Article Figures & Data
Figures
- Figure 1.
Activity of AZD8186. A, AZD8186. B, inhibition of ADP-induced platelet aggregation, representative curve, and mean IC50 186 ± 36 nmol/L (SEM; n = 3). C, LPA (10 mmol/L) activation of serum-starved MDA-MB-468 and PC3 cells in the presence or absence of 250 nmol/L AZD8186 as indicated. Western blots for pAKT (Ser473), pERK, total AKT, and total ERK, with vinculin as a loading control. Representative of three similar experiments.
- Figure 2.
AZD8186 inhibits phosphorylation of AKT and downstream pathway proteins inducing nuclear translocation of FOXO3a in vitro. A, Western blot analysis of protein phosphorylation reduction by AZD8186 in three prostate cancer cell lines. Inhibition of proliferation shown as GI50 (μmol/L ± SEM). B, Western blot analysis of protein phosphorylation reduction by AZD8186 in three breast cancer cell lines. Inhibition of proliferation shown as GI50 (μmol/L ± SEM). C, nuclear accumulation of FOXO3a in HCC70, LNCAP, and BT474 cells. Representative images show Hoescht (blue) and FOXO3a (green) localization at ×20 magnification in control cells and cells treated with 0.1 and 0.5 μmol/L AZD8186 for 2 hours. A representative dose–response plot (of three experiments) of relative nuclear translocation for each line is shown [mean relative Foxo translocation (± SD)]. D, AZD8186 activity in a 72-hour MTS cell proliferation assay; relationship of most sensitive lines (classified as <1 μmol/L GI50) with PTEN genetic status (green, WT; red, MT; blue, increased copy number; gray, incomplete data). E and F, sensitivity of AZD8186 and AZD5363 (AKTi) in MTS cell proliferation assay and relationship to SGK-1 mRNA expression in a panel of breast cancer cell lines (E) and prostate cancer cell lines (F). SGK expression: blue, low; and red, high expression. GI50 data: red bars, resistant; green bars, sensitive to compound. Western blot analysis of PTEN protein expression for each cell line is shown; NT, not tested.
- Figure 3.
In vivo activity of AZD8186. A, HCC70 tumors treated with vehicle (closed circles), 50 mg/kg (closed triangles), and 25 mg/kg (open inverted triangles) AZD8186 twice a day (b.i.d.). Modulation of the biomarkers pAKT Ser473 (B), pAKT Thr308 (C), and pPRAS40 (D) in HCC70 tumors by 50 and 25 mg/kg AZD8186 the times indicated. The mean percentage of inhibition (± SD) is shown. E, MDA-MB-468 tumors treated with vehicle (closed circles), 50 mg/kg (closed triangles), and 25 mg/kg (open inverted triangles) AZD8186 twice a day. Modulation of the biomarkers pAKT Ser473 (F), pAKT Thr308 (G), and pPRAS40 (H) in MDA-MB-468 tumors by 50 and 25 mg/kg AZD8186 at times indicated. The mean percentage of inhibition (±SD) is shown. I, PC3 tumors treated with vehicle (closed circles), AZD8186 at 25 mg/kg (open triangles), and 50 mg/kg (closed triangles) twice a day. J, HID28 tumors were treated with vehicle (closed circles) and AZD8186 at 50 mg/kg (closed triangles) twice a day. K, a representative PK profile of AZD8186 dosed twice a day with and without ABT (once in a 24 hours dosing cycle). L, PC3 tumors treated with vehicle + ABT (closed circles) and 30 mg/kg AZD8186 + ABT (open diamonds). M, HCC70 tumors treated with vehicle (closed circles), AZ8186 at 50 mg/kg twice a day (closed triangles), 50 mg/kg twice a day (4 days on, 3 days off; inverted closed triangles); or (N) treated with vehicle (closed circles), AZ8186 at 25 mg/kg twice a day (open inverted triangles), 25 mg/kg twice a day (5 days on, 2 days off; closed triangles). Geometric mean tumor volume ± SEM are shown. Statistical significance relative to control *, P < 0.05; ** P < 0.01; ***, P < 0.001.
- Figure 4.
