BLT2-IL-8 Cascade in Aggressive Breast Cancer Figure 4. Nox1 catalyzes ROS generation associated with BLT2 signaling in MDA-MB-231 and MDA-MB-435 cells. (A) Semiquantitative RT-PCR analysis of Nox1 and Nox4 mRNAs in MCF-10A, MDA-MB-231, and MDA-MB-435 cells. Data are representative of three independent experiments. (B) MDA-MB-231 and MDA-MB-435 cells were incubated with U75302 (1 mM), LY255283 (10 mM), or DMSO vehicle for 1 h or were transfected with BLT2 or control siRNAs for 48 h before determination of the abundance of Nox1 and Nox4 mRNAs by semiquantitative RT-PCR analysis. Data are representative of three independent experiments. (C and D) MDA-MB-231 and MDA-MB-435 cells were transfected with a vector for Nox1 siRNA (pSUPER-siNox1) or the corresponding empty vector for 48 h and were then analyzed for the intracellular level of ROS (C) or invasiveness (D). Scale bars, 50 mm. Data are expressed relative to the corresponding value for cells transfected with the empty vector and are means6SD from five independent experiments. *P,0.05, {P,0.01, {P,0.005. doi:10.1371/journal.pone.0049186.g004 possibility that activation of this BLT2–Nox1–ROS–NF-kB–IL-8 cascade also contributes to other cancer-promoting activities such as angiogenesis, especially in vivo. IL-8 also functions as an angiogenic factor, and BLT2-dependent IL-8 production may thus contribute to angiogenesis in the tumor microenvironment and thereby promote cancer progression. In addition to its potential role in angiogenesis, BLT2-ROS signaling was recently shown to promote survival in estrogen receptor (ER)–negative, nonmetastatic breast cancer cells, such as MDA-MB-468 and MDA-MB-453 cells, although the downstream effectors of this BLT2-ROS pathway were not determined [13]. However, we did not detect a role for BLT2 in the survival of MDA-MB-231 and MDA-MB-435 cells (Figure S1), which are ER-negative and highly aggressive, suggesting that BLT2 regulates the invasiveness but not the survival of these cells. The biological functions of BLT2 signaling may therefore differ even among cells that are ER- negative and may vary with breast cancer stage. We previously observed that BLT2 levels were higher in ER-positive cells such as MCF-7 than in MDA-MB-231 ER-negative cells [13]. Nonethe- less, MCF-7 cells are considered less invasive. And, we speculate that the biological function of BLT2 may differ among breast cancer cells. For instance, we have observed that the proliferation, not the invasiveness, of MCF-7 cells was severely attenuated by BLT2 inhibition, pointing to BLT2 as a mediator of the proliferation, not the invasiveness, in MCF-7 cells (data not shown). Indeed, many G protein–coupled receptors implicated in tumorigenesis have been shown to have pleiotropic effects in this process [32]. Further studies will be required to elucidate the potential pleiotropic actions of BLT2 in cancer progression, including its roles in the various stages of breast cancer development. PLOS ONE | www.plosone.org 6 November 2012 | Volume 7 | Issue 11 | e49186