BLT2 Up-Regulates Interleukin-8 Production and Promotes the Invasiveness of Breast Cancer Cells Hyunju Kim, Jung-A Choi, Geun-Soo Park, Jae-Hong Kim* School of Life Sciences and Biotechnology, Korea University, Seoul, Korea Abstract Background: The elevated production of interleukin (IL)–8 is critically associated with invasiveness and metastatic potential in breast cancer cells. However, the intracellular signaling pathway responsible for up-regulation of IL-8 production in breast cancer cells has remained unclear. Methodology/Principal Findings: In this study, we report that the expression of BLT2 is markedly up-regulated in the highly aggressive human breast cancer cell lines MDA-MB-231 and MDA-MB-435 compared with MCF-10A immortalized human mammary epithelial cells, as determined by RT-PCR, real-time PCR and FACS analysis. Blockade of BLT2 with BLT2 siRNA knockdown or BLT2 inhibitor treatment downregulated IL-8 production and thereby diminished the invasiveness of aggressive breast cancer cells, analyzed by Matrigel invasion chamber assays. We further characterized the downstream signaling mechanism by which BLT2 stimulates IL-8 production and identified critical mediatory roles for the generation of reactive oxygen species (ROS) and the consequent activation of the transcription factor NF-kB. Moreover, blockade of BLT2 suppressed the formation of metastatic lung nodules by MDA-MB-231 cells in both experimental and orthotopic metastasis models. Conclusions/Significance: Taken together, our study demonstrates that a BLT2–ROS–NF-kB pathway up-regulates IL-8 production in MDA-MB-231 and MDA-MB-435 cells, thereby contributing to the invasiveness of these aggressive breast cancer cells. Our findings provide insight into the molecular mechanism of invasiveness in breast cancer. Citation: Kim H, Choi J-A, Park G-S, Kim J-H (2012) BLT2 Up-Regulates Interleukin-8 Production and Promotes the Invasiveness of Breast Cancer Cells. PLoS ONE 7(11): e49186. doi:10.1371/journal.pone.0049186 Editor: Dennis J. Templeton, University of Virginia, United States of America Received May 8, 2012; Accepted October 3, 2012; Published November 7, 2012 Copyright: 2012 Kim 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 work was supported by the grants from the Senior Researcher Project (No. 2012R1A2A2A01044526), the General Researcher Project (2012- 0001877), and the Bio & Medical Technology Development Program (2012M3A9C5048709) of the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology (MEST), Republic of Korea, and also by the grant from the National R&D Program for Cancer Control (1220020), Ministry for Health and Welfare, Republic of Korea. 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: jhongkim@korea.ac.kr Introduction Cancer invasion is a fundamental aspect of metastasis and is the major cause of death in cancer patients [1]. The acquisition of invasive capacity is required for cancer cells to invade surrounding tissues, to cross anatomic barriers, and to travel through the bloodstream or lymphatic system [2], all of which are properties of aggressive metastatic cancer cells [3]. An understanding of the molecular mechanisms that regulate the invasion process is thus key for development of therapeutic interventions to prevent tumor metastasis. Interleukin (IL)-8 has recently been suggested to promote the invasive and metastatic potential of breast cancer cells. In particular, increased levels of IL-8 were detected in breast cancer cells with a highly invasive phenotype [4], and the expression of IL-8 was found to correlate with the progression of metastasis in breast cancer cells [5,6]. Clinical studies have also shown that the levels of IL-8 are higher in breast tumor tissue than in normal breast tissue, and an increased serum concentration of IL-8 has been suggested to be associated with advanced stages of breast cancer [7]. Despite the importance of IL-8 in cancer invasion and metastasis, however, the cellular signals required for the production of IL-8 in breast cancer cells have remained unknown. Leukotriene B4 (LTB4) is a potent chemoattractant and pro- inflammatory lipid mediator that plays a role in the pathogenesis of several inflammatory diseases including asthma [8]. However, recent studies have also suggested that LTB4 and its receptors may regulate tumor progression by promoting cell proliferation and survival [9,10,11,12,13]. For instance, an increased abundance of LTB4 and its receptors has been observed in many types of tumor, including neuroblastoma as well as pancreatic, colon, and ovarian cancers [9,10]. In addition, LY293111, an antagonist of the LTB4 receptor BLT1, inhibited the growth of and induced apoptosis in human pancreatic cancer and lymphoma cells in vitro. Moreover, LY293111 treatment inhibited the growth of tumor xenografts and reduced the incidence of metastasis in athymic mice [11]. Expression of the LTB4 receptor BLT2 in ovarian, bladder and breast cancer tissue was also found to be increased at advanced cancer stages and to be associated with a poor clinical outcome [9,13,14,15]. Similarly, BLT2 was detected in infiltrating tumor cells of pancreatic adenocarcinoma tissue and in lymph node metastases [12]. Despite these various observations implicating BLT2 as a potential marker for aggressive metastatic cancer, the PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49186