Estrogen Related Receptor Alpha (ERRα) a Bridge between Metabolism and Adrenocortical Cancer Progression

Abstract

The aim of this study was to investigate the metabolic changes that occur in adrenocortical cancer (ACC) cells in response to the modulation of Estrogen Related Receptor (ERR)α expression and the impact on ACC progression. Proteomics analysis and metabolic profiling highlighted an important role for ERRα in the regulation of ACC metabolism. Stable ERRα overexpression in H295R cells promoted a better mitochondrial fitness and prompted toward a more aggressive phenotype characterized by higher Vimentin expression, enhanced cell migration and spheroids formation. By contrast, a decrease in ERRα protein levels, by molecular (short hairpin RNA) and pharmacological (inverse agonist XCT790) approaches modified the energetic status toward a low energy profile and reduced Vimentin expression and ability to form spheroids. XCT790 produced similar effects on two additional ACC cell lines, SW13 and mitotane-resistant MUC-1 cells. Our findings show that ERRα is able to modulate the metabolic profile of ACC cells, and its inhibition can strongly prevent the growth of mitotane-resistant ACC cells and the progression of ACC cell models to a highly migratory phenotype. Consequently, ERRα can be considered an important target for the design of new therapeutic strategies to fight ACC progression.

Keywords: ERRα; XCT790; adrenocortical cancer; cancer progression; metabolic changes; mitochondria.

Figure 1

Functional enrichment analysis of differentially expressed proteins (a): up-regulated; (b): down-regulated proteins) between untreated and XCT790-treated cells. KEGG database and selected metabolism-related pathways were used. Only significant enriched pathways are reported (FDR-adjusted p-value, padj < 0.05, i.e., −log10(padj) > 1.3).

Figure 2

Metabolic changes in H295R cells related to ERRα expression levels. The metabolic profiles of H295R wild type (WT), shCTR, shERRα−/− and ERRα+/+ cells were assessed by Seahorse XFe96 Analyzer. (a,b) ATP Rate Assay was evaluated as indicated in “Materials and Methods”. Graphs represent the mean ± SD of three independent experiments of Total ATP Production Rate (pmol/min) (a) and ATP production (%) (b) deriving from glycolysis and oxidative phosphorylation after the sequential addition of specific inhibitors; (* p < 0.05 vs. WT). (c–e) Mitochondrial Stress Analysis was performed as indicated in “Materials and Methods”. Graphs represent the mean ± SD of three independent experiments of real-time oxygen consumption (OCR) rate (pmol/min/cells); (* p < 0.05 vs. shCTR). Mitochondrial Respiration (c), Basal Respiration (d), Maximal Respiration (e) were measured from OCR after the addition of specific inhibitors. (f–h) Glycolytic Stress Analysis was performed as indicated in “Materials and Methods”. Graph represents the mean ± SD of three independent experiments of Real-time extracellular acidification (ECAR) rate (mpH/min/cells); (* p < 0.05 vs. shCTR). Glycolitic function (f), Glycolysis (g) and Glycolytic Capacity (h) were measured from ECAR after the addition of specific inhibitors.

Figure 3

Effect of XCT790 treatment on H295R cell metabolism. The metabolic profiles of H295R cells untreated (0) or treated with XCT790 (1, 5, 10 µM) for 18h were assessed using the Seahorse XFe96 analyzer. (a,b) ATP Rate Assay was evaluated as indicated in “Materials and Methods”. Graphs represent the mean ± SD of three independent experiments of Total ATP Production Rate (pmol/min) (a) and ATP production (%) (b) derived from glycolysis and oxidative phosphorylation after the sequential addition of specific inhibitors; (* p < 0.05 vs. 0). (c–e) Mitochondrial Stress Analysis was performed as indicated in “Materials and Methods”. Graphs represent the mean ± SD of three independent experiments of real-time oxygen consumption (OCR) rate (pmol/min/cells); (* p < 0.05 vs. 0). Mitochondrial Respiration(c), Basal Respiration (d), Maximal Respiration (e) were measured from OCR after the addition of specific inhibitors. (f–h) Glycolytic Stress Analysis was performed as indicated in “Materials and Methods”. Graphs represent the mean ± SD of three independent experiments of real-time extracellular acidification (ECAR) rate (mpH/min/cells); (* p < 0.05 vs. 0). Glycolitic function (f), Glycolysis (g) and Glycolytic Capacity (h) were measured from ECAR after the addition of specific inhibitors.

