ARRB1 Regulates Metabolic Reprogramming to Promote Glycolysis in Stem Cell-Like Bladder Cancer Cells

Abstract

β-arrestin 1 (ARRB1) is a scaffold protein that regulates signaling downstream of G protein-coupled receptors (GPCRs). In the current work, we investigated the role of ARRB1 in regulating the metabolic preference of cancer stem cell (CSC)-like cells in bladder cancer (BC). We show that ARRB1 is crucial for spheroid formation and tumorigenic potential. Furthermore, we measured mitochondrial respiration, glucose uptake, glycolytic rate, mitochondrial/glycolytic ATP production and fuel oxidation in previously established ARRB1 knock out (KO) cells and corresponding controls. Our results demonstrate that depletion of ARRB1 decreased glycolytic rate and induced metabolic reprogramming towards oxidative phosphorylation. Mechanistically, the depletion of ARRB1 dramatically increased the mitochondrial pyruvate carrier MPC1 protein levels and reduced the glucose transporter GLUT1 protein levels along with glucose uptake. Overexpression of ARRB1 in ARRB1 KO cells reversed the phenotype and resulted in the upregulation of glycolysis. In conclusion, we show that ARRB1 regulates the metabolic preference of BC CSC-like cells and functions as a molecular switch that promotes reprogramming towards glycolysis by negatively regulating MPC1 and positively regulating GLUT1/ glucose uptake. These observations open new therapeutic avenues for targeting the metabolic preferences of cancer stem cell (CSC)-like BC cells.

Keywords: bladder cancer; cancer system cells; glucose transporter; metabolic reprograming; mitochondrial pyruvate carrier; β-arrestin 1.

Figure 1

ARRB1 depletion affects spheroid formation and tumor growth: ARRB1 KO clones were established using the CRISPR/Cas9 technology as described previously [19]. (a) ARRB1-depleted HT1376 cells (KO cl.9 and KO cl. 10) and corresponding ARRB1 wild type controls (NT) were seeded at 2000 cells/well in 96-well plates for spheroid formation. Cells were placed on SeedEZ scaffolds to prevent attachment on plates and to allow spheroid formation as described in Material and Methods. The Cell Titer-Glo 3D Cell Viability assay was utilized to determine spheroid viability. Data: means ± SEM, n = 4; *** p < 0.001 (one-way ANOVA). (b) 1 × 10⁶ ARRB1-depleted HT1376 cells (KO cl.10) (n = 6) and corresponding controls (NT) (n = 6) were transplanted subcutaneously in nude mice. Tumor growth was monitored weekly. Data: means ± SEM; * p = 0.0459 (two-way ANOVA). (c) Detection of CSC markers Bmi-1 and CD44 in ARRB1-KO HT1376 tumor xenografts and corresponding controls (NT) using immunofluorescence. Scale bar: 50 µm.

Figure 2

Depletion of ARRB1 alters mitochondrial respiration: (a) oxygen consumption rate (OCR) of ARRB1-depleted HT1376 cells (KO cl.9) and corresponding controls (NT) prior to and after injection of respiration modulators oligomycin (OLIG.), FCCP, and rotenone/antimycin A (ROT and A.A). Data: means ± SEM, n = 6; ** p < 0.01 (Two-way RM ANOVA). (b) Basal respiration in ARRB1-depleted HT1376 cells (KO cl. 9) and corresponding controls (NT). Data: means ± SEM, n = 6; *** p < 0.001 (two-tailed unpaired t test). (c) Mitochondrial ATP production in ARRB1-depleted HT1376 cells (KO cl. 9) and corresponding controls (NT) (calculated using the Seahorse XF Cell Mito Stress Test Generator). Data: means ± SEM, n = 6; *** p < 0.001 (two-tailed unpaired t test).

