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MnSOD upregulation sustains the Warburg effect via mitochondrial ROS and AMPK-dependent signalling in cancer.

Hart PC, Mao M, de Abreu AL, Ansenberger-Fricano K, Ekoue DN, Ganini D, Kajdacsy-Balla A, Diamond AM, Minshall RD, Consolaro ME, Santos JH, Bonini MG - Nat Commun (2015)

Bottom Line: Here we demonstrate that MnSOD upregulation in cancer cells establishes a steady flow of H2O2 originating from mitochondria that sustains AMP-activated kinase (AMPK) activation and the metabolic shift to glycolysis.Restricting MnSOD expression or inhibiting AMPK suppresses the metabolic switch and dampens the viability of transformed cells indicating that the MnSOD/AMPK axis is critical to support cancer cell bioenergetics.Recapitulating in vitro findings, clinical and epidemiologic analyses of MnSOD expression and AMPK activation indicated that the MnSOD/AMPK pathway is most active in advanced stage and aggressive breast cancer subtypes.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medicine, University of Illinois at Chicago, 909 South Wolcott Avenue, COMRB 1131, Chicago, Illinois 60612, USA [2] Department of Pathology, University of Illinois at Chicago, 909 South Wolcott Avenue, COMRB 1131, Chicago, Illinois 60612, USA.

ABSTRACT
Manganese superoxide dismutase (MnSOD/SOD2) is a mitochondria-resident enzyme that governs the types of reactive oxygen species egressing from the organelle to affect cellular signalling. Here we demonstrate that MnSOD upregulation in cancer cells establishes a steady flow of H2O2 originating from mitochondria that sustains AMP-activated kinase (AMPK) activation and the metabolic shift to glycolysis. Restricting MnSOD expression or inhibiting AMPK suppresses the metabolic switch and dampens the viability of transformed cells indicating that the MnSOD/AMPK axis is critical to support cancer cell bioenergetics. Recapitulating in vitro findings, clinical and epidemiologic analyses of MnSOD expression and AMPK activation indicated that the MnSOD/AMPK pathway is most active in advanced stage and aggressive breast cancer subtypes. Taken together, our results indicate that MnSOD serves as a biomarker of cancer progression and acts as critical regulator of tumour cell metabolism.

No MeSH data available.


Related in: MedlinePlus

Calmodulin kinase II (CaMKII) is a redox sensitive kinase upstream of AMPK(A) CaMKII oxidation was examined by Western blot using an antibody recognizing the oxidation of M281/M282 to methionine sulfoxide on CaMKII. Panel to the right shows quantification by densitometry of oxidized CaMKII detected in three independent experiments. (B) Impact of KN93 (a bona fide CaMKII inhibitor) on lactate production and steady ATP levels in neo, Mn44, Mn11. Water soluble KN93 (5 μM) was diluted into complete media. Cells were harvested at 36 h for assessments of lactate production and steady state levels of ATP as described in methods. (C) The impact of CaMKII inhibition with KN93 on AMPK phosphorylation and activity (pACC was used as a surrogate) was examined. Cells exposed to KN93 (5 μM) were harvested at the indicated time points after KN93 addition to full media, lysed and analyzed for AMPK(Thr172p) and pACC (Ser79) content by Western blot. *p<0.05; **p<0.05; ***p<0.01. Statistical analysis was performed using 6 independent replicates.
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Figure 7: Calmodulin kinase II (CaMKII) is a redox sensitive kinase upstream of AMPK(A) CaMKII oxidation was examined by Western blot using an antibody recognizing the oxidation of M281/M282 to methionine sulfoxide on CaMKII. Panel to the right shows quantification by densitometry of oxidized CaMKII detected in three independent experiments. (B) Impact of KN93 (a bona fide CaMKII inhibitor) on lactate production and steady ATP levels in neo, Mn44, Mn11. Water soluble KN93 (5 μM) was diluted into complete media. Cells were harvested at 36 h for assessments of lactate production and steady state levels of ATP as described in methods. (C) The impact of CaMKII inhibition with KN93 on AMPK phosphorylation and activity (pACC was used as a surrogate) was examined. Cells exposed to KN93 (5 μM) were harvested at the indicated time points after KN93 addition to full media, lysed and analyzed for AMPK(Thr172p) and pACC (Ser79) content by Western blot. *p<0.05; **p<0.05; ***p<0.01. Statistical analysis was performed using 6 independent replicates.

