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RRAD inhibits the Warburg effect through negative regulation of the NF-κB signaling.

Liu J, Zhang C, Wu R, Lin M, Liang Y, Liu J, Wang X, Yang B, Feng Z - Oncotarget (2015)

Bottom Line: However, the mechanism by which RRAD inhibits the Warburg effect remains unclear.Mechanically, RRAD directly binds to the p65 subunit of the NF-κB complex and inhibits the nuclear translocation of p65, which in turn negatively regulates the NF-κB signaling to inhibit GLUT1 translocation and the Warburg effect.Blocking the NF-κB signaling largely abolishes the inhibitory effects of RRAD on the translocation of GLUT1 to the plasma membrane and the Warburg effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Rutgers Cancer Institute of New Jersey, Rutgers, State University of New Jersey, New Brunswick, NJ, USA.

ABSTRACT
Cancer cells preferentially use aerobic glycolysis to meet their increased energetic and biosynthetic demands, a phenomenon known as the Warburg effect. Its underlying mechanism is not fully understood. RRAD, a small GTPase, is a potential tumor suppressor in lung cancer. RRAD expression is frequently down-regulated in lung cancer, which is associated with tumor progression and poor prognosis. Recently, RRAD was reported to repress the Warburg effect, indicating that down-regulation of RRAD expression is an important mechanism contributing to the Warburg effect in lung cancer. However, the mechanism by which RRAD inhibits the Warburg effect remains unclear. Here, we found that RRAD negatively regulates the NF-κB signaling to inhibit the GLUT1 translocation and the Warburg effect in lung cancer cells. Mechanically, RRAD directly binds to the p65 subunit of the NF-κB complex and inhibits the nuclear translocation of p65, which in turn negatively regulates the NF-κB signaling to inhibit GLUT1 translocation and the Warburg effect. Blocking the NF-κB signaling largely abolishes the inhibitory effects of RRAD on the translocation of GLUT1 to the plasma membrane and the Warburg effect. Taken together, our results revealed a novel mechanism by which RRAD negatively regulates the Warburg effect in lung cancer cells.

No MeSH data available.


Related in: MedlinePlus

RRAD interacts with p65 through the N-terminus(A) Schematic representation of RRAD deletion mutants. pCMV-Flag vectors expressing WT RRAD or two deletion mutants were constructed. (B) RRAD-ΔC249-Flag but not RRAD-ΔN88-Flag interacted with p65-HA. H1299 cells were transfected with WT or mutant RRAD-Flag vectors together with p65-HA vectors followed by Co-IP. Co-IP assays were performed with the antibodies against Flag (Left panel) or HA (Right panel). (C) Ectopic expression of RRAD-ΔC249 but not RRAD-ΔN88 inhibited the transcriptional activity of NF-κB. Cells transfected with indicated vectors together with the NF-κB luciferase reporter vectors. At 24 h after transfection, cells were treated with or without TNF-α (10 ng/ml) for 6 h before luciferase activities were measured. (D) Ectopic expression of RRAD-ΔC249 but not RRAD-ΔN88 inhibited glucose uptake in H1299 and H460 cells. Cells were transfected with indicated vectors for 24 h before glucose uptake assays. Data are presented as mean ± S.D. (n=3). *p < 0.05; **p < 0.01 (student's t test).
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Figure 4: RRAD interacts with p65 through the N-terminus(A) Schematic representation of RRAD deletion mutants. pCMV-Flag vectors expressing WT RRAD or two deletion mutants were constructed. (B) RRAD-ΔC249-Flag but not RRAD-ΔN88-Flag interacted with p65-HA. H1299 cells were transfected with WT or mutant RRAD-Flag vectors together with p65-HA vectors followed by Co-IP. Co-IP assays were performed with the antibodies against Flag (Left panel) or HA (Right panel). (C) Ectopic expression of RRAD-ΔC249 but not RRAD-ΔN88 inhibited the transcriptional activity of NF-κB. Cells transfected with indicated vectors together with the NF-κB luciferase reporter vectors. At 24 h after transfection, cells were treated with or without TNF-α (10 ng/ml) for 6 h before luciferase activities were measured. (D) Ectopic expression of RRAD-ΔC249 but not RRAD-ΔN88 inhibited glucose uptake in H1299 and H460 cells. Cells were transfected with indicated vectors for 24 h before glucose uptake assays. Data are presented as mean ± S.D. (n=3). *p < 0.05; **p < 0.01 (student's t test).

