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. Author manuscript; available in PMC: 2021 Apr 24.
Published in final edited form as: Mol Pharm. 2020 Dec 8;18(1):338–346. doi: 10.1021/acs.molpharmaceut.0c00978

Targeting Triple Negative Breast Cancer with a Nucleus-Directed p53 Tetramerization Domain Peptide

Gu Xiao 1, George K Annor 2, Kimberly Fung 3, Outi Keinänen 4, Brian M Zeglis 5, Jill Bargonetti 6
PMCID: PMC8068092  NIHMSID: NIHMS1687620  PMID: 33289569

Abstract

Triple negative breast cancer (TNBC) has no targeted detection or treatment method. Mutant p53 (mtp53) is overexpressed in >80% of TNBCs, and the stability of mtp53 compared to the instability of wild-type p53 (wtp53) in normal cells makes mtp53 a promising TNBC target for diagnostic and theranostic imaging. We generated Cy5p53Tet, a novel nucleus-penetrating mtp53-oligomerization-domain peptide (mtp53ODP) to the tetramerization domain (TD) of mtp53. This mtp53ODP contains the p53 TD sequence conjugated to a Cy5 fluorophore for near-infrared fluorescence imaging (NIRF). In vitro co-immunoprecipitation and glutaraldehyde cross-linking showed a direct interaction between mtp53 and Cy5p53Tet. Confocal microscopy and flow cytometry demonstrated higher uptake of Cy5p53Tet in the nuclei of TNBC MDA-MB-468 cells with mtp53 R273H than in ER-positive MCF7 cells with wtp53. Furthermore, depletion of mtp53 R273H caused a decrease in the uptake of Cy5p53Tet in nuclei. In vivo analysis of the peptide in mice bearing MDA-MB-468 xenografts showed that Cy5p53Tet could be detected in tumor tissue 12 min after injection. In these in vivo experiments, significantly higher uptake of Cy5p53Tet was observed in mtp53-expressing MDA-MB-468 xenografts compared with the wtp53-expressing MCF7 tumors. Cy5p53Tet has clinical potential as an intraoperative imaging agent for fluorescence-guided surgery, and the mtp53ODP scaffold shows promise for modification in the future to enable the delivery of a wide variety of payloads including radionuclides and toxins to mtp53-expressing TNBC tumors.

Keywords: mutant p53, peptides, molecular imaging, TNBC, preclinical

Graphical Abstract

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INTRODUCTION

Breast cancer is the second leading cause of death from cancer among women.1 Invasive primary breast cancers often express mutant p53 (mtp53) proteins that are stabilized and appear to have tumor-driving properties.2,3 In contrast to a very short half-life of wild-type p53 (wtp53) in normal cells, the mtp53 protein required for “gain of function” (GOF) activities is highly stable and results in cancerous phenotypes, including altered metabolism and increased proliferation, migration, invasion, and chemoresistance.4 The TP53 gene is mutated in approximately 80% of triple negative breast cancers (TNBCs), cancers which lack detectable estrogen receptor (ER) and progesterone receptor expression and HER2 gene amplification.5 The high frequency of p53 mutations in TNBC and the stability of the mtp53 protein suggest a diagnostic and therapeutic (theranostic) strategy for shifting some TNBC to be categorized as a subclass of mtp53-positive breast cancers. Targeting mtp53 could form the basis for a theranostic approach in this subset of breast cancer.

One strategy for targeting mtp53 has focused on degrading the stable GOF mtp53 with Hsp90 inhibitors and statins.6 The heat shock protein HSP90/HDAC6 chaperone machinery is a major determinant of highly stable mtp53.7 Another mtp53-targeting strategy has focused on using molecules to restore mtp53 to wtp53 functionality with normal transcriptional activity, such as PRIMA-1, PRIMA-1MET (APR-246), PK11007, and COTI-2.8 Several peptides have been found that restore wtp53 functions in mouse cancer models.9,10 A series of peptides were identified that allow for proper p53 folding and transcriptional activity that can promote apoptosis in tumor cells.9 A peptide designed to inhibit p53 amyloid formation (called ReACp53) rescues p53 function in cancer cell lines.10

The high stability of mtp53 has not yet been leveraged to target cancers. What has also been underappreciated is that the mtp53 protein contains an intact tetramerization domain (TD). Interestingly, a p53 TD peptide bearing cell-penetrating and nuclear localization signals was shown to interact with wild-type p53 (wtp53) and thereby inhibit p21 expression via hetero-tetramerization.11 The mtp53 protein consists of the same five functional domains as wtp53: a transactivation domain (residues 1–42), a proline-rich domain (residues 63–97), an often-mutated sequence-specific DNA binding domain (residues 98–292), an oligomerization domain that confers the tetrameric structure necessary for p53 function (TD, residues 325–355), and a C-terminal domain (CTD, residues 363–393) that interacts with DNA in a sequence-nonspecific manner.12 The p53TD consists of a β-strand (Glu326-Arg333), a tight turn (Gly334), and an α-helix (Arg335-Gly356).13 Wild-type p53 binds to DNA site-specifically as a homotetramer regulated by the p53 TD and CTD.14 The mtp53 protein interacts with chromatin nonspecifically, and this interaction is potentially also regulated by the TD and CTD interactions.15

