Ubiquinol-mediated suppression of mitochondria-associated ferroptosis is a targetable function of lactate dehydrogenase B in cancer

Abstract

Lactate dehydrogenase B (LDHB) fuels oxidative cancer cell metabolism by converting lactate to pyruvate. This study uncovers LDHB's role in countering mitochondria-associated ferroptosis independently of lactate's function as a carbon source. LDHB silencing alters mitochondrial morphology, causes lipid peroxidation, and reduces cancer cell viability, which is potentiated by the ferroptosis inducer RSL3. Unlike LDHA, LDHB acts in parallel with glutathione peroxidase 4 (GPX4) and dihydroorotate dehydrogenase (DHODH) to suppress mitochondria-associated ferroptosis by decreasing the ubiquinone (coenzyme Q, CoQ) to ubiquinol (CoQH2) ratio. Indeed, supplementation with mitoCoQH2 (mitochondria-targeted analogue of CoQH2) suppresses mitochondrial lipid peroxidation and cell death after combined LDHB silencing and RSL3 treatment, consistent with the presence of LDHB in the cell fraction containing the mitochondrial inner membrane. Addressing the underlying molecular mechanism, an in vitro NADH consumption assay with purified human LDHB reveals that LDHB catalyzes the transfer of reducing equivalents from NADH to CoQ and that the efficiency of this reaction increases by the addition of lactate. Finally, radiation therapy induces mitochondrial lipid peroxidation and reduces tumor growth, which is further enhanced when combined with LDHB silencing. Thus, LDHB-mediated lactate oxidation drives the CoQ-dependent suppression of mitochondria-associated ferroptosis, a promising target for combination therapies.

Fig. 1. Silencing of LDHB induces mitochondrial lipid peroxidation in cancer cells.

Measurement of mitochondrial lipid peroxidation in A549, MSTO-211H, HT1080 and PANC-1 cells after 72 h of transfection with siRNA (siCTRL) or with LDHB-targeted siRNAs (siLDHB) (a, b), n = 6, n = 5, n = 3 independent replicates for A549, MSTO-211H, HT1080, and PANC-1 respectively. Mitochondrial lipid peroxidation (c, d scale bar, 20 µm), 4-HNE, and TOM20 colocalization (e, f scale bar, 20 µm) were examined by immunofluorescence in A549 siRNAs cells after 72 h of transfection, n = 3 independent replicates. Transmission electron microscopy images of A549 siRNAs cells after 72 h of transfection (g). Images of the clonogenic assays of A549, PANC-1 siRNAs cells at day 10 after treatment with DMSO or 2 µM ferrostatin-1 (FER1) or 20 µM mitoTEMPO (h). Analysis of colony numbers of A549, PANC-1 siRNAs cells at day 10 after treatment with DMSO or 2 µM ferrostatin-1 (FER1) or 20 µM mitoTEMPO, n = 9 independent replicates for DMSO and FER1 treated groups, n = 3 independent replicates for mitoTEMPO treated groups for A549 cell line. n = 3 independent replicates for PANC-1 cell line (i). Data were presented as mean ± SD. Unpaired, two-tailed t-test. ns no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are provided as a Source Data file.

Fig. 2. LDHB suppresses mitochondria-associated ferroptosis in cancer cells.

Heatmap of cell viability and images of the clonogenic assay of siRNAs cells treated with RSL3 alone or in combination with 5 µM FER1 for 48 h after pretreatment with vehicle or 5 µM FER1 for 24 h (a, b). Assessment of cell viability with PrestoBlue in A549, HT1080, PANC-1, MSTO-211H siRNAs cells treated with DMSO or RSL3 alone or in combination with 20 µM mitoTEMPO, 20 µM TEMPO, 5 µM FER1 for 48 h after pretreatment with vehicle, 20 µM mitoTEMPO, 20 µM TEMPO, 5 µM FER1 for 24 h, n = 3 independent replicates (c). Assessment of mitochondrial lipid peroxidation by flow cytometry in A549, HT1080, PANC-1, MSTO-211H siRNAs cells treated with DMSO or RSL3 alone (1 µM for A549, 0.75 µM for HT1080, and MSTO-211H cells, 0.5 µM PANC-1) or in combination with 20 µM mitoTEMPO, 20 µM TEMPO, 5 µM FER1 for 1 h after pretreatment with vehicle, 20 µM mitoTEMPO, 20 µM TEMPO, 5 µM FER1 for 24 h, n = 4 independent replicates for A549, PANC-1, MSTO-211H cell lines treated with DMSO and n = 3 independent replicates for the rest groups, n = 3 independent replicates for HT1080 cell line (d, e). Analysis of PI staining by flow cytometer in A549, HT1080, PANC-1, MSTO-211H cells after 72 h of transfection with siRNAs treated with DMSO or RSL3 alone (1 µM for A549, 500 nM for HT1080, 250 nM for MSTO-211H, 50 nM for PANC-1) or in combination with 5 µM FER1, 20 µM mitoTEMPO, 20 µM TEMPO for 24 h. n = 3 independent replicates (f). Data were presented as mean ± SD. Two-way ANOVA (c), Unpaired, two-tailed t-test (e, f). ns no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are provided as a Source Data file.