Mentions: To define the binding region of RRAD with p65, two Flag-tagged deletion mutants of RRAD were constructed and cloned into pCMV-Flag vectors, including ΔN88 (deletion of the N-terminal 88 amino acids) and ΔC249 (deletion of the C-terminal 59 amino acids) (Figure 4A). Both N-terminus and C-terminus of RRAD have been reported to be important for interacting with other proteins [18-20]. These two mutant vectors or wild-type (WT) RRAD-Flag expression vectors were transfected into H1299 cells together with the pCMV-p65-HA vector, and their binding to p65-HA protein were analyzed by Co-IP assays. As shown in Figure 4B, deletion of C-terminal residues 249-308 (ΔC249 mutant) did not significantly affect the ability of RRAD-Flag to bind to p65-HA. In contrast, deletion of N-terminal residues (ΔN88 mutant) largely abolished the RRAD binding to p65-HA. Furthermore, deletion of C-terminal residues 249-308 (ΔC249 mutant) did not significantly reduce the inhibitory effects of RRAD on the luciferase activities of the NF-κB (Figure 4C) and glucose uptake (Figure 4D) in H1299 and H460 cells. In contrast, deletion of N-terminal residues (ΔN88 mutant) largely abolished the inhibitory effects of RRAD on luciferase activities of the NF-κB (Figure 4C) and glucose uptake (Figure 4D) in cells. These results indicate that the N-terminus of RRAD is essential for the interaction of RRAD with p65, as well as for the function of RRAD in inhibiting GLUT1 translocation and the Warburg effect.


RRAD inhibits the Warburg effect through negative regulation of the NF-κB signaling.

Liu J, Zhang C, Wu R, Lin M, Liang Y, Liu J, Wang X, Yang B, Feng Z - Oncotarget (2015)

RRAD interacts with p65 through the N-terminus(A) Schematic representation of RRAD deletion mutants. pCMV-Flag vectors expressing WT RRAD or two deletion mutants were constructed. (B) RRAD-ΔC249-Flag but not RRAD-ΔN88-Flag interacted with p65-HA. H1299 cells were transfected with WT or mutant RRAD-Flag vectors together with p65-HA vectors followed by Co-IP. Co-IP assays were performed with the antibodies against Flag (Left panel) or HA (Right panel). (C) Ectopic expression of RRAD-ΔC249 but not RRAD-ΔN88 inhibited the transcriptional activity of NF-κB. Cells transfected with indicated vectors together with the NF-κB luciferase reporter vectors. At 24 h after transfection, cells were treated with or without TNF-α (10 ng/ml) for 6 h before luciferase activities were measured. (D) Ectopic expression of RRAD-ΔC249 but not RRAD-ΔN88 inhibited glucose uptake in H1299 and H460 cells. Cells were transfected with indicated vectors for 24 h before glucose uptake assays. Data are presented as mean ± S.D. (n=3). *p < 0.05; **p < 0.01 (student's t test).
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Related In: Results  -  Collection

