Inhibition of AKT results in feedback upregulation of RTKs. 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. Average fold changes over control (Con) were calculated and used to generate a heatmap. B) LTED cells were treated for 24 hours in 10% DCC-FBS ± 15 µM AZD5363. Protein lysates were analyzed by immunoblot. Densitometric analysis was performed and the ratio of InsR:actin is shown below the InsR blots. C) LTED cells in 10% DCC-FBS were treated ± 2 µM AZD5363 for 0 to 24 hours. Cell lysates (0.5 mg) were prepared and analyzed by phospho-RTK arrays. D) Mice bearing MCF-7 xenografts ≥150 mm3 were treated with vehicle or AZD5363 (150 mg/kg bid p.o.) for one or three days. Xenografts were harvested four hours after the last dose; tumor lysates were prepared and analyzed by immunoblot. Densitometric analysis was performed and a graphical representation of the average RTK:actin levels is shown below the blot. E) Xenografts from D) were homogenized and RNA was extracted and analyzed by real-time PCR as in A. Data are presented as fold versus control; each bar, mean ± SEM (n = 3; *P <0.05 versus Con, t-test). DCC-FBS, dextran/charcoal-treated fetal bovine serum; LTED, long-term estrogen deprivation; RTK, receptor tyrosine kinase; SEM, standard error of the mean.