Abstract 2215: Transcriptional co-regulation of Nox4 by p53 and SMAD3

Recent studies suggest p53 plays an important role in TGF-beta-mediated cell signaling and migration. Previously, we showed that wild-type (WT) and mutant forms of p53 differentially regulate reactive oxygen species (ROS) generation by NADPH oxidase-4 (Nox4). We found that WT-p53 is a potent suppressor of TGF-beta-induced Nox4, ROS production, and cell migration, whereas tumor-associated mutant p53 proteins enhanced Nox4 mRNA and protein expression and cell migration by both TGF-beta-dependent and independent mechanisms (Boudreau et al. Br J Cancer. 2014 May 13;110(10):2569-82). In this study, we examined the basis of human Nox4 promoter regulation by p53 and SMAD3. By constructing a deletion series of Nox4 promoter-reporter constructs, we discovered two regions (-4.76kb to -3.9kb and -1.8kb to - 0.2kb) upstream of the transcription start site that induce promoter activity upon activation of the TGF-beta/SMAD3 pathway. Interestingly, TGF-beta/SMAD3-mediated Nox4 promoter activity was abolished by heterologous expression of p53-WT in p53-null cell lines Hep3B (hepatocytes) and H1299 (lung epithelial). This activity was repressed by wild-type p53 in a dose-dependent manner. In contrast, expression of common tumor-associated mutant-p53 proteins (R175H, R248H, R249Q, R273H) did not repress but enhanced Nox4 promoter activity. We observed this enhancement of Nox4 promoter activity by mutant p53 (R273H) in H1299 cells expressing T204D, a constitutively active TGF-beta receptor. When individual p53 response elements were mutated, however, both untreated and R273H-expressing cells showed 50% reduction in promoter activity. These results indicate that the Nox4 promoter region between -3.97kb and -4.76kb, containing candidate p53 response elements, plays a role in modulating Nox4 expression. Preliminary chromatin immunoprecipitation results suggest that endogenous mutant p53 R249S in PLC/PRF/5 hepatoma cells complexes with the Nox4 promoter region between -3.97kb and -4.76kb. Furthermore, constructs lacking this region show significantly reduced Nox4 promoter activity in response to TGF-beta. Our results provide a basis for Nox4 co-regulation by SMAD3 and mutant p53 that may be involved in tumor progression and metastasis. Citation Format: Howard E. Boudreau, Jonathan J. Park, Thomas L. Leto. Transcriptional co-regulation of Nox4 by p53 and SMAD3. [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 2215. doi:10.1158/1538-7445.AM2015-2215