Tumor antigen PRAME is a potential therapeutic target of p53 activation in melanoma cells

Abstract

Upregulation of PRAME (preferentially expressed antigen of melanoma) has been implicated in the progression of a variety of cancers, including melanoma. The tumor suppressor p53 is a transcriptional regulator that mediates cell cycle arrest and apoptosis in response to stress signals. Here, we report that PRAME is a novel repressive target of p53. This was supported by analysis of melanoma cell lines carrying wild-type p53 and human melanoma databases. mRNA expression of PRAME was downregulated by p53 overexpression and activation using DNA-damaging agents, but upregulated by p53 depletion. We identified a p53-responsive element (p53RE) in the promoter region of PRAME. Luciferase and ChIP assays showed that p53 represses the transcriptional activity of the PRAME promoter and is recruited to the p53RE together with HDAC1 upon etoposide treatment. The functional significance of p53 activationmediated PRAME downregulation was demonstrated by measuring colony formation and p27 expression in melanoma cells. These data suggest that p53 activation, which leads to PRAME downregulation, could be a therapeutic strategy in melanoma cells. [BMB Reports 2024; 57(6): 299-304].

Fig. 1

Negative correlation between PRAME and p53. (A) Negative correlation between p53 and PRAME expression in melanoma cell lines. Protein and mRNA levels were measured by Western blot and RT-PCR, respectively (n = 1). (B) Correlation coefficient between p53 and PRAME. The band densities of p53 and PRAME in Fig. 1A were quantified utilizing ImageJ software. (C, D) Comparison of p53 and PRAME expression. Using the GSE4570 data set, mRNA levels of p53 and PRAME were assessed in normal melanocytes (n = 2) and melanoma (n = 6). (E) Correlation of p53 and PRAME mRNA levels assessed by calculating the Pearson correlation coefficient using the GSE4570 data set. All Pearson correlation tests and linear regression analyses were performed using MedCalc software.

Fig. 2

Downregulation of PRAME by p53 activation. PRAME mRNA expression was assessed via RT-qPCR, and the data were displayed as means ± SD (n = 3, *P < 0.05; **P < 0.01; ***P < 0.005). Proteins were visualized by WB using indicated antibodies (n = 1). (A) Negative effect of p53 on PRAME expression. PRAME protein was visualized by WB in HCT116p53^−/− and p53-null H1299 cells transfected with Flag-p53 expression vector (2 and 5 μg) (n = 3). (B) Effect of p53 knockdown on PRAME expression. HCT116 cells carrying wild-type p53 were transfected with a luciferase control or p53 shRNA. Twenty-four hours after transfection, the protein levels of PRAME and p27 were measured by WB (n = 1). PRAME mRNA expression was measured by RT-qPCR (n = 3, **P < 0.01). (C) Effect of ETO on PRAME expression. A375P cells were treated with 20 μM etoposide (ETO) for indicated times. Proteins were visualized by WB. PRAME band intensity was measured by ImageJ software and normalized to β-actin. The PRAME mRNA level was quantified by RT-qPCR and normalized to GAPDH (n = 3, *P < 0.05). (D) Effects of UV-C and MMS on PRAME expression. A375P cells were treated with 50 J/m² UV-C or 2 mM methyl methane sulfonate (MMS). Proteins were visualized by WB using indicated antibodies (n = 3). PRAME mRNA levels were quantified using RT-qPCR and normalized to GAPDH (n = 3, *P < 0.05; ***P < 0.005).

Fig. 3

p53 binds to the PRAME promoter and recruits HDAC1. (A, B) Mapping of functional p53RE. Five deletion constructs carrying the luciferase gene (A) were introduced into HCT116p53^−/− cells with the Flag-p53 vector for luciferase assays (B). Luciferase data are means ± SD (n = 3). *P < 0.05; **P < 0.01. (C) Nucleotide sequences of p53RE1 and RE2. Two mutants are displayed: mutRE, base substitution; ΔRE1, RE1 deletion. (D) Effect of RE1 mutation on p53-dependent transcriptional repression of the PRAME promoter. HCT116p53^−/− cells were transfected with mutRE (left), ΔRE1 (right) with the Flag-p53 vector. Cell lysates were subjected to luciferase assays. Luciferase data are means ± SD (n = 3). **P < 0.01. (E) p53 binding to the RE1 of the PRAME promoter. H1299 cells were transfected with Flag or Flag-p53 for ChIP using an anti-Flag antibody. qPCR was performed using primer sets covering p53RE1 and RE2 of PRAME. The p53RE in p21 was used as the positive control. Quantification of ChIP-qPCR data; means ± SD (n = 3). **P < 0.01; ***P < 0.005. (F) HDAC1 recruitment to the RE1 of the PRAME promoter. A375 cells were treated with ETO and subjected to ChIP assay using antibodies against p53, histone deacetylase 1 (HDAC1), and acetylated histone H3 at lysine 9 (H3K9ac). Quantification was performed by qPCR using a primer set covering the RE1 of PRAME. Data are means ± SD (n = 3). *P < 0.05; ***P < 0.005.

Fig. 4

PRAME knockdown and ETO treatment suppress melanoma cell proliferation and p27 induction. (A) Effects of PRAME depletion and ETO treatment. A375SM or A375P cells were transfected with siPRAME (left) or treated with 20 μM ETO (right) and subjected to colony formation assay (n = 3). Control siRNA (siCtrl) is an unspecific scrambled siRNA purchased from Bioneer (Korea). Colonies were fixed and stained with crystal violet. (B) The number of colonies was counted from three independent experiments and the relative numbers are shown. Data are mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.005. (C) Upregulation of p27 by PRAME depletion and ETO treatment. Cell lysates prepared as described above were subjected to WB using the indicated antibodies (n = 1). β-Actin was used as the internal control. (D) Hypothetical model for the p53-mediated PRAME downregulation through HDAC1 recruitment.