Abstract 4785: S-nitrosylation of p53 in melanoma alters p53-DNA binding and downstream gene expression

Abstract Aberrant expression of inducible nitric oxide synthase (iNOS) and chronic nitrosative stress is correlated with growth, metastasis, resistance to therapy, and poor patient survival of metastatic melanoma. One physiological influence of nitric oxide (NO) is exerted directly through the post-translational modification of many proteins, including the unique S-nitrosylation (SNO). To date, the role of SNO in melanoma development has not been fully characterized. Herein, we focus on the mechanism of how nitrosative stress regulates the tumor suppressor p53 in melanoma cells. We first examined the effects of different levels of NO donors, S-nitrosoglutathione (GSNO) and diethylenetriamine (DETA) NONOate, on the growth of melanoma cells by the MTT assay. Low levels of nitrosative stress (NO donors ≤ 20 µM) did not suppress the growth of human melanoma A375 and SB2 cells, but high levels of nitrosative stress (NO donors ≥ 50 µM) inhibited melanoma cell growth. Our biotin switch assays (BSA) showed that the treatment of NO donors significantly increased total S-nitrosylated proteins in A375 and SB2 cells. Markedly, p53 is confirmed to be one of the S-nitrosylated proteins derived from BSA. Our mass spectrometry analysis of SNO proteins in melanoma cells confirms that p53 is S-nitrosylated in A375 cells under nitrosative stress. Further proteomic characterization identifies Cys242, Cys275, and Cys277 as the SNO sites of p53. We conducted a molecular dynamics (MD) simulation of p53-DNA binding under SNO modification. The structural analysis shows that the SNO of Cys277 reduces the hydrogen bonds between p53 and the targeted DNA, interfering with the recognition and binding of p53 to DNA. Moreover, the chelating ability of Cys242 for Zn2+ is weakened after SNO, indicating the SNO of p53 reduces p53 binding to Zn2+ and affects its active form. The electrophoretic mobility shift assay also confirmed that the treatment of 100 µM of DETA NONOate, significantly decreased the nuclear p53 of A375 and SB2 cells binding to the consensus p53-binding DNA. The induction of nitrosative stress by NO donors also significantly increased mouse double minute 2 homolog (MDM2), p21, and p53 expression but decreased p53 upregulated modulator of apoptosis (PUMA) expression.This study confirms that the crucial tumor suppressor, p53, is S-nitrosylated in melanoma cells under nitrosative stress. Notably, for the first time, the SNO sites of p53 and the effects of SNO on p53 functions were characterized. Our results describe an important mechanism of how nitrosative stress modifies crucial cysteines of p53, alters p53 DNA-binding activities, and regulates downstream gene expression through SNO. Our study provides much-needed insights into identifying novel NO-driven SNO proteins in melanoma as new biomarkers and targets for drug development. Citation Format: Jordan Winfield, Mariana Grigoruta, Yiliang Li, Fancui Meng, Yong Qin, Elizabeth Grimm, Leyuan Chen, Kevin Rosenblatt, Li Li. S-nitrosylation of p53 in melanoma alters p53-DNA binding and downstream gene expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4785.