Cross-talk between SIM2s and NFB regulates cyclooxygenase 2 expression in breast cancer.
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BACKGROUND: Breast cancer is a leading cause of cancer-related death for women in the USA. Thus, there is an increasing need to investigate novel prognostic markers and therapeutic methods. Inflammation raises challenges in treating and preventing the spread of breast cancer. Specifically, the nuclear factor kappa b (NFB) pathway contributes to cancer progression by stimulating proliferation and preventing apoptosis. One target gene of this pathway is PTGS2, which encodes for cyclooxygenase 2 (COX-2) and is upregulated in 40% of human breast carcinomas. COX-2 is an enzyme involved in the production of prostaglandins, which mediate inflammation. Here, we investigate the effect of Singleminded-2s (SIM2s), a transcriptional tumor suppressor that is implicated in inhibition of tumor growth and metastasis, in regulating NFB signaling and COX-2. METHODS: For in vitro experiments, reporter luciferase assays were utilized in MCF7 cells to investigate promoter activity of NFB and SIM2. Real-time PCR, immunoblotting, immunohistochemistry, and chromatin immunoprecipitation assays were performed in SUM159 and MCF7 cells. For in vivo experiments, MCF10DCIS.COM cells stably expressing SIM2s-FLAG or shPTGS2 were injected into SCID mice and subsequent tumors harvested for immunostaining and analysis. RESULTS: Our results reveal that SIM2 attenuates the activation of NFB as measured using NFB-luciferase reporter assay. Furthermore, immunostaining of lysates from breast cancer cells overexpressing SIM2s showed reduction in various NFB signaling proteins, as well as pAkt, whereas knockdown of SIM2 revealed increases in NFB signaling proteins and pAkt. Additionally, we show that NFB signaling can act in a reciprocal manner to decrease expression of SIM2s. Likewise, suppressing NFB translocation in DCIS.COM cells increased SIM2s expression. We also found that NFB/p65 represses SIM2 in a dose-dependent manner, and when NFB is suppressed, the effect on the SIM2 is negated. Additionally, our ChIP analysis confirms that NFB/p65 binds directly to SIM2 promoter site and that the NFB sites in the SIM2 promoter are required for NFB-mediated suppression of SIM2s. Finally, overexpression 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. CONCLUSION: Our findings identify a novel role for SIM2s in NFB signaling and COX-2 expression.
