Forkhead Box Protein P3 (FOXP3), a transcription factor of the FOX protein family, is essentially involved in the development of regulatory T (Treg) cells, and functions as a tumor suppressor. Although FOXP3 has been widely studied in immune system and cancer development, its function in the regulation of the UBC9 gene (for the sole E2 enzyme of SUMOylation) is unknown. Herein, we find that the overexpression of FOXP3 in human MCF7 breast cancer cells increases the level of UBC9 mRNA. Moreover, the level of UBC9 protein dose-dependently increases in the FOXP3-Tet-off MCF7 cells. Notably, the promoter activity of the UBC9 is activated by FOXP3 in a dose-dependent manner in both the MCF7 and HEK293 cells. Next, by mapping the UBC9 promoter as well as the site-directed mutagenesis and ChIP analysis, we show that the FOXP3 response element at the −310 bp region, but not the −2182 bp region, is mainly required for UBC9 activation by FOXP3. Finally, we demonstrate that the removal of phosphorylation (S418A and Y342F) and the removal of acetylation/ubiquitination (K263R and K263RK268R) of the FOXP3 result in attenuated transcriptional activity of UBC9. Taken together, FOXP3 acts as a novel transcriptional activator of the human UBC9 gene, suggesting that FOXP3 may have physiological functions as a novel player in global SUMOylation, as well as other post-translational modification systems.

Activating transcription factor 3 (ATF3), a transcription factor and acute stress sensor, is rapidly induced by a variety of pathophysiological signals and is essential in the complex processes in cellular stress response. FOXP3, a well-known breast and prostate tumor suppressor from the X chromosome, is a novel transcriptional repressor for several oncogenes. However, it remains unknown whether ATF3 is the target protein of FOXP3. Herein, we demonstrate that ATF3 expression is regulated by FOXP3. Firstly, we observed that overexpression of FOXP3 reduced ATF3 protein level. Moreover, knockdown FOXP3 by siRNA increased ATF3 expression. Secondly, FOXP3 dose-dependently reduced ATF3 promoter activity in the luciferase reporter assay. Since FOXP3 is regulated by post-translational modifications (PTMs), we next investigated whether PTMs affect FOXP3-mediated ATF3 expression. Interestingly, we observed that phosphorylation mutation on FOXP3 (Y342F) significantly abolished FOXP3-mediated ATF3 expression. However, other PTM mutations on FOXP3, including S418 phosphorylation, K263 acetylation and ubiquitination, and K268 acetylation and ubiquitination, did not alter FOXP3-mediated ATF3 expression. Finally, the FOXP3 binding site was found on ATF3 promoter region by deletion and mutagenesis analysis. Taken together, our results suggest that FOXP3 functions as a novel regulator of ATF3 and that this novel event may be involved in tumor development and progression.

Steroidogenic Factor 1 (SF-1/NR5A1), an orphan nuclear receptor, is important for sexual differentiation and the development of multiple endocrine organs, as well as cell proliferation in cancer cells. Activating transcription factor 3 (ATF3) is a transcriptional repressor, and its expression is rapidly induced by DNA damage and oncogenic stimuli. Since both NR5A1 and ATF3 can regulate and cooperate with several transcription factors, we hypothesized that NR5A1 may interact with ATF3 and plays a functional role in cancer development. First, we found that NR5A1 physically interacts with ATF3. We further demonstrated that ATF3 expression is up-regulated by NR5A1. Moreover, the promoter activity of the ATF3 is activated by NR5A1 in a dose-dependent manner in several cell lines. By mapping the ATF3 promoter as well as the site-directed mutagenesis analysis, we provide evidence that NR5A1 response elements (−695 bp and −665 bp) are required for ATF3 expression by NR5A1. It is well known that the transcriptional activities of NR5A1 are modulated by post-translational modifications, such as small ubiquitin-related modifier (SUMO) modification and phosphorylation. Notably, we found that both SUMOylation and phosphorylation of NR5A1 play roles, at least in part, for NR5A1-mediated ATF3 expression. Overall, our results provide the first evidence of a novel relationship between NR5A1 and ATF3.

Programmed Cell Death Protein 4 (PDCD4), a well-known tumor suppressor, acts to inhibit translation initiation and cap-dependent translation by inhibiting the helicase activity of EIF4A. EIF4A tends to target mRNAs with structured 5'-UTR. PDCD4 can also prevent tumorigenesis by inhibiting tumor promoter-induced neoplastic transformation, and studies indicate that PDCD4 may inhibit translation of certain mRNAs via directly binding. A previous study has demonstrated that PDCD4 inhibits translation of p53 mRNA and treatment with DNA-damaging agents down-regulates PDCD4 expression but activates p53 expression. The study further demonstrated that treatment with DNA-damaging agents resulted in down-regulation of PDCD4 expression and an increase in p53 expression, suggesting a potential mechanism in which p53 regulates expression of Pdcd4. However, whether p53 directly regulates PDCD4 remains unknown. Herein, we demonstrate for the first time that p53 regulates PDCD4 expression. First, we found that overexpression of p53 in p53-null cells (H1299 and Saos2 cells) decreased PDCD4 protein level. Secondly, p53 dose-dependently decreased PDCD4 promoter activity in gene reporter assays. Moreover, we demonstrated that mutations on p53 (R273H: contact mutant, hotspot and R175H: conformational mutant, hotspot) abolished p53-mediated PDCD4 repression. Furthermore, using Tet-on expression system, PDCD4 protein level was decreased when WT p53, but not hotspot mutants of p53, was expressed in H1299 cells. Finally, mutations on DNA-binding domain, but not C-terminal regulatory domain, of p53 disrupted p53-mediated PDCD4 repression. Taken together, our results suggest that p53 functions as a novel regulator of PDCD4 and the relationship between of p53 and PDCD4 may be involved in tumor development and progression. Citation Format: Wei-Hsiung Yang, Andrew P. George, William H. Yang, Chiung-Min Wang, Richard H. Yang. Tumor suppressor p53 down-regulates programmed cell death protein 4 (PDCD4) expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2160.