Abstract 4438: NaBt can regulate the expression of COX-2 post-transcriptionally in the presence of chemically induced stress in colon epithelial cells
Authors: Banerjee Sreeparna , Enayat Shabnam , Tuncer Sinem , Ulgen Doğukan H. , Torun Aydan 
Published: August 2018

Abstract
Sodium butyrate (NaBt) is a histone deacetylase inhibitor (HDACi) produced in the colon by commensal microbiota-mediated fermentation of dietary fibers. It is regarded to have tumor suppressive and anti-inflammatory effects. NaBt can regulate gene expression through chromatin remodeling and altered transcription. However, it has not been addressed adequately whether inflammatory genes may also be regulated by NaBt in a post transcriptional manner via AU rich elements (ARE) in their 3'UTR. Overexpression of Cyclooxygenase-2 (COX-2) is observed in chronic inflammatory diseases and colorectal cancer (CRC). The aim of this study was to explore whether NaBt regulates COX-2 expression via post-transcriptional mechanisms. Pre-confluent Caco-2 and HT-29 cells were treated with different concentrations (1-5mM) of NaBt for short (3-6h) and long (24-48h) time points. Gene expression was determined by qPCR or Western blot. mRNA stability was determined with an Actinomycin D (ActD) chase assay. 3'UTR activity was determined in an RPSM30 vector containing the ARE rich and corresponding mutated regions of the 3'UTR from COX-2. NaBt strongly reduced COX-2 mRNA and protein expression with both short and long-term treatments. This reduction was not through NF-κB, since short-term treatment of cells with NaBt could not alter NF-κB activity in a luciferase reporter assay. An Act D chase assay indicated that in the absence of new mRNA synthesis, NaBt treatment induced stability of COX-2 mRNA in Caco-2 cells while it enhanced COX-2 mRNA decay in HT-29 cells. However, we did not observe any change in the 3'UTR activity of COX-2 in the presence of NaBt alone, indicating that the presence of a stress factor (such as ActD) was necessary for mRNA stabilization. Treatment of Caco-2 cells with NaBt and ActD together resulted in enhanced activation of the stress-related protein kinase MAPKAPK2 (MK2) compared to ActD alone, which can induce mRNA stabilization through nucleo-cytoplasmic shuttling of the ARE binding protein HuR. This was also reflected in increased COX-2 mRNA and protein levels. In HT-29 cells, simultaneous (NaBt+ActD) treatment resulted in a decrease in p-MK2, but an increase in p-Chk2 compared to ActD alone. p-Chk2 can preserve Cdk1 in a hyperphosphorylated state where it may promote retention of HuR in the nucleus, facilitating COX2 mRNA degradation and lower protein levels. No change in p-Chk2 was observed in Caco-2 cells in a similar experimental set up. These findings suggest that in the presence of a stress-inducing agent like ActD, NaBt can alter mRNA stability of COX-2 in a contextual manner, dependent on upstream signaling. Thus, while the rate of COX-2 mRNA degradation in HT-29 was enhanced, it slows down the same in Caco-2 cells. Future studies will indicate how NaBt can affect the dichotomy in Chk2/MK2 activation in the presence of stress.
Citation Format: Sreeparna Banerjee, Shabnam Enayat, Sinem Tuncer, Doğukan H. Ulgen, Aydan Torun. NaBt can regulate the expression of COX-2 post-transcriptionally in the presence of chemically induced stress in colon epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4438.