We found that mtp53 R273H, R280K, and L194F proteins are tightly associated with chromatin.16 Therefore, we reasoned that the delivery of the labeled-p53 TD peptides could be designed to enter cancer cells and hijack the stable mtp53 as a targeting device for TNBC detection. In light of this, we designed a nucleus-penetrating, fluorophore-labeled mtp53-oligomerization-domain peptide (mtp53ODP) to the TD of mtp53, which we call Cy5p53Tet, for the detection of TNBC chromatin-associated mtp53. Cy5p53Tet is a peptide derived from the p53 TD modified with both a Cy5 fluorophore and a HIV-1 Tat Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg sequence for cell and nuclear penetration. In addition to nuclear penetration, HIV-1 Tat also binds to the p53TD without inhibiting p53 tetramerization, which indicates that the p53TD interacts with Tat facing away from the p53 tetramerization interface.17 Herein, we validate that Cy5p53Tet is able to bind to mtp53 and enables the visualization of mtp53-expressing cells both in vitro and in vivo. Ultimately, this mtp53ODP technology has the potential to facilitate the delivery of a wide variety of diagnostic and therapeutic payloads to tissues expressing stable mtp53 (while avoiding the detection of wtp53 due to its short half-life), thereby providing a new approach to the imaging and treatment of TNBC and other malignancies.

MATERIALS AND METHODS

Materials.

The mutant p53 oligomerization domain peptide (mtp53ODP) called Cy5p53Tet was purchased from JPT peptide (Germany) at a purity >95%. The mtp53ODP is 35 amino acids long with an N-Terminal Cy5 fluorophore conjugation: H-CysCy5-RKKRRQRRGEYFTLQIRGRER-FEMFRELNEALELK-OH.

Solvents and reagents including dimethyl sulfoxide (DMSO), glutaraldehyde (GA), and anti-β-actin antibody (Cat# A2066) were obtained from Sigma-Aldrich. Anti-p53 antibody (DO-1, Cat# sc-126), anti-PARP1 antibody (Cat# sc-7150), and normal mouse IgG (Cat# sc-2025) were purchased from Santa Cruz. Magnetic beads were purchased from Cell Signaling. Anti-MDM2 antibody (Cat# AF1244) was obtained from the R&D System.

Ethics.

All animal experiments were done in accordance with protocols approved by the Institutional Animal Care and Use Committees (IACUC) of Hunter College, Weill Cornell Medical College, and Memorial Sloan Kettering Cancer Center and followed the National Institutes of Health guidelines for animal welfare.

Cell Culture.

Human breast cancer cell lines MCF7, MDA-MB-468, MDA-MB-231, HCC70, and SK-BR-3 and normal human mammary epithelial cell MCF10A were purchased from American Type Culture Collection (ATCC). We have authenticated all the cell lines by short tandem repeat technology (Genetica DNA Laboratories). Cells were tested for mycoplasma using the Universal Mycoplasma Detection Kit from ATCC. Cells were maintained at 5% CO2 in a 37 °C humidified incubator. MCF7, MDA-MB-468, MDA-MB-231, and HCC70 cells were grown in DMEM (Invitrogen) and supplemented with 10% FBS (Gemini). SK-BR-3 cells were cultured in McCoy’s 5a Medium and supplemented with 10% FBS (Gemini). MCF10A cells were grown in MEGM Mammary Epithelial Cell Growth Medium SingleQuots Kit without gentamycin–amphotericin B mix (Lonza) with 100 ng/mL cholera toxin. All cells were supplemented with 50 U/mL penicillin, 50 μg/mL streptomycin (Mediatech), and 5 μg/mL plasmocin (InvivoGen). MDA-MB-468 shp53 cells generated with mir30 short hairpin RNA can induce knockdown of mtp53 with 8 μg/mL doxycycline for 7 days.3,16

Cy5p53Tet Cellular Uptake by Live Cell Imaging.

Cells were seeded at 2 × 105 per well in a 12-well glass bottom plate 1 day before imaging (MatTek). Cells were incubated with 100 or 500 nM Cy5p53Tet at 37 °C for the indicated time. Cy5p53Tet was then removed, and the cells were washed three times with phosphate-buffered saline (PBS) at room temperature and costained with 1 μg/mL Hoechst 33342 (Thermo-Fisher) in PBS for 5 min. Z-stack images of stained cells were taken by confocal microscopy using a Nikon A1 confocal microscope with a 60× objective.

In Vitro Cy5p53Tet Cellular Uptake by Flow Cytometry.

