Figure 3. Targeting NRG1 signaling overcomes resistance to trametinib in UM cells.

(A) ERBB3 antibody, U3-1287, blocks NRG1-induced activation of ERBB3 and AKT. UM001 cells were treated with 1 μg/ml or 10 μg/ml of U3-1287 for either 1 hr or 24 hr followed by 10 ng/ml NRG1 stimulation for 15 min. Levels of ERBB3, AKT and ERK1/2 phosphorylation were evaluated by Western blotting with the indicated antibodies. (B) NRG1-induced resistance to trametinib is reversed by U3-1287. UM001 cells were first treated with 100 nM trametinib for 24 hr. Cells were then washed and treated with 10 μg/ml U3-1287 for 45 min, followed by 10 ng/ml NRG1 and 100 nM trametinib for 72 hr. Cells were stained with crystal violet. Representative microscopic images are shown (200 × magnifications). Scale bar is equal to 50 μm (left). (C) UM001 cells were treated as in B. Cell viability was assessed by AlamarBlue® staining. *P<0.05, **P<0.01, ***P<0.001 based on two-tail Student's t-test assuming unequal variance. (D) NRG1-induced resistance was overcome by lapatinib. UM001 cells were treated with 100 nM trametinib, 10 ng/ml of NRG1 or together with 1 μM lapatinib, as indicated for 72 hr. Cells were stained and representative images shown. (E) UM001 cells were treated as in D. Cell viability was assessed by AlamarBlue® staining. *P<0.05, **P<0.01, ***P<0.001, based on two-tail Student's t-test assuming unequal variance. (F) UM003 cells were treated with 100 nM trametinib, 10 ng/ml NRG1 or together with 1 μM lapatinib, as indicated for a total of 5 days. Culture medium was changed and new drug and growth factors were added on day 3. Cell viability was assessed by AlamarBlue® staining. *P<0.05, **P<0.01, ***P<0.001, based on two-tail Student's t-test assuming unequal variance.