Figure 4

Effect of XCT790 treatment on SW13 cell metabolism. The metabolic profiles of SW13 cells untreated (0) or treated with XCT790 (1, 5, 10 µM) for 18 h were assessed by Seahorse XFe96 analyzer. (a,b) ATP Rate Assay was evaluated as indicated in “Materials and Methods”. Graphs represent the mean ± SD of three independent experiments of Total ATP Production Rate (pmol/min) (a) and ATP production (%) (b) derived from glycolysis and oxidative phosphorylation after the sequential addition of specific inhibitors (* p < 0.05 vs. 0). (c–e) Mitochondrial Stress Analysis was performed as indicated in “Materials and Methods”. Graphs represent the mean ± SD of three independent experiments of real-time oxygen consumption (OCR) rate (pmol/min/cells) (* p < 0.05 vs. 0). Mitochondrial Respiration(c), Basal Respiration (d), Maximal Respiration (e) were measured from OCR after the addition of specific inhibitors. (f–h) Glycolytic Stress Analysis was performed as indicated in “Materials and Methods”. Graphs represent the mean ± SD of three independent experiments of real-time extracellular acidification (ECAR) rate (mpH/min/cells); (* p < 0.05 vs. 0). Glycolitic function (f), Glycolysis (g) and Glycolytic Capacity (h) were measured from ECAR after the addition of specific inhibitors.

Figure 5

Effect of XCT790 treatment on MUC-1 cell metabolism. The metabolic profiles of MUC-1 cells untreated (0) or treated with XCT790 (1, 5, 10 µM) for 18 h were assessed by Seahorse XFe96 analyzer. (a,b) ATP Rate Assay was evaluated as indicated in “Materials and Methods”. Graphs represent the mean ± SD of three independent experiments of Total ATP Production Rate (pmol/min) (a) and ATP production (%) (b) derived from glycolysis and oxidative phosphorylation after the sequential addition of specific inhibitors (* p < 0.05 vs. 0). (c–e) Mitochondrial Stress Analysis was performed as indicated in “Materials and Methods”. Graphs represent the mean ± SD of three independent experiments of real-time oxygen consumption (OCR) rate (pmol/min/cells) (* p < 0.05 vs. 0). Mitochondrial Respiration (c), Basal Respiration (d), Maximal Respiration (e) were measured from OCR after the addition of specific inhibitors. (f–h) Glycolytic Stress Analysis was performed as indicated in “Materials and Methods”. Graphs represent the mean ± SD of three independent experiments of real-time extracellular acidification (ECAR) rate (mpH/min/cells); (* p < 0.05 vs. 0). Glycolitic function (f), Glycolysis (g) and Glycolytic Capacity (h) were measured from ECAR after the addition of specific inhibitors.

Figure 6

ERRα modulates H295R cell motility and Vimentin expression. (a,b) H295R (WT), H295R clones, knock in (ERRα+/+) or knock out (shERRα−/−) for ERRα gene, and H295R cell stably transfected with control plasmid (shCTR) were used in Wound Healing (a) and Boyden Chamber (b) assays as reported in “Materials and Methods”. Images are from a representative experiment. (c,d) H295R cells were treated with vehicle (0) or XCT790 (1, 5, 10 μM) for 18 h and Wound Healing (c) and Boyden Chamber (d) assays were performed as reported in “Materials and Methods”. Images are from a representative experiment. (c) The wounds were observed under an inverted microscope immediately (0 h) and 18 h after the scratch (100× magnification). (b,d) Migrated cells were photographed under an inverted microscope and counted (see Material and Methods), 20× magnification. Graphs represent the mean ± SD of three independent experiments. The number of untreated cells (0) was set as 100% (* p < 0.05 vs. 0). (e) Total proteins from H295R clones (shCTR, shERRα−/−, ERRα+/+) were analyzed by western Blotting (WB) using antibodies against ERRα and Vimentin. GAPDH was used as a loading control. Blots are representative of three independent experiments with similar results. (f) H295R were transfected for 48 h with pcDNA3.1 non containing (EV) or containing ERRα coding sequence (pcDNA3.1-ERRα). After transfection cells were left untreated (−) or treated (+) for 24 h with XCT790 (10 μM). Total proteins were analyzed by WB using antibodies against ERRα and Vimentin. GAPDH was used as a loading control. (g) Cells were untreated (0) or treated with XCT790 (1, 5, 10 μM) for 24 h. Total proteins were analyzed by WB using antibodies against ERRα and Vimentin. GAPDH was used as a loading control. Original image of western blot can be found at File S1.

