Fig. 9

NUAK1 inhibition synergies with Akt or mTOR blockage. A, B Effect of co-targeting NUAK1 and Akt on cell viability of spheroids (3D culture) from MDA-MB-231 (A) and U87 (B) for 96 h. Cell viability measurements were described in METHODS. Each bar represents the mean ± SD, n = 5. Data were analyzed by one-way ANOVA (P < 0.0001 for A, B) followed by Tukey’s test (MK-2206 compared to MK-2206 plus HTH-01-015, P < 0.0001 for A, B). C, D Effect of co-targeting NUAK1 and Akt or mTOR on cell viability of spheroids from MDA-MB-231 cells EGF-stimulated for 48 h. HTH-01-015, 10 µM (C) or HTH-01-015, 5 µM (D). Each bar represents the mean ± SD, n = 5. Data from C were analyzed by one-way ANOVA (P < 0.0001 for Akt and NUAK1 Co-targeting; P < 0.0001 for mTOR and NUAK1 Co-targeting; P < 0.0001 for mTORC1 and NUAK1 Co-targeting) followed by Tukey’s test (MK-2206 compared to MK-2206 plus HTH-01-015, P < 0.0001; Torin 1 compared to Torin 1 plus HTH-01-015, P < 0.0001; Rapamycin compared to Rapamycin plus HTH-01-015, P < 0.0001). Data from D were analyzed by one-way ANOVA (P = 0.0016 for Akt and NUAK1 Co-targeting; P < 0.0001 for mTOR and NUAK1 Co-targeting; P < 0.0001 for mTORC1 and NUAK1 Co-targeting) followed by Tukey’s test (MK-2206 compared to MK-2206 plus HTH-01-015, P = 0.0051; Torin 1 compared to Torin1 plus HTH-01-015, P = 0.0111; Rapamycin compared to Rapamycin plus HTH-01-015, P < 0.0001). E Effect of co-targeting NUAK1 and Akt or mTOR on cell viability of spheroids from U87 cells EGF-stimulated by 48 h. Each bar represents the mean ± SD, n = 5. Data from E were analyzed by one-way ANOVA (P < 0.0001 for Akt and NUAK1 Co-targeting; P < 0.0001 for mTOR and NUAK1 Co-targeting; P < 0.0001 for mTORC1 and NUAK1 Co-targeting) followed by Tukey’s test (MK-2206 compared to MK-2206 plus HTH-01-015, P < 0.0001; Torin 1 compared to Torin 1 plus HTH-01-015, P < 0.0001; Rapamycin compared to Rapamycin plus HTH-01-015 (10 µM), P < 0.0001). F Soft-agar colony formation assays in MDA-MB-231 cells (Zoom ×4). G Quantification of number of colonies per well from F. Each bar represents the mean ± SD, n = 3. Data from G were analyzed by one-way ANOVA (P < 0.0001) followed by Tukey’s test (MK-2206 compared to MK-2206 plus HTH-01-015, P = 0.0003). H Soft-agar colony formation assays in U87 cells (Zoom ×4). I Quantification of number of colonies per well from H. Each bar represents the mean ± SD, n = 3. Data from I were analyzed by one-way ANOVA (P = 0.0001 for Akt and NUAK1 Co-targeting; P < 0.0001 for mTOR and NUAK1 Co-targeting; P = 0.0002 for mTORC1 and NUAK1 Co-targeting) followed by Tukey’s test (MK-2206 compared to MK-2206 plus HTH-01-015, P = 0.026; Torin 1 compared to Torin1 plus HTH-01-015, not significant (ns); Rapamycin compared to Rapamycin plus HTH-01-015, P = 0.0173). J Correlation between NUAK1 and EGFR expression in TNBC (Brown n = 198, MAS5.0 u133p2) from R2: Genomics Analysis and Visualization Platform. K Correlation between NUAK1 and EGFR expression, and NUAK1 expression and Akt Ser-473 phosphorylation in Breast Invasive carcinoma (n = 874), COAD (n = 636), Prostate Adenocarcinoma (n = 498), STAD (n = 440), and Kidney Renal Clear Cell Carcinoma (n = 537) from c-Bioportal. L Hazard Ratio (HR) plot for NUAK1, Akt1, Akt2, Akt3, mTOR, and Rictor in BRCA (Breast Carcinoma), COAD, GBM (Glioblastoma Multiforme), PRAD (Prostate Adenocarcinoma), STAD (Stomach Adenocarcinoma) and OV (Ovarian cancer) from Gepia2