The AMPK-related kinase NUAK2 suppresses glutathione peroxidase 4 expression and promotes ferroptotic cell death in breast cancer cells

Abstract

Ferroptosis is a caspase-independent form of regulated cell death strongly linked to the accumulation of reactive lipid hydroperoxides. Lipid hydroperoxides are neutralized in cells by glutathione peroxidase 4 (GPX4) and inhibitors of GPX4 are potent ferroptosis inducers with therapeutic potential in cancer. Here we report that siRNA-mediated silencing of the AMPK-related kinase NUAK2 suppresses cell death by small-molecule inducers of ferroptosis but not apoptosis. Mechanistically we find that NUAK2 suppresses the expression of GPX4 at the RNA level and enhances ferroptosis triggered by GPX4 inhibitors in a manner independent of its kinase activity. NUAK2 is amplified along with MDM4 in a subset of breast cancers, particularly the claudin-low subset, suggesting that this may predict vulnerability to GPX4 inhibitors. These findings identify a novel pathway regulating GPX4 expression as well as ferroptotic sensitivity with potential as a biomarker of breast cancer patients that might respond to GPX4 inhibition as a therapeutic strategy.

Fig. 1. Silencing NUAK2, but not NUAK1, partially protects TNBC cells from cell death by ferroptosis inducers.

a Western blot showing YAP1 protein levels in BT-549 cells 72 hours after transfection with a pool of YAP1-targeting siRNA or non-targeting siRNA. β-actin is the loading control for this and the subsequent western blots unless otherwise specified. (n = 1 independent experiment). Normalized YAP1 protein levels are show beneath the band. b Quantitative RT-PCR of YAP1 mRNA from YAP1-siRNA or nontargeting siRNA in BT-549 cells similar to (a). n = 3 independent experiments and a one sample t test was used to test the difference from control YAP1 mRNA levels (p-value shown above the comparator bar). Error bars, here and in the subsequent panels denote standard deviation centered on the mean. c Relative viability of BT-549 cells 72 h after transfection with the pool of YAP1-targeting or non-targeting siRNA followed by 48-h treatment with the indicated dose of ML162. p-values above comparator bars in this and subsequent panels are from two-sided Student’s t-tests unless noted. (n = 3 independent experiments). d Western blot showing NUAK2 protein level in BT-549 cells 72 h after transfection with a pool of NUAK2-targeted siRNA or nontargeting siRNA (n = 3 independent experiments). The right panel shows the quantitation of relative NUAK2 protein levels from the 3 experiments and the p-value from a one sample t test. e Western blot showing NUAK1 protein level in BT-549 cells after NUAK1 silencing as in (d). n = 1 f Quantitative RT-PCR of NUAK1 mRNA from NUAK1-siRNA or nontargeting siRNA in BT-549 cells similar to (e). n = 2 independent experiments and a one sample t test was used to test the difference from control NUAK1 mRNA levels. g Relative viability of BT-549 cells 72 h after transfection with either NUAK1-targeting, NUAK2-targeting, or nontargeting siRNA followed by 48-h treatment with indicated dose of (g) ML162 or (h) RSL3 (n = 3 independent experiments). i Cell viability dose-response curves in BT-549 cells 72 h after transfection with a pool of NUAK2-targeted siRNA or nontargeting siRNA followed by 48 h incubation with the indicated dose of staurosporine, calculated EC₅₀ values were 3.2 nM (95% CI, 2–4 nM) for nontargeting siRNA, 3.4 nM (95% CI, 2–5 nM for NUAK2-targeted siRNA (n = 3 independent experiments).

Fig. 2. Overexpression of NUAK2 enhances ferroptosis on GPX4 inhibition.

a Western blot of BT-549 cells transfected with vectors encoding wild-type NUAK2 or control cell line expressing eGFP (representative of n = 3 independent experiments). Right panel shows quantification of relative NUAK2 protein levels from the 3 experiments. Error bars, here and below, denote standard deviation centered on the mean. b Relative viability of these cells after 72 h incubation with the indicated dose of ML162 (n = 3). p-values from two-tailed Student’s t-tests are shown here and below. c Bar chart showing the relative viability of MDA-MB-231 cells expressing either eGFP or NUAK2 following incubation for 72 h with 62.5 nM RSL3 and co-treatment with either DMSO vehicle (-) or 2 µM ferrostatin-1 (+fer-1) (n = 3 independent experiments). d Relative viability of NUAK2 overexpressing MDA-MB-231 cells following incubation for 72 h with 125 nM RSL3 and co-treatment with either DMSO vehicle, 2 µM ferrostatin-1, 50 µM deferoxamine, 50 µM Necrostatin-1s or 20 µM Z-VAD-FMK.