Fluorescence-activated cell-sorting (FACS) was used to determine the cellular uptake of Cy5p53Tet. MCF7 and MDA-MB-468 cells were seeded in six-well plates at a density of 5 × 105 cells/well and incubated at 37 °C overnight. On the following day, media were replaced with fresh media containing vehicle control or 100 or 500 nM Cy5p53Tet and further incubated at 37 °C for 2 h. Cells were washed two times with PBS and trypsinized at 37 °C for 5 min. Trypsin was neutralized by adding media, and the cell suspension was spun down. Cell pellets were washed with PBS and resuspended in PBS. FACS was performed on a FACScan (BD Biosciences), processing 2 × 104 events for each sample.

Peptide Cytotoxixity Assay.

A total of 1.25 × 105 cells were seeded in a 12-well plate the day before and grown at 37 °C. Cells were treated with 500 nM Cy5p53Tet for 24 h, and 0.1 mL MTT solution [5 mg mL−1 (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] was added to the cells and incubated at 37 °C for 1 h. The cells were then resuspended in 0.04 N hydrochloric acid diluted in isopropanol and incubated in the dark on a shaker for 5 min at room temperature. The absorbance was quantified at 550 nm, and the background absorbance was subtracted at 620 nm.

Co-immunoprecipitation Assay.

Magnetic beads were used for co-immunoprecipitation assays, and 50 μL of the bead suspension was placed in each sample. The beads were washed twice with 1X PBS-0.1% Tween-20 by vortexing for 10 s. The beads were resuspended in 1X PBS-0.1% Tween-20 with either 1 μg of anti-p53 DO1 antibody or 1 μg of normal mouse IgG at a final volume of 100 μL. The tubes were incubated at room temperature for 10 min with continuous mixing. The beads were washed three times with 1X PBS-0.1% Tween-20, and 1 μg of purified mtp53 R273H and 100 ng of Cy5p53Tet peptide were incubated with the immobilized antibody at 4 °C for 150 min with constant rotation. Beads were pelleted and then washed with 1 mL 1X PBS-0.1% Tween-20 four times at room temperature. Bound proteins were eluted by incubating in 2x SDS Laemmli sample buffer containing 0.2 M DTT, heated at 95 °C for 10 min, and loaded on 15 or 10% polyacrylamide gel.

Protein Extraction.

Whole-cell extraction and protein extraction from xenograft models were conducted as previously described.18

Glutaraldehyde Cross-Link Assay.

Cells were treated with vehicle control or Cy5p53Tet for 2 or 4 h and lysed with phosphate lysis buffer (PBS, 10% glycerol, 10 mM EDTA, 0.5% NP-40, 0.1 M KCl, 1 mM PMSF, 8.5 μg/mL aprotinin, 2 μg/mL leupeptin, and phosphatase inhibitor cocktail). Glutaraldehyde was added to 100 μg of lysate to final concentrations of 0.0025, 0.005, or 0.01%. After incubating with rotation for 20 min at room temperature, the reactions were stopped by adding 2X SDS Laemmli sample buffer containing 0.2 M DTT, the samples were heated for 5 min at 100 °C, and 25 μg of sample was resolved by 8% SDS-PAGE.

NIRF Imaging of Cy5p53Tet in Mice Bearing Bilateral MCF7/MDA-MB-468 Xenografts.

Female athymic nude mice (6–10 weeks old, 01B74-Athymic NCr-nu/nu;) were obtained from Charles River Laboratories. Animals were supplemented with 17β-estradiol with a dose of 0.72 mg/pellet (60-day release) into the neck 7 days before MCF7 cells were subcutaneous implanted. 5 × 106 cells/mouse MCF7 cells were suspended in 100 μL of 1:1 media/matrigel basement membrane matrix (Corning) and injected subcutaneously on the left flank of each mouse (n ≥ 3/group). After 4 weeks, 5 × 106 cells/mouse MDA-MB-468 cells were subcutaneously implanted in the right flank of the mouse in 100 μL of 1:1 media/matrigel basement membrane matrix. Imaging experiments were performed when the tumors reached a volume of ∼50–250 mm3 (after approximately 3 weeks). Cy5p53Tet (10 nmol) was injected into the tail vein of each mouse. Prior to in vivo imaging, the mice were anesthetized with 1.5–2.0% isoflurane (Baxter Healthcare). Images were collected using an IVIS Spectrum (Perkin Elmer) 12 min, 30 min, and 3 h following the administration of Cy5p53Tet. Epifluorescence exposure time on each side was identical, with multiple exposures ranging from 0.2 to 2 s. Fluorescence imaging was carried out with excitation and emission wavelengths of 640 and 680 nm, respectively. Animals were sacrificed 40 min, 80 min, or 3 h after the injection of Cy5p53Tet, and epifluorescence images of the excised MCF7 and MDA-MB-468 xenografts were obtained using the same condition as mentioned above. Semiquantitative analysis of the Cy5p53Tet signal was conducted by measuring the average radiant efficiency [p/s/cm2/sr]/[μW/cm2] in regions of interest.

