BLT2-IL-8 Cascade in Aggressive Breast Cancer Figure 3. ROS production occurs downstream of BLT2 and contributes to the invasiveness of breast cancer cells. (A) MCF-10A, MDA- MB-231, and MDA-MB-435 cells were incubated in the presence of 0.5% serum for 3 h and then loaded with the diacetate form of DCF (20 mM) for 20 min. Intracellular ROS levels were then determined by flow cytometric analysis of DCF fluorescence. (B) MDA-MB-231 and MDA-MB-435 cells were incubated with U75302 (1 mM), LY255283 (10 mM), or DMSO vehicle for 1 h before determination of the intracellular level of ROS as in (A). (C and D) MDA-MB-231 and MDA-MB-435 cells were incubated with NAC (5 mM), DPI (0.5 mM), or DMSO vehicle for 1 h and then monitored for ROS levels (C) or assayed for invasiveness in the continued presence of the test agents (D). All data are expressed relative to the corresponding control value and are means6SD from five independent experiments. *P,0.05, {P,0.01, {P,0.005. doi:10.1371/journal.pone.0049186.g003 implicated BLT2 in metastasis of highly aggressive MDA-MB-231 breast cancer cells in vivo. Discussion In the present study, we have found that the expression level of BLT2 is markedly increased in the highly aggressive breast cancer cell lines MDA-MB-231 and MDA-MB-435. Selective attenuation of BLT2 function by LY255283 treatment or RNAi-mediated knockdown resulted in suppression of the invasiveness of MDA- MB-231 and MDA-MB-435 cells, implicating BLT2 as a key determinant of the invasive phenotype of breast cancer. In addition, we characterized the BLT2 signaling pathway respon- sible for the regulation of cell invasiveness, finding that the Nox1- dependent generation of ROS and NF-kB activation function downstream of BLT2 to induce up-regulation of IL-8, a cytokine previously implicated in breast cancer invasiveness and metastasis. Finally, we showed that the formation of metastatic lung nodules by MDA-MB-231 cells was greatly suppressed by BLT2 blockade in both experimental and orthotopic metastasis models. Together, our findings suggest that a BLT2–Nox1–ROS–NF-kB signaling cascade up-regulates IL-8 expression and thereby contributes to the invasiveness of aggressive breast cancer cells. BLT2 is a G protein–coupled receptor that is expressed on the cell surface and interacts with specific ligands such as LTB4, 12(S)- HETE and 12-HHT [18,19,27]. BLT1, another LTB4 receptor, is expressed mainly on inflammatory cells, such as leukocytes, and plays a role in inflammatory processes [8]. Unlike BLT1, BLT2 has a low affinity for LTB4 and is expressed in a wide variety of tissues. Whereas various inflammatory functions of BLT1 have been extensively characterized, few biological functions of BLT2 have been identified, although recent studies have suggested that it plays a role in inflammatory diseases [8] and cancer progression [9,10,11,12,13,14,20]. Consistent with our findings, the level of IL-8, which is the most effective chemotactic factor known for neutrophils [28], is highly correlated with the invasiveness of breast cancer, with increased amounts of IL-8 having been detected in breast tumors with a high invasive potential [4]. High serum concentrations of IL-8 have also been associated with advanced stages of cancer [29]. The IL-8 produced by cancer cells is thought to act in an autocrine or paracrine manner through interaction with its cell surface receptors to promote invasiveness. Indeed, several lines of evidence have shown that the autocrine action of IL-8 drives the invasiveness of cancer cells [30,31]. In the present study, we found that a BLT2 signaling pathway regulates the invasive phenotype of MDA-MB-231 and MDA- MB-435 breast cancer cells. However, we cannot exclude the PLOS ONE | www.plosone.org 5 November 2012 | Volume 7 | Issue 11 | e49186