Stress-induced epinephrine enhances lactate dehydrogenase A and promotes breast cancer stem-like cells

Abstract

Chronic stress triggers activation of the sympathetic nervous system and drives malignancy. Using an immunodeficient murine system, we showed that chronic stress-induced epinephrine promoted breast cancer stem-like properties via lactate dehydrogenase A-dependent (LDHA-dependent) metabolic rewiring. Chronic stress-induced epinephrine activated LDHA to generate lactate, and the adjusted pH directed USP28-mediated deubiquitination and stabilization of MYC. The SLUG promoter was then activated by MYC, which promoted development of breast cancer stem-like traits. Using a drug screen that targeted LDHA, we found that a chronic stress-induced cancer stem-like phenotype could be reversed by vitamin C. These findings demonstrated the critical importance of psychological factors in promoting stem-like properties in breast cancer cells. Thus, the LDHA-lowering agent vitamin C can be a potential approach for combating stress-associated breast cancer.

Keywords: Breast cancer; Metabolism; Oncology.

Figure 1. Chronic stress promotes ADRB2-dependent cancer stem cell–like properties in vivo.

(A) Tumor growth of MDA-MB-231 tumors in control (Ctrl) and stressed mice; n = 5 (1-way ANOVA). (B–D) Primary MDA-MB-231 tumors from the Ctrl and stress groups were subjected to immunoblot (C, control; S, stressed) (B), immunohistochemical staining (scale bar: 50 μm; original magnification, ×20, ×40, ×96 [insets]) (C), and primary and secondary spheroid formation; n = 5 (1-way ANOVA) (D). (E) Concentrations (pg/ml) of cortisol (Cort), norepinephrine (NE), and epinephrine (Epi) in serum of Ctrl and stress mice after the last day of stress; n = 5 (Student’s t test). (F) Immunoblot analysis of indicated antibodies in MDA-MB-231 cells treated with indicated concentrations of Epi. (G) Growth of Ctrl, propranolol (Pro), stress, and stress-induced propranolol-treated (Pro + stress) MDA-MB-231 tumors in mice; n = 6 (1-way ANOVA). (H) MDA-MB-231 cells were transfected with siADRB2 and then treated with Epi for 5 days. Expression of proteins was determined by immunoblot analysis. (I) Growth of MDA-MB-231 tumors in Ctrl and stress mice in the presence or absence of ICI118,551 (ICI); n = 5 (1-way ANOVA). (J) Model of chronic stress–mediated cancer stem-like traits mediated by β₂-adrenergic receptor (ADRB2) signaling. Data are representative of at least 3 independent experiments. Data represent mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. Chronic stress–induced MYC activates SLUG transcription to stimulate CSCs.

(A) A cluster heatmap of expression profiles of mRNAs in PBS- and Epi-treated MDA-MB-231–derived tumors; n = 3. (B) Comparison of array data (fold change >2, Q < 0.05, 54 genes) with stem-like cell genes (405 genes). Common genes were verified by Epi treatment and are listed in the column according to fold change. n = 3. (C) Distribution patterns and number (d > 50 μm) of mammospheres from the negative control (shNC) or shSLUG MDA-MB-231 cells after treatment with PBS or Epi for 5 days; n = 3 (1-way ANOVA). (D) Growth of shNC or shSLUG-3 MDA-MB-231 tumors in mice with or without stress treatment; n = 6 (1-way ANOVA). (E) Dual-luciferase reporter assays of MDA-MB-231 cells transfected with SLUG truncated promoters or empty vector (EV) in the presence or absence of Epi for 5 days; n = 3 (1-way ANOVA). (F) Dual-luciferase analysis in shNC or shMYC MDA-MB-231 cells treated with PBS or Epi for 5 days and transfected with EV or SLUG promoter (–496 to 0); n = 3 (1-way ANOVA). (G) Dual-luciferase reporter assays of MDA-MB-231 cells treated with Epi for 5 days and transfected with SLUG WT, mutant 1 (Mut1, –57 to –54), mutant 2 (Mut2, –104 to –101), or mutant 3 (Mut3, –412 to –408) promoters; n = 3 (1-way ANOVA). (H) ChIP-PCR analysis in MDA-MB-231 cells of MYC occupancy on the SLUG promoter. (I) Model of Epi-induced cancer stem-like traits through MYC/SLUG signaling. Data are representative of at least 3 independent experiments. Data represent mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Chronic stress–induced USP28 stabilizes MYC and promotes CSCs.

