Inhibition of ERRα Prevents Mitochondrial Pyruvate Uptake Exposing NADPH-Generating Pathways as Targetable Vulnerabilities in Breast Cancer

Abstract

Most cancer cells exhibit metabolic flexibility, enabling them to withstand fluctuations in intratumoral concentrations of glucose (and other nutrients) and changes in oxygen availability. While these adaptive responses make it difficult to achieve clinically useful anti-tumor responses when targeting a single metabolic pathway, they can also serve as targetable metabolic vulnerabilities that can be therapeutically exploited. Previously, we demonstrated that inhibition of estrogen-related receptor alpha (ERRα) significantly disrupts mitochondrial metabolism and that this results in substantial antitumor activity in animal models of breast cancer. Here we show that ERRα inhibition interferes with pyruvate entry into mitochondria by inhibiting the expression of mitochondrial pyruvate carrier 1 (MPC1). This results in a dramatic increase in the reliance of cells on glutamine oxidation and the pentose phosphate pathway to maintain nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis. In this manner, ERRα inhibition increases the efficacy of glutaminase and glucose-6-phosphate dehydrogenase inhibitors, a finding that has clinical significance.

Keywords: ERR⍺; MPC1; NADPH; breast cancer; glutaminase; mitochondrial metabolism; nuclear receptor; oxidative stress; pentose phosphate pathway; pyruvate carrier.

Figure 1.. Metabolic Changes in Response to ERRα Inhibitor Cpd29 in Glucose-Deprived Conditions

(A) Relative abundance of indicated metabolites in MDA436 cells cultured in glucose and glutamine-free DMEM (10% dialyzed fetal bovine serum [FBS]) supplemented with 10 mM lactate + 2 mM glutamine (glucose-deprived conditions) and treated with DMSO or 5 μM Cpd29 for 24 h (left). A schematic representation of metabolic pathways upregulated in response to Cpd29. Red circles represent increased metabolites (right). (B) A schematic representation of ¹³C-glutamine incorporation into metabolites of the TCA cycle. Black and red circles are ¹²C and ¹³C, respectively (left). Percent of M+4 succinate, fumarate, malate, and aspartate in MDA436 cells following steady-state tracing (24-h labeling) with 10 mM lactate + 2 mM [U-¹³C]-glutamine in the presence or absence of 5 μM Cpd29 (right). (C) Mass isotopomer distribution of pyruvate in MDA436 cells grown in the same conditions as in (B). The levels of metabolites in Cpd29-treated group were normalized with respect to the relevant metabolites in DMSO controls. The error bars represent SD (n = 3). The p value was calculated using two-tailed Student’s t test, **p < 0.05. See also Figure S1.

Figure 2.. Upon ERRα Inhibition, Glutamine Flux into Mitochondria Is Increased to Generate NADPH

(A) A schematic representation of mitochondrial NADPH synthesis pathway. (B) NADP⁺ and NADPH levels were determined over time in MDA436 cells cultured in glucose and glutamine-free DMEM (10% dialyzed FBS) supplemented with 10 mM lactate + 2 mM glutamine in the treatment of DMSO or 5 μM Cpd29. (C) MDA436 cells were incubated for 48 h in glucose and glutamine-free DMEM (10% dialyzed FBS) supplemented with 10 mM lactate + 2 mM glutamine or 10 mM lactate + 10 mM glutamine in the presence of Cpd29, and cells were then incubated with CM-H₂DCFDA for 60 min and the intensity of fluorescence was measured using flow cytometry. (D) MDA436 cells were cultured for 3 days in the same conditions as in (C), and the levels of NADP⁺ and NADPH were measured.(E and F) MDA436 cells were cultured in the indicated media in the presence of Cpd29 or BPTES (1 μM) and harvested at the time points shown, and cell numbers were determined. (G) MDA436 cells were transfected with control (siCtrl) or ERRα (siERRα) siRNAs for 48 h. Cells were then switched to the indicated media. Cells were harvested at the time points shown, and cell numbers were determined. (H) MDA436 cells were cultured in the indicated media in the presence of GSH (10 mM), and cell numbers were determined. (I) ROS levels were measured in the presence of indicated treatments for 48 h. (J) MDA436 cells were transfected with control (siCtrl) or ERRα (siERRα) siRNAs for 48 h. Cells were then switched to the indicated media in the absence or presence of GSH. Cells were harvested at the time points shown, and cell numbers were determined. Cell numbers were determined by staining with the DNA dye Hoechst 33258. The error bars represent SD (n = 3). The p value was calculated using two-tailed Student’s t test, **p < 0.05. See also Figure S2.

