NDRG1 regulates neutral lipid metabolism in breast cancer cells

Abstract

Background: Altered lipid metabolism is an emerging hallmark of aggressive breast cancers. The N-myc downstream regulated gene (NDRG1) gene plays a critical role in peripheral nervous system myelination, as inactivating mutations cause severe demyelinating neuropathy. In breast cancer, elevated NDRG1 expression has been linked to clinical outcomes, but its functional role in breast cancer physiology has remained unclear.

Methods: A meta-analysis of NDRG1 expression in multiple large publicly available genomic databases was conducted. Genome-wide expression correlation and Cox proportional hazards and Kaplan-Meier modeling of clinical outcomes associated with elevated expression were assessed. To study NDRG1 function, gene silencing and overexpression phenotypic studies were carried out in a panel of cell lines representing all major breast cancer molecular subtypes. Changes in cell proliferation, morphology, and neutral lipid accumulation due to altered NDRG1 expression were assessed by high throughput, quantitative microscopy. Comprehensive lipidomics mass spectrometry was applied to characterize global changes in lipid species due to NDRG1 silencing. Labeled fatty acids were used to monitor cellular fatty acid uptake and subcellular distribution under nutrient replete and starvation culture conditions.

Results: NDRG1 overexpression correlated with glycolytic and hypoxia-associated gene expression, and was associated with elevated rates of metastasis and patient mortality. Silencing NDRG1 reduced cell proliferation rates, causing lipid metabolism dysfunction including increased fatty acid incorporation into neutral lipids and lipid droplets. Conversely, NDRG1 expression minimized lipid droplet formation under nutrient replete and starvation conditions.

Conclusions: Here we report that NDRG1 contributes to breast cancer aggressiveness by regulating the fate of lipids in cells that exhibit an altered lipid metabolic phenotype. In line with its role in promoting myelination and its association with altered metabolism in cancer, our findings show that NDRG1 is a critical regulator of lipid fate in breast cancer cells. The association between NDRG1 and poor prognosis in breast cancer suggests it should play a more prominent role in patient risk assessment. The function of NDRG1 in breast cancer lipid metabolism may represent a promising therapeutic approach in the future.

Keywords: Aggressiveness; Lipogenic; Marker; Metabolism; Metabolomics.

Fig. 1

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)

Fig. 2

NDRG1 expression level and localization variability in breast cancer cell lines. a Cancer Cell Line Encyclopedia (CCLE)-analyzed breast cancer cell lines ordered by NDRG1 expression level - cell lines selected for in vitro analysis are indicated. b Immunoblots of NDRG1 and GAPDH loading control from crude lysates (10 μg total protein/lane). c Immunofluorescence analysis of NDRG1 in breast cancer cell lines, scale = 25 μm: i MCF7, ii BT474, iii MDA-MB-231, iv MDA-MB-468, v SKBR3, vi HCC1569. d, e NDRG1 pT346 and pS330 immunofluorescence and localization in MDA-MB-231 and MDA-MB-468 cells treated with 100 μM deferoxamine (DFO): nucleus:cytoplasm ratio is shown: N = 3/group. f pNDRG1 T346 immunofluorescence localization to puncta in SKBR3 cells: thapsigargin (Thap) (1 μM) but not tunicamycin (Tun) treatment (5 μg/ml) disperses puncta. Data are represented as mean ratio +/− SD. *p < 0.001, ***p < 1 × 10^− 10. CRTL, control; NS, not significant