Statistical Analysis.

Statistical analyses were conducted in Graphpad Prism 7. Results are expressed as mean + SEM. Statistical significance for hypothesis testing was performed by two-tailed Student’s t-test of unknown variance.

RESULTS

Cy5p53Tet Penetrates MDA-MB-468 TNBC Cells Expressing mtp53 R273H.

The mtp53ODP called Cy5- p53Tet designed to monitor the expression of mtp53 in the nuclei of cancer cells contained three essential components: (1) a p53TD (residues 325–351) sequence to facilitate the recognition of the TD of mtp53, (2) a Cy5 fluorophore to enable NIRF imaging, and (3) an N-terminal HIV-1 TAT nuclear localization sequence to ensure the correct subcellular targeting (Figure 1A). Following the synthesis and purification of Cy5p53Tet, we performed preliminary in vitro uptake, specificity, and toxicity assays to gauge the potential of mtp53ODPs as imaging agents. First, we performed live cell imaging to compare the ability of Cy5p53Tet to stain ER+ MCF7 breast cancer cells that express wtp53 and MDA-MB-468 TNBC cells which express stable missense mtp53 R273H. We expected Cy5p53Tet to detect both wtp53 and mtp53, but because of the higher stability of mtp53, we predicted that MDA-MB-468 cells would have a higher signal. The intensity of the Cy5p53Tet signal was clearly higher in the MDA-MB-468 cells compared to the MCF7 cells (Figure 1B), a difference that correlated to the level of p53 expression as determined by Western blot (Figure 1C). Merged fluorescent images of the cell staining with Cy5p53Tet and Hoechst revealed the significant nuclear localization of Cy5p53Tet in the MDA-MB-468 cells (see Figure S1 for vehicle control and 3D images). Quantification of the uptake of Cy5p53Tet via Nikon Element analysis demonstrated a twofold higher uptake of the mtp53ODP in MDA-MB-468 cells compared to MCF7 cells (Figure 1D). We also observed (by confocal microscopy) that the Cy5p53Tet did not colocalize with the stable transfected cell GFP marker in the cytoplasm. However, after a 2 h incubation with Cy5p53Tet, the colocalization was clearly evident with the Hoechst-stained DNA (Figure S3).

Figure 1.

Figure 1.

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Cy5p53Tet penetrates into MDA-MB-468 TNBC cells expressing mtp53 R273H. (A) Schematic of the structure of Cy5p53Tet. (B) Live cell imaging staining of MCF7 and MDA-MB-468 cells after 2 h of incubation with 500 nM Cy5p53Tet (red). Hoechst staining (blue) was used to stain the nuclei. Two independent experiments with biological replicates were performed. (C) p53 protein levels in MCF7 and MDA-MB-468 cells determined by Western blot analysis before carrying out live cell imaging. (D) Quantification of Cy5p53Tet uptake in MCF7 and MDA-MB-468 cells via Nikon Element analysis. At least 200 cells per sample were measured by fluorescence microscopy. (E) Flow cytometry of MCF7 and MDA-MB-468 cells after incubation with 100 or 500 nM Cy5p53Tet for 2 h at 37 °C. FlowJo software was used to analyze the cytometric data. (F) Geometric MFI from the FACS experiments in (E). (G) MTT assay conducted in MCF7, MDA-MB-468, HCC70, SK-BR-3, and MCF10A cells to measure mitochondrial dehydrogenase activity in response to 500 nM Cy5p53Tet treatment for 24 h. Three independent experiments with biological replicates were performed for all ± SEM *p-value ≤ 0.05, **p-value ≤ 0.01, ***p-value ≤ 0.001.

The cellular uptake of Cy5p53Tet in these two cell lines was also measured using flow cytometry. The data from flow cytometry of MCF7 and MDA-MB-468 treated with vehicle or Cy5p53Tet at 100 or 500 nM for 2 h showed increased uptake in the mtp53-expressing cells (Figure 1E). The geometric mean fluorescence intensity (MFI) values showed that the uptake of Cy5p53Tet was significantly elevated in MDA-MB-468 cells with mtp53 compared to MCF7 cells with wtp53, with 1.58-fold and 1.65-fold more in the mtp53-expressing cells at 100 and 500 nM Cy5p53Tet, respectively (Figure 1F).

Toxicity to normal cells and tissues represents an important barrier for drug delivery, and a crucial advantage of cell-penetrating peptide-based therapies is their low toxicity compared to most drug carriers.19 To evaluate the toxicity of Cy5p53Tet to breast cancer cells and normal breast mammary epithelial cells, the viability of cells was evaluated following peptide treatment (Figure 1G). No significant reduction of mitochondrial activity was detected in nonmalignant human mammary epithelial cells MCF-10A with wtp53 after 24 h of treatment with 500 nM Cy5p53Tet. The breast cancer cells treated with 500 nM Cy5p53Tet demonstrated that mitochondrial activity reduced by 9.8% in MCF7 cells (wtp53), 10.6% in MDA-MB-468 cells (mtp53 R273H), and 13.6% in HCC70 (mtp53 R248Q). Overall, Cy5p53Tet exhibited no cytotoxicity to normal cells and low cytotoxicity to cancer cells in vitro which paved the way for subsequent in vivo investigations with the imaging agent.

