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2-Methoxyestradiol Induces Mammary Gland Differentiation through Amphiregulin-Epithelial Growth Factor Receptor-Mediated Signaling: Molecular Distinctions from the Mammary Gland of Pregnant Mice

Laboratories of Cell Regulation and Carcinogenesis (J.-I.H., T.H.Q., R.C., J.E.G.), Biosystems and Cancer (G.V.R.C.), and Receptor Biology and Gene Expression (M.W., G.L.H.), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; Division of Oncology (R.C., A.C.), Center for Applied Medical Research (CIMA), and Department of Histology and Pathology, University of Navarra, Pamplona, 31080 Spain; EntreMed, Inc. (T.M.L.), Rockville, Maryland 20850; and California Pacific Medical Center (P.-Y.D.), Cancer Research Institute, San Francisco, California 94143

Address all correspondence and requests for reprints to: Jeffrey E. Green, Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, National Institutes of Health, Building 41, Room C629, 41 Medlars Drive, Bethesda, Maryland 20892. E-mail: jegreen{at}nih.gov.

Levels of 2-methoxyestradiol (2ME₂), an endogenous metabolite of estradiol, are highly elevated during late stages of pregnancy when mammary glands have differentiated with the formation of alveolar structures producing milk proteins. Based upon our previous demonstration that 2ME₂ induces mammary ductal dilation associated with expression of mammary differentiation markers when administered to transgenic mice that spontaneously develop mammary cancer, we studied the effects of 2ME₂ on normal mammary gland development. The results of this study demonstrate that 2ME₂ can induce a partial differentiation of normal mammary glands in virgin mice, as evidenced by the appearance of limited numbers of alveolar cells and significantly increased expression of the differentiation markers ß-casein and whey acidic protein. 2ME₂-induced differentiation is associated with inhibition of expression of inhibitor of differentiation 1 (Id-1) in normal mammary epithelial cells through elements in the 5'-flanking region of the Id-1 gene. Microarray analysis revealed that 2ME₂-induced differentiation of the mammary gland shares some significant similarities in gene expression with that of mammary glands from late-stage pregnancy, including elevated expression of many milk protein differentiation markers. However, several genes are differentially regulated between 2ME₂-treated mammary glands and differentiated mammary glands through pregnancy. Significantly, amphiregulin, ATF3, serpine2, and SOX6 were up-regulated in 2ME₂-treated mammary glands but not in mammary glands from pregnant mice. Using the SCp2 differentiation cell line system, we demonstrate that 2ME₂ induces differentiation through the down-regulation of Id-1 and up-regulation of amphiregulin. Administration of amphiregulin to SCp2 cells induced differentiation, whereas inhibition of 2ME₂-induced expression of amphiregulin by small interfering RNA blocked differentiation. Estrogen receptor-negative SCp2 cells differentiate in response to 2ME₂, but not estradiol, suggesting that 2ME₂ operates through an estrogen receptor-independent mechanism. These data demonstrate that 2ME₂ can induce a partial differentiation of the mammary gland through mechanisms that differ from those normally used during pregnancy.