Limits...
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 inhibits GLUT1 translocation to the plasma membrane through down-regulating NF-κB signaling(A) The NF-κB signaling promoted the translocation of endogenous GLUT1 to the plasma membrane in H1299 cells detected by Western-blot assays. Cells were transfected with pCMV-p65-HA expression vectors (left panel) or p65 siRNA oligos to knock down p65 (right panel), and the endogenous GLUT1 levels in the isolated plasma membrane fraction and whole cell lysates were analyzed by Western-blot assays. (B) Ectopic expression of p65 promoted the translocation of Myc-GLUT1 to the plasma membrane (left panel), whereas p65 knockdown inhibited the translocation of Myc-GLUT1 to the plasma membrane (right panel) in H1299 and H460 cells. Cells were transfected with p65 expression vectors or p65-siR together with Myc-GLUT1 vectors. The levels of Myc-GLUT1 on cell surface were analyzed in a flow cytometer and normalized with the total Myc-GLUT1 levels in cells. (C) GLUT1 knockdown greatly abolished the promoting effects of p65 on glucose uptake, the glycolytic rate and lactate production in H1299 and H460 cells. Cells were pre-transfected with 2 different GLUT1 siRNAs (GLUT1-siR) before transfection with p65 expression vectors. Left panel: Western-blot analysis of knockdown of GLUT1 by siRNA in cells. (D) p65 knockdown largely abolished the inhibitory effects of RRAD overexpression on Myc-GLUT1 translocation to cell surface in cells measured by flow cytometry. H1299 and H460 cells with stable RRAD overexpression (RRAD) and control cells (Con) were pre-transfected with p65-siR before transfection of Myc-GLUT1 vectors. (E) p65 knockdown largely abolished the promoting effects of RRAD knockdown on Myc-GLUT1 translocation to cell surface. H1299 and H460 cells stably transduced with 2 different RRAD shRNA vectors (RRAD-shR) and control shRNA (Con-shR) were pre-transfected with p65-siR before transfection of Myc-GLUT1 vectors. Data are presented as mean value ± SD (n=3). *p < 0.05; **p < 0.01 (student's t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4558130&req=5

Figure 3: RRAD inhibits GLUT1 translocation to the plasma membrane through down-regulating NF-κB signaling(A) The NF-κB signaling promoted the translocation of endogenous GLUT1 to the plasma membrane in H1299 cells detected by Western-blot assays. Cells were transfected with pCMV-p65-HA expression vectors (left panel) or p65 siRNA oligos to knock down p65 (right panel), and the endogenous GLUT1 levels in the isolated plasma membrane fraction and whole cell lysates were analyzed by Western-blot assays. (B) Ectopic expression of p65 promoted the translocation of Myc-GLUT1 to the plasma membrane (left panel), whereas p65 knockdown inhibited the translocation of Myc-GLUT1 to the plasma membrane (right panel) in H1299 and H460 cells. Cells were transfected with p65 expression vectors or p65-siR together with Myc-GLUT1 vectors. The levels of Myc-GLUT1 on cell surface were analyzed in a flow cytometer and normalized with the total Myc-GLUT1 levels in cells. (C) GLUT1 knockdown greatly abolished the promoting effects of p65 on glucose uptake, the glycolytic rate and lactate production in H1299 and H460 cells. Cells were pre-transfected with 2 different GLUT1 siRNAs (GLUT1-siR) before transfection with p65 expression vectors. Left panel: Western-blot analysis of knockdown of GLUT1 by siRNA in cells. (D) p65 knockdown largely abolished the inhibitory effects of RRAD overexpression on Myc-GLUT1 translocation to cell surface in cells measured by flow cytometry. H1299 and H460 cells with stable RRAD overexpression (RRAD) and control cells (Con) were pre-transfected with p65-siR before transfection of Myc-GLUT1 vectors. (E) p65 knockdown largely abolished the promoting effects of RRAD knockdown on Myc-GLUT1 translocation to cell surface. H1299 and H460 cells stably transduced with 2 different RRAD shRNA vectors (RRAD-shR) and control shRNA (Con-shR) were pre-transfected with p65-siR before transfection of Myc-GLUT1 vectors. Data are presented as mean value ± SD (n=3). *p < 0.05; **p < 0.01 (student's t test).

