Fig. 2. Activating ERBB2 mutations generate resistance to anti-ER therapies.

(A) Isogenically modified MCF7 cells with incorporated HER2^WT (targeted WT), ERBB2^G309A, ERBB2^L755S and ERBB2^V777L into the host genome, were grown in estrogen deprived medium [5% charcoal-stripped serum (CSS), phenol-red free]. Cell counts were taken on day 8. Data represent the average ± SD of 3 replicate wells. Statistical comparisons were between HER2 mutant and HER WT cells (****p<0.0001, ANOVA). Experiment was repeated 3 times. (B) Growth in estrogen-deprived medium (60 days) was carried out in 100-mm dishes. Passage number was recorded for each cell line. (C) Representative crystal violet-stained monolayers of cells grown for 28 days in estrogen-deprived medium. (D) Cells were treated with 1 μM fulvestrant in medium containing 10% FBS. Cell counts were taken on day 8 and are shown as % of vehicle-treated controls for each cell line. Data represent the average ± SD of 3 replicate wells. Statistical comparisons were between HER2 mutant and HER WT cells (****p<.0001, ANOVA). Experiment was repeated 3 times. (E) Cells were plated in estrogen deprived medium and transfected with pERE (Estrogen Responsive Elements)-luciferase and pCMV-Renilla plasmids; 24 h post transfection, cells were treated ± 1 nM estradiol ± 1 μM fulvestrant. Luciferase activity was measured 24 h later using the Dual Luciferase Kit (Promega) as described in Methods. Data represent the average ± SD of 4 replicate wells. The experiment was repeated three times. (F) Cells were grown in full medium ± 1 μM fulvestrant for 24 h. Cell lysates were prepared and subjected to immunoblot analyses with the indicated antibodies.