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, 293 (12), 4262-4276

The Curcumin Analog HO-3867 Selectively Kills Cancer Cells by Converting Mutant p53 Protein to Transcriptionally Active Wildtype p53

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The Curcumin Analog HO-3867 Selectively Kills Cancer Cells by Converting Mutant p53 Protein to Transcriptionally Active Wildtype p53

Esha Madan et al. J Biol Chem.

Abstract

p53 is an important tumor-suppressor protein that is mutated in more than 50% of cancers. Strategies for restoring normal p53 function are complicated by the oncogenic properties of mutant p53 and have not met with clinical success. To counteract mutant p53 activity, a variety of drugs with the potential to reconvert mutant p53 to an active wildtype form have been developed. However, these drugs are associated with various negative effects such as cellular toxicity, nonspecific binding to other proteins, and inability to induce a wildtype p53 response in cancer tissue. Here, we report on the effects of a curcumin analog, HO-3867, on p53 activity in cancer cells from different origins. We found that HO-3867 covalently binds to mutant p53, initiates a wildtype p53-like anticancer genetic response, is exclusively cytotoxic toward cancer cells, and exhibits high anticancer efficacy in tumor models. In conclusion, HO-3867 is a p53 mutant-reactivating drug with high clinical anticancer potential.

Keywords: apoptosis; breast cancer; cancer; cancer therapy; drug discovery; p53; transcription regulation.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
HO-3867 exhibits differential cytotoxicity to cancer cells with p53MT compared with healthy (noncancerous) cells. a, clinically relevant models were used to analyze the safety of HO-3867 toward normal body cells while simultaneously observing its anticancer efficiency in human cancer-derived cell populations. The graphical representation shows the isolation of heterogeneous cell populations from breast, colon, and liver cancer samples. In addition, noncancerous healthy cells such as fibroblasts from stromal tissue adjacent to breast cancer and radio- and chemosensitive cells from lymphoid and GI tract tissue were used. All of these cells were treated with HO-3867, cisplatin, or vehicle alone. b, a cancer-specific pro-apoptotic effect of HO-3867 was observed in all cells depicted in panel a. Cells were treated with HO-3867 (10 μm), cisplatin (10 μm), or vehicle (DMSO) alone, and apoptosis was measured using annexin V flow cytometry. HO-3867 selectively induced apoptosis in tumor-derived cells and minimal apoptosis in primary culture from normal tissue of different origins as well as tumor-adjacent stroma-derived fibroblasts and radiosensitive lymphoid and GI tract tissue. Cisplatin nonspecifically killed a significantly higher percentage of cells derived from normal tissues (n = 3 for all experiments; p values are as indicated, ANOVA was used for p value calculations, and error bars indicate standard deviation). c, p53 mutational analysis of breast, colon, and liver cancer samples used for the cell cultures shown in b confirms the presence of DNA-binding domain mutations. The exact nucleotide sequence point mutations and resulting amino acid sequence changes are depicted. d, the ability of HO-3867 to induce apoptosis in cancer cells with a p53MT genotype was determined using annexin V flow cytometry. p53MT cells (A431, MDA-MB-468, WRO, and DU-145) and p53−/− cells (MCF-7p53−/− and HCT7p53−/−) were used in the analysis. Cellular apoptosis was not observed in untreated p53MT and p53−/− cells (bars 1–6). shRNA-mediated p53 knockdown and the exogenous addition of p53MT cDNA were used as controls in untreated cells (bars 7–18). In the experimental set, all cell lines