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. 2018 Dec;11(6):1343-1349.
doi: 10.1016/j.tranon.2018.08.009. Epub 2018 Sep 6.

The Mutant p53-Targeting Compound APR-246 Induces ROS-Modulating Genes in Breast Cancer Cells

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Free PMC article

The Mutant p53-Targeting Compound APR-246 Induces ROS-Modulating Genes in Breast Cancer Cells

Naoise C Synnott et al. Transl Oncol. .
Free PMC article

Abstract

TP53 is the most frequently mutated gene in human cancer and thus an attractive target for novel cancer therapy. Several compounds that can reactive mutant p53 protein have been identified. APR-246 is currently being tested in a phase II clinical trial in high-grade serous ovarian cancer. We have used RNA-seq analysis to study the effects of APR-246 on gene expression in human breast cancer cell lines. Although the effect of APR-246 on gene expression was largely cell line dependent, six genes were upregulated across all three cell lines studied, i.e., TRIM16, SLC7A11, TXNRD1, SRXN1, LOC344887, and SLC7A11-AS1. We did not detect upregulation of canonical p53 target genes such as CDKN1A (p21), 14-3-3σ, BBC3 (PUMA), and PMAIP1 (NOXA) by RNA-seq, but these genes were induced according to analysis by qPCR. Gene ontology analysis showed that APR-246 induced changes in pathways such as response to oxidative stress, gene expression, cell proliferation, response to nitrosative stress, and the glutathione biosynthesis process. Our results are consistent with the dual action of APR-246, i.e., reactivation of mutant p53 and modulation of redox activity. SLC7A11, TRIM16, TXNRD1, and SRXN1 are potential new pharmacodynamic biomarkers for assessing the response to APR-246 in both preclinical and clinical studies.

Figures

Figure 1
Figure 1
Fold-change in mRNA expression of CDKN1A (A), 14-3-3σ (B), BBC3 (C), or PMAIP1 (D) in three breast cancer cell lines. Cells were treated with 25 or 50 μM APR-246 or DMSO for 12 hours. Data were analyzed using paired t test. All experiments were carried out in triplicate. Values are means ± SEM, *P > .01, **P > .001, ***P > .0001.
Figure 2
Figure 2
Fold-change in mRNA expression of SLC7A11 (A), TRIM16 (B), SRXN1 (C), or TXNRD1 (D) in a panel of nine breast cell lines. Cells were treated with 50 μM APR-246 or DMSO for 12 hours. Data were analyzed using paired t test. All experiments were carried out in triplicate. Values are means ± SEM, *P > .01, **P > .001, ***P > .0001; p53-mut, p53 mutated; P53-WT; p53 wild-type.
Figure 3
Figure 3
Image representative of SKBR3 cells treated with APR-246 and stained with PAb1620 (A) to detect wild-type-p53 or PAb240 (B) to detect mutant p53. DAPI nuclear stain was used as a control. All experiments were carried out in triplicate. (C) Histogram representatives of SKBR3 cells treated with APR-246 and stained with PAb1620 to detect WT-p53. (D) Bar chart representation of Pab1620 staining measured by flow cytometry and analyzed using FlowJo v.10 software. Data were analyzed using paired t test. (E) Bar chart representation of absolute p53 protein levels quantified using the PathScan p53 ELISA kit.

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