Combined inhibition of AKT and ER suppresses hormone-independent tumor growth. A) LTED cells were treated with 10% DCC-FBS ± 2 µM AZD5363 for 24 hours. RNA was extracted, reverse transcribed to cDNA and analyzed by real-time PCR. Threshold cycle values were normalized for actin. Data are presented as fold versus control; each bar, mean ± SEM (n = 2; *P <0.0001 versus Con, two-way ANOVA). B) LTED cells were treated with 10% DCC-FBS ± 2 µM AZD5363, 1 µM fulvestrant or 1 nM E2. Media and drugs were replenished every three days. Cells were counted after five days. Data are presented as percent of control; each bar, mean ± SEM (n = 3; *P <0.0001 versus Con or E2; #P <0.01 versus AZD or fulvestrant; ^P <0.01 versus E2 + fulvestrant, one-way ANOVA). C) MCF-7 cells were injected s.c. into athymic mice supplemented with 14-day release 17β-estradiol pellets. Mice bearing tumors ≥150 mm3 were randomized to vehicle, AZD5363 (150 mg/kg/day bid), fulvestrant (5 mg/wk), or AZD5363 + fulvestrant for six weeks. Data are presented as mean tumor volume ± SEM (*P <0.01 versus vehicle; #P <0.001 versus AZD or fulvestrant, t-test). D) Xenografts from C) were homogenized one hour after the last dose and tumor lysates were analyzed by immunoblot using the indicated antibodies. ANOVA, analysis of variance; DCC-FBS, dextran/charcoal-treated fetal bovine serum; LTED, long-term estrogen deprivation; SEM, standard error of the mean.