Figure 3.. ERRα Inhibition Leads to a Compensatory Upregulation of Glucose Flux into Pentose Phosphate Pathway and OCM and Glutamine Flux into Mitochondria

(A) Relative abundance of indicated metabolites in MDA436 cells cultured in glucose and glutamine-free DMEM (10% dialyzed FBS) supplemented with 12.5 mM glucose + 2 mM glutamine in the treatment of DMSO or 5 μM Cpd29 for 24 h. (B) A schematic representation of ¹³C-glucose or ¹³C-glutamine incorporation into metabolites. Black and red circles are ¹²C and ¹³C, respectively. (C and D) Mass isotopomer distribution of pyruvate (C) and lactate (D) in MDA436 cells grown in glucose and glutamine-free DMEM (10% dialyzed FBS) supplemented with 12.5 mM [U-¹³C] glucose + 2 mM glutamine or 12.5 mM glucose + 2 mM [U-¹³C] glutamine in the presence of DMSO or Cpd29 for 24 h. (E) Mass isotopomer distribution of aconitate and α-ketoglutarate from 12.5 mM [U-¹³C] glucose + 2 mM glutamine in the presence of DMSO or Cpd29 for 24 h. (F) Relative abundance of M+0 or M+4 succinate, fumarate, malate, and aspartate in MDA436 cells cultured in glucose-free media supplemented with 12.5 mM [U-¹³C] glucose + 2 mM glutamine or 12.5 mM glucose + 2 mM [U-¹³C] glutamine for 24 h in the presence or absence of 5 μM Cpd29. (G) Mass isotopomer distribution of glucose-6-phosphate (G6P) and serine from cells cultured in 12.5 mM [U-¹³C] glucose + 2 mM glutamine or 12.5 mM glucose + 2 mM [U-¹³C] glutamine for 24 h in the presence or absence of 5 μM Cpd29. In all isotopomer analyses, the levels of metabolites in Cpd29-treated group were normalized with respect to the relevant metabolites in DMSO controls. (H) MDA436 cells were transfected with control (siCtrl), ERRα (siERRα), or MDH1 (siMDH1) siRNAs in media with 12.5 mM glucose + 2 mM glutamine (glucose-replete media), and NADPH/NADP⁺ ratio was determined. (I) MDA436 cells were cultured in the indicated media in the presence of 10 mM GSH, and cell numbers were determined by staining with the DNA dye Hoechst 33258. The error bars represent SD (n = 3). The p value was calculated using two-tailed Student’s t test, **p < 0.05. See also Figure S3.

Figure 4.. MPC1 Is a Direct Transcriptional Target of ERRα

(A and B) MDA436 cells were transfected with control (siCtrl) or ERRα (siERRα) siRNAs (A) or treated with DMSO or Cpd29 (B) in glucose-replete media. RNA and protein were harvested over time, and the expression of MPC1 was analyzed by qRT-PCR and immunoblotting. (C and D) MDA436 cells were transfected with either control (siCtrl) or ERRα (siERRα-A and siERRα-B) siRNAs for 24 h, followed by infection of adenoviruses expressing β-gal (negative control), wild-type PGC-1α, ERRα-selective PGC-1α (2X9), or the nuclear-receptor-binding-deficient PGC-1α (L2L3M) for 48 h. RNA(C) and protein (D) were harvested, and the expression of MPC1 was analyzed by qRT-PCR and immunoblotting. (E) Tracks are retrieved from ENCODE ChIP-seq datasets and shown for MPC1 genomic regions with ERRα occupancy. (F) ChIP was performed to validate ERRα recruitment to MPC1 genomic regions in MDA436 cells transfected with either control (siCtrl) or ERRα (siERRα-A and siERRα-B) siRNAs for 24 h, followed by infection of adenoviruses expressing β-gal (negative control), wild-type PGC-1α, ERRα-selective PGC-1α (2X9), or the nuclear-receptor-binding-deficient PGC-1α (L2L3M) for 48 h. The error bars represent SD (n = 3). The p value was calculated using two-tailed Student’s t test, **p < 0.05. See also Figure S4.