Fig. 3

NDRG1 is required for normal breast cancer cell proliferation, viability and morphology. a Immunoblots of NDRG1 and GAPDH loading control after selection of four stable lentiviral transduced shRNA expressing cell lines. A green fluorescent protein (GFP) targeting shRNA serves as negative control, and silencing was verified at 1% O₂. b Cell proliferation assays: cell number was compared by comparing NDRG1 silenced cell numbers to control at the times indicated: ***p < 0.0001, N = 3/group - see Additional file 1: Figure S5. c Cleaved caspase 3 (CC3) immunofluorescence analysis of stable shRNA expressing SKBR3 cells at 1 week post selection. CC3 positive area was normalized to total cell area and each population compared: shNDRG1_1 p = 2.5 × 10^− 6, shNDRG1_2 p = 1.2 × 10^− 4, scale = 100 μm, N = 12/group. d Individual frames from a 48 h live cell microscopy analysis of SKBR3 cells expressing the indicated shRNAs: N = 3/group, see Additional files 3, 4, 5, 6, 7 and 8: Movies S1–S6. e Mitotic cell fractions comparing the percentage of histone H3 phsopho-serine10 positive cells in the indicated shRNA expressing cell lines: shNDRG1_1 p = 0.02, shNDRG1_2 p = 0.06, N = 12/group. f Comparison of individual cell two-dimensional area based on phalloidin stain (p < 0.001, Mann-Whitney U test, N = > 650 cells each group). Log2 two-dimensional area is shown. All bar graphs represent means +/− SD. Comparisons were by two-sided Student’s t test unless indicated otherwise

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 depletion increases neutral lipid storage in lipid droplets. a Boron-dipyrromethene (BODIPY) 493/503 and Hoechst-stained stable SKBR3 cells expressing NDRG1 or green fluorescent protein (GFP) targeting shRNA at 2 weeks post selection. Scale bar = 25 μm. b Comparison of BODIPY-positive lipid droplet specific signal/cell in stable SKBR3, MCF7 1% O₂, MDA-MB-231, MDA-MB-468, and HCC1569 expressing the indicated shRNAs: all N = 6 and ***p < 0.00001. See Additional file 1: Figure S6 for related data. c Comparison of lipid droplet signal in stable SKBR3 cells expressing FLAG tagged full-length NDRG1 and N and C terminal truncations as indicated. N = 12, **p < 0.01, ***p < 0.00000001, scale bar = 25 μm. d Electron microscopy analysis of Hs578T cells +/− NDRG1 depletion. Arrows indicate lipid droplets. Scale bar = 0.5 μm. e-f Analysis of thapsigargin and tunicamycin and survival by counting Hoechst-stained nuclei 48 h post stable SKBR3 +/− NDRG1 depletion. Plots represent surviving cell fraction compared to untreated cells, N = 3. All bar graphs represent mean +/− SD and comparisons were by the two-sided t test. Scale bars = 25 μm

Fig. 6

Fluorescent fatty acid tracers reveal NDRG1 suppresses fatty acid incorporation Into neutral lipids and storage in lipid droplets. a Fluorescence micrographs of stable NDRG1 SKBR3 +/− NDRG1 depletion fed the 16-carbon boron-dipyrromethene (BODIPY) palmitate (BODIPY C16–100 μM, 16 h), fixed. Lipid droplets and nuclei counterstained with LipidTox Deep Red and Hoechst, respectively. b Quantification of total BODIPY C16 uptake in SKBR3 cells +/− NDRG1 depletion. Graphs represent average total signal per cell at indicated tracer concentrations. c Quantification of lipid droplet specific BODIPY C16 by segmentation of small granules. d Relative distribution of BODIPY C16 signal representing droplet/whole cell signal. e LipidTox stained lipid droplet numbers represented as objects per cell. f Comparison of lipid droplet intensities of starved and fed controls at day 3 in stable BODIPY 558/568 C12 loaded MCF7 cells. Complete medium versus Hank’s buffered saline (HBSS). g BODIPY 493/503 counterstaining of NDRG1-FLAG overexpressing and control cells fed complete medium or HBSS for 3 days. h Analysis of cell survival due to starvation in stable BODIPY 558/568 C12-fed MCF7 cells transduced with NDRG1-FLAG or empty vector after 3 days of culture in HBSS. i, j Time course of changes in lipid droplet formation and cell count due to HBSS starvation in stable MCF7 cells transduced with the indicated constructs. Values represent HBSS/complete medium. All bar graphs represent mean +/− SD and comparisons were by two-sided t test. N = 12/group for all. Scale bars = 25 μm. CTRL, control