Figure 6
Identification of a HER2 mutation as a potential driver genetic alteration in the HER2-negative component of a HER2 heterogeneous breast cancer. (A) Cell-free in vitro kinase assay determining the tyrosine kinase activity of the Poly(Glu4-Tyr) substrate and the autophosphorylation activity of wild-type (WT) HER2 (dark gray) and I767M mutant HER2 (light gray) in the presence and absence of neuregulin-1 (NRG1). Tyrosine kinase activity was assessed using the ADP Hunter HS Assay (DiscoveRx, left). Western blot analysis of representative elutes post-kinase assay of HER2-tagRFP, HER2(I767M)-tagRFP and tagRFP control proteins. The amounts of HER2 wild-type and I767M mutant HER2 enzymes used in the DiscoveRx kinase assay were similar as confirmed using antibodies against total HER2 (top panel) and tagRFP (bottom panel). ****P < 0.0001, Holm-Å Ãdák-correction, multiple t-test. Error bars represent standard deviation of mean. (B) The Tyrosine Kinase Assay Kit (Millipore) confirmed the significantly higher transphosphorylation of the tyrosine kinase substrate Poly(Glu4-Tyr) by I767M mutant HER2 (light gray) compared with wild-type HER2 (dark gray). ***P < 0.001, Holm-Å Ãdák-correction, multiple t-test. Error bars represent standard deviation of mean. (C) Foci formation assay of NIH3T3 cells stably expressing empty vector, wild-type HER2 or I767M mutant HER2 protein. Cells were fixed and stained with crystal violet 5 and 12 days after plating. Quantification was performed at day 5. Note that at day 12, the wild-type HER2 resulted in increased foci formation. ***P < 0.001, unpaired t-test. Error bars represent standard deviation of mean. (D) Anchorage-independent growth of MCF10A cells stably expressing empty vector, wild-type HER2 or I767M mutant HER2 protein. Quantification was performed using an MTT assay (left) or by defining the number and size of colonies (right). *P < 0.05, **P < 0.01, ****P < 0.0001, unpaired t-test. N.s., not significant. Error bars represent standard deviation of mean. (E) Effect of stable expression of wild-type HER2 (blue) and I767M mutant HER2 (red) on survival and growth of ER-positive MCF7 (PIK3CA mutant) and T47D (PIK3CA and TP53 mutant) cells in growth media with or without neuregulin-1 (NRG1, 10 ng/ml). **P < 0.01, ***P < 0.001, Holm-Å Ãdák-correction, multiple t-test. Error bars represent standard deviation of mean.