were treated with HO-3867, and p53MT cells showed a significant increase in cellular apoptosis (bars 19–22). HO-3867–treated p53null cells did not show a marked increase in apoptosis (bars 23–24). shRNA-mediated p53MT knockdown abolished the HO-3867–induced increase in apoptosis (bars 25–30). The exogenous addition of p53MT cDNA alongside HO-3867 treatment significantly increased apoptosis in both p53MT and p53−/− cells (bars 31–36) (n = 3; mean ± S.D. shown). p values are shown on the figure; standard ANOVA test). Inset, the efficiency of lentiviral particles coding for p53MT cDNA or p53 shRNA was demonstrated using immunoblotting of MCF-7 p53−/− or MCF-7 cells. Untreated MCF-7 p53−/− samples showed no expression of p53 (lane 1). Overexpression of increasing amounts of p53MT cDNA led to increased p53 protein levels. p53 shRNA treatment showed effective knockdown of p53 expression (a representative image from n = 3 replicates is shown).
Figure 2.
Figure 2.
HO-3867 covalently binds to p53MT in the DNA-binding domain. a, 1H-15N HSQC NMR spectrum of the p53MT-Y220C core domain (75 μm) after incubation with (green) or without (red) 1000 μm HO-3867 for 70 min. Chemical shift perturbations were observed for residues in proximity to the solvent-exposed cysteine 277. b, an NMR-generated model depicting the putative sites of interaction between p53MT and HO-3867. c, 1H-15N-HSQC NMR spectrum of the p53MT-Y220C core domain (75 μm) after incubating with (green) or without (red) 430 μm HO-3867 for 20 or 150 min. Chemical shift perturbations were time-dependent, suggesting a covalent binding mode. d, ESI (ES+) mass spectra of the p53MT-Y220C core domain (50 μm) after incubation without (left) or with HO-3867 (right) for 4 h at room temperature. HO-3867 treatment led to a mass increase of 713 or 1426 Da, confirming covalent binding to the p53 core domain by HO-3867. e, ESI (ES+) mass spectra of p53 DBD (50 μm) C182S/C277S, C124S/C277S, and C124S/C182S mutants after incubation without (left) or with HO-3867 (right) for 4 h at room temperature. HO-3867 treatment led to a mass increase of 713 Da only for the C124S/C277S and C124S/C182S mutants, confirming selective covalent modification of Cys-277 and Cys-182 by HO-3867.
Figure 3.
Figure 3.
HO-3867 shows anticancer efficacy in both p53MT and p53WT tumor xenografts by inducing p53MT–RE interaction and induces expression of p53 downstream effectors. a, the anticancer effect of HO-3867 on genetically tractable tumor xenografts of p53WT (MCF-7), p53MT (A-431), and p53−/− (MCF7 p53−/−) cells was observed (n = 3). In row 1, the excised tumors for untreated p53WT, p53MT, and p53−/− xenografts after 4 weeks are shown. In row 2, all of the tumors were treated with HO-3867 along with lentivirus-assisted overexpression of p53WT. A reduction in the tumor volumes of all tumor types was observed in row 2 when compared with the control (row 1). In row 3, tumors were treated with vehicle (DMSO) and lentiviral transfections. The tumor volumes in the vehicle-treated group remained unaltered. In row 4, all tumors were treated with lentivirus coding for p53 shRNA. In row 5, all tumors were treated with HO-3867, and p53WT tumors and p53MT tumors showed a decrease in tumor volume for all biological replicates. Interestingly, in p53 knockdown tumors, HO-3867 did not exhibit very high anticancer efficacy. These data suggest a role for p53 in HO-3867-mediated anticancer activity that appears to be independent of p53 mutational status. In row 6, p53WT and p53MT tumors were treated with HO-3867 along with lentiviral particles coding for p53 shRNA. p53 knockdown in these tumors reversed the anticancer effect of HO-3867, and all biological replicates in both experimental groups showed larger tumor volumes. In rows 6 and 7, p53 