Fig. 3. NUAK2 does not enhance ferroptosis by depleting glutathione.

a Relative glutathione levels (oxidized plus reduced forms) from BT-549 cells 72 h after transfection with either NUAK1-targeting, NUAK2-targeting, or nontargeting siRNA (control) and 24 h treatment with either vehicle or 20 µM buthionine sulfoximine (BSO) (n = 3 independent experiments). b Relative glutathione levels from BT-549 cells exogenously expressing NUAK2 or eGFP (controls) 24 h after treatment with either vehicle (untreated) or 20 µM BSO. (n = 4 technical replicates). Glutathione levels were normalized to total viable cells (CellTiter-Glo). Error bars denote standard deviation centered on the mean. The numbers above the brackets are p values from Student’s t-tests (two-sided), ns denotes not significant.

Fig. 4. NUAK2 suppresses GPX4 expression at the RNA level.

a Western blot showing GPX4 protein levels in BT-549 cells 72 h after transfection with a pool of NUAK1 or NUAK2-targeting siRNA or non-targeting siRNA. β-actin is the loading control. (representative of n = 3 independent experiments). Relative GPX4 protein levels are show beneath the band. b Relative mRNA expression of NUAK1, NUAK2 and GPX4 by qPCR in BT-549 cells 72 h after transfection with the indicated pool of NUAK1, NUAK2 siRNA or non-targeting siRNA (control) (n = 3 independent experiments). Error bars denote standard deviation centered on the mean. The numbers above the brackets are p values from Student’s t-tests (two-sided), ns denotes not significant. c Western blot showing GPX4 protein levels in (c) BT-549 and (d) MDA-MB-231 cells, stably over-expressing NUAK2 compared to the control line expressing eGFP (representative of n = 2 independent experiments for BT-549 and n = 3 for MDA-MB-231). Relative GPX4 protein levels are show beneath the band.

Fig. 5. NUAK2 regulation of GPX4 is independent of its kinase activity.

a Western blot of BT-549 cells transfected with cDNA encoding wild-type NUAK2 (NUAK2 OE) or kinase-dead NUAK2 (NUAK2 OE^K81R) compared to control cells expressing eGFP. β-actin is the loading control (representative of n = 2 independent experiments). Quantification of normalized NUAK2 protein levels are shown in the right panel. b Relative mRNA expression of GPX4 by RT-PCR in BT-549 cells overexpressing either wild-type NUAK2 or NUAK2 ^K81R compared to eGFP-expressing controls (n = 2 independent experiments). Numbers above the brackets are p values from Student’s t-tests (one-sided). c Cell viability for these cells treated with the indicated dose of ML162 for 72 h. Calculated EC₅₀ values were 94 nM (95% CI, 83−105 nM) for controls (eGFP), 18 nM (95% CI, 15–22 nM for NUAK2 OE and 26 nM (95% CI, 22-30 nM for NUAK2^K81R OE (n = 3 independent experiments).

Fig. 6. NUAK2 does not affect GPX4 expression via YAP/TAZ.

a Relative mRNA expression of the indicated genes quantified by qPCR in BT-549 cells 72 h after transfection with the indicated siRNA pool (n = 3 independent experiments). Error bars denote standard deviation centered on the mean. p values from Student’s t-tests (two-sided) are shown over brackets, ns denotes not significant. b qPCR quantitation of the indicated genes in BT-549 cells 72 hours after transfection with the indicated siRNA pool (n = 2 independent experiments). Error bars denote standard deviation centered on the mean. The numbers above the brackets are p values from Student’s t-tests (one-sided). c Western blot showing GPX4 protein levels in BT-549 cells 72 h after silencing of TEAD4 or YAP1. β-actin is the loading control, (n = 1). Relative GPX4 protein levels are show beneath the band.

Fig. 7. NUAK2 is frequently amplified in breast cancers, is highly expressed in the claudin-low subtype and is associated with sensitivity to GPX4 inhibition.

a 100 human cancer cell lines were treated with ML162 or RSL3 at multiple doses for 48 h and cell viability was measured. Cell lines sensitive to GPX4 inhibitors (13 cell lines; blue) were defined based on an area under the dose-response curve (AUC) of >0.15. Resistant cell lines (17 cell lines; red) were defined on the basis of an AUC of 0. NUAK2 expression was extracted from the Cancer Cell Line Encyclopedia database and is plotted for each GPX4 inhibitor-sensitive or resistant cell line. The p value for the between-group comparison (Student’s t-test) is p < 0.00001. b Alterations in the NUAK2 gene across 10,953 cancer patients from TCGA Pan Cancer Data c Oncoprint of 2,173 breast cancer patients from the METABRIC data set [27] showing amplification of NUAK2 and/or MDM4. In (d) mRNA expression (microarray) of NUAK2 in these patients is broken down according to PAM50 breast cancer subtype.