Figure 3

Depletion of ARRB1 decreases glycolysis and glycolytic ATP production rate: (a) total ATP per 10,000 cells in ARRB1-depleted HT176 clones (KO cl. 9 and cl.10) compared to control cells (NT). Data: means ± SEM, n = 3; *** p < 0.001 (One-way ANOVA). (b) Calculated glycolytic and mitochondrial ATP production rate in ARRB1-depleted HT1376 cells (KO cl. 9 and cl.10) compared to corresponding controls (NT). Data: means ± SEM, n = 3 or 4; *** p < 0.001 (glycoATP, KO vs. NT), *** p < 0.001 (mitoATP, KO vs. NT) (Two-way ANOVA). Total ATP: (NT vs. Cl.9, ** p < 0.01; NT vs. Cl.10, *** p < 0.001) (c) Basal glycolytic proton efflux rate (PER) in ARRB1 depleted HT1376 cells (KO cl. 9 and cl.10) compared to corresponding controls (NT). Data: means ± SEM, n = 3 or 4; *** p < 0.001, (One-way ANOVA). (d) Glycolytic proton efflux rate (glycoPER) following treatment with inhibitors of oxidative phosphorylation (Rotenone and Antimycin A). Data: means ± SEM, n = 4; * p < 0.05; *** p < 0.001 (one-way ANOVA). (e) Quantification of lactate secreted by ARRB1-depleted cells (KO cl. 9 and cl.10) and corresponding controls (NT). Data: means ± SEM, n = 7 or 8; *** p < 0.001 (One-way ANOVA).

Figure 4

ARRB1 regulates mitochondrial translocation of pyruvate and glucose uptake: (a) immunoblot for detection of glycolytic markers, MPC1 and GLUT1 in bladder cancer cell lines (253J, HT1376, 5637) and nonmalignant cells (UROTSA), as well as ARRB1-depleted HT1376 cells (KO cl. 9 and cl. 10). (b) Mitochondria of ARRB1-depleted HT1376 cells (KO cl. 9) and corresponding controls (NT) were collected, and mitochondria/total protein ratio was calculated. Data: means ± SEM, n = 2. The experiment was repeated once (c) Mitochondrial and cytoplasmic extracts were collected for colorimetric detection of pyruvate. Data: means ± SEM, n = 3; *** p < 0.001 (two-tailed unpaired t test). The experiment was repeated once (d) Secreted Lactate (glycolysis) in presence or absence of the MPC1-inhibitor UK-5099. Data: means ± SEM, n = 4; *** p < 0.001 (two-way ANOVA). (e) Glucose uptake in ARRB1-depleted HT1376 cells (KO cl. 9 and cl.10) compared to corresponding controls (NT). Data: means ± SEM, n = 4; *** p < 0.001, * p < 0.05 (One-way ANOVA). (f) Measurement of mitochondrial fuel usage dependency of ARRB1-depleted HT1376 cells to oxidize glucose/pyruvate, glutamine/glutamate, and long-chain fatty acids. Data: means ± SEM, n = 4 to 8; * p < 0.05, (two-way ANOVA). NS: not significant.

Figure 5

Re-expression of ARRB1 in ARRB1 depleted cells restores the metabolic program: (a) HT1376 ARRB1 KO cells (KO cl. 9 and KO cl. 10) were transfected with ARRB1 for re-expression/overexpression (OE) of ARRB1. Protein extracts were isolated for detection of ARRB1 and MPC1 by Western blot analysis. (b) ARRB1 KO cells (KO cl. 9) were transfected with ARRB1, and stable clones overexpressing ARRB1 were generated (OE cl.2 and cl.3). The Metabolic preference of clones was analyzed by measuring the oxygen consumption rate. Data: means ± SEM, n = 8; *** p < 0.001, ** p < 0.01,* p < 0.05 (two-way ANOVA). (c,d) Basal respiration and mitochondrial ATP production in clones generated following re-expression/overexpression of ARRB1. Data: means ± SEM, n > 10; *** p < 0.001 (one-way ANOVA).