Mutations in the p53 gene occur in a variety of human cancers with remarkably high frequencies (as high as 80%), depending on the type and stage of the tumors (www-p53.iarc.fr). The majority of mutations are missense that are localized to six “hotspot” residues, which play a role either in the structural integrity (structural mutants, e.g., R175H) or in the DNA binding domain (contact mutants, e.g., R273H). Previously, we showed that phenethyl isothiocyanate (PEITC), a naturally occurring dietary constituent present abundantly in watercress, selectively depletes p53 mutant protein, but not the WT p53, and induces apoptosis in tumor cells expressing p53 mutants. In this study we demonstrated that PEITC can reactivate p53R175 mutant protein and inhibit tumor growth in a mutant p53R175H -dependent manner.
PEITC displayed a 175 allele preference of inhibition of cell proliferation as compared to other hotspot mutants as detected by WST-1 assay and induced apoptosis in a p53R175 mutant -dependent manner. Immunofluorescence and co-immunoprecipitation assay on PEITC treated p53R175 cells using conformation-specific antibodies (PAB1620 and PAB240) showed restoration of the “WT-like” conformation to p53 mutant. Reactivation of the WT like functions to the p53R175 mutant was established by its ability to bind DNA, transactivate p53 regulated downstream target genes (p21, PUMA, MDM2, NOXA, BAX and BCL2), and overexpression of p21 protein. PEITC treatment induced DNA damage response as detected by pATM-S1981 and pCHK2-Thr68, G2/M and S-phase cell-cycle arrest and oxidative stress as measured by decrease in the GSH levels in the cells. In contrast, co-treatment of cells with PEITC and reducing agents alleviated the inhibitory effects of PEITC on cell proliferation. Elevated oxidative stress due to the combined effect of PEITC and elevated ROS levels in p53 mutant cells might be responsible for the activation of restored “WT-like” p53R175 mutant protein and induction of apoptosis. PEITC induced proteasomal degradation and autophagy of the p53R175 mutant. Animals fed with PEITC diet showed a statistically significant reduction in xenograft tumor volumes, proliferation marker Ki67, and mutant p53 stained cells. Elevated mRNA levels of p53 regulated genes from animals fed PEITC diet provide in vivo evidence for the p53R175H mutant reactivation and inhibition of xenograft growth in a mutant p53-dependent manner. These are important findings because they demonstrated that a dietary compound can inhibit the growth of tumor cells in vivo by restoring the tumor suppressor functions to mutant p53, and thus provide a target for the development of novel chemopreventive strategies through dietary interventions.
Citation Format: Monika Aggarwal, Elizabeth Sinclair, Anna Jacobs, Ying Fu, Marcin Dyba, Xiantao Wang, Idalia M. Cruz, Deborah Berry, Kallakury Bhaskar, Susette C. Mueller, Maria Laura Avantaggiati, Fung-Lung Chung. Reactivation of p53R175 mutant by dietary phenethyl isothiocyanate (PEITC) impairs tumor growth in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1910. doi:10.1158/1538-7445.AM2015-1910