Cy5p53Tet Accumulates in mtp53 Xenograft Tumors.

To investigate whether Cy5p53Tet could be used for the imaging of tumors with mtp53, in vivo NIRF imaging experiments were performed in mice bearing bilateral MCF7 and MDA-MB-468 xenografts that express wtp53 and mtp53 R273H (Figure 2). Importantly, a Western blot confirmed the expression profiles of ER and p53 in the two cell lines prior to implantation, with the MCF7 cells exhibiting high levels of ER and low wtp53 and the MDA-MB-468 cells producing high levels of mtp53 (Figure 2A). The NIRF signal was clearly observed in the MDA-MB-468 tumors, after the intravenous administration of Cy5p53Tet, whereas no signal was present in the MCF7 (Figure 2B). At 40, 80, and 180 min after injection, mice were sacrificed, and the xenografts were resected and imaged ex vivo (Figure 2C). At each time point, the MDA-MB-468 mtp53-expressing tumors exhibited higher radiant efficiency than the MCF7 xenografts (Figure 2D). More specifically, the ratios of the Cy5 signal in the MDA-MB-468 xenografts to that in the MCF7 tumors at 40, 80, and 180 min were 1.95:1, 1.63:1, and 1.75:1 respectively (Figure 2D).

Figure 2.

Figure 2.

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Cy5p53Tet accumulates in mtp53 xenograft tumors. (A) ER and p53 protein levels in MCF7 and MDA-MB-468 cells were determined by Western blot analysis before implantation. (B) In vivo optical imaging of Cy5p53Tet uptake in mice bearing bilateral MCF7 and MDA-MB-468 xenograft models. A representative image acquired 30 min after injection is shown. The tumor is marked by a “T.” (C) Epifluorescence imaging of MCF7 and MDA-MB-468 tumors excised 80 min after the administration of Cy5p53Tet. (D) Epifluorescence intensity quantification of the tumors resected at 40, 80, and 180 min after injection. (E) SDS-PAGE analysis of extracts collected from the tumors in the in vivo imaging experiment. Protein (25 μg) from each sample was run on a 12% polyacrylamide gel, and the fluorescence signal from Cy5p53Tet was interrogated and quantified. The expression levels of p53 and MDM2 in the same tumor samples were determined by Western blot analysis.

We also investigated the accretion of Cy5p53Tet in tumor tissue by extracting proteins from the xenografts excised at 40, 80, and 180 min after injection and examined them on a 12% SDS polyacrylamide gel (Figure 2E). The amount of Cy5p53Tet in each sample was interrogated by scanning on the Cy5 channel by a Typhoon FLA 7000 laser scanner and quantifying the signal intensity. In this analysis, the ratios of the Cy5 signal derived from the MDA-MB-468 cells to that in the MCF7 cells at 40, 80, and 180 min were 5.14:1, 1.65:1, and 1.05:1 respectively. The expression levels of p53 and MDM2 following Cy5p53Tet treatment were probed by Western blot. The levels of both oncogenic proteins mtp53 and MDM2 were higher in the TNBC MDA-MB-468 cells (Figure 2E). Interestingly, in MCF7 cells during the Cy5p53Tet uptake, wtp53 and MDM2 levels demonstrated the classic oscillation pattern (Figure 2E, lanes 2, 4, and 6). This is logical, as cellular wtp53 is an unstable protein, with a half-life ranging from 5 to 30 min in the absence of an activation signal.20 The short half-life time is due to MDM2, a transcriptional target of wtp53 that acts as an E3 ubiquitin ligase that binds to p53 for proteosomal degradation.20 Oscillations in p53 and Mdm2 protein levels are required to keep wtp53 levels low.21 Taken together, the elevated uptake of Cy5p53Tet in the mtp53-expressing tumors of the mice indicates that Cy5p53Tet preferentially targets tumors expressing mtp53. The permeation properties of Cy5p53Tet hold great potential as in vivo TNBC detection agents and could be a delivery vehicle for cancer treatments.

Cy5p53Tet Uptake Is Reduced by Depletion of mtp53 R273H, Indicating a Complex with mtp53 R273H.

