Targeting Cancer Metabolism Breaks Radioresistance by Impairing the Stress Response

Abstract

The heightened energetic demand increases lactate dehydrogenase (LDH) activity, the corresponding oncometabolite lactate, expression of heat shock proteins (HSPs) and thereby promotes therapy resistance in many malignant tumor cell types. Therefore, we assessed the coregulation of LDH and the heat shock response with respect to radiation resistance in different tumor cells (B16F10 murine melanoma and LS174T human colorectal adenocarcinoma). The inhibition of LDH activity by oxamate or GNE-140, glucose deprivation and LDHA/B double knockout (LDH^-/-) in B16F10 and LS174T cells significantly diminish tumor growth; ROS production and the cytosolic expression of different HSPs, including Hsp90, Hsp70 and Hsp27 concomitant with a reduction of heat shock factor 1 (HSF1)/pHSF1. An altered lipid metabolism mediated by a LDHA/B double knockout results in a decreased presence of the Hsp70-anchoring glycosphingolipid Gb3 on the cell surface of tumor cells, which, in turn, reduces the membrane Hsp70 density and increases the extracellular Hsp70 levels. Vice versa, elevated extracellular lactate/pyruvate concentrations increase the membrane Hsp70 expression in wildtype tumor cells. Functionally, an inhibition of LDH causes a generalized reduction of cytosolic and membrane-bound HSPs in tumor cells and significantly increases the radiosensitivity, which is associated with a G2/M arrest. We demonstrate that targeting of the lactate/pyruvate metabolism breaks the radioresistance by impairing the stress response.

Keywords: LDHA/B double knockout; lactate/pyruvate metabolism; membrane heat shock protein 70 (Hsp70); radiosensitivity; stress response.

Figure 1

Lactate dehydrogenase (LDH) inhibition by oxamate (Oxa) and GNE-140 (GNE) decreases the expression of Hsp90, Hsp70 and Hsp27. (a) The effect of Oxa (60 mM, 48 h) and GNE (10 µM, 24 h) on LDH activity in LS174T cells (*: p ≤ 0.05 and **: p ≤ 0.01). (b) Representative immunoblot showing the intracellular expression of Hsp90, Hsp70 and Hsp27 in untreated (Ctrl) and Oxa- or GNE-treated LS174T cells. β-Actin was used as a loading control. The quantification of the heat shock protein (HSP) expression levels is shown in the adjacent bar chart. Error bars show the standard deviations (SD) of at least three biological replicates (*: p ≤ 0.05, **: p ≤ 0.01 and ***: p ≤ 0.001). Full Western blot images are available in Figure S4.

Figure 2

LDHA/B double knockout and its effect on lactate dehydrogenase (LDH) activity. (a) Representative immunoblot showing the successful double knockout of LDHA and LDHB (referred to as LDH^−/−) of mouse B16F10 and human LS174T tumor cell lines. β-Actin was used as a loading control. (b) The effect of LDHA/B double knockout on LDH activity in B16F10 and LS174T cells (***: p ≤ 0.001). (c) Flow cytometric analysis of the lactate transporter MCT1 using MCT1-APC mAb on LS174T WT and LDH^−/− cells. The proportion of positively stained cells is shown. Error bars show the standard deviations of at least three biological replicates. Full Western blot images are available in Figure S5.

Figure 3

LDHA/B double knockout (LDH^−/−) inhibits the expression of heat shock proteins (HSP) Hsp90, Hsp70 and Hsp27 and inhibits the proliferative activity and reactive oxygen species (ROS) production in tumor cells. (a,b) Representative immunoblot showing the expression of intracellular Hsp90, Hsp70 and Hsp27 of B16F10 (a) and LS174T (b) cells. Quantification of the HSP expression levels are shown in the adjacent bar chart. Error bars show the standard deviations (SD) of at least four biological replicates (*: p ≤ 0.05, **: p ≤ 0.01 and ***: p ≤ 0.001). (c) A cell proliferation assay (CCK-8) demonstrated that LDHA/B double knockout inhibited the proliferation in LS174T cells (**: p ≤ 0.01 and ***: p ≤ 0.001). (d) The DCFDA assay was used to determine the intracellular ROS levels in LS174T cells, with fluorescence being measured using a microplate reader. Error bars show the SD of four biological replicates (**: p ≤ 0.01). Full Western blot images are available in Figures S6 and S7.

