Hyperactivation of MAPK induces loss of ERα expression in breast cancer cells

ER alpha -negative breast tumors tend to overexpress growth factor receptors such as epidermal growth factor receptor or c-erbB-2. Raf-1 is a key intermediate in the signal transduction pathways of these receptors. High levels of constitutive Raf kinase (Delta raf) activity imparts ER alpha- positive MCF-7 breast cancer cells with the ability to grow in the absence of estrogen. Delta raf transfectants maintained in estrogen-depleted media showed greatly diminished responses to 17 beta -estradiol or the pure antiestrogen ICI 182,780. Western blotting, ligand binding, and immunohistochemistry assays revealed a loss of ER alpha protein expression, and ribonuclease protection assays indicated that this correlated with loss of ER alpha message. In examining the basal expression of estrogen-induced genes in the stable transfectants or in transient cotransfection assays with an estrogen-response element-reporter construct and Delta raf or constitutively active MAPK kinase (Delta MEK), no ligand- independent activation of ER alpha was observed. Transient expression of Delta raf and double-label immunostaining showed ER alpha was lost in those cells that transiently expressed Delta raf. Abrogation of Raf signaling via treatment with the MEK inhibitors PD 098059 or U0126 resulted in reexpression of ERa. Similar studies performed with MCF-7 cells overexpressing epidermal growth factor receptor or c-erbB-2 confirmed that hyperactivation of MAPK resulted in down-regulation of ER alpha that was reversible by MEK inhibition or transfection with dominant negative ERK1 and ERK2 constructs. These data suggest that the hyperactivation of MAPK in epidermal growth factor receptor- or c-erbB-2-overexpressing breast cancer cells is directly responsible for generation of an ER alpha -negative phenotype and, more importantly, that this process may be abrogated by inhibiting these pathways, thus restoring ER alpha expression.