TY - JOUR
T1 - Epigenetic reprogramming governs EcSOD expression during human mammary epithelial cell differentiation, tumorigenesis and metastasis
AU - Teoh-Fitzgerald, M. L.
AU - Fitzgerald, M. P.
AU - Zhong, W.
AU - Askeland, R. W.
AU - Domann, F. E.
N1 - Funding Information:
The authors thank Dr James Crapo (National Jewish Medical and Research Center, Denver, CO, USA) for providing antibodies to EcSOD and Dr Michael Henry (Department of Molecular Physiology and Biophysics, University of Iowa, IA, USA) for providing the 4T1.luc cells. We also thank Justin Fishbaugh from the Flow Cytometry Core Facility for assisting with the DCFH assays as well as the Central Microscopy Research Facility. This study was supported by NIH grant R01 CA073612 and R01 CA115438 (FE Domann), Susan G Komen for the Cure grant KG080437 (M Teoh-Fitzgerald), SFRBM Research Mini-Fellowship (M Teoh-Fitzgerald), Oberley Seed Grant (M Teoh-Fitzgerald), and Carver Research Program of Excellence in Redox Biology and Medicine, and by the Holden Comprehensive Cancer Center Breast Cancer Research Fund.
PY - 2014/1/16
Y1 - 2014/1/16
N2 - Expression of the antioxidant enzyme EcSOD in normal human mammary epithelial cells was not recognized until recently. Although expression of EcSOD was not detectable in non-malignant human mammary epithelial cells (HMEC) cultured in conventional two-dimensional (2D) culture conditions, EcSOD protein expression was observed in normal human breast tissues, suggesting that the 2D-cultured condition induces a repressive status of EcSOD gene expression in HMEC. With the use of laminin-enriched extracellular matrix (lrECM), we were able to detect expression of EcSOD when HMEC formed polarized acinar structures in a 3D-culture condition. Repression of the EcSOD-gene expression was again seen when the HMEC acini were sub-cultured as a monolayer, implying that lrECM-induced acinar morphogenesis is essential in EcSOD-gene activation. We have further shown the involvement of DNA methylation in regulating EcSOD expression in HMEC under these cell culture conditions. EcSOD mRNA expression was strongly induced in the 2D-cultured HMEC after treatment with a DNA methyltransferase inhibitor. In addition, epigenetic analyses showed a decrease in the degree of CpG methylation in the EcSOD promoter in the 3D versus 2D-cultured HMEC. More importantly, >80% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA and protein expression levels compared with normal mammary tissues and there is an inverse correlation between the expression levels of EcSOD and the clinical stages of breast cancer. Combined bisulfite restriction analysis analysis of some of the tumors also revealed an association of DNA methylation with the loss of EcSOD expression in vivo. Furthermore, overexpression of EcSOD inhibited breast cancer metastasis in both the experimental lung metastasis model and the syngeneic mouse model. This study suggests that epigenetic silencing of EcSOD may contribute to mammary tumorigenesis and that restoring the extracellular superoxide scavenging activity could be an effective strategy for breast cancer treatment.
AB - Expression of the antioxidant enzyme EcSOD in normal human mammary epithelial cells was not recognized until recently. Although expression of EcSOD was not detectable in non-malignant human mammary epithelial cells (HMEC) cultured in conventional two-dimensional (2D) culture conditions, EcSOD protein expression was observed in normal human breast tissues, suggesting that the 2D-cultured condition induces a repressive status of EcSOD gene expression in HMEC. With the use of laminin-enriched extracellular matrix (lrECM), we were able to detect expression of EcSOD when HMEC formed polarized acinar structures in a 3D-culture condition. Repression of the EcSOD-gene expression was again seen when the HMEC acini were sub-cultured as a monolayer, implying that lrECM-induced acinar morphogenesis is essential in EcSOD-gene activation. We have further shown the involvement of DNA methylation in regulating EcSOD expression in HMEC under these cell culture conditions. EcSOD mRNA expression was strongly induced in the 2D-cultured HMEC after treatment with a DNA methyltransferase inhibitor. In addition, epigenetic analyses showed a decrease in the degree of CpG methylation in the EcSOD promoter in the 3D versus 2D-cultured HMEC. More importantly, >80% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA and protein expression levels compared with normal mammary tissues and there is an inverse correlation between the expression levels of EcSOD and the clinical stages of breast cancer. Combined bisulfite restriction analysis analysis of some of the tumors also revealed an association of DNA methylation with the loss of EcSOD expression in vivo. Furthermore, overexpression of EcSOD inhibited breast cancer metastasis in both the experimental lung metastasis model and the syngeneic mouse model. This study suggests that epigenetic silencing of EcSOD may contribute to mammary tumorigenesis and that restoring the extracellular superoxide scavenging activity could be an effective strategy for breast cancer treatment.
KW - DNA methylation
KW - acinar morphogenesis
KW - experimental lung metastasis
KW - laminin-enriched extracellular matrix
KW - spontaneous metastasis
KW - three-dimensional culture
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U2 - 10.1038/onc.2012.582
DO - 10.1038/onc.2012.582
M3 - Article
C2 - 23318435
AN - SCOPUS:84892665190
SN - 0950-9232
VL - 33
SP - 358
EP - 368
JO - Oncogene
JF - Oncogene
IS - 3
ER -