Hepatitis B Virus X Protein Drives Multiple Cross-Talk Cascade Loops Involving NF-kB, 5-LOX, OPN and Capn4 to Promote Cell Migration Xuan Zhang1., Xiaona You1., Qi Wang1., Tao Zhang 1 , Yumei Du 1 , Na Lv1, Zhao Zhang2, Shuai Zhang 1 , Changliang Shan1, Lihong Ye2*, Xiaodong Zhang 1 * 1 Department of Cancer Research, Key Laboratory of Molecular Microbiology and Technology of Ministry of Education, Institute for Molecular Biology, College of Life Sciences, Nankai University, Tianjin, People’s Republic of China, 2 Department of Biochemistry, College of Life Sciences, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, People’s Republic of China Abstract Hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). However, the mechanism remains unclear. Recently, we have reported that HBx promotes hepatoma cell migration through the upregulation of calpain small subunit 1 (Capn4). In addition, several reports have revealed that osteopontin (OPN) plays important roles in tumor cell migration. In this study, we investigated the signaling pathways involving the promotion of cell migration mediated by HBx. We report that HBx stimulates several factors in a network manner to promote hepatoma cell migration. We showed that HBx was able to upregulate the expression of osteopontin (OPN) through 5-lipoxygenase (5- LOX) in HepG2-X/H7402-X (stable HBx-transfected cells) cells. Furthermore, we identified that HBx could increase the expression of 5-LOX through nuclear factor-kB (NF-kB). We also found that OPN could upregulate Capn4 through NF-kB. Interestingly, we showed that Capn4 was able to upregulate OPN through NF-kB in a positive feedback manner, suggesting that the OPN and Capn4 proteins involving cell migration affect each other in a network through NF-kB. Importantly, NF-kB plays a crucial role in the regulation of 5-LOX, OPN and Capn4. Thus, we conclude that HBx drives multiple cross-talk cascade loops involving NF-kB, 5-LOX, OPN and Capn4 to promote cell migration. This finding provides new insight into the mechanism involving the promotion of cell migration by HBx. Citation: Zhang X, You X, Wang Q, Zhang T, Du Y, et al. (2012) Hepatitis B Virus X Protein Drives Multiple Cross-Talk Cascade Loops Involving NF-kB, 5-LOX, OPN and Capn4 to Promote Cell Migration. PLoS ONE 7(2): e31458. doi:10.1371/journal.pone.0031458 Editor: Wang-Shick Ryu, Yonsei University, Korea, Republic of Received June 17, 2011; Accepted January 8, 2012; Published February 15, 2012 Copyright: 2012 Zhang 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: The work was funded by National Basic Research Program of China (973 Program, No. 2007CB914804, No. 2007CB914802, No. 2009CB521702) and National Natural Science Foundation of China (No. 81071624). 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: zhangxd@nankai.edu.cn (XZ); yelihong@nankai.edu.cn (LY) . These authors contributed equally to this work. Introduction Hepatitis B virus (HBV) has oncogenic potential in the development of hepatocellular carcinoma (HCC). The HBV genome is a partially double-stranded DNA molecule with four open reading frames (ORFs), in which S ORF encodes hepatitis surface antigen (HBs) and C ORF encodes hepatitis core antigen (HBc). Hepatitis B virus X protein (HBx) is a 17 kDa protein encoded by the X ORF, which plays a crucial role in hepatocarcinogenesis [1]. HBx has multiple biological functions, including interaction with other proteins, mediation of cell proliferation and apoptosis [2,3]. Recent studies have shown that HBx is associated with cell migration, implicating HBx in HCC metastasis. For example, HBx may promote tumor spreading by facilitating integrin-mediated cell migration and regulating the adhesion-deadhesion balance of the cells in the primary tumor site [4], enhancing CD44-mediated HA-interaction efficiency and modifying the migratory properties of transformed hepatocytes [5] and inducing matrix metalloproteinase (MMP) activation [6,7,8]. It has been reported that 5-lipoxygenase (5-LOX) is a key regulator of malignant mesothelial cell proliferation and survival via a VEGF- related circuit [9]. Our laboratory previously found that cycloox- ygenase-2 (COX-2) and 5-LOX were highly expressed in breast cancer LM-MCF-7 cells and MDA-MB-231 cells, which were related to breast cancer metastasis [10]. Moreover, our have found that HBx could upregulate the levels of cyclooxygenase-2 (COX-2) and 5-lipoxygenase in liver cells [11]. Accordingly, nuclear factor- kB (NF-kB) plays an instrumental role in carcinogenesis and in the regulation of immune and inflammatory responses [12]. NF-kB induces the expression of various target genes related to proliferation, apoptosis, angiogenesis and metastasis. HBx protein activates the transcription factor NF-kB by acting on two distinct cytoplasmic NF-kB inhibitor pathways [13]. Furthermore, HBx can induce the expression of various target genes through activation of NF-kB, such as cyclin D1 through the NF-kB2(p52)/BCL-3 complex in the nucleus [14]. HBx induces expression of the CXC chemokine IP-10 and MIG and increases migration of leukocytes through the activation of NF-kB [15,16]. Previous studies demonstrated that tumor cell invasion and metastasis after liver transplantation for HCC was highly PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e31458