Figure 4

The lactate dehydrogenase (LDH) inhibition by oxamate (Oxa) and GNE-140 (GNE) and a LDHA/B double knockout (LDH^−/−) results in a reduced heat shock protein 70 (Hsp70) and globotriaosylceramide (Gb3) expression on the cell membrane. (a,b) Membrane-bound Hsp70 on untreated (Ctrl) and Oxa- or GNE-treated LS174T tumor cells (a) and membrane-bound Hsp70 on B16F10 and LS174T cells (b), as determined by flow cytometry using cmHsp70.1-FITC mAb. The proportion of positively stained cells is shown. Error bars show the standard deviations (SD) of at least three biological replicates (*: p ≤ 0.05, **: p ≤ 0.01 and ***: p ≤ 0.001). (c) LS174T cells stained for the expression of membrane Hsp70 (cmHsp70.1-FITC mAb, green) and Gb3 (CD77-PE mAb, red) were analyzed by confocal microscopy. The colocalization of Hsp70 and Gb3 is visualized in yellow as a merge of the red and green staining. Scale bar: 5 µm. (d) Extracellular Hsp70 levels in the supernatant of the LS174T cells were measured by ELISA and the data normalized to 1 × 10⁶ viable tumor cells. Error bars show the SD of five biological replicates (***: p ≤ 0.001). (e) Proteome profiling of LS174T WT and LDH^−/− cells. The most significant canonical pathways and top molecular functions altered by LDH depletion are illustrated. The analyses were generated through the use of Ingenuity Pathway Analysis (IPA) (QIAGEN Inc., https://www.qiagenbio-informatics.com/products/ingenuity-pathway-analysis, accessed on 22 April 2021). Bars indicate the canonical pathways, and the y-axis displays the −(log p) enrichment significance. NRF2: nuclear factor erythroid 2–related factor 2, PPARα: peroxisome proliferator-activated receptor alpha.

Figure 5

Inhibition of the LDHA/B activity significantly increases radiosensitivity in LS174T and B16F10 cells. (a) Colony-forming assay of untreated (Ctrl) and Oxa-treated LS174T cells after irradiation with 0 Gray (Gy, sham), 2 Gy, 4 Gy and 6 Gy (*: p ≤ 0.05). (b,c) Colony-forming assay of LS174T (b) and B16F10 (c) and wildtype (WT) and LDHA/B double knockout LDH^−/− cells after irradiation with 0 Gy (sham), 2 Gy, 4 Gy and 6 Gy (**: p ≤ 0.01 and ***: p ≤ 0.001). (d) Percentage of LS174T WT and LDH^−/− cells expressing membrane heat shock factor 70 (Hsp70) 24 h after irradiation with 0 Gy (sham), 2 Gy, 4 Gy and 6 Gy. Bars represent the mean value and the corresponding standard deviations (SD) of four independent experiments (***: p ≤ 0.001). (e) Representative immunoblot showing cytosolic Hsp70 expression by LS174T WT and LDH^−/− cells 24 h after irradiation with 0 Gy (sham), 2 Gy, 4 Gy and 6 Gy. β-Actin was used as a loading control. (f) LS174T WT and LDH^−/− cells were irradiated with 6 Gy. Twenty-four hours after irradiation, cells were fixed, and the cell cycle distribution was determined by flow cytometry. The mean value and the corresponding SD of three independent experiments is shown (***: p ≤ 0.001). (g) Schematic illustration of the major findings: An impaired LDH activity induced by an LDHA/B double knock out (LDH^−/−) or by the LDH inhibitors (Oxa and GNE) increases the radiosensitivity of tumor cells by a generalized downregulation of cytosolic pro-survival stress proteins, a reduction in plasma membrane-bound Hsp70 and reduced lactate levels-although the lactate transporter MCT1 remains unaffected-and an impaired lipid metabolism. Elevated extracellular Hsp70 levels are associated with a reduction in Hsp70 membrane-anchoring glycosphingolipid Gb3. Full Western blot images are available in Figure S8.