The specificity of the interaction between Cy5p53Tet and mtp53 R273H was further investigated by studying the uptake of the peptide in MDA-MB-468 cells with and without the depletion of mtp53 (Figure 3A upper panel and Figures S2 and S3). To this end, a miR30-based doxycycline-inducible shRNA mtp53 knockdown cell line MDA-MB-468.shp53 was employed. The cells were incubated with 500 nM Cy5p53Tet for 30 min, 2 h, 4 h, and 24 h under both knockdown and control conditions (Figure 3B). The imaging of the cells with the shRNA-mediated knockdown of the mtp53R273H message and protein also showed a corresponding reduction in the Cy5p53Tet uptake (Figure S3). Cy5p53Tet could be detected as early as 30 min and was localized predominantly to the nucleus. The signal from the peptide increased up to 4 h, after which it was sustained until its decrease after 24 h. Most importantly, the depletion of mtp53 clearly correlates with reduced uptake of Cy5p53Tet (Figure 3A,B, and Figures S2 and S3). More specifically, the relative region of interest intensity values showed a statistically significant reduction of 61, 63, and 67% of the Cy5p53Tet signal at 30 min, 2 h, and 4 h, respectively. By 24 h, however, the overall Cy5p53Tet signal was greatly reduced, and no statistically significant difference could be observed between the knockdown and control cells (Figure 3B).

Figure 3.

Figure 3.

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Cy5p53Tet forms a complex with mtp53 R273H. (A) (Top) Live cell imaging of Cy5p53Tet (red) in MDA-MB-468 shp53 cells with or without shRNA induction. Cells were imaged by confocal microscopy after 30 min, 2 h, 4 h, and 24 h incubation of 500 nM Cy5p53Tet. Hoechst staining (blue) was used to stain the nucleus. Three independent experiments with biological replicate were performed. A representative picture acquired after 2 h of incubation is shown. (Bottom) Mtp53 protein levels in MDA-MB-468 shp53 cells with or without shRNA induction were determined by Western blot analysis before carrying out live cell imaging. Whole-cell extract (50 μg) was loaded on 10% SDS-PAGE gel. (B) Quantification of Cy5p53Tet uptake in MDA-MB-468 shp53 cells with or without mtp53 depletion. *p-value ≤ 0.05, **p-value ≤ 0.01, ***p-value ≤ 0.001. (C) Co-immunoprecipitation assay carried out with purified mtp53 R273H and Cy5p53Tet in a molar ratio of 1:1. Anti-p53 DO1 antibody-coupled magnetic beads were used to pull down Cy5p53Tet/mtp53 complex, and normal mouse IgG-coupled magnetic beads were used as a control.

To determine if the Cy5p53Tet interacts with the p53TD, we first evaluated the p53TD and Cy5p53Tet complex using the protein–peptide globe docking method CABS-dock22 and obtained a high-quality prediction (see Figure S4). To experimentally determine if Cy5p53Tet binds to mtp53 R273H, we performed a co-immunoprecipitation assay using purified mtp53 mixed with Cy5p53Tet in a molar ratio of 1:1 (Figure 3C). The co-immunoprecipitation was carried out using anti-p53 antibody or nonspecific IgG. We found that mtp53 R273H was enriched by the anti-p53 antibody and not enriched by immunoprecipitation with IgG. Cy5p53Tet was significantly pulled down with mtp53 R273H in the anti-p53 antibody sample (see arrow), but not by the normal IgG sample (Figure 3C).

Cy5p53Tet Binds to Tetrameric mtp53.

Wtp53 can form a tetramer, and its oligomerization regulates its transcriptional activity.23 Tetramerization is important for both wtp53 and mtp53, as both are preferentially degraded by MDM2 when present as dimers rather than tetramers.24 We examined the oligomerization states of mtp53 and investigated the interactions by Cy5p53Tet and mtp53 using glutaraldehyde (GA) cross-linking assays (Figure 4). MDA-MB-468 cells were treated with vehicle or 1.5 μM of Cy5p53Tet for 4 h. Whole-cell lysates were cross-linked with glutaraldehyde concentrations of 0, 0.0025, or 0.005%, and the samples were analyzed by Western blot to detect p53 oligomerization forms (Figure 4A). In cells without glutaraldehyde, monomers of mtp53 were detected; at 0.0025% glutaraldehyde, monomers, dimers, and tetramers were visible; and at 0.005% glutaraldehyde, the predominant form was tetramers. We also evaluated the presence of Cy5p53Tet-containing mtp53 polypeptides using the Cy5 channel (Figure 4B). Cy5p53Tet was detected at a low molecular weight: <12 kDa in the absence of glutaraldehyde (Figure 4B, lane 4). Remarkably, in the presence of glutaraldehyde, Cy5p53Tet was observed at the molecular weight larger than the mtp53 tetramer: >225 kDa in a very obvious form (Figure 4B, lanes 5 and 6). The merged mtp53 and Cy5p53Tet images demonstrated that this high-molecular-weight species (yellow, indicated with arrow) was a p53/Cy5p53Tet complex. As a control, we examined the glutaraldehyde cross-linking of Cy5p53Tet without mtp53 and found no such signal (Figure 4D). Similar results were observed in MDA-MB-468 cells treated with Cy5p53Tet for 2 or 4 h with higher glutaraldehyde levels (Figure S5). Ultimately, the high-molecular-weight complex that was >225 kDa is larger than the p53 tetramer detected in the MDA-MB-468 cells treated with Cy5p53Tet and could represent the hetero-tetramerization of mtp53 and Cy5p53Tet.