(A) Immunoblots of MYC in MDA-MB-231 and MCF-7 cells treated for 5 days with PBS or Epi. (B) MDA-MB-231 cells were treated with Epi for 5 days followed by treatment with cycloheximide (CHX) for the indicated times. The intensity of MYC expression for each time point was quantified by densitometry and plotted against time. (C) Immunoblots of MDA-MB-231 cells treated with Epi for 5 days and then incubated with or without MG132 for 6 hours. (D) Fold change of immunoblots of MDA-MB-231 cells transfected with indicated plasmids followed by treatment with Epi for 5 days; n = 3 (Student’s t test). (E) Immunoblot analysis of MDA-MB-231 cells transfected with USP28 siRNA-2 followed by treatment with PBS or Epi for 5 days. (F) Immunoprecipitation of USP28 constructs and MYC (amino acids 1–95) in 293T cells. (G) Free-energy surface of the USP28^WT-MYC^46–74 complex and USP28^Mut-MYC^46–74 complex (top panel). Gray cartoons, USP28^WT/Mut structures; red spheres, side chain of His⁶⁰⁰ and Cys/Ala¹⁷¹; green cartoons, MYC motifs; blue spheres, Lys⁵¹ and Lys⁵² in MYC motif. (H) Ubiquitin assays of 293T cells transfected with MYC and WT or a C171A mutant (Mut) of USP28 followed by treatment with Epi. (I) Distribution patterns and number (d > 50 μm) of mammospheres from cells in E; n = 3 (1-way ANOVA). (J) Model of Epi-induced cancer stem-like traits through USP28/MYC/SLUG signaling. Data are representative of at least 3 independent experiments. Data represent mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. Chronic stress elevates LDHA to enhance glycolysis.

(A) Glucose uptake, lactate production, and cellular ATP were measured in MCF-7 and MDA-MB-231 cells treated with PBS or Epi for 5 days; n = 3 (Student’s t test). (B) Representative heatmap of metabolome profiles (top panel). Heatmap colors represent relative metabolite levels as indicated in the color key. Average fold change of glycolytic metabolites was measured by capillary electrophoresis–mass spectrometry (bottom). G-6-P, glucose-6-phosphate; F-6-P, fructose-6-phosphate; FBP, fructose-1,6-phosphate. n = 3 (Student’s t test). (C) Relative mRNA expression of indicated genes in 4 GEO databases analyzed by GEO2R. (D) Immunoblot analysis of MDA-MB-231 cells treated with PBS or Epi for 5 days. (E) Immunoblot analysis of MDA-MB-231 and MCF-7 cells transfected with siLDHA in the presence or absence of Epi for 5 days. (F) Model of Epi-induced USP28/MYC signaling through LDHA-mediated metabolic rewiring. Data are representative of at least 3 independent experiments. Data represent mean ± SEM; *P < 0.05, **P < 0.01.

Figure 5. LDHA generating lactate enhances the USP28 signaling.

