Abstract 1219: ID4 and p53 cross-talk promotes restoration of mutant-p53 transcriptional activity
Authors: Morton Derrick Jerone , Patel Divya , Joshi Jugal , Sharma Pankaj , Knowell Ashley , Hunt Aisha , Chaudhary Jaideep 
Published: August 2015

The physiological mechanisms that can restore biological activity of mutant p53 is an area of high interest given that mutant p53 expression is observed in one third of prostate cancer and more than 50% of all cancers. Here we demonstrate that ID4 (inhibitor of differentiation-4) a dominant negative regulator of bHLH transcription factors can restore mutant p53 transcriptional activity in prostate cancer cells. ID4 is highly expressed in the normal prostate and decreased in prostate cancer due to promoter hypermethylation. Prostate cancer cell lines: DU145 harbors mutant p53 and also lacks ID4 expression; LNCaP cells express wild-type p53 and ID4; whereas PC3 cells are null for p53 and express low levels of ID4. p53 mutants (P223L and V274F) in DU145 cells are within the DNA binding domain and abrogate p53 transcriptional activity due to structural de-stabilization and/or DNA interactions. Ectopic expression of ID4 in DU145 cells resulted in increased apoptosis and expression of BAX, PUMA and p21, transcriptional targets of p53. DNA binding, p53 luciferase reporter studies and ChIP analysis demonstrated that mutant p53 gains ID4 dependent DNA binding and transcriptional activity in part due to CBP/p300 dependent acetylation of p53 at lysine 373. Loss of ID4 in LNCaP cells also abrogated wild-type p53 DNA binding and transcriptional activity with concomitant loss of CBP/p300 requirement and decreased acetylation of p53. To further elucidate ID4 dependent restoration of biological activity of p53 we stably transfected p53-null cell line PC3, which has endogenous ID4 with mutant p53 mimicking DU145 cells. mRNA, protein levels and apoptosis assays were used to determine the effects on cell death and to determine if mutant p53 had the ability to transactivate upstream/downstream targets (MDM2, PUMA, and p21). To further validate our gene expression profile of p53 responsive targets, we performed DNA binding assays as well as p53 luciferase reporter assays to demonstrate wild-type and mutant p53 transcriptional activity in PC3 cells. Also, to elucidate the mechanism by which ID4 promotes apoptosis, we performed acetylation studies in LNCaP and DU145 xenografts (+/-) ID4 as well as p53 co-immunoprecipation studies to demonstrate interaction of both lysine residues 320 and 373. Our results indicate that transfected mutant p53 in PC3 cells were able to induce expression of downstream targets as well as effect biological processes such as apoptosis and cell cycle arrest. Also, post-translational modifications on residues lys320 and lys373 indicated activation of mutant p53. Collectively, based on our studies we demonstrate that ID4 promotes post-translational modifications of p53, which in turn regulates p53 transcriptional activity. NIH grant #R01CA128914

Citation Format: Derrick Jerone Morton, Divya Patel, Jugal Joshi, Pankaj Sharma, Ashley Knowell, Aisha Hunt, Jaideep Chaudhary. ID4 and p53 cross-talk promotes restoration of mutant-p53 transcriptional activity. [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 1219. doi:10.1158/1538-7445.AM2015-1219