Fig. 3. LDHB acts in parallel with GPX4 to suppress mitochondria-associated ferroptosis.

Immunoblot analysis of LDHB, GPX4, DHODH, LDHA, SLC7A11, and FSP1 in HT1080, MSTO-211H, A549, PANC-1 siRNAs cells, n = 3–6 independent repeats, and the statistical analysis was shown in Supplementary Fig. 3a, b (a). Colocalization analysis by immunofluorescence in HT1080 siRNAs cells after 72 h of transfection (b, scale bar, 20 µm). Images of microscopic analysis (c, 20x objective) and assessment of mitochondrial lipid peroxidation by flow cytometry in HT1080 sgNTC and sgGPX4 cells after 72 h of transfection with siRNAs, n = 3 independent replicates (d, e). Clonogenic assays of parental HT1080 (HT1080-P) and HT1080 cells treated with 4 µM RSL3 after ten cycles (HT1080-R) (f). Immunoblot analysis of LDHB, GPX4, DHODH, and LDHA in HT1080 parental and RSL3 resistant cells. The experiment was repeated three times, yielding consistent results (g). Cell viability of HT1080-P and HT1080-R cells after transfection with siRNAs treated with DMSO or RSL3 alone or in combination with 5 µM FER1 for 48 h, following pretreatment with vehicle, 5 µM FER1 for 24 h, n = 3 independent replicates (h). Immunoblot analysis of LDHB, GPX4, LDHA in HT1080 control and LDHB overexpression cells (i). Cell viability assays of HT1080 cells and HT1080 LDHB ORF cells treated with RSL3 with or without 5 µM FER1 for 48 h, following pretreatment with vehicle, 5 µM FER1 for 24 h. n = 3 independent replicates (j). Clonogenic assay of HT1080 siRNAs cells treated with DMSO or RSL3 alone or in combination with 2 mM glutathione reduced ethyl ester (GSH-mee) for 48 h after pretreatment with vehicle, 2 mM GSH-mee for 24 h (k). Tumor volume and weight of A549 sgNTC shCTRL (n = 12), sgNTC shLDHB (n = 11), sgGPX4 shCTRL (n = 12), sgGPX4 shLDHB (n = 12), sgGPX4 shLDHB treated with liproxstatin-1 (n = 12) xenograft tumors from different mice (l, m), tumor volume and weight of HT1080 sgNTC shCTRL (n = 10), sgNTC shLDHB (n = 10), sgGPX4 shCTRL (n = 10), sgGPX4 shLDHB (n = 12), sgGPX4 shLDHB treated with liproxstatin-1 (n = 12) xenograft tumors from different mice (o, p), 4-HNE expression of A549 and HT1080 sgNTC shCTRL, sgNTC shLDHB, sgGPX4 shCTRL, and sgGPX4 shLDHB xenograft tumors treated with or without liproxstatin-1, n = 10 or n = 12 different regions from different tumors from different mice respectively (n, q). Data were presented as mean ± SEM (l, o) or mean ± SD (e, h, j, m, n, p, q). Two-way ANOVA (h, j, l, o), Unpaired, two-tailed t-test (e, m, n, p, q). ns no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are provided as a Source Data file.

Fig. 4. LDHB suppresses ferroptosis by regulating the reduction of CoQH2 in mitochondria.

