Inhibition of IGF-IR/InsR enhances the anti-tumor effect of the AKT inhibitor AZD5363. A) MCF-7 cells were transfected with siRNA specific for a non-silencing control (siCon), InsR, IGF-IR or HER3 and re-seeded the next day for assessment of growth in monolayer (A) or immunoblot analyses (B). In A, cells were treated with 10% DCC-FBS ± 2 µM AZD5363 and counted after five days. Data are presented as % of control; mean ± SEM (n = 3; *P <0.0001 versus each Con, #P <0.01 versus siCon + AZD; two-way ANOVA). In B, cells were grown in 10% DCC-FBS and harvested three days later; lysates were analyzed by immunoblot using the indicated antibodies. C) MCF-7/LTED cells in 10% DCC-FBS were treated ± 2 µM AZD5363 or 1 µM AZD9362. Media and drugs were replenished every three days. Cells were counted after five days. Data are presented as % of control; mean ± SEM (n = 2; *P <0.0001 versus Con, #P <0.05 versus 5363 or 9362; one-way ANOVA). D) Mice bearing MCF-7 xenografts ≥150 mm3 were randomized to the indicated treatments. Data are presented as mean tumor volume ± SEM (*P <0.05 versus vehicle; #P = 0.0041 versus AZD5363, t-test). ANOVA, analysis of variance; DCC-FBS, dextran/charcoal-treated fetal bovine serum; IGF, insulin-like growth factor; LTED, long-term estrogen deprivation; SEM, standard error of the mean.