Mentions: A recent study reported that the activated NF-κB signaling promotes the translocation of GLUT1 to the plasma membrane to facilitate glucose uptake, which could be an important mechanism by which NF-κB activates the Warburg effect [27]. Together with our finding that RRAD negatively regulates the NF-κB signaling, these findings raised a possibility that RRAD inhibits the GLUT1 translocation to the plasma membrane through its negative regulation of the NF-κB signaling, which could be an important mechanism by which RRAD inhibits GLUT1 translocation. Consistent with this previous report [27], our results clearly showed that the NF-B activation promoted the GLUT1 translocation to the plasma membrane and therefore enhanced the Warburg effect in lung cancer cells. Ectopic expression of p65 by the pCMV-p65 expression vector greatly promoted the translocation of endogenous GLUT1 to the plasma membrane as shown by Western-blot assays using the isolated plasma membrane fraction of H1299 and H460 cells (Figure 3A). Furthermore, knockdown of p65 by siRNA reduced the translocation of endogenous GLUT1 to the plasma membrane in cells (Figure 3B). To confirm this result, cells with p65 overexpression or knockdown were transduced with pLPCX-Myc-GLUT1 vectors that express GLUT1 with Myc tag in its first exofacial loop, and the levels of Myc-GLUT1 on the cell surface or in the whole cell were measured by immunofluorescence (IF) staining with an anti-Myc antibody followed by flow cytometry analysis. While p65 overexpression or knockdown did not affect the total levels of Myc-GLUT1 in cells, p65 overexpression significantly increased the levels of Myc-GLUT1 protein on the cell surface (Figure 3B, left panel), whereas p65 knockdown significantly reduced the levels of Myc-GLUT1 protein on the cell surface (Figure 3B, right panel). Our results further showed that knockdown of the endogenous GLUT1 by siRNA largely abolished the promoting effects of NF-κB activation on glucose uptake, the glycolytic rate and lactate production in H1299 and H460 cells with overexpression of p65 (Figure 3C). These results together indicate that activated NF-κB signaling promotes the translocation of GLUT1 to the plasma membrane to facilitate glucose uptake, which is an important mechanism for NF-κB to activate the Warburg effect in cancer cells.


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 inhibits GLUT1 translocation to the plasma membrane through down-regulating NF-κB signaling(A) The NF-κB signaling promoted the translocation of endogenous GLUT1 to the plasma membrane in H1299 cells detected by Western-blot assays. Cells were transfected with pCMV-p65-HA expression vectors (left panel) or p65 siRNA oligos to knock down p65 (right panel), and the endogenous GLUT1 levels in the isolated plasma membrane fraction and whole cell lysates were analyzed by Western-blot assays. (B) Ectopic expression of p65 promoted the translocation of Myc-GLUT1 to the plasma membrane (left panel), whereas p65 knockdown inhibited the translocation of Myc-GLUT1 to the plasma membrane (right panel) in H1299 and H460 cells. Cells were transfected with p65 expression vectors or p65-siR together with Myc-GLUT1 vectors. The levels of Myc-GLUT1 on cell surface were analyzed in a flow cytometer and normalized with the total Myc-GLUT1 levels in cells. (C) GLUT1 knockdown greatly abolished the promoting effects of p65 on glucose uptake, the glycolytic rate and lactate production in H1299 and H460 cells. Cells were pre-transfected with 2 different GLUT1 siRNAs (GLUT1-siR) before transfection with p65 expression vectors. Left panel: Western-blot analysis of knockdown of GLUT1 by siRNA in cells. (D) p65 knockdown largely abolished the inhibitory effects of RRAD overexpression on Myc-GLUT1 translocation to cell surface in cells measured by flow cytometry. H1299 and H460 cells with stable RRAD overexpression (RRAD) and control cells (Con) were pre-transfected with p65-siR before transfection of Myc-GLUT1 vectors. (E) p65 knockdown largely abolished the promoting effects of RRAD knockdown on Myc-GLUT1 translocation to cell surface. H1299 and H460 cells stably transduced with 2 different RRAD shRNA vectors (RRAD-shR) and control shRNA (Con-shR) were pre-transfected with p65-siR before transfection of Myc-GLUT1 vectors. Data are presented as mean value ± SD (n=3). *p < 0.05; **p < 0.01 (student's t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4558130&req=5

