Figure 9: ErbB2 homodimers stimulate RAS-mediated AKT reactivation.

(a–c) HRAS/KRAS knockdown in combination with TZB was sufficient to abolish the p-AKT rebound (a), to block proliferation (b) and to induce apoptosis (c) cf. (Supplementary Fig. 5E–H). Analysis was done 72 h after knockdown of HRAS/KRAS by siRNA cocktail in combination with 100 nM of TZB or 6L1G (a–c). Note that HRAS/KRAS knockdown robustly blocks p-ERK in the presence of an active ErbB3/PI3K/AKT pathway (cf. Supplementary Fig. 5G) (*P value in b Mann–Whitney test; data in c are mean±s.e.m.,*P value in c two-sided, unpaired Student's t-test). (d) ErbB2 homodimers depend on HRAS/KRAS expression to mediate the p-AKT rebound in the absence of ErbB3 expression. Double knockdown of ErbB3 and RAS results in effective p-AKT and p-ERK inhibition and blocks GL4G-induced p-AKT and p-ERK stimulation completely after 72 h of treatment. (e–g) Grb2 knockdown in combination with TZB-induced apoptosis by inhibition of the p-AKT rebound. BT474 cells were treated for 72 h with 100 nM TZB or 6L1G in combination with Grb2 knockdown by siRNA. Immunoblot detection (e), XTT cell proliferation assays (f) and cell cycle (g) (*P value in e Mann–Whitney test). (h) Detection of RAS-GTP by pull-down assay with GST-RBD. BT474 cells were treated for 72 h with 100 nM TZB or 6L1G in combination with siGrb2 knockdown or 12.5 μM ARRY-380 (top), or 50 nM GL4G or zH2-DHLX in combination with either 5 μM lonafarnib or 12.5 μM ARRY-380 (bottom).