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Figure 4: RRAD interacts with p65 through the N-terminus(A) Schematic representation of RRAD deletion mutants. pCMV-Flag vectors expressing WT RRAD or two deletion mutants were constructed. (B) RRAD-ΔC249-Flag but not RRAD-ΔN88-Flag interacted with p65-HA. H1299 cells were transfected with WT or mutant RRAD-Flag vectors together with p65-HA vectors followed by Co-IP. Co-IP assays were performed with the antibodies against Flag (Left panel) or HA (Right panel). (C) Ectopic expression of RRAD-ΔC249 but not RRAD-ΔN88 inhibited the transcriptional activity of NF-κB. Cells transfected with indicated vectors together with the NF-κB luciferase reporter vectors. At 24 h after transfection, cells were treated with or without TNF-α (10 ng/ml) for 6 h before luciferase activities were measured. (D) Ectopic expression of RRAD-ΔC249 but not RRAD-ΔN88 inhibited glucose uptake in H1299 and H460 cells. Cells were transfected with indicated vectors for 24 h before glucose uptake assays. Data are presented as mean ± S.D. (n=3). *p < 0.05; **p < 0.01 (student's t test).
Mentions: To define the binding region of RRAD with p65, two Flag-tagged deletion mutants of RRAD were constructed and cloned into pCMV-Flag vectors, including ΔN88 (deletion of the N-terminal 88 amino acids) and ΔC249 (deletion of the C-terminal 59 amino acids) (Figure 4A). Both N-terminus and C-terminus of RRAD have been reported to be important for interacting with other proteins [18-20]. These two mutant vectors or wild-type (WT) RRAD-Flag expression vectors were transfected into H1299 cells together with the pCMV-p65-HA vector, and their binding to p65-HA protein were analyzed by Co-IP assays. As shown in Figure 4B, deletion of C-terminal residues 249-308 (ΔC249 mutant) did not significantly affect the ability of RRAD-Flag to bind to p65-HA. In contrast, deletion of N-terminal residues (ΔN88 mutant) largely abolished the RRAD binding to p65-HA. Furthermore, deletion of C-terminal residues 249-308 (ΔC249 mutant) did not significantly reduce the inhibitory effects of RRAD on the luciferase activities of the NF-κB (Figure 4C) and glucose uptake (Figure 4D) in H1299 and H460 cells. In contrast, deletion of N-terminal residues (ΔN88 mutant) largely abolished the inhibitory effects of RRAD on luciferase activities of the NF-κB (Figure 4C) and glucose uptake (Figure 4D) in cells. These results indicate that the N-terminus of RRAD is essential for the interaction of RRAD with p65, as well as for the function of RRAD in inhibiting GLUT1 translocation and the Warburg effect.

Bottom Line: However, the mechanism by which RRAD inhibits the Warburg effect remains unclear.Mechanically, RRAD directly binds to the p65 subunit of the NF-κB complex and inhibits the nuclear translocation of p65, which in turn negatively regulates the NF-κB signaling to inhibit GLUT1 translocation and the Warburg effect.Blocking the NF-κB signaling largely abolishes the inhibitory effects of RRAD on the translocation of GLUT1 to the plasma membrane and the Warburg effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Rutgers Cancer Institute of New Jersey, Rutgers, State University of New Jersey, New Brunswick, NJ, USA.

ABSTRACT
Cancer cells preferentially use aerobic glycolysis to meet their increased energetic and biosynthetic demands, a phenomenon known as the Warburg effect. Its underlying mechanism is not fully understood. RRAD, a small GTPase, is a potential tumor suppressor in lung cancer. RRAD expression is frequently down-regulated in lung cancer, which is associated with tumor progression and poor prognosis. Recently, RRAD was reported to repress the Warburg effect, indicating that down-regulation of RRAD expression is an important mechanism contributing to the Warburg effect in lung cancer. However, the mechanism by which RRAD inhibits the Warburg effect remains unclear. Here, we found that RRAD negatively regulates the NF-κB signaling to inhibit the GLUT1 translocation and the Warburg effect in lung cancer cells. Mechanically, RRAD directly binds to the p65 subunit of the NF-κB complex and inhibits the nuclear translocation of p65, which in turn negatively regulates the NF-κB signaling to inhibit GLUT1 translocation and the Warburg effect. Blocking the NF-κB signaling largely abolishes the inhibitory effects of RRAD on the translocation of GLUT1 to the plasma membrane and the Warburg effect. Taken together, our results revealed a novel mechanism by which RRAD negatively regulates the Warburg effect in lung cancer cells.

No MeSH data available.


Related in: MedlinePlus