Analysis of mitochondrial lipid peroxidation in HT1080 sgNTC, sgDHODH cells 72 h after transfection with siRNAs, n = 3 independent replicates (a, b). Cell viability of A549 and HT1080 sgNTC, sgDHODH or sgFSP1 cells after transfection with siRNAs treated with DMSO or RSL3 alone or in combination with 5 µM FER1 for 48 h, following pretreatment with vehicle, 5 µM FER1 for 24 h, n = 3 or n = 4 independent replicates for A549 sgDHODH cells and HT1080sgDHODH, A549sgFSP1, and HT1080sgFSP1 cells, respectively (c, d). Cell viability of PANC-1 transfected cells treated with DMSO or FIN56 alone or in combination with 5 µM FER1 for 48 h, following pretreatment with vehicle, 5 µM FER1 for 24 h, or treated with DMSO or RSL3 alone or in combination with 5 mM 4-carboxybenzaldehyde (4CBA) for 48 h, following pretreatment with vehicle, 5 mM 4CBA for 24 h, n = 3 (e) or 4 independent replicates (f). CoQ and CoQH2 analysis of A549 cells 72 h after transfection with siRNAs, n = 3 independent samples (g). NADH consumption assay (A340 nm) in TBS buffer containing different concentrations of CoQ10 with or without recombinant human LDHB. Representative curves from an independent test with three technical replicates are shown. Tests were performed independently three times (h). NADH development assay (A340 nm) in glycine buffer containing 1 mM lactate, 216 mM hydrazine, and 500 µM NAD with different concentrations of CoQ10. Representative curves from an independent test with three technical replicates are shown. Tests were performed independently three times (i). Diagram illustrating how LDHB inhibits mitochondrial lipid peroxidation (j). Assessment of mitochondrial lipid peroxidation (k) and PI staining (l) by flow cytometry in A549, HT1080, PANC-1, and MSTO-211H cells after 72 h of transfection with siRNAs treated with DMSO or RSL3 alone (1 µM for A549, 0.75 µM for HT1080 and MSTO-211H cells, 0.5 µM PANC-1) or in combination with 500 nM mitoCoQH2 for 1 h (k) or 5–6 h (l), n = 3 independent replicates. Cell viability of HT1080 and MSTO-211H transfected cells treated with DMSO or RSL3 alone or in combination with 100 nM mitoCoQH2 or 100 nM mitoCoQ or 10 µM CoQ10, for 48 h, following pretreatment with vehicle, 100 nM mitoCoQH2 or 100 nM mitoCoQ or 10 µM CoQ10 for 24 h, n = 3 independent replicates (m). Data were presented as mean ± SD. Two-way ANOVA (c, d). Unpaired, two-tailed t-test (b, g, k, l). ns no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are provided as a Source Data file.

Fig. 5. Inhibition of LDHB sensitizes tumor cells to radiotherapy by enhancing mitochondria-associated ferroptosis.

Mitochondrial lipid peroxidation was assessed by flow cytometry in MSTO-211H after 24 h of irradiation with 6 Gy, following pretreatment with 5 µM FER1, 20 µM mitoTEMPO, 500 nM mitoCoQH2 for 5 h, n = 3 independent replicates (a, b). Mitochondrial lipid peroxidation was assessed by immunofluorescence in A549 siRNAs cells after 24 h of irradiation with 6 Gy, n = 3 random fields (c scale bar, 20 µm). Clonogenic survival curves for A549 siRNAs cells after irradiation with doses from 0 to 6 Gy, data were normalized to the corresponding unirradiated control group, n = 3 independent replicates (d). Representative images and analysis of clonogenic survival assay of A549 shCTRL, shLDHB cells irradiated with doses at 0, 4, 6 Gy alone or in combination with 20 µM mitoTEMPO, following pretreatment with vehicle, 20 µM mitoTEMPO for 24 h, n = 3 independent replicates (e, f). Volume of A549 shCTRL 0 Gy (n = 10), shLDHB-10 Gy (n = 8), shLDHB-20 Gy (n = 12) and 10 Gy irradiated xenograft tumors (n = 10) (g). Volume (h) and representative microCT images (i) of Ldhb^+/+; Kras^LSL-G12D/+;p53^fl/fl (Ldhb WT) and Ldhb^-/-; Kras^LSL-G12D/+;p53^fl/fl (Ldhb KO) lung tumors (red) at different time points after local irradiation with 0 Gy or 24 Gy, Ldhb WT 0 Gy (n = 9), Ldhb KO 0 Gy (n = 8), Ldhb WT 24 Gy (n = 10), Ldhb KO 10 Gy (n = 12). The log2 fold change in tumor volume after 27 days of local irradiation with 0 Gy or 24 Gy, Ldhb WT 0 Gy (n = 9), Ldhb KO 0 Gy (n = 8), Ldhb WT 24 Gy (n = 10), Ldhb KO 10 Gy (n = 12) (j). Kaplan–Meier overall survival analysis of high (n = 31) and low (n = 34) LDHB groups in lung cancer patients who received radiotherapy (k). Data were presented as mean ± SD (b, d–f, j) or as mean ± SEM (g, h). Unpaired, two-tailed t-test. ns no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are provided as a Source Data file.