Figure 5.. MPC1 Overexpression Restores Pyruvate Flux into Mitochondria in ERRα-Inhibited Cells

(A) Western blot analysis of MDA436 cell lines with transduction of lentiviral targeting vector (Ctrl) or MPC1/2. MPC1/2 overexpressing cell lines were generated by fluorescence sorting three times. (B) Relative abundance of indicated metabolites in control (Ctrl) and MPC1/2-expressing MDA436 cells cultured in glucose and glutamine-free DMEM (10% dialyzed FBS) supplemented with 10 mM lactate + 2 mM glutamine in the treatment of DMSO or 5 μM Cpd29 for 24 h. (C and D) Mass isotopomer distribution of pyruvate (C), citrate, and aconitate (D) in Ctrl and MPC1/2-expressing MDA436 cells grown in glucose and glutamine-free DMEM (10% dialyzed FBS) supplemented with 10 mM [U-¹³C] lactate + 2 mM glutamine in the presence of DMSO or Cpd29 for 24 h. The levels of metabolites were normalized with respect to the relevant metabolites in Ctrl-DMSO. (E–G) Control (MDA436_IG2/IC2) and MPC1/2-expressing (MDA436_MPC1/2) cells were cultured in glucose-deprived (E) or glucose-replete media (F) in the presence of Cpd29 or MPC inhibitor UK5099 (5 μM) (G) and harvested at the time points shown, and cell numbers were determined. Cell numbers were determined by staining with the DNA dye Hoechst 33258. The error bars represent SD (n = 3). The p value was calculated using two-tailed Student’s t test, **p < 0.05. See also Figure S5.

Figure 6.. Cpd29 in Combination with Inhibitors Targeting NADPH Synthesis Pathways Is More Effective to Suppress Breast Cancer Cell Proliferation In Vitro

(A) A schematic representation of metabolite changes in response to Cpd29 in glucose-replete conditions. Red circle represents increased metabolites, and blue circle represents decreased metabolites. (B) MDA436 cells were treated with ERRα antagonist Cpd29 (5 μM), GLS1 inhibitor CB-839 (1 μM), G6PD inhibitor DHEA (30 μM), or DHFR inhibitor MTX (25 nM) alone or in combination for 8 days. Cell numbers were determined by staining with Hoechst 33258. (C) MDA436 cells were cultured in glucose-replete media with the indicated pharmacological inhibitors in the presence or absence of dNTP mix (100 μM each). Cells were harvested at the time points shown, and cell numbers were determined by staining with the DNA dye Hoechst 33258. See also Figure S6.

Figure 7.. Analysis of ERRα Antagonist and CB-839 Combinations in an Animal Model of Breast Cancer

(A) MDA436 xenografts were treated with Cpd29 (30 mg/kg/day), CB-839 (400 mg/kg/day), or the combination of the two drugs and tumor volumes were measured throughout the experiment. (B) Body weight was also monitored during the course of the experiment. Data are the mean of tumor volumes in each experimental group (Ctrl, n = 15; Cpd29, n = 14; CB-839, n = 9; Cpd29+CB-839, n = 8). Error bars represent SEM. Statistical significance from single agent alone. The p value was calculated using two-way ANOVA followed by a Bonferroni multiple comparison test, **p < 0.05. See also Figure S7.