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

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 β-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.