BLT2-IL-8 Cascade in Aggressive Breast Cancer Figure 2. BLT2 is required for the invasiveness of MDA-MB-231 and MDA-MB-435 breast cancer cells. (A and B) MDA-MB-231 (A) and MDA-MB-435 (B) cells were transfected with BLT2 (siBLT2) or control (siCont) siRNAs for 48 h in medium supplemented with 0.5% FBS, after which the amounts of BLT1 and BLT2 mRNAs were determined by semiquantitative RT-PCR (left panel) and quantitative real-time PCR (center and right panels) analysis. Data are representative of five independent experiments. (C) Cells treated as in (A) and (B) were assayed for invasiveness. Representative invading cells stained with H&E are shown, and quantitative data are expressed relative to the value for cells transfected with the control siRNA. Scale bars, 50 mm. (D) MDA-MB-231 and MDA-MB-435 cells were incubated with U75302 (0.5 or 1 mM), LY255283 (5 or 10 mM), or DMSO vehicle for 30 min and were then assayed for invasiveness in the continued presence of the corresponding test agent. Scale bars, 50 mm. All quantitative data are means6SD from five independent experiments. *P,0.05, {P,0.01, {P,0.005. doi:10.1371/journal.pone.0049186.g002 Together, we propose that a LTB4-BLT2 signaling pathway is likely to up-regulate IL-8 expression and thereby increase the invasiveness of breast cancer cells. To evaluate further the role of the BLT2–ROS–NF-kB cascade in the up-regulation of IL-8, we examined the effects of Bay11-7082, a specific inhibitor of IkB kinase. Inhibition of NF-kB signaling by this agent resulted in down-regulation of IL-8 mRNA (Figure S3B) and protein (Figure 6C) in MDA-MB-231 and MDA-MB-435 cells. Similarly, treatment with DPI or NAC markedly reduced the amount of IL-8 mRNA in these cells (Figure S3C). In addition, knockdown of Nox1 resulted in depletion of IL-8 mRNA (Figure S3C) and protein (Figure 6D). These observations thus suggested that the BLT2–Nox1–ROS–NF-kB cascade up-regulates IL-8 expression in highly invasive breast cancer cells. To evaluate whether this BLT2-dependent IL-8 expression contributes to the invasiveness of MDA-MB-231 and MDA-MB-435 cells, we examined the effect of an IL-8 antisense oligonucleotide. The marked decrease in the abundance of IL-8 mRNA and protein induced by transfection of MDA-MB-231 and MDA-MB-435 cells with this antisense oligonucleotide (Figure 6E) was accompanied by significant inhibition of cell invasiveness (Figure 6F) but no effect on cell growth (Figure S3D), suggesting that IL-8 indeed contributes to the invasive phenotype of these cells. Collectively, these results indicated that the BLT2–ROS–NF-kB cascade is a previously unrecognized regulator of IL-8 production, and that such regulation controls the invasiveness of highly aggressive breast cancer cells. Role of BLT2 in Metastasis of Breast Cancer Cells in vivo Cell invasiveness is closely associated with metastatic potential [3]. We therefore finally examined the role of BLT2 in the metastatic potential of MDA-MB-231 cells in vivo. To this end, we first investigated the effect of LY255283 in an in vivo experimental metastasis assay in which cancer cells are injected into the tail vein of nude mice [26]. MDA-MB-231 cells were pretreated with 10 mM LY255283 or DMSO for 24 h prior to tail vein injection. Mice injected with MDA-MB-231 cells and treated with DMSO vehicle manifested large metastatic nodules in the lungs, whereas the number of such nodules was reduced by ,80% by treatment with LY255283 (Figure 7A and 7B). We also examined the effect of LY255283 on the metastatic potential of aggressive breast cancer cells in an orthotopic metastasis assay in which MDA-MB- 231 cells were implanted into the mammary fat pad. MDA-MB- 231 cells pretreated for 24 h with 10 mM LY255283 or DMSO were implanted into the mammary fat pad of mice, and the animals were then administrated intraperitoneally with LY255283 or DMSO three times at 5-day intervals. Administration of LY255283 resulted in a marked reduction in the extent of metastasis to the lungs compared with that apparent in mice treated with DMSO (Figure 7C and 7D). Together, these results PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e49186