AZD8186 modulates pAKT and nuclear translocation of FOXO3a. Representative images of pAKT Ser473 and FOXO3a staining in HCC70, MDA-MB-468, PC3, and HID-28 tumors. Vehicle-treated and AZD8186-treated samples are shown. PC3 samples, vehicle control (8 hours after dose) and 30 mg/kg AZD8186 (30 minutes after dose). For HID28, vehicle and 50 mg/kg AZD8186 (2 hours after dose). For HCC70, vehicle and 50 mg/kg AZD8186 (30 minutes after dose). For MDA-MB-468, vehicle and 50 mg/kg AZD8186 (2 hours after dose). Images were taken at ×20 magnification. A, pAKT Ser473 staining. B, Foxo3a staining.
- Figure 5.
AZD8186 combines with docetaxel. A, HCC70 tumors treated with control (closed circles), single dose 15 mg/kg docetaxel (closed star), and single dose (arrow) 15 mg/kg docetaxel with 50 mg/kg (closed triangles) or 25 mg/kg (inverted open triangles) twice a day (b.i.d.). Right-hand graph, single-agent AZD8186 50 mg/kg (closed triangles) and 25 mg/kg (inverted open triangles). B, PC3 tumors treated with control (closed circles), single dose (arrow) 15 mg/kg docetaxel (closed star) and single dose 15 mg/kg docetaxel with 30 mg/kg (closed diamonds) and 10 mg/kg (open diamonds) AZD8186 twice a day (plus ABT). The single-agent effect for 30 mg/kg (closed diamonds) and 10 mg/kg (open diamonds; plus ABT) AZD8186 is shown in the right hand graph. ##, P < 0.01; ###, P < 0.005. C, HCC70 tumors treated with vehicle (closed circles) and 25 mg/kg AZD8186 twice a day (inverted open triangles); D, with vehicle (closed circles), single dose (arrow) docetaxel (15 mg/kg; closed stars) alone or in combination with 25 mg/kg AZD8186 (twice a day; inverted closed triangles), or 25 mg/kg 2 days on 5 days off (inverted open triangles); E, with vehicle (closed circles), single dose (arrow) docetaxel (15 mg/kg; closed stars) alone or in combination with 25 mg/kg AZD8186 (twice a day; inverted closed triangles), or 25 mg/kg 5 days on 2 days off (inverted open triangles). Geometric mean tumor volume ± SEM are shown. Statistical significance: on combination studies * indicates comparison with the docetaxel alone control and # indicates comparison with the corresponding AZD8186 monotherapy control; *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, P < 0.05; ##, P < 0.01; ###, P < 0.001.
Tables
- Table 1.
Potency of AZD8186 in biochemical and cell based assays
Biochemical assay IC50μmol/L ± SEM (n) Cell isoform selectivity: cell line Modulation of pAKT IC50μmol/L ± SEM PI3Kα 0.035 ± 0.005 PI3Kα: BT474 0.752 ± 0.293 PI3Kβ 0.004 ± 0.001 PI3Kβ: MDA-MB-468 0.003 ± 0.001 PI3Kδ 0.012 ± 0.002 PI3Kδ: JEKO 0.017 ± 0.004 PI3Kγ 0.675 ± 0.114 PI3Kγ NT
Supplementary Data
Files in this Data Supplement:
- Supplementary Tables 1-3 and Figure 1-7 - Supplementary Tables 1-3 and Figure 1-7. Supp Table 1 Summary of cell line origins and verification. Supp Table 2 Summary of antibodies used in all experimental analysis Supp Fig 1 AZD8186 inhibits PI3K pathway in PTEN null but not PTEN WT PIKC3A MT cells Supp Fig 2 AZD8186 inhibits LPA, EGF and rac dependent Ser473 AKT phosphorylation in serum starved prostate and breast cancer cells Supp Fig 3 Western blot analysis of showing induction of FOXO3a association with the chromatin fraction following treatment with AZD8186 Supp Fig 4 AZD8186 inhibits proliferation in a subset of cancer cell lines Supp Table 3 GI50 of all cell lines showing sensitivity to AZD8186 Supp Fig 5 Western blot protein analysis of PTEN status across a panel of breast and prostate cell lines Supp Fig 6 Pathway biomarker suppression in PC3 and HID28 tumours Supp Fig 7 AZD8186 selectively suppresses pathway biomarkers in tumour versus lung
- Supplementary Materials text - Supplementary Materials text. Supplementary text to support supplementary data.
Volume 14, Issue 1
