Abstract 3784: Role of OBG like ATPase 1 (OLA1) in F-actin polymerization and cell adhesion.

Cell adhesion and migration, driven by actin cytoskeletal dynamics, are the fundamental phenomenon in cancer metastasis. However, the molecular nature that underlies the coordination of actin cytoskeleton at sites of focal adhesions remains elusive. Obg like ATPase (OLA1) belongs to the YchF subfamily of Obg like P-loop GTPases. YchF proteins are highly conserved from yeast to humans, and believed to be regulatory proteins that interact with their client protein(s) by the conformational switch between the ADP and ATP bound forms. However, the actual physiological functions of OLA1 are poorly understood. In our previous studies we reported that OLA1 functions as a negative regulator in antioxidant response and knockdown of OLA1 inhibits migration and invasion of breast cancer cells in vitro. In this study, we have examined the mechanism underlying the anti-migratory role of OLA1. Expression of OLA1 was silenced by siRNA transfection (siOLA1) in human breast cancer cells MDA-MB-231 and human embryonic lung fibroblast cells WI-38, and the rate of attachment of suspended cells was evaluated against control siRNA-transfected cells. After plating into fibronectin or laminin coated plates, siOLA1 cells were attached and spread significantly faster than the control cells. At the end of 60 min siOLA1 MDA-MB-231 cells showed profound F-actin fibers on fibronectin coated plates. Immunofluorescence staining further revealed the co-localization of vinculin with actin at the spherical leading edges in control siRNA-transfected cells, which proclaimed that these cells are still trying to spread. In contrast, siOLA1 cells were fully spread and immensely polymerized actin fibers were completely dissociated from vinculin. Due to the augmented actin polymerization, siOLA1 cells are rendered with impaired depolymerization, a process required for an efficient actin disassembly. Furthermore, levels of S-glutathionylation, a form of redox sensitive and reversible posttranslational modification, were measured in these cells by immunoblot analysis. Consistently, glutathionylation of actin was found increased significantly in siOLA1 MDA-MB-231 cells at various adherent growth conditions, as well as in the form of single cell suspension immediately before the cells were plated. Since earlier studies have established the impacts of actin glutathionylation in impaired actin depolymerization and cell adhesion processes, we reason that heavier glutathionylation on actin attributes to the altered actin dynamics in siOLA1 cells. These studies provide evidence that focal adhesions are more stable in the absence of OLA1 and suggest that OLA1 regulates cell adhesion and migration in part by actin polymerization and S-glutathionylation of actin. Further studies are warranted to explore molecular mechanisms by which OLA1 modulates protein S-glutathionylation either globally or specifically on cytoskeleton molecules such as actin. Citation Format: Prince V. Jeyabal, Valentina Rubio, Jiawei Zhang, Zheng-Zheng Shi. Role of OBG like ATPase 1 (OLA1) in F-actin polymerization and cell adhesion. [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 3784. doi:10.1158/1538-7445.AM2013-3784