ERa-negative breast tumors tend to overexpress growth factor receptors such as epidermal growth factor receptor or c-erbB-2. Raf-1 is a key inter- mediate in the signal transduction pathways of these receptors. High levels of constitutive Raf ki- nase (Draf) activity imparts ERa- positive MCF-7 breast cancer cells with the ability to grow in the absence of estrogen. Draf transfectants main- tained in estrogen-depleted media showed greatly diminished responses to 17b-estradiol or the pure antiestrogen ICI 182,780. Western blotting, ligand binding, and immunohistochemistry assays re- vealed a loss of ERa protein expression, and ribo- nuclease protection assays indicated that this cor- related with loss of ERa message. In examining the basal expression of estrogen-induced genes in the stable transfectants or in transient cotransfec- tion assays with an estrogen-response element- reporter construct and Draf or constitutively active MAPK kinase (DMEK), no ligand- independent acti- vation of ERa was observed. Transient expression of Draf and double-label immunostaining showed ERa was lost in those cells that transiently ex- pressed Draf. Abrogation of Raf signaling via treat- ment with the MEK inhibitors PD 098059 or U0126 resulted in reexpression of ERa. Similar studies performed with MCF-7 cells overexpressing epi- dermal growth factor receptor or c-erbB-2 con- firmed that hyperactivation of MAPK resulted in down-regulation of ERa that was reversible by MEK inhibition or transfection with dominant neg- ative ERK1 and ERK2 constructs. These data sug- gest that the hyperactivation of MAPK in epidermal growth factor receptor- or c-erbB-2-overexpressing breast cancer cells is directly responsible for gener- ation of an ERa-negative phenotype and, more im- portantly, that this process may be abrogated by inhibiting these pathways, thus restoring ERa ex- pression. (Molecular Endocrinology 15: 1344-1359, 2001)