The S-Nitrosylation-Mediated Modification Of P53 Leads To Altered Dna Binding And Associates With Inactivity Of P53 In Human Melanoma Cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Melanoma represents one of the cancers most resistant to radiation, DNA-damaging chemotherapy, and biotherapy. Notably, melanoma expresses high levels of wild-type tumor suppressor p53, while mutational inactivation of p53 is very rare. The mechanism for inactivating wild-type p53 function in melanoma remains unknown. It is known that melanoma secrets high levels of proinflammatory cytokines and produces reactive oxygen (ROS) and nitrogen species (RNS). Chronic inflammation is strongly associated with the progression of melanoma. Our previous studies showed that constitutive expression of inducible nitric oxide synthase (iNOS) has been found in human melanoma, and was highly correlated with poor patient survival. Further studies showed that iNOS inhibitor L-NIL could significantly inhibit melanoma growth and extended the survival of tumor-bearing mice. Moreover, in melanoma A375 cells, which express wild-type p53, the depletion of nitric oxide (NO) by scavenger c-PTIO enhanced cisplatin-induced apoptosis in a p53-dependent manner. Thus, we hypothesize that the aberrant high levels of NO in melanoma cells inhibit p53 transcriptional activity through posttranslational modification of important cysteines of p53 by S-nitrosylating the thiol group. Our biotin switch assay data confirmed that p53 was highly S-nitrosylated in A375 cells. Furthermore, the NOS inhibitor, L-NAME, significantly reduced S-nitrosylated p53 levels in A375 cells as compared with controls. ChIP assay studies showed that manipulation of NO levels by NOS inhibitor or NO donor substantially affected p53 binding to the promoters of downstream genes, such as p21, Pig3, and Mdm2, in A375 cells. In the initial Mass spectrometry studies on protein samples derived from biotin switch assays, we identified that Cys277 of p53, which directly contacts DNA in the DNA-binding domain, was highly S-nitrosylated in A375 cells. Molecular modeling to simulate p53-DNA complex was performed based on p53 crystal structure. The structural analysis showed that the S-nitrosylation of Cys277 reduced three hydrogen bonds between p53 and the conserved DNA site of p21Waf/Cip1 as compared with non-S-nitrosylated p53. However, a new hydrogen bond was also observed between Lys120 of S-nitrosylated p53 with DNA. It indicates that the S-nitrosylation of Cys277 can reduce the affinity and specificity of p53 binding to p21Waf/Cip1 promoter resulting in inhibiting transcription. Therefore, our studies represent a novel mechanism for S-nitrosylation-mediated regulation of wild-type p53 activity in melanoma cells as well as other cancer cells with aberrant high levels of RNS. Supported by MDACC SPORE in Melanoma P50CA093459. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4988. doi:1538-7445.AM2012-4988