Cross-talk between SIM2s and NFkB regulates cyclooxygenase 2 expression in breast cancer

Abstract

AbstractBackgroundBreast cancer is a leading cause of cancer-related death for women in the United States. Thus, there a need to investigate novel prognostic markers and therapeutic strategies. Inflammation raises challenges to both treating and preventing the spread of breast cancer. Specifically, the nuclear factor kappa b (NFkB) pathway contributes to cancer progression by stimulating proliferation and preventing apoptosis. One target gene of this pathway is PTGS2, the gene that encodes for cyclooxygenase 2 (COX-2), which is upregulated in 40% of human breast carcinomas. COX-2 is an enzyme involved in inflammation. Here we investigate the effect of Singleminded 2s, a transcriptional tumor suppressor that is implicated in inhibition of tumor growth and metastasis, in regulating NFkB and COX-2.MethodsWe utilized in vitro reporter assays, immunoblot analyses, qPCR and immunohistochemical analysis to dissect the relationship between NFκB, SIM2s, and COX-2. Furthermore, we utilized COX-2 targeting strategies to determine tumor suppressive activities.ResultsOur results reveal that SIM2s attenuates the activation of a NFκB via luciferase reporter assays. Furthermore, immunostaining of lysates from breast cancer cells over expressing SIM2s showed reduction in various NFκB signaling proteins, whereas knockdown of SIM2 revealed increases in the same NFκB signaling proteins. Additionally, by increasing NFκB translocation to the nucleus in DCIS.COM cells, we show that NFκB signaling can act in a reciprocal manner to decrease expression of SIM2s. Likewise, suppressing NFκB translocation in DCIS.COM cells increases SIM2s expression. We also found that NFκB/p65 represses SIM2 in via dose-dependent manner and when NFκB is suppressed the effect on the SIM2 is negated. Additionally, our CHIP analysis confirms that NFκB/p65 binds directly to SIM2 promoter site and that the NFκB sites in the SIM2 promoter are required for NFkB-mediated suppression of SIM2s. Finally, over expression of SIM2s decreases PTGS2 in vitro and COX-2 staining in vivo while decreasing PTGS2 and/or Cox-2 activity results in re-expression of SIM2. Our findings identify a novel role for SIM2s in NFκB signaling and COX-2 expression.ConclusionsThese findings provide evidence for a mechanism where SIM2s may represses COX-2 expression to provide an overall better prognosis for breast cancer patients.