null tumor xenografts were treated with HO-3867 and lentiviral particles coding for p53MT cDNA (p53R175H (row 6); p53R273H (row 7)). Interestingly, HO-3867 reduced tumor growth in the presence of p53MT cDNA (compare tumor volumes in row 5 with rows 6 and 7) (n = 3) (HO-3867 treatment started at week 0 in the plot). b, tumor growth curves showing the volume of MCF-7 p53WT, A-431 p53MT, and MCF-7 p53−/− tumors in the eight treatment groups over the course of 4 weeks. In both MCF-7 p53WT and A-431 p53MT tumors, treatment with HO-3867 and HO-3867+ p53WT cDNA led to the greatest reduction in tumor volume. Treatment of MCF-7 p53−/− tumors with HO-3867, HO-3867+p53R175H cDNA, and HO-3867+p53R273H cDNA led to a significant reduction in tumor volume compared with control. In the insets, the efficiency of lentiviral particles coding for p53 shRNA, p53WT cDNA, or p53MT cDNA was demonstrated in MCF-7 p53WT or A-431 p53MT cells using immunoblotting with the indicated antibodies. MCF-7 p53WT and A-431 p53MT samples treated with p53 shRNA showed no expression of p53 (lane 2). p53 shRNA showed effective knockdown of p53 expression. MCF-7 p53−/− cells were treated with p53WT, p53R175H, and p53R273H cDNA and blotted with anti-p53 antibody or anti-GAPDH antibody (loading control). Overexpression of p53WT cDNA or p53MT cDNA led to increased p53 protein expression in MCF-7 p53−/− cells (HO-3867 treatment started at week 0 in the plot. n = 3 for all experiments; p values are labeled on the figure, and two-factor ANOVA with repeated measures was performed for p value calculations).
Figure 4.
Figure 4.
HO-3867 converts mutant p53 conformation to its wildtype form. a, model depicting sites of mutagenesis in the p53 gene in a panel of 29 cell lines. All mutations are present in the p53 DNA-binding domain. b, a Fluidigm digital qPCR-based gene expression analysis of a panel of 14 genes (Fig. S3) was conducted in a panel of 29 control and HO-3867–treated cell lines. Consistent with qChIP analysis, p53-regulated genes were overexpressed in all HO-3867–treated p53MT cell lines; this effect was reversed upon p53 shRNA treatment. Cisplatin (10 μm) was used as a positive control for p53 activation. (n = 5 for all experiments; p values are labeled on the figure, and ANOVA was performed for p value calculations). c, ChIP analysis was conducted in a genetically tractable system of p53MT (A-431) and p53−/− (MCF-7p53−/−) cell lines to measure the binding of p53MT to its REs at the bax (left) and p21 (right) promoters. The results were analyzed using the QIAxcel advanced instrument platform (Qiagen). Input (lane 1), no antibody (lane 2), actin antibody (lane 3), and p53 shRNA (lanes 5 and 11) were used as controls. The data show the presence of p53 on the bax and p21 promoters in HO-3867–treated p53WT and p53MT cell lines but not p53−/− cell lines (lane 8). Exogenous addition of either p53WT (lane 12) or p53MT (lane 13) cDNA resulted in significant binding of p53 at its respective REs in HO-3867–treated p53MT and p53−/− cell lines. d, up-regulation of two important p53 target genes, p21 and Noxa, was confirmed at the protein level by Western blotting. A genetically tractable system of p53WT (MCF-7), p53MT (A-431), and p53−/− (MCF-7 p53−/−) cells was used to study the effect of HO-3867 treatment (10 μm) in p53MT cells (lanes 1–6). Lane 7, both p21 and Noxa Western blotting show less expression in MCF-7 p53−/− cells transfected with p53MT cDNA. However, the same combination in the presence of HO-3867 significantly increases p21 and Noxa expression (lane 8).
Figure 5.
Figure 5.