Figure 3: RRAD inhibits GLUT1 translocation to the plasma membrane through down-regulating NF-κB signaling(A) The NF-κB signaling promoted the translocation of endogenous GLUT1 to the plasma membrane in H1299 cells detected by Western-blot assays. Cells were transfected with pCMV-p65-HA expression vectors (left panel) or p65 siRNA oligos to knock down p65 (right panel), and the endogenous GLUT1 levels in the isolated plasma membrane fraction and whole cell lysates were analyzed by Western-blot assays. (B) Ectopic expression of p65 promoted the translocation of Myc-GLUT1 to the plasma membrane (left panel), whereas p65 knockdown inhibited the translocation of Myc-GLUT1 to the plasma membrane (right panel) in H1299 and H460 cells. Cells were transfected with p65 expression vectors or p65-siR together with Myc-GLUT1 vectors. The levels of Myc-GLUT1 on cell surface were analyzed in a flow cytometer and normalized with the total Myc-GLUT1 levels in cells. (C) GLUT1 knockdown greatly abolished the promoting effects of p65 on glucose uptake, the glycolytic rate and lactate production in H1299 and H460 cells. Cells were pre-transfected with 2 different GLUT1 siRNAs (GLUT1-siR) before transfection with p65 expression vectors. Left panel: Western-blot analysis of knockdown of GLUT1 by siRNA in cells. (D) p65 knockdown largely abolished the inhibitory effects of RRAD overexpression on Myc-GLUT1 translocation to cell surface in cells measured by flow cytometry. H1299 and H460 cells with stable RRAD overexpression (RRAD) and control cells (Con) were pre-transfected with p65-siR before transfection of Myc-GLUT1 vectors. (E) p65 knockdown largely abolished the promoting effects of RRAD knockdown on Myc-GLUT1 translocation to cell surface. H1299 and H460 cells stably transduced with 2 different RRAD shRNA vectors (RRAD-shR) and control shRNA (Con-shR) were pre-transfected with p65-siR before transfection of Myc-GLUT1 vectors. Data are presented as mean value ± SD (n=3). *p < 0.05; **p < 0.01 (student's t test).
Mentions: A recent study reported that the activated NF-κB signaling promotes the translocation of GLUT1 to the plasma membrane to facilitate glucose uptake, which could be an important mechanism by which NF-κB activates the Warburg effect [27]. Together with our finding that RRAD negatively regulates the NF-κB signaling, these findings raised a possibility that RRAD inhibits the GLUT1 translocation to the plasma membrane through its negative regulation of the NF-κB signaling, which could be an important mechanism by which RRAD inhibits GLUT1 translocation. Consistent with this previous report [27], our results clearly showed that the NF-B activation promoted the GLUT1 translocation to the plasma membrane and therefore enhanced the Warburg effect in lung cancer cells. Ectopic expression of p65 by the pCMV-p65 expression vector greatly promoted the translocation of endogenous GLUT1 to the plasma membrane as shown by Western-blot assays using the isolated plasma membrane fraction of H1299 and H460 cells (Figure 3A). Furthermore, knockdown of p65 by siRNA reduced the translocation of endogenous GLUT1 to the plasma membrane in cells (Figure 3B). To confirm this result, cells with p65 overexpression or knockdown were transduced with pLPCX-Myc-GLUT1 vectors that express GLUT1 with Myc tag in its first exofacial loop, and the levels of Myc-GLUT1 on the cell surface or in the whole cell were measured by immunofluorescence (IF) staining with an anti-Myc antibody followed by flow cytometry analysis. While p65 overexpression or knockdown did not affect the total levels of Myc-GLUT1 in cells, p65 overexpression significantly increased the levels of Myc-GLUT1 protein on the cell surface (Figure 3B, left panel), whereas p65 knockdown significantly reduced the levels of Myc-GLUT1 protein on the cell surface (Figure 3B, right panel). Our results further showed that knockdown of the endogenous GLUT1 by siRNA largely abolished the promoting effects of NF-κB activation on glucose uptake, the glycolytic rate and lactate production in H1299 and H460 cells with overexpression of p65 (Figure 3C). These results together indicate that activated NF-κB signaling promotes the translocation of GLUT1 to the plasma membrane to facilitate glucose uptake, which is an important mechanism for NF-κB to activate the Warburg effect in cancer cells.

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