PCR analysis. Data are representative of three independent experiments. (TIF) Figure S3 BLT2 regulates the production of IL-8. (A) MDA-MB-231 and MDA-MB-435 cells were incubated with U75302 (1 mM), LY255283 (10 mM), or DMSO vehicle for 48 h or were transfected with BLT2 or control siRNAs for 48 h, after which the amount of IL-8 mRNA were assessed by semiquanti- tative RT-PCR analysis. (B) MDA-MB-231 and MDA-MB-435 cells were incubated with a specific IkB kinase inhibitor Bay11- 7082 (10 mM), a specific NF-kB inhibitor Bay11-7085 (1 mM), or DMSO vehicle for 48 h and then assayed for IL-8 mRNA by semiquantitative RT-PCR analysis. (C) MDA-MB-231 and MDA- MB-435 cells were incubated with DPI (0.5 mM), NAC (5 mM), or DMSO vehicle for 48 h or were transfected with a vector for Nox1 siRNA (pSUPER-siNox1) or the corresponding empty vector for 48 h, after which the abundance of IL-8 mRNA was determined by semiquantitative RT-PCR analysis. Semiquantitative RT-PCR data are representative of three independent experiments. (D) Cells transfected with sense (S) or antisence (AS) IL-8 oligonucle- otides for 24 h were incubated for the incubated times before determining cell growth by trypan blue staining. Data are means6SD from three independent experiments. (TIFF) References 1. Christofori G (2006) New signals from the invasive front. Nature 441: 444–450. 2. Sahai E (2007) Illuminating the metastatic process. Nat Rev Cancer 7: 737–749. 3. Gotzmann J, Mikula M, Eger A, Schulte-Hermann R, Foisner R, et al. (2004) Molecular aspects of epithelial cell plasticity: implications for local tumor invasion and metastasis. Mutat Res 566: 9–20. 4. Sheridan C, Kishimoto H, Fuchs RK, Mehrotra S, Bhat-Nakshatri P, et al. (2006) CD44+/CD24– breast cancer cells exhibit enhanced invasive properties: an early step necessary for metastasis. Breast Cancer Res 8: R59. 5. Wu K, Katiyar S, Li A, Liu M, Ju X, et al. (2008) Dachshund inhibits oncogene- induced breast cancer cellular migration and invasion through suppression of interleukin-8. Proc Natl Acad Sci U S A 105: 6924–6929. 6. De Larco JE, Wuertz BR, Rosner KA, Erickson SA, Gamache DE, et al. (2001) A potential role for interleukin-8 in the metastatic phenotype of breast carcinoma cells. Am J Pathol 158: 639–646. 7. Benoy IH, Salgado R, Van Dam P, Geboers K, Van Marck E, et al. (2004) Increased serum interleukin-8 in patients with early and metastatic breast cancer correlates with early dissemination and survival. Clin Cancer Res 10: 7157– 7162. 8. Funk CD (2001) Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 294: 1871–1875. 9. Rocconi RP, Kirby TO, Seitz RS, Beck R, Straughn JM Jr, et al. (2008) Lipoxygenase pathway receptor expression in ovarian cancer. Reprod Sci 15: 321–326. 10. Sveinbjornsson B, Rasmuson A, Baryawno N, Wan M, Pettersen I, et al. (2008) Expression of enzymes and receptors of the leukotriene pathway in human neuroblastoma promotes tumor survival and provides a target for therapy. FASEB J 22: 3525–3536. 11. Hennig R, Ventura J, Segersvard R, Ward E, Ding XZ, et al. (2005) LY293111 improves efficacy of gemcitabine therapy on pancreatic cancer in a fluorescent orthotopic model in athymic mice. Neoplasia 7: 417–425. 12. Hennig R, Osman T, Esposito I, Giese N, Rao SM, et al. (2008) BLT2 is expressed in PanINs, IPMNs, pancreatic cancer and stimulates tumour cell proliferation. Br J Cancer 99: 1064–1073. 13. Choi JA, Lee JW, Kim H, Kim EY, Seo JM, et al. (2010) Pro-survival of estrogen receptor-negative breast cancer cells is regulated by a BLT2-reactive oxygen species-linked signaling pathway. Carcinogenesis 31: 543–551. 14. Kim EY, Seo JM, Kim C, Lee JE, Lee KM, et al. (2010) BLT2 promotes the invasion and metastasis of aggressive bladder cancer cells through a reactive oxygen species-linked pathway. Free Radic Biol Med 49: 1072–1081. 15. Seo JM, Park S, Kim JH (2012) The leukotriene B4 receptor-2 promotes invasiveness and metastasis of ovarian cancer cells through STAT3-dependent up-regulation of matrix metalloproteinase 2. J Biol Chem 288: 13840–13849. 16. Cicek M, Fukuyama R, Welch DR, Sizemore N, Casey G (2005) Breast cancer metastasis suppressor 1 inhibits gene expression by targeting nuclear factor- kappaB activity. Cancer Res 65: 3586–3595. 17. Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson SA, et al. (1987) A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res 47: 3239–3245. BLT2-IL-8 Cascade in Aggressive Breast Cancer Figure S4 The addition of LTB4 induces the invasive- ness. (A and B) MDA-MB-231 and MDA-MB-435 cells were incubated for 48 h with LTB4 (50, 100, 300 or 300 nM) (A) or MK886 (5 mM) (B). Thereafter, the levels of IL-8 were assessed for mRNA and protein by semiquantitative RT-PCR (left panel) and ELISA (right panel), respectively. Data are representative of three independent experiments. (C and D) MDA-MB-231 and MDA- MB-435 cells were incubated with LTB4 (300 nM) (C) or MK886 (5 mM) (D) for 30 min and then assayed for the invasiveness. Scale bars, 50 mm. All quantitative data are mean6SD from five independent experiments. *P,0.05, {P,0.01, {P,0.005. (TIF) Acknowledgments We thank to Dr. Aree Moon (Duksung Women’s University, Seoul, Korea) for the MCF-10A cell line. Author Contributions Conceived and designed the experiments: HK JAC JHK. Performed the experiments: HK JAC. Analyzed the data: HK GSP. Contributed reagents/materials/analysis tools: HK JAC. Wrote the paper: HK JAC JHK. 18. Yokomizo T, Izumi T, Chang K, Takuwa Y, Shimizu T (1997) A G-protein- coupled receptor for leukotriene B4 that mediates chemotaxis. Nature 387: 620– 624. 19. Yokomizo T, Kato K, Terawaki K, Izumi T, Shimizu T (2000) A second leukotriene B(4) receptor, BLT2. A new therapeutic target in inflammation and immunological disorders. J Exp Med 192: 421–432. 20. Kim EY, Seo JM, Cho KJ, Kim JH (2010) Ras-induced invasion and metastasis are regulated by a leukotriene B4 receptor BLT2-linked pathway. Oncogene 29: 1167–1178. 21. Choi JA, Kim EY, Song H, Kim C, Kim JH (2008) Reactive oxygen species are generated through a BLT2-linked cascade in Ras-transformed cells. Free Radic Biol Med 44: 624–634. 22. Nakshatri H, Bhat-Nakshatri P, Martin DA, Goulet RJ Jr, Sledge GW Jr (1997) Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth. Mol Cell Biol 17: 3629–3639. 23. Richmond A (2002) Nf-kappa B, chemokine gene transcription and tumour growth. Nat Rev Immunol 2: 664–674. 24. Baud V, Karin M (2009) Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls. Nat Rev Drug Discov 8: 33–40. 25. De Larco JE, Wuertz BR, Yee D, Rickert BL, Furcht LT (2003) Atypical methylation of the interleukin-8 gene correlates strongly with the metastatic potential of breast carcinoma cells. Proc Natl Acad Sci U S A 100: 13988– 13993. 26. Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, et al. (2005) Genes that mediate breast cancer metastasis to lung. Nature 436: 518–524. 27. Okuno T, Iizuka Y, Okazaki H, Yokomizo T, Taguchi R, et al. (2008) 12(S)- Hydroxyheptadeca-5Z, 8E, 10E-trienoic acid is a natural ligand for leukotriene B4 receptor 2. J Exp Med 205: 759–766. 28. Waugh DJ, Wilson C (2008) The interleukin-8 pathway in cancer. Clin Cancer Res 14: 6735–6741. 29. Rao SK, Pavicevic Z, Du Z, Kim JG, Fan M, et al. (2010) Pro-inflammatory genes as biomarkers and therapeutic targets in oral squamous cell carcinoma. J Biol Chem 285: 32512–32521. 30. Yao C, Lin Y, Chua MS, Ye CS, Bi J, et al. (2007) Interleukin-8 modulates growth and invasiveness of estrogen receptor-negative breast cancer cells. Int J Cancer 121: 1949–1957. 31. Inoue K, Slaton JW, Kim SJ, Perrotte P, Eve BY, et al. (2000) Interleukin 8 expression regulates tumorigenicity and metastasis in human bladder cancer. Cancer Res 60: 2290–2299. 32. Dorsam RT, Gutkind JS (2007) G-protein-coupled receptors and cancer. Nat Rev Cancer 7: 79–94. 33. Song H, Ki SH, Kim SG, Moon A (2006) Activating transcription factor 2 mediates matrix metalloproteinase-2 transcriptional activation induced by p38 in breast epithelial cells. Cancer Res 66: 10487–10496. 34. Kim GY, Lee JW, Ryu HC, Wei JD, Seong CM, et al. (2010) Proinflammatory cytokine IL-1beta stimulates IL-8 synthesis in mast cells via a leukotriene B4 receptor 2-linked pathway, contributing to angiogenesis. J Immunol 184: 3946– 3954. PLOS ONE | www.plosone.org 12 November 2012 | Volume 7 | Issue 11 | e49186