Hyperactivation of MAPK induces loss of ER alpha expression in breast cancer cells

ER alpha -negative breast tumors tend to overexpress growth factor receptor s such as epidermal growth factor receptor or c-erbB-2. Raf-1 is a key inte rmediate in the signal transduction pathways of these receptors. High level s of constitutive Raf kinase (Delta raf) activity imparts ER alpha- positiv e MCF-7 breast cancer cells with the ability to grow in the absence of estr ogen. 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 as says revealed a loss of ER alpha protein expression, and ribonuclease prote ction assays indicated that this correlated with loss of ER alpha message. In examining the basal expression of estrogen-induced genes in the stable t ransfectants or in transient cotransfection assays with an estrogen-respons e element-reporter construct and Delta raf or constitutively active MAPK ki nase (Delta MEK), no ligand- independent activation of ER alpha was observe d. Transient expression of Delta raf and double-label immunostaining showed ER alpha was lost in those cells that transiently expressed Delta raf. Abr ogation 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 confirm ed that hyperactivation of MAPK resulted in down-regulation of ER alpha tha t was reversible by MEK inhibition or transfection with dominant negative E RK1 and ERK2 constructs. These data suggest that the hyperactivation of MAP K in epidermal growth factor receptor- or c-erbB-2-overexpressing breast ca ncer cells is directly responsible for generation of an ER alpha -negative phenotype and, more importantly, that this process may be abrogated by inhi biting these pathways, thus restoring ER alpha expression.