Activation of mitogen-activated protein kinase in xenografts and cells during prolonged treatment with aromatase inhibitor letrozole

Ovariectomized mice bearing tumor xenografts grown from aromatase-transfected estrogen receptor (ER)-positive human breast cancer cells (MCF-7Ca) were injected s.c. with 10 mu g/d letrozole for up to 56 weeks. Western blot analysis of the tumors revealed that ERs (ER alpha) were increased at 4 weeks but decreased at weeks 28 and 56. Expression of erbB-2 and p-Shc increased throughout treatment, whereas growth factor receptor binding protein 2 (Grb2) increased only in tumors proliferating on letrozole (weeks 28 and 56). In cells isolated from tumors after 56 weeks and maintained as a cell line (LTLT-Ca) in 1 mu mol/L letrozole, ER alpha was also decreased whereas erbB-2, adapter proteins (p-Shc and Grb2), and the signaling proteins in the mitogen-activated protein kinase (MAPK) cascade were increased compared with MCF-7Ca cells. Growth was inhibited in LTLT-Ca cells but not in MCF-7Ca cells treated with MAPK kinase 1/2 inhibitors U0126, and PD98059 (IC50 similar to 25 mu mol/L). PD98059 (5 mu mol/L) also reduced MAPK activity and increased ER alpha to the levels in MCF-7Ca cells. Epidermal growth factor receptor kinase inhibitor, gefitinib (ZD1839) inhibited growth of LTLT-Ca cells (IC50 similar to 10 mu mol/L) and restored their sensitivity to tamoxifen and anastrozole. In xenografts, combined treatment with ER down-regulator fulvestrant and letrozole, prevented increases in erbB-2 and activation of MAPK and was highly effective in inhibiting tumor growth throughout 29 weeks of treatment. These results indicate that blocking both ER- and growth factor-mediated transcription resulted in the most effective inhibition of growth of ER-positive breast cancer cells.