Fig. 3. Dual blockade of ER and HER2 in ER+/HER2-mutant cells.

(A) The indicated cells were grown in complete medium ± 1 μM fulvestrant ± 200 nM neratinib. Cell counts were taken on day 12. Data represent the average ± SD of 3 replicate wells. Statistical comparison between HER2 mutant cells treated with single drugs and DMSO-treated controls are shown as percent reduction (**p<0.01, ****p<0.0001, ANOVA with Tukey’s multiple comparison). For the combination of neratinib/fulvestrant (red), statistical comparisons were to fulvestrant (green) (*p<0.05, **p<0.01, ANOVA with Tukey’s multiple comparison). Experiment was repeated 3 times. (B) Synergistic growth assay. MCF7^V777L were treated with a dose range (0, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, and 1000 nM) of fulvestrant and neratinib alone and in combination. Data represent the average of 3 replicate plates. The combination Index value 1 μM fulvestrant and 250 nM neratinib (indicated by the bold box) was .22 and considered to be synergistic. (C) The indicated cells were plated in 3D matrigel. Estrogen deprived media ± 1 nM estradiol ± 1 μM fulvestrant ± 200 nM neratinib, was used as indicated. Media and growth factors were changed every 3 days. Colonies (≥50 μM) were imaged on Day 12. Representative images shown. Assay was carried out in duplicate wells in 3 separate experiments. (D) 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 ± 200 nM neratinib. 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. (E) Gene expression analysis using Reverse Transcription (RT) Profiler of genes regulated by ERα in MCF^WT and MCF7^V777L cells as described in Methods. Bar graphs are an average of 3 separate wells. Fold change in gene expression between MCF7^V777L and MCF^WT cells is plotted in the Y axis. Genes with a change in expression ≥2 are labeled.