Figure 4.

Figure 4.

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Cy5p53Tet binds to tetrameric mtp53. MDA-MB-468 cells incubated with 1.5 μM Cy5p53Tet for 4 h, and 100 μg of the resultant cell lysates was treated with increasing amounts of glutaraldehyde (0, 0.0025, and 0.005%) for 20 min at room temperature. (A) Western blot carried out to detect mtp53 oligomer using anti-p53 DO1 antibody. (B) Cy5p53Tet fluorescence signal detected using the Cy5 channel. (C) Merged mtp53 (green) and Cy5p53Tet (red) image demonstrating a high-molecular-weight signal (yellow, indicate with arrow) that is likely an mtp53/Cy5p53Tet complex. (D) Cy5p53Tet treated with glutaraldehyde without MDA-MB-468 cell lysate.

DISCUSSION

Our studies demonstrate that the mtp53 oligomerization domain peptide (mtp53ODP) called Cy5p53Tet is capable of cellular uptake and tissue penetration and detects mtp53 in TNBC cancer cells and tumor tissues. This newly designed mtp53ODP facilitates the selective targeting of mtp53 in TNBC. We used a biochemical pull-down assay to validate that Cy5p53Tet interacts with mtp53. The in vivo behavior of Cy5p53Tet was evaluated in mouse models of TNBC, and the imaging agent accumulated in the target tissue. We propose that mtp53ODPs like Cy5p53Tet can be developed into NIRF fluorescence imaging agents to visualize TNBC tissue during surgery. Indeed, intraoperative NIRF imaging has been applied previously to improve the visualization of tumor tissue and thus facilitate more complete resections and reduce injuries to healthy tissue.25 Advances to detect TNBC by fluorescent intraoperative imaging of mtp53 positive tissue using pseudocolor would be a breakthrough.

Previously, we discovered that the activation of apoptosis by PARP inhibitors (PARPi) in TNBC is mitigated by the knockdown of mtp53.3 PARP inhibitors have been approved for the clinical treatment of breast cancers that have aberrations in DNA repair by homologous recombination.26 The ability of PARPi to sensitize tumor cells to DNA-damaging chemotherapies is caused by either PARP1 trapping on the chromatin or the catalytic inhibition of PARP1.26 The close association of mtp53 with chromatin and the different intracellular distributions of Cy5p53Tet in TNBC and normal mammary epithelial cells (see supplementary Figure S6) suggest that mtp53ODPs could be developed as a TNBC-specific chromatin targeting vector. Indeed, peptides have been used to carry a wide variety of cargoes to tumor tissue, including toxins, vaccines, hormones, and radionuclides.27 For example, a doxorubicin-conjugated Tat peptide induced cell death in human breast cancer cell lines.28 Furthermore, a peptide conjugated to doxorubicin effectively suppressed tumor proliferation with fewer side effects.29 In particular, one cell-penetrating peptide azurin-p28 (NSC745104) has completed phase I in clinical trials in cancer treatment.19,30 p28-mediated targeted delivery of apoptin inhibits angiogenesis and cancer cell growth with a selective cytotoxicity toward cancer cells, not normal cells.31,32 While normal cells have low levels of steady state of p53 cancer cells, they can have high levels of inactive p53 and Cy5p53Tet may also be able to target the chromatin of such cancer cells. High cancer cell proliferation rates, nutrient deficiency, and hypoxia lead to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum.33 High levels of misfolded and aggregated conformation of wtp53 oligomers are also associated with cancer.34

This is the first disclosure of our novel idea for targeting dysfunctional p53. The use of Cy5p53Tet as a theranostic still faces several obstacles. The whole-body images and the images of the resected tissues confirm the specific uptake of the peptide in the MDA-MB-468 xenografts―particularly at early time points―but also reveal significantly high levels of the signal in several healthy tissues, including the liver, large intestines, and small intestines (Figure S7). These data with three mice clearly illustrate that Cy5p53Tet is not yet an optimized in vivo imaging agent, despite its promising selectivity for mtp53-expressing tumor tissue. Consequently, we have already begun the development of a series of “second-generation” mtp53-targeting imaging agents with improved pharmacokinetic profiles. One of the highest priorities of this effort will be shifting the clearance of the agents from the hepatobiliary system to the renal system by modifying their charge and size. Additionally, achieving selectivity for mtp53 over wtp53 and other members of the p53 family may be required. There remains the possibility that aberrant alternatively spliced p53 and/or p53 family members or S100 proteins that are also highly expressed in cancers35 might also be targets of Cy5p53Tet. S100 proteins are small calcium-binding proteins that regulate multiple cellular processes such as proliferation, migration and/or invasion, and differentiation.36 These proteins possess structural regions that are similar to that recognized by Cy5p53Tet. The interaction of S100 proteins with the TDs of p63 and p73 was discovered after the ability of S100 proteins to form complexes with p53 had been firmly established both in vitro and in vivo.37,38 S100 proteins bind only to monomers or dimers of p53 and thus regulate p53 protein oligomerization by displacing the equilibrium of its tetrameric form.39 Thus, while Cy5p53Tet can detect tumor tissue, it will be important to determine whether the peptide has molecular targets beyond mtp53. A more detailed structural and cellular analysis would prove valuable in this regard. Furthermore, the structure-guided optimization of Cy5p53Tet will also provide an opportunity to increase the affinity and selectivity of the peptide for mtp53. The goal is to optimize the sensitivity of next-generation Cy5p53Tet and mtp53ODPs so that we can detect single metastatic circulating tumor cells.

