Double-Stranded RNA-Dependent Protein Kinase Regulates the Motility of Breast Cancer Cells Mei Xu 1 , Gang Chen 1 , Siying Wang1,2, Mingjun Liao1, Jacqueline A. Frank 1 , Kimberly A. Bower1, Zhuo Zhang 3 , Xianglin Shi3, Jia Luo 1 * 1 Department of Internal Medicine, University of Kentucky College of Medicine, Lexington, Kentucky, United States of America, 2 Pathophysiological Department, School of Basic Medicine, Anhui Medical University, Hefei, Anhui, China, 3 Graduate Center for Toxicology, University of Kentucky College of Medicine, Lexington, Kentucky, United States of America Abstract Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is an interferon-induced protein kinase that plays a central role in the anti-viral process. Due to its pro-apoptotic and anti-proliferative action, there is an increased interest in PKR modulation as an anti-tumor strategy. PKR is overexpressed in breast cancer cells; however, the role of PKR in breast cancer cells is unclear. The expression/activity of PKR appears inversely related to the aggressiveness of breast cancer cells. The current study investigated the role of PKR in the motility/migration of breast cancer cells. The activation of PKR by a synthesized dsRNA (PIC) significantly decreased the motility of several breast cancer cell lines (BT474, MDA-MB231 and SKBR3). PIC inhibited cell migration and blocked cell membrane ruffling without affecting cell viability. PIC also induced the reorganization of the actin cytoskeleton and impaired the formation of lamellipodia. These effects of PIC were reversed by the pretreatment of a selective PKR inhibitor. PIC also activated p38 mitogen-activated protein kinase (MAPK) and its downstream MAPK-activated protein kinase 2 (MK2). PIC-induced activation of p38 MAPK and MK2 was attenuated by the PKR inhibitor and the PKR siRNA, but a selective p38 MAPK inhibitor (SB203580) or other MAPK inhibitors did not affect PKR activity, indicating that PKR is upstream of p38 MAPK/MK2. Cofilin is an actin severing protein and regulates membrane ruffling, lamellipodia formation and cell migration. PIC inhibited cofilin activity by enhancing its phosphorylation at Ser3. PIC activated LIM kinase 1 (LIMK1), an upstream kinase of cofilin in a p38 MAPK-dependent manner. We concluded that the activation of PKR suppressed cell motility by regulating the p38 MAPK/MK2/LIMK/cofilin pathway. Citation: Xu M, Chen G, Wang S, Liao M, Frank JA, et al. (2012) Double-Stranded RNA-Dependent Protein Kinase Regulates the Motility of Breast Cancer Cells. PLoS ONE 7(10): e47721. doi:10.1371/journal.pone.0047721 Editor: Chryso Kanthou, University of Sheffield, United Kingdom Received April 24, 2012; Accepted September 14, 2012; Published October 24, 2012 Copyright: 2012 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research is supported by a NIH grant (AA017226). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jialuo888@uky.edu Introduction Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is a 551 amino acid serine/threonine protein kinase and is ubiquitously expressed in mammalian cells. PKR was initially identified as an essential element in interferon-induced anti-viral processes. PKR is activated by viral dsRNA intermediates during infection via a mechanism involving autophosphorylation. Once activated, the enzyme phosphorylates the a-subunit of protein synthesis initiation factor eIF2, thereby inhibiting translation [1]. PKR also mediates the activation of signal transduction pathways by proinflammatory stimuli, including bacterial lipopolysaccharide (LPS), tumor necrosis factor-a (TNF-a) and interleukin 1 (IL-1) [1]. PKR can be directly activated by its cellular activator PACT [2]. In addition, serum deprivation, disruption of intracellular Ca2+ homeostasis, oxidative stress and endoplasmic reticulum (ER) stress also stimulate PKR activity [3,4]. Active PKR regulates diverse downstream substrates and signaling pathways, such as NFkB, p53, protein phosphatase 2A (PP2A), MAPK and STAT1/ STAT3 signaling [3]. PKR has been implicated in the regulation of cell proliferation, apoptosis, differentiation and transformation [5,6]. In general, activation of PKR results in the inhibition of cell proliferation or the induction of apoptosis and translational suppression; therefore, PKR is considered a ‘‘tumor suppressor’’ and considerable attention has been paid to the PKR pathway for its anti-tumor potential [3,7]. In contrast to its potential role as a tumor suppressor, PKR is over-expressed in a number of human cancers including breast cancers [8–11]. A higher level of PKR is observed in human invasive ductal breast carcinomas than surrounding normal mammary tissues [9]. In addition, much more PKR is expressed in mammary carcinoma cell lines compared to non-transformed mammary epithelial cell lines [4,11]. However, it appears the expression levels of PKR are inconsistent with its activity detected in mammary tumor cells and epithelial cells. The role of PKR in breast cancer cells is unclear. Among human breast cancer cell lines, it appears that the aggressiveness is inversely related to PKR expression/activity [8]. For example, MCF-7, a minimally invasive breast cancer cell line expresses high levels/activity of PKR, while MDA-MB231, a highly invasive breast cancer cell line, has low levels/activity of PKR [8]. We therefore hypothesized that PKR plays a role in the aggressiveness in breast cancer cells. Tumor cell motility/migration is the hallmark of invasion and an essential step in metastasis. Cell motility/migration is a complex biological process that is regulated by a myriad of molecular/cellular PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e47721