Fig 2: MNK inhibition regulates the phosphorylation and overall expression of NDRG1 in MDA-MB-231 breast cancer cellsBreast cancer cells were treated for different times with MNK-I1 (5 µM). Levels of specific proteins in (A) MDA-MB-231 and (B) MDA-MB-453 cells were analysed by Western blot with indicated antibodies (the latter display two distinct bands for P-eIF4E). Ratio of p-NDRG1/NDRG1 was calculated using Image J. NDRG1 mRNA levels in (C) MDA-MB-231 and (D) MDA-MB-453 cells were analysed by RT-qPCR. (E) MDA-MB-231 cells were pretreated with MNK-I1 (5 µM) for 4 h and followed by 100 ng/ml actinomycin D for 16 h, where indicated. NDRG1 mRNA were analysed by qPCR. Data are shown as mean ± S.E.M. from three replicates. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 3: Roles of SGK1 and MNKs in modulating the phosphorylation of NDRG1(A) Where indicated, MDA-MB-231 cells were starved of glucose for 4 h in the presence, where shown, of 5 μM MNK-I1 and/or 10 μM SGKi. Lysates were analysed by Western blot. (B) MDA-MB-231 cells were treated with 5 μM MG132 and 5 μM MNK-I1 for 16 h and lysates were analysed by Western blot. (C) MDA-MB-231 cells were pretreated with 5 μM MNK-I1 for 1 h and then with 5 μM MG132 for the indicated times. Cell lysates were analysed by Western blot. (D) MDA-MB-231 cells were transfected with a vector for HA-tagged NDRG1 and then treated with 5 μM MG132 and 5 μM MNK-I1 for 12 h. HA-IP was performed to isolate NDRG1. Beads were then incubated with Antarctic phosphatase (AnP) at 37°C for 30 or 60 min. Reaction products were analysed by Western blot.

Fig 4: NDRG1 depletion causes increased levels of major lipid species. a Semi-quantitative shotgun LC MS/MS of major lipid species in SKBR3 cells. Heatmap represents fold differences in species detected in all samples standardized relative to row means. Color range 0.01–8-fold relative to row mean, N = 5 per group. See Additional file 1: Figure S5 for quantification of an independent analysis (b) in micrograms of triacylglycerol/mg protein. Individual species and sums are plotted separately (c) in nanograms of cholesterol ester per million cells. Sums of individual species and cholesterol ester (CE) species are plotted separately: N = 6 for TAG and N = 5 for CE analysis. Data represent mean +/− SD, comparisons were by the two-sided t test: **p < 0.01, ***p < 0.00001. TAG, triacylglycerols; GFP, green fluorescent protein

Fig 5: NDRG1 expression is associated with adverse outcomes and altered metabolism in breast cancer. a Meta-analysis of 23 distinct breast cancer cohorts (n = 3554 subjects). Kaplan-Meier curve of recurrence-free survival analysis of NDRG1 mRNA expression (upper quartile (Q) versus lower three quartiles). Post-DX, Post-diagnosis. b Metastasis-free survival of NDRG1 quartiles in the Van’t Veer cohort (n = 295). c NDRG1 expression in estrogen receptor (ER)+/− groups of the the Van’t Veer cohort. d The distribution of total Van’t Veer cohort NDRG1 expression quartiles in ER+ and ER- tumors. The unequal distribution further reflects the data in (c). e, f Metastasis-free survival using the 42-gene signature in The Cancer Genome Atlas breast cancer cohort (n = 962), and the Van’t Veer breast cancer cohort (n = 295)