The rapid clearance of Cy5p53Tet from tumors indicated that the pharmacokinetic profile of the mtp53ODPs must be improved. A variety of methods have been used to improve the in vivo performance of peptides. For example, the conjugation of peptides to small molecules capable of binding transthyretin can protect peptides against serum proteases and thus lengthen their biological residence times.40 PEGylation can likewise extend the serum half-life of peptides. In addition, the creation of a “stapled” mtp53ODP similar to Cy5p53Tet could lock the peptide in a bioactive conformation through the site-specific introduction of a hydrocarbon brace, a chemical “staple,” and thus increase its binding affinity and biological half-life.41

CONCLUSIONS

In the end, the results presented here underscore the potential of mtp53ODPs like Cy5p53Tet to work as molecular imaging agents that could be applied to the detection of mtp53 in TNBC as well as other cancers. Our research provides an important and promising proof of principle for the creation of mtp53-specific targeted treatment strategies using mtp53ODPs that could benefit TNBC and other cancers that express mtp53. Cy5p53Tet has clinical potential as an intraoperative imaging agent for fluorescence-guided surgery, and the mtp53ODP scaffold can be modified in the future to enable the delivery of a wide variety of payloads including radionuclides and toxins to mtp53-expressing TNBC tumors.

Supplementary Material

supporting materials

ACKNOWLEDGMENTS

We thank Bargonetti lab members for their helpful discussions, Dr. Viola Ellison for help with cytometric data analysis, Dr. Delphine Vivier for animal estrogen pellets implantation, and Dr. Brendon Cook for peptide intravenous injections. We also thank the Flow Cytometry Facility at Hunter College and Small-Animal Imaging Core Facility at Memorial Sloan Kettering Cancer Center.

Funding

This work was supported by the Breast Cancer Research Foundation (JB, BCRF-19–011), the TUFCCC/HC Regional Comprehensive Cancer Health Disparity Partnership (GX, U54 CA221704), and the NIH (JB: R01CA239603 and BZ: R01CA240963, U01CA221046, and R01CA204167).

Footnotes

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.0c00978.

Higher uptake of Cy5p53Tet in MDA-MB-468 TNBC cells than in ER-positive MCF7 cells, Cy5p53Tet signal reduction upon depletion of mtp53 R273H, nuclear penetration of Cy5p53Tet in TNBC, prediction of p53TD and Cy5p53Tet complex by the protein–peptide globe docking method CABS-dock, glutaraldehyde cross-link assay-validated Cy5p53Tet binding to tetrameric mtp53 R273H, different intracellular distributions of the Cy5p53Tet between TNBC and normal mammary epithelial cells, and the Cy5p53Tet signal in whole animal and healthy organs (PDF).

The authors declare the following competing financial interest(s): There is a patent pending for the Cy5p53Tet.

Contributor Information

Gu Xiao, Department of Biological Sciences Hunter College, City University of New York, New York, New York 10021, United States.

George K. Annor, Department of Biological Sciences Hunter College, City University of New York, New York, New York 10021, United States; The Graduate Center Biochemistry PhD Program of City University of New York, New York, New York 10016, United States

Kimberly Fung, Department of Chemistry Hunter College of the City University of New York, New York, New York 10021, United States; Ph.D. Program in Chemistry, The Graduate Center of the City University of New York, New York, New York 10016, United States.

Outi Keinänen, Department of Chemistry Hunter College of the City University of New York, New York, New York 10021, United States.

Brian M. Zeglis, Department of Chemistry Hunter College of the City University of New York, New York, New York 10021, United States; Ph.D. Program in Chemistry, The Graduate Center of the City University of New York, New York, New York 10016, United States; Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York 10021, United States.

Jill Bargonetti, Department of Biological Sciences Hunter College, City University of New York, New York, New York 10021, United States; The Graduate Center Biochemistry PhD Program of City University of New York, New York, New York 10016, United States; Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, New York 10021, United States.

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Supplementary Materials

supporting materials
NIHMS1687620-supplement-supporting_materials.pdf (16.9MB, pdf)

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