Figure 6.

Brk confers resistance of breast cancer cells to EGFR inhibition. (a) Knockdown of Brk reduces breast cancer cell response to EGF stimulation. SUM102 cells were subjected to treatment with Brk-specific siRNA or control siRNA for 48 h prior to serum starvation for overnight. The next day, the cells were treated with 10 nM EGF or not for 30 min and harvested for Western blotting with the indicated antibodies. (b) Knockdown of Brk sensitizes breast cancer cells to cetuximab. SUM102 cells were subjected to Brk knockdown as in (a) and then treated with 20 nM cetuximab or not for 24 h. Cell lysates were prepared for apoptosis enzyme-linked immunosorbent assay and Western blotting with the indicated antibodies. O.D., optical density. (c) Overexpression of Brk confers resistance to cetuximab-induced inhibition of cell signaling. SUM102 cells selected for overexpression of wild-type Brk and corresponding control cells were treated with the indicated concentrations of cetuximab in 0.5% FBS culture medium for 16 h. Whole cell lysates were subjected to Western blotting with the indicated antibodies. (d) Overexpression of Brk confers resistance to cetuximab-induced growth inhibition. SUM102 cells selected for overexpression of wild-type Brk or Brk-Y447F and corresponding control cells were treated with the indicated concentrations of cetuximab for 5 days. Cell survival and proliferation after cetuximab treatment were measured with an MTT assay and plotted as a percentage of the optical density at 570 nm of the untreated cells. ■ = Control vector-transfected cells; ● = wild-type-Brk-transfected cells; ▲ = Brk-Y447F-transfected cells.