Abstract 332: The role of OLA1, a novel obg-like ATPase, in epithelial-mesenchymal transition

Abstract

Abstract Human OLA1 is a 45 kDa cytosolic protein that belongs to the YchF subfamily of Obg-like P-loop GTPases and is ubiquitously expressed in most tissues. YchF proteins are highly conserved from yeast to humans and all have a greater affinity for ATP than GTP. Based on structure analysis, OLA1 is predicted to be a regulatory protein that interacts with its client protein(s) and exerts its downstream functions by the conformational switch between the ADP- and ATP-bound forms. However, the actual physiological functions of OLA1 are poorly understood. Our earlier studies have suggested that OLA1 is a negative regulator of cellular antioxidant response and functions through post-transcriptional mechanisms (PNAS 2009; 106:15356). Oxidative stress, mediated by reactive oxygen species (ROS), has been implicated in epithelial-mesenchymal transition (EMT), a fundamental phenotypic alteration closely associated with cancer progression and metastasis. In this study we investigated the effect of OLA1 on EMT and the possible underlying mechanisms. We first demonstrated that the human lung epithelial carcinoma A549 cells, after exposure to transforming growth factor (TGF)-β1 for 3 days, underwent EMT as evidenced by acquisition of a mesenchymal-like morphology, down-regulation of epithelial-specific marker E-cadherin, and induction of mesenchymal marker vimentin. However, knockdown of OLA1 with sequence-specific siRNA in A549 cells resulted in a significant attenuation of the TGF-β1-induced EMT phenotypes, including those in cellular morphology and the EMT markers E-cadherin and vimentin, as compared with the cells transfected with the control non-targeting siRNA. Next, we questioned if this inhibitory effect on EMT was through inhibiting the Akt/GSK3β/Snail pathway. When treated with TGF-β1, OLA1-knockdown cells showed impaired GSK-3β phosphorylation (indicating an overactivation of GSK-3β), and subsequently reduced abundance of the transcription factor Snail that functions as the suppressor of E-cadherin gene expression. Concurrently, OLA1-knockdown cells exhibited significantly decreased TGF-β1-mediated intracellular ROS production assessed by DCFDA staining and flow cytometry analysis. Furthermore, we found that treatment of H2O2 abrogated resistance of OLA1-knockdown cells to TGF-β1-induced EMT, evidenced by phosphorylation of GSK-3β, upregulation of Snail and changes in expression of EMT markers. These results suggest that OLA1 plays an important regulatory role in EMT of human cancer cells, and inhibition of OLA1 may have a negative effect on EMT, probably through down-regulation of the ROS-dependent TGF-β1 signaling pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 332. doi:1538-7445.AM2012-332