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. 2024 Jul 18;84(19):3250–3266. doi: 10.1158/0008-5472.CAN-24-0970

Figure 3.

Figure 3.

AKT inhibitors synergize with pitavastatin to induce TNBC cytotoxicity. A, TNBC cell lines (SUM159 and BT20) were treated with DMSO, AZD5363 (SUM159, 5 µmol/L; BT20, 1.25 µmol/L), pitavastatin (SUM159, 4 µmol/L; BT20, 2 µmol/L), or a combination of AZD5363 and pitavastatin, and the total cell number (rapid red nuclear dye) and number of dead cells (cleaved caspase-3/7 dye) were measured every 2 hours for 72 hours by Incucyte Live-Cell Analysis. Data are represented as mean ± SD of percent cleaved caspase-3/7 signal (N = 4 technical replicates). B–D, HCC70 cells were injected subcutaneously into NSG mice, and tumors were allowed to grow for 21 days before starting treatments. Mice were switched to a low geranylgeraniol chow diet 3 days before starting treatments and were treated once daily with vehicle (0.5% carboxymethylcellulose, N = 10), 100 mg/kg AZD5363 (4 days on, 3 days off, N = 12), 100 mg/kg pitavastatin (daily, N = 12), or both (N = 13) for 24 days. B, Tumor size (mm3) was measured every 3 to 4 days, starting 10 days after injection of cells. C, Tumor size (mm3) was measured at the endpoint. D, Tumor weight and mouse body weight were measured at the endpoint, and the tumor percent body weight was calculated by dividing tumor weight by mouse body weight. For B–D, statistical analysis was performed using two-way ANOVA with Tukey multiple comparison test (*, significant differences; in B, significant differences compared with the AZD5363 and pitavastatin combination treatment at the endpoint). E, A panel of breast cancer PDOs were treated with DMSO, 1 µmol/L AZD5363, 5 µmol/L pitavastatin, or the combination of AZD5363 and pitavastatin for 96 hours and then pulsed with EdU and stained with a cleaved caspase-3 antibody. A representative image for each PDO in each treatment condition is shown. Scale bars, 40 µm. F, Immunoblots of NPC1, HMGCR, PARP, pAKTSer473, pPRAS40Thr246, cleaved caspase-3, unprenylated RAP1A, β-actin, and vinculin in an ER-low (patient 10) and ER-positive (patient 26) organoid treated with DMSO, 1 µmol/L AZD5363, 5 µmol/L pitavastatin, or the combination for 24 hours. *, P = 0.0332; **, P = 0.0021; ***, P = 0.0002; ****, P < 0.0001; ns, not significant.