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Cross-talk between SIM2s and NFκB regulates cyclooxygenase 2 expression in breast cancer.
Breast Cancer Res 2019; 21(1):131BC

Abstract

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 (NFκB) 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 NFκB signaling and COX-2.

METHODS

For in vitro experiments, reporter luciferase assays were utilized in MCF7 cells to investigate promoter activity of NFκB 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 NFκB as measured using NFκB-luciferase reporter assay. Furthermore, immunostaining of lysates from breast cancer cells overexpressing SIM2s showed reduction in various NFκB signaling proteins, as well as pAkt, whereas knockdown of SIM2 revealed increases in NFκB signaling proteins and pAkt. Additionally, we show that NFκB signaling can act in a reciprocal manner to decrease expression of SIM2s. Likewise, suppressing NFκB translocation in DCIS.COM cells increased SIM2s expression. We also found that NFκB/p65 represses SIM2 in a 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 NFκB-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 NFκB signaling and COX-2 expression.

Authors+Show Affiliations

Department of Integrative Biosciences, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.Department of Medicine, Division of Medical Oncology, University of Colorado Anschutz Medical Campus, Aurora, USA. The University of Colorado Cancer Center Young Women's Breast Cancer Translational Program, Aurora, CO, USA.Department of Medicine, Division of Medical Oncology, University of Colorado Anschutz Medical Campus, Aurora, USA. The University of Colorado Cancer Center Young Women's Breast Cancer Translational Program, Aurora, CO, USA.Department of Medicine, Division of Medical Oncology, University of Colorado Anschutz Medical Campus, Aurora, USA. The University of Colorado Cancer Center Young Women's Breast Cancer Translational Program, Aurora, CO, USA.Department of Integrative Biosciences, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.Department of Integrative Biosciences, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.Department of Integrative Biosciences, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.Department of Nutrition, Texas A&M University, College Station, TX, USA.Department of Nutrition, Texas A&M University, College Station, TX, USA.Department of Integrative Biosciences, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA. wporter@cvm.tamu.edu.Department of Medicine, Division of Medical Oncology, University of Colorado Anschutz Medical Campus, Aurora, USA. traci.lyons@ucdenver.edu. The University of Colorado Cancer Center Young Women's Breast Cancer Translational Program, Aurora, CO, USA. traci.lyons@ucdenver.edu.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

31783895

Citation

Wyatt, Garhett L., et al. "Cross-talk Between SIM2s and NFκB Regulates Cyclooxygenase 2 Expression in Breast Cancer." Breast Cancer Research : BCR, vol. 21, no. 1, 2019, p. 131.
Wyatt GL, Crump LS, Young CM, et al. Cross-talk between SIM2s and NFκB regulates cyclooxygenase 2 expression in breast cancer. Breast Cancer Res. 2019;21(1):131.
Wyatt, G. L., Crump, L. S., Young, C. M., Wessells, V. M., McQueen, C. M., Wall, S. W., ... Lyons, T. R. (2019). Cross-talk between SIM2s and NFκB regulates cyclooxygenase 2 expression in breast cancer. Breast Cancer Research : BCR, 21(1), p. 131. doi:10.1186/s13058-019-1224-y.
Wyatt GL, et al. Cross-talk Between SIM2s and NFκB Regulates Cyclooxygenase 2 Expression in Breast Cancer. Breast Cancer Res. 2019 11 29;21(1):131. PubMed PMID: 31783895.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cross-talk between SIM2s and NFκB regulates cyclooxygenase 2 expression in breast cancer. AU - Wyatt,Garhett L, AU - Crump,Lyndsey S, AU - Young,Chloe M, AU - Wessells,Veronica M, AU - McQueen,Cole M, AU - Wall,Steven W, AU - Gustafson,Tanya L, AU - Fan,Yang-Yi, AU - Chapkin,Robert S, AU - Porter,Weston W, AU - Lyons,Traci R, Y1 - 2019/11/29/ PY - 2019/05/09/received PY - 2019/11/07/accepted PY - 2019/12/1/entrez PY - 2019/12/1/pubmed PY - 2019/12/1/medline KW - Breast cancer KW - COX-2 KW - Inflammation KW - NFκB KW - Prostaglandin E2 KW - SIM2 SP - 131 EP - 131 JF - Breast cancer research : BCR JO - Breast Cancer Res. VL - 21 IS - 1 N2 - 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 (NFκB) 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 NFκB signaling and COX-2. METHODS: For in vitro experiments, reporter luciferase assays were utilized in MCF7 cells to investigate promoter activity of NFκB 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 NFκB as measured using NFκB-luciferase reporter assay. Furthermore, immunostaining of lysates from breast cancer cells overexpressing SIM2s showed reduction in various NFκB signaling proteins, as well as pAkt, whereas knockdown of SIM2 revealed increases in NFκB signaling proteins and pAkt. Additionally, we show that NFκB signaling can act in a reciprocal manner to decrease expression of SIM2s. Likewise, suppressing NFκB translocation in DCIS.COM cells increased SIM2s expression. We also found that NFκB/p65 represses SIM2 in a 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 NFκB-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 NFκB signaling and COX-2 expression. SN - 1465-542X UR - https://www.unboundmedicine.com/medline/citation/31783895/Cross-talk_between_SIM2s_and_NFκB_regulates_cyclooxygenase_2_expression_in_breast_cancer L2 - https://breast-cancer-research.biomedcentral.com/articles/10.1186/s13058-019-1224-y DB - PRIME DP - Unbound Medicine ER -