precise mechanism by which BLT2 promotes invasion and metastasis has not been definitively identified, especially in breast cancer cells. We have now found that BLT2 expression is markedly increased in highly metastatic MDA-MB-231 and MDA-MB- 435 human breast cancer cells and that BLT2 plays a key role in the invasiveness of these cells. We also show that reactive oxygen species (ROS) produced by NADPH oxidase 1 (Nox1) act as downstream mediators of BLT2 signaling and induce up- regulation of IL-8 through activation of nuclear factor (NF)–kB, thereby promoting the invasiveness of these cells. In addition, the formation of metastatic lung nodules by MDA-MB-231 cells was greatly suppressed by BLT2 inhibition in both experimental and orthotopic metastatic settings. Together, our results implicate a BLT2–Nox1–ROS–NF-kB cascade in up-regulation of IL-8 production and the consequent promotion of invasiveness in highly aggressive breast cancer cells. These finding will provide insight into the molecular mechanisms of invasion and metastasis in breast cancer cells. Results BLT2 Contributes to the Invasiveness of Aggressive Breast Cancer Cells To elucidate the role of BLT2 in the invasiveness of breast cancer, we first analyzed BLT2 expression in the highly invasive breast cancer cell lines MDA-MB-231 and MDA-MB-435, both of which have been studied extensively with regard to the charac- terization of metastatic processes [5,16]. Both semiquantitative RT-PCR and quantitative real-time PCR analysis revealed that the amount of BLT2 mRNA, but not that of BLT1 mRNA, was markedly increased (about twofold) in MDA-MB-231 and MDA- MB-435 cells compared with that in immortalized mammary epithelial MCF-10A cells (Figure 1A and 1C). Consistent with these results, the abundance of BLT2 on the cell surface as determined by flow cytometry was significantly higher for MDA- MB-231 and MDA-MB-435 cells than for MCF-10A cells (Figure 1B and 1D). Together, these results indicated that BLT2 expression is markedly up-regulated in highly invasive breast cancer cells. We next determined the effect of BLT2 depletion by RNAi on the invasiveness of MDA-MB-231 and MDA-MB-435 cells with the use of a modified Boyden chamber assay [17]. Transfection of the cells with a BLT2 siRNA resulted in the knockdown of BLT2 mRNA but did not affect the amount of BLT1 mRNA (Figure 2A and 2B). Furthermore, BLT2 knockdown suppressed the invasive activity of MDA-MB-231 and MDA-MB-435 cells (Figure 2C). Depletion of BLT2 had no significant inhibitory effect on the proliferation (Figure S1A) or survival (Figure S1B) of these cells, suggesting that its effect on invasiveness was specific. Exposure of MDA-MB-231 and MDA-MB-435 cells to LY255283, a selective pharmacological inhibitor of BLT2 [18,19], also attenuated cell invasiveness (Figure 2D) without affecting cell proliferation (Figure S1C) or survival (Figure S1D), whereas the BLT1 inhibitor U75302 had no such effect. Together, these results suggested that BLT2 is necessary for the invasiveness of MDA-MB-231 and MDA-MB-435 cells. ROS Produced by a BLT2-Nox1 Pathway are Necessary for the Invasiveness of Breast Cancer Cells Our previous observations implicated ROS production down- stream of BLT2 signaling in the mechanism of Ras transformation or bladder cancer progression [14,20,21]. We therefore next examined whether ROS contribute to the invasive phenotype of BLT2-IL-8 Cascade in Aggressive Breast Cancer MDA-MB-231 and MDA-MB-435 cells. With the use of the redox-sensitive fluorescent probe dichlorofluorescein (DCF), we found that the intracellular level of ROS was increased in MDA- MB-231 and MDA-MB-435 cells compared with that in MCF- 10A cells (Figure 3A). The increase in ROS levels apparent in MDA-MB-231 and MDA-MB-435 cells was markedly attenuated by exposure of the cells to the BLT2 inhibitor LY255283, whereas it was not affected by the BLT1 inhibitor U75302 (Figure 3B), suggesting that increased production of ROS in these cells is dependent on BLT2. Depletion of ROS by addition of the antioxidant N-acetylcysteine (NAC) (Figure 3C) resulted in significant inhibition of the invasiveness of MDA-MB-231 and MDA-MB-435 cells (Figure 3D), implicating the increased ROS production in the invasive phenotype of these cells. We next examined the role of Nox in this phenotype, given that we previously found that Nox catalyzes BLT2-dependent ROS production in Ras-transformed fibroblasts [21]. Treatment with DPI, an inhibitor of flavoenzymes such as Nox, largely abolished the increase in ROS levels in MDA-MB-231 and MDA-MB-435 cells (Figure 3C), suggesting that Nox may mediate ROS production in these cells. Diphenyleneiodonium (DPI) also greatly inhibited the invasiveness of MDA-MB-231 and MDA-MB-435 cells (Figure 3D). Under these experimental conditions, treatment of NAC or DPI had no inhibitory effects on cell proliferation or survival in these breast cancer cells (data not shown). To examine the extent to which Nox activity contributes to BLT2-dependent ROS generation, we first determined the abundance of Nox mRNAs in MDA-MB-231 and MDA-MB- 435 cells. The amounts of both Nox1 and Nox4 mRNAs were much higher in MDA-MB-231 and MDA-MB-435 cells than in MCF-10A cells (Figure 4A). In addition, the level of Nox1 mRNA, but not that of Nox4 mRNA, in both MDA-MB-231 and MDA- MB-435 cells was greatly reduced by exposure to LY255283, whereas U75302 had no such effect (Figure 4B). Similarly, the amount of Nox1 mRNA in both these cell lines was markedly reduced by transfection with BLT2 siRNA (Figure 4B), suggesting that Nox1 may function downstream of BLT2 in the regulation of cell invasiveness. Consistent with the proposed role of Nox1 in ROS production in MDA-MB-231 and MDA-MB-435 cells, the intracellular ROS level was significantly reduced by RNAi- mediated depletion of Nox1 (Figure 4C and S2). In addition, the invasiveness of MDA-MB-231 and MDA-MB-435 cells was significantly inhibited by transfection with Nox1 siRNA (Figure 4D). Together, these results suggested that a BLT2- Nox1-ROS pathway regulates the invasiveness of aggressive breast cancer cells. BLT2-ROS Signaling Activates NF-kB in MDA-MB-231 and MDA-MB-435 Cells The activity of NF-kB is markedly increased in invasive breast cancer cells [16,22,23], suggesting that NF-kB signaling plays a key role in regulation of the invasiveness of breast cancer. Increased production of ROS has also been shown to activate NF- kB [23]. We therefore next examined whether NF-kB might function downstream of BLT2-ROS signaling in regulation of the invasiveness of MDA-MB-231 and MDA-MB-435 cells. Immu- noblot analysis revealed that LY255283, but not U75302, markedly inhibited the phosphorylation of the endogenous NF- kB inhibitor IkB-a (Figure 5A), whose phosphorylation by the IkB kinase complex results in NF-kB activation [24]. Depletion of BLT2 by RNAi also inhibited the phosphorylation of IkB-a in these cells (Figure 5B). To examine whether ROS contribute to NF-kB activation, we determined the effects of the Nox inhibitor DPI and the ROS scavenger NAC. Both NAC and DPI inhibited PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e49186