HO-3867 converts mutant p53 conformation to its wildtype form. a, the p53MT and p53WT forms were immunoprecipitated using Ab 240 or Ab 1620, respectively, and immunoblotted using a polyclonal anti-p53 antibody (FL393) in p53MT (A-431), p53WT (MCF-7), and MCF-7p53−/− tumors. Input (lane 1), actin antibody (lane 2), and p53 shRNA (lanes 5 and 6) were used as controls for all tumors. In untreated MCF-7 tumors, p53 was recognized by Ab 1620 (lane 3) and to a minor extent by Ab 240 (lane 4). In untreated A-431 tumors, p53 was exclusively recognized by Ab 240 (lane 4). No signal was detected in MCF-7p53−/− tumors (third row). Overexpression of p53WT and p53MT cDNA in all three tumors resulted in a strong signal for Ab 1620 (lane 7) and Ab 240 (lane 10), respectively. HO-3867 treatment in MCF-7 tumors significantly increased detection by Ab 1620 (compare lanes 3 with 11). HO-3867 treatment in A-431 tumors resulted in a change in the p53 conformation from an Ab 1620–recognized form to an Ab 1620–recognized form (compare lanes 4 and 11). HO-3867 had no effect on MCF-7p53−/− tumors. Exogenous addition of p53WT cDNA in HO-3867–treated A-431, MCF-7, and MCF-7p53−/− tumors showed the strong presence of p53 in the Ab 1620–recognized form (lanes 15 and 16). Exogenous addition of p53MT cDNA in HO-3867–treated A-431, MCF-7, and MCF-7p53−/− tumors again showed the strong presence of p53 in the Ab 1620–recognized form (lanes 17 and 18) (n = 3). b, wildtype and mutant forms of p53 were immunoprecipitated using Ab 1620 and Ab 240, respectively, and immunoblotted for p53 protein (FL393) in p53WT (MCF-7 and HCT) or p53MT (A-431, DU-145, and MDA-MB-231) cell lines. Input (lane 1) and actin antibody (lane 2) were used as controls. In untreated p53WT cells, p53 was recognized by Ab 1620 (lane 3, rows 1 and 3). p53−/− (MCF-7p53−/− and HCTp53−/−) cells served as negative controls and showed no p53 signal (rows 2 and 4). In untreated p53MT cells, p53 existed exclusively in an Ab 240–recognized form (lane 4, rows 5–7), which upon HO-3867 treatment converted to an Ab 1620–recognized form (compare conversion from 240 to 1620 form, lanes 4 and 5) (n = 3). c, graphical representation of the experimental design for conducting in vitro transcription assays (top). The synthetic DNA template consisted of a poly(6)-p53 DNA-binding site followed by an adenovirus major late core promoter, a transcription start site, a G-less cassette as the coding region, and a poly(A) tail coding region (for qPCR-based detection) followed by a CCT stop signal. Nuclear extracts from p53null (H1299) cells were the source of the RNA polymerase machinery. Lack of reverse transcriptase to convert synthetic transcripts to a qPCR-detectable form in the reaction mix served as a negative control (No RT, bars 1 and 7). p53 immunoprecipitated from untreated MCF-7 cells in combination with H1299 nuclear extracts showed basal transcript synthesis (second bar). p53 from p53MT cell lines in combination with H1299 nuclear extract resulted in minimal transcript synthesis (bars 3–6). p53 immunoprecipitated from HO-3867–treated p53WT and p53MT cell lines in combination with H1299 nuclear extracts successfully generated RNA transcripts from the synthetic DNA template (blue) (n = 3 for all experiments; p values are labeled on the figure, and ANOVA was performed for p value calculations). d, luciferase-based reporter transcription assay (Cignal) was used to analyze p53-dependent transcription in HO-3867–treated p53MT cell lines in vivo. Empty vector (bars 1 and 7) was used as a negative control. Standard p53-dependent transcription was observed in p53WT MCF-7 cells. Results showed minimal p53-dependent transcription in a variety of p53MT cell lines. The effect of HO-3867 on p53-induced transcription was observed in treated p53WT and p53MT cells (n = 3 for all experiments; p values are labeled on the figure, and ANOVA was performed for p value calculations).

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