Purpose: Previous study identified E2F1 as a key mediator of non-muscle-invasive bladder cancer (NMIBC) progression. The aim of this study was to identify the E2F1-related genes associated with poor prognosis and aggressive characteristics of bladder cancer.Experimental Design: Microarray analysis was performed to find E2F1-related genes associated with tumor progression and aggressiveness in the gene expression data from 165 primary patients with bladder cancer. The biologic activity of E2F1-related genes in tumor progression and aggressiveness was confirmed with experimental assays using bladder cancer cells and tumor xenograft assay.Results: The expression of E2F1 was significantly associated with EZH2 and SUZ12. The overexpression of E2F1, EZH2, and SUZ12 enhanced cancer progression including cell colony formation, migration, and invasiveness. Knockdown of these genes reduced motility, blocked invasion, and decreased tumor size in vivo. E2F1 bound the proximal EZH2 and SUZ12 promoter to activate transcription, suggesting that E2F1 and its downstream effectors, EZH2 and SUZ12, could be important mediators for the cancer progression. In addition, we confirmed an association between these genes and aggressive characteristics. Interestingly, the treatment of anticancer drugs to the cells overexpressing E2F1, EZH2, and SUZ12 induced the expression of CD44, KLF4, OCT4, and ABCG2 known as cancer stem cell (CSC)-related genes.Conclusions: The link between E2F1, EZH2, and/or SUZ12 revealed that E2f1 directly regulates transcription of the EZH2 and SUZ12 genes. The signature of E2F1--EZH2--SUZ12 shows a predictive value for prognosis in bladder tumors and the E2F1-EZH2-SUZ12-driven transcriptional events may regulate the cancer aggressiveness and chemo-resistance, which may provide opportunity for development of new treatment modalities. Clin Cancer Res; 21(23); 5391-403. Ó2015 AACR.

The transcription factor E2F1 is active during G1 to S transition and is involved in the cell cycle and progression. A recent study reported that increased E2F1 is associated with DNA damage and tumor development in several tissues using transgenic models. Here, we show that E2F1 expression is regulated by tristetraprolin (TTP) in prostate cancer. Overexpression of TTP decreased the stability of E2F1 mRNA and the expression level of E2F1. In contrast, inhibition of TTP using siRNA increased the E2F1 expression. E2F1 mRNA contains three AREs within the 3'UTR, and TTP destabilized a luciferase mRNA that contained the E2F1 mRNA 3'UTR. Analyses of point mutants of the E2F1 mRNA 3'UTR demonstrated that ARE2 was mostly responsible for the TTP-mediated destabilization of E2F1 mRNA. RNA EMSA revealed that TTP binds directly to the E2F1 mRNA 3'UTR of ARE2. Moreover, treatment with siRNA against TTP increased the proliferation of PC3 human prostate cancer cells. Taken together, these results demonstrate that E2F1 mRNA is a physiological target of TTP and suggests that TTP controls proliferation as well as migration and invasion through the regulation of E2F1 mRNA stability.

Edited by Ivan Sadowski

Tristetraprolin (TTP) is an AU-rich element binding protein that destabilizes ARE-containing mRNAs. We previously reported that TTP suppresses several target genes such as those encoding proto-oncogenes and cytokines in various cancer cells. However, the signaling pathway to induce TTP expression is not well known. In this study, we investigate that Tymoquinone (TQ) which is an active ingredient derived from the medicinal plant Nigella satia induces the TTP mRNA and protein expression in AGS and MDA-MB231. TQ treatment destabilized a luciferase mRNA containing cIAP2, VEGF and MUC4 ARE. Transfected cells were treated with various kinds of MAPK inhibitors, treatment with Sorafenib (RAF inhibitor) and U0126 (MEK-1/2) reduced the TTP induction activity of TQ in a dose-dependent manner. We confirmed that co-treatment with sorafenib/U0126 and TQ also reduced TTP expression. Following TQ downregulated the expression of MUC4 in previous study, we next examined whether the changes of TTP expression affects the expression of MUC4. Overexpression of TTP decreased the stability of MUC4 mRNA and expression level of MUC4. On the contrary, inhibition of TTP using siRNA increased the MUC4 expression. MUC4 mRNA contains two AREs within the 3′UTR and TTP destabilized a luciferase mRNA containing MUC4 mRNA 3′UTR. Analyses of point mutants of MUC4 mRNA 3′UTR demonstrated that ARE2 were mostly responsible for the TTP mediated destabilization of MUC4 mRNA. Our data suggest that TQ enhances the mRNA and protein level of TTP via the modulation of the Raf-MEK MAPK signaling and suppressed cancer cell growth and promotion through destabilizing MUC4 mRNA as TTP novel target gene. Citation Format: Hyun Hee Lee, Se-Ra Lee, Sun-Hee Leem. Thymoquinone induces the MUC4 mRNA-destabilizing activity of Tristetraprolin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2335. doi:10.1158/1538-7445.AM2014-2335

Background. The aryl hydrocarbon receptor repressor (AHRR) inhibits the transcription activity of the aryl hydrocarbon receptor (AHR) by competing for dimerization with the AHR nuclear translocator (ARNT) and subsequently binding to XRE. Interestingly, the AHRR has two AU-rich elements (AREs) in its 3’UTR which relates to the post-transcriptional regulation by ARE-binding proteins including Tristetraprolin (TTP), BRF1, HuR and so on. In previous study, we determined that TTP plays a critical role in the decaying of VEGF, LATS2, cIAP2, COX2 and Pim-1 through binding these AREs in 3’ UTR. Methodology/principal Findings. We investigated the post-transcriptional regulation of the AHRR expression by TTP. Our results show that the expression level of AHRR is inversely correlated with TTP expression in human breast cancer cell lines using the real-time PCR and western blot. The overexpression of TTP decreased the expression level of AHRR and inhibited the proliferation of MDA-MB435 human breast cancer cells. On the contrary, the treatment with a small interfering RNA against TTP increased the expression of AHRR mRNA and protein. AHRR mRNA contains two AREs within the 3’UTR and TTP destabilized a luciferase mRNA containing AHRR ARE. Moreover, RNA electrophoretic mobility shift assay revealed that TTP binds directly to the ARE of AHRR mRNA. Conclusions/Significance. These studies demonstrated that the TTP expression is inversely correlated with AHRR expression in several human breast cancer cell lines using the real-time PCR and western blot. We suggest that TTP acts as a negative regulator of the AHRR gene expression and it may have an effect on tumor development through inducing AHR activation and the expression of tumor suppressors containing XRE in human breast cancer cells. Citation Format: Hyun Hee Lee, Won Tae Kim, Dong Hee Kim, Sun-Hee Leem. Tristetraprolin downregulates AHRR expression through mRNA destabilization. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1791. doi:10.1158/1538-7445.AM2013-1791