Figure 7

ERRα promotes H295R spheroids formation. (a) H295R were transfected for 48 h with pcDNA3.1 non containing (EV) or containing ERRα coding sequence (pcDNA3.1-ERRα) and then grown as 3D spheroids for 5 days. Spheroids were counted under an inverted microscope and results were expressed as fold change over control (EV) ± SD (TSFE, tumor spheroids formation efficiency); (* p < 0.05 vs. EV). Insert confirms ERRα overexpression. (b) H295R cells were left untreated (0) or treated with XCT790 (1, 5, 10 μM) for 24 h and TSFE was evaluated 5 days later (* p < 0.05 vs. 0). Images below graph are from a representative experiment (20× magnification). (c) Wild type H295R (WT) and H295R clones (shCTR, shERRα−/−, ERRα+/+) were used to evaluate 3D spheroids formation. TSFE was evaluated 5 days later (* p < 0.05 vs. WT). Images below graph are from a representative experiment (20× magnification). (d) H295R spheroids (H295R Sph-5) were allowed to grow for 5 days and then trypsinized and reseeded weekly in spheroid media for 5 weeks. Boyden Chamber Assay was performed as reported in the “Materials and Methods”. Migrated cells were randomly photographed and counted with ImageJ software (* p < 0.05 vs. WT). (e) H295R (WT) cells and H295R grown as spheroids for 5 weeks (H295R Sph-5), were analyzed by WB using antibody against Vimentin. GAPDH was used as a loading control. Blots are representative of three independent experiments with similar results. Original image of western blot can be found at File S1.

Figure 8

Effects of XCT790 treatment on MUC-1 cells. (a) MUC-1 cells were left untreated (0) or treated with XCT790 (1, 5, 10 μM) for 24 h. Total proteins were analyzed by WB using antibodies against ERRα. GAPDH was used as a loading control. Blots are from one experiment representative of three with similar results. (b) MUC-1 cells were seeded in 12-well plates and allowed to grow in the absence or presence of different XCT790 (1, 5, 10 μM) doses for 14 days. Colonies were stained with 0.05% Coomassie Blue in methanol/water/acetic acid (45:45:10, v/v/v). Colony number (relative colony formation rate) was assessed using Image J software and normalized to untreated cells (0). (c,d) MUC-1 cells were seeded in the Boyden insert and vehicle (0), XCT790 (1, 5, 10 μM) (C) or mitotane (2.5, 25, 40 μM) (d) were added in the upper chamber; cells were allowed to migrate across the membrane for 18 h. Migrated cells were photographed under an inverted microscope and counted (see Material and Methods), with 20× magnification. The number of untreated cells (0) was set as 100% (* p < 0.05 vs. 0). Images below are from a representative experiment (20× magnification). (e) MUC-1 cells were untreated (0) or treated with XCT790 (1, 5, 10 μM) for 24 h. TSFE was evaluated 5 days later. Results were expressed as fold change over untreated cells (0) ±SD; (* p < 0.05 vs. 0). Images are from a representative experiment (20× magnification). Original image of western blot can be found at File S1.

Figure 9

ERRα expression levels and cholesterol influence H295R cell migration. (a) H295R clones (shCTR, ERRα+/+, shERRα−/−) were maintained in 5% FBS or 5% LpFS containing medium. Cells were used in Wound Healing (a) and Boyden Chamber (b) assays performed as reported in “Materials and Methods”. (a) Images are from a representative experiment (100× magnification). (b) Migrated cells were photographed under an inverted microscope (20× magnification) and counted with ImageJ software. Graphs represent the mean ± SD of three independent experiments (* p < 0.05 vs. FBS).