HER2 Phosphorylation Is Maintained by a PKB Negative Feedback Loop in Response to Anti-HER2 Herceptin in Breast Cancer
Figure 5
Combination of Herceptin with ADAM inhibitors decreases HER2 phosphorylation and is additive in cell viability inhibition.
(A) SKBR3 cells were treated with 100 µM ADAM17 inhibitor (inh), 100 µM ADAM10/17 inhibitor, 100 µM ADAM inhibitor TAPI-1, or a combination of TAPI-1 and 40 µg/ml Herceptin (Herc) for 1 h. Cells were then lysed, and equal amounts of protein were loaded on an SDS gel. Membrane was probed for phosphorylated HER2 and actin. (B) FRET experiments to assess HER2 phosphorylation in SKBR3 cells. The cells were treated with 40 µg/ml Herceptin for 1 h with or without TAPI-1. The medians of the lifetimes were compared with the basal condition using the Mann-Whitney test. (C) SKBR3 cells were transfected with siRNA against ADAM17 or a control sequence. Three days after transfection, cells were treated with 40 µg/ml Hercpetin for 1 h. Lysates were loaded on an SDS gel. The membrane was probed for pHER2 and actin. (D) BT474 cells were grown in 24-well plates and left to grow for at least 24 h before being treated for different durations with 40 µg/ml Herceptin, 10 µM TAPI-1, 10 µM ADAM17 inhibitor, or a combination of ADAM inhibitors with Herceptin, as illustrated. The viable cells were counted using a cell counter.