Figure 6. ADAM10 inhibition or knockdown decreases activation of HER receptors and cell viability in trastuzumab resistant cell lines.

All resistant cells were continuously treated with 40μg/ml trastuzumab unless otherwise stated. (A) Resistant SKBR3 cells were treated with 5μM ADAM10 inhibitor INCB8765 (ADAM10i) for 24h in serum-free media or (B) were transfected with 20nM of siRNAs against ADAM10 for 72h before western blotting and quantification of three blots is shown next to the respective blot (phosphorylated proteins relative to the respective total proteins). (C) For FACS analysis, resistant BT474 cells were treated with 5μM ADAM10 inhibitor INCB8765 (ADAM10i) in serum-reduced media for 5 days. Camptothecin (6μM) was used as positive control. Cells were stained for Annexin V and percentage of positive cells analysed by FACS. (D) For the clonogenic assay, resistant SKBR3 cells were treated as in (C) and re-plated in duplicate for colony-formation for 12 days. (E) For cell counting studies, trastuzumab was withdrawn overnight from resistant SKBR3 cell lines. For the “withdrawal group”, trastuzumab remained withdrawn whereas the “continuous group” was co-treated with 40μg/ml trastuzumab. Both groups were treated in as in (C), or were transfected with 20nM of specific siRNAs against ADAM10 for 5 days (right panel). (F) BT474 and SKBR3 cells were treated in triplicate with 40μg/ml trastuzumab, 5μM of the ADAM10 inhibitor INCB8765 (ADAM10i), the ADAM17 inhibitor INCB4298 (ADAM17i), the ADAM10/17 inhibitor INCB3619 (ADAM10/17i), or their combination for 5 days before cell counting. (G) BT474 resistant cells were treated as in (F) with continuous 40μg/ml trastuzumab for 5 days before cell counting. Graphs represent data from three independent experiments and show means ± SD.