p53 mutation by PRIMA-1. Our approach has identified Hsp90α as a partner protein that is associated, in part, with the restoration of p53 transcriptional transactivation function by PRIMA-1. We also showed that the α isoform of Hsp90 protein is associated with the nuclear translocation of p53 protein after treatment of breast tumor cells with PRIMA-1. Our results that PFTα inhibits the induction of p21 expression after treatment with PRIMA-1, and the recent work of Murphy and colleagues  indicating that PFTα inhibits the signaling of p53-mediated gene transcription, strongly suggest that PRIMA-1 facilitates the interaction of Hsp90α and the restoration of mutant p53 to wild type followed by the translocation of both proteins into the nucleus. This may result in the transactivation of genes containing p53-binding sites as reported by Walerych and colleagues .
- DAPI = 4':
heat shock protein 90, α isoform
heat shock protein 90, β isoform
matrix-assisted laser desorption ionization-time-of-flight
nuclear localization signal
whole cell extracts.
We thank Dr Heiko T Jansen (Washington State University, Pullman, WA) for help with the immunocytochemistry studies. This work is supported in part by grant no. BCTR0402398 to SSD from The Susan G Komen Foundation for Breast Cancer Research.
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