Re-expression of estrogen receptor alpha in estrogen receptor alpha-negative MCF-7 cells restores both estrogen and insulin-like growth factor-mediated signaling and growth

Estrogen can increase insulin-like growth factor-I receptor (IGF-IR) and in sulin receptor substrate-1 (IRS-1) expression, two key components of IGF-I- mediated signaling. The result is sensitization of breast cancer cells to I GF-I and synergistic growth in the presence of estrogen and IGF-L We hypoth esized that loss of estrogen receptor alpha (ER alpha) would result in redu ced IGF-mediated signaling and growth. To test this hypothesis, we examined IGF-I effects in MCF-7 breast cancer cell sublines that have been selected for loss of ER alpha (C4 and C4-12 cells are ER alpha -negative) by long-t erm estrogen withdrawal. C4 and C4-12 cells had reduced IGF-IR and IRS-1 mR NA and protein expression (compared with MCF-7 cells) that was not inducibl e by estrogen. Furthermore, C4 and C4-12 cells showed reduced IGF-I signali ng and failed to show any growth response to either estrogen or IGF-I. To p rove that loss of IGF and estrogen-mediated signaling and growth was a cons equence of loss of ER alpha, we re-expressed ER alpha in C4-12 cells by sta ble transfection with HA-tagged ER alpha. Three independent C4-12 ER alpha -HA clones expressed a functional ER alpha that (a) was down-regulated by e strogen, (b) conferred estrogen-induction of cyclin DI expression, and (c) caused estrogen-mediated increase in the number of cells in S phase. All of the effects were completely blocked by antiestrogens. Interestingly, ER al pha -HA expression in C4-12 cells did not restore estrogen induction of pro gesterone receptor expression. However, ER alpha -positive C4-12 cells now exhibited estrogen-induction of IGF-IR and IRS-1 levels and responded mitog enically to both estrogen and IGF-I. These data show that ER alpha is a cri tical requirement for IGF signaling, and to our knowledge this is the first report of functional ER alpha expression that confers estrogen-mediated gr owth of an ER-negative breast cancer cell line.