(A) Immunoblots of siNC and siLDHA MDA-MB-231 cells treated with CHX for the indicated times. Intensity of USP28 expression for each time point was quantified by densitometry and plotted against time (right panel). (B) Immunoblots of MCF-7 cells treated for 5 days with Epi and/or siLDHA followed by incubation with or without MG132 for 6 hours. (C) Ubiquitin assays of 293T cells transfected with ubiquitin (Ubi) and siLDHA followed by treatment with Epi. (D) Immunoblots of MDA-MB-231 cells treated with the indicated concentrations of sodium oxamate (Oxa) for 48 hours. (E) Immunoblots of MDA-MB-231 and MCF-7 cells treated with lactate (Lac) for 72 hours. (F) Immunoblots of MDA-MB-231 cells transfected with USP28 WT or C171A and then treated with lactate for 72 hours. (G) Immunoblots of immunoprecipitation experiments of 293T cells transfected with USP28 WT or C171A and then treated with lactate for 72 hours. (H) MDA-MB-231 cells were treated with lactate, hydrochloric acid (HCl), and acetic acid (HAC) for 72 hours. Expression of the indicated proteins was examined by immunoblotting. (I) Snapshot structures of USP28 interacting with MYC motif extracted from constant-pH MD simulations at 2 representative pH conditions. Gray cartoons, USP28; green cartoons, MYC; blue spheres, Lys⁵¹ and Lys⁵² on MYC motif; red spheres and yellow spheres, His⁶⁰⁰ and Cys¹⁷¹ of USP28, respectively. (J) Lactate levels in serum of Ctrl (n = 15) or stress (n = 20) mice (Student’s t test). (K) Model of chronic stress–mediated USP28 stabilization through decreased pH caused by LDHA generating lactate. Data are representative of at least 3 independent experiments. Data represent mean ± SEM; ***P < 0.001.

Figure 6. Clinical relevance of LDHA expression under chronic stress.

(A) Representative immunohistochemistry photomicrographs of tissues stained with indicated antibodies in patients with breast cancer (Epi^lo, n = 42; and Epi^hi, n = 41). Scale bar: 50 μm; original magnification, ×20, ×40, ×96 (enlarged insets). (B) Immunoblot analysis of proteins in breast cancer tissues (T) and adjacent normal breast tissues (N); n = 5. (C) Expression of mRNA for the indicated genes in MDA-MB-231-2D cells or spheres was measured by quantitative reverse transcriptase PCR; n = 3 (Student’s t test). (D) Kaplan-Meier estimates of overall survival and disease-free survival of patients with breast cancer, according to the serum Epi concentrations (Epi^lo, n = 42; and Epi^hi, n = 41). Eighty-three patients were in the data set (log-rank test). (E) Kaplan-Meier estimates of overall survival and disease-free survival of patients with breast cancer, according to LDHA expression (LDHA^lo, n = 30; and LDHA^hi, n = 41). Seventy-one patients were in the data set (log-rank test). (F) Kaplan-Meier estimates of overall survival and disease-free survival of patients with breast cancer expressing high or low LDHA together with high or low serum Epi. Seventy-one patients were in the data set (log-rank test). Data are representative of at least 3 independent experiments. Data represent mean ± SEM; ***P < 0.001.

Figure 7. Vitamin C reverses chronic stress–induced breast cancer stem-like properties in vivo and in vitro.

(A) US drug collection screening: MDA-MB-231 cells stably expressed with pEGFP-LDHA were treated with 1280 drugs. Fluorescence intensities were examined. n = 3. (B) Representative heatmap of LDHA expression after treatment with the 18 candidate compounds; n = 3. Heatmap colors represent relative LDHA protein levels as indicated by the color key. (C) Immunoblot analysis of MDA-MB-231 and MCF-7 cells treated with vitamin C and/or Epi for 5 days. (D) Lactate levels were examined in the cell culture media of MDA-MB-231 cells in C; n = 3 (Student’s t test). (E) Representative spheroid images formed by single cells with vitamin C and/or Epi; n = 3. Scale bar: 50 μm. Bottom left panel shows distribution patterns of sphere diameter. Bottom right panel shows the number of spheres (d > 50 μm) (1-way ANOVA). (F) Tumor growth curves of indicated treatments of mice; n = 5 (1-way ANOVA). (G) Model of targeting chronic stress–mediated cancer stem-like traits by vitamin C. Data are representative of at least 3 independent experiments. Data represent mean ± SEM; *P < 0.05, **P < 0.01.