Mentions: Data presented above indicated that the constitutive elevation of cellular H2O2 maintained AMPK activation thereby promoting the irreversible conversion of the cellular metabolism to glycolytic. The mechanism of MnSOD-dependent AMPK constitutive activation was next investigated with a focus on CaMKII, a kinase responsive to H2O2-driven oxidative activation upstream of AMPK [43,44]. Oxidation of CaMKII at residues M281 and M282 had previously been shown to result in chronic activation, and this constitutive activation of CaMKII was apparent even in cells overexpressing low levels of MnSOD (2 – 3 fold over basal, Mn1), Figure 7A. Consistent with the hypothesis that AMPK is critical for maintaining increased glycolysis and steady state ATP levels in MnSOD overexpressing cells; the CaMKII inhibitor KN93 dampened glycolysis and reduced the steady state ATP levels in Mn44 and Mn11 cells (Figure 7B). Importantly, inhibition of CaMKII resulted in robust dephosphorylation of AMPK at the active site Thr172 (Figure 7C). Decreased AMPK phosphorylation was accompanied, as expected, by a reduction in AMPK activity as indicated by lower levels of phosphorylated ACC (Figure 7C), a downstream target of AMPK. This observation indicates that CaMKII oxidation by mtH2O2 leads to its constitutive activation and consequentially the activation of AMPK. Relevantly, inhibition of CaMKII markedly reduced the viability of cells expressing high MnSOD levels (Figure 8). The potential clinical importance of this finding was furthered by comparing the effect of CaMKII inhibitor KN93 with two commonly used breast cancer chemotherapeutic compounds, paclitaxel (a mitotic inhibitor) and 5-fluorouracil (a thymidylate synthase inhibitor). Exposure to KN93 resulted in 60–70% cell death of Mn44 and Mn11 cells while only minimally reducing the viability of MCF7/neo cells by 20% approximately, which was in clear contrast to the effect of paclitaxel and 5-flurouracil which were far more efficient in killing MCF7/neo (Figure 8). This observation indicated that cells expressing high MnSOD levelsare far more sensitive to CaMKII inhibition than to paclitaxel or 5-fluorouracil.


MnSOD upregulation sustains the Warburg effect via mitochondrial ROS and AMPK-dependent signalling in cancer.

Hart PC, Mao M, de Abreu AL, Ansenberger-Fricano K, Ekoue DN, Ganini D, Kajdacsy-Balla A, Diamond AM, Minshall RD, Consolaro ME, Santos JH, Bonini MG - Nat Commun (2015)

Calmodulin kinase II (CaMKII) is a redox sensitive kinase upstream of AMPK(A) CaMKII oxidation was examined by Western blot using an antibody recognizing the oxidation of M281/M282 to methionine sulfoxide on CaMKII. Panel to the right shows quantification by densitometry of oxidized CaMKII detected in three independent experiments. (B) Impact of KN93 (a bona fide CaMKII inhibitor) on lactate production and steady ATP levels in neo, Mn44, Mn11. Water soluble KN93 (5 μM) was diluted into complete media. Cells were harvested at 36 h for assessments of lactate production and steady state levels of ATP as described in methods. (C) The impact of CaMKII inhibition with KN93 on AMPK phosphorylation and activity (pACC was used as a surrogate) was examined. Cells exposed to KN93 (5 μM) were harvested at the indicated time points after KN93 addition to full media, lysed and analyzed for AMPK(Thr172p) and pACC (Ser79) content by Western blot. *p<0.05; **p<0.05; ***p<0.01. Statistical analysis was performed using 6 independent replicates.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4319569&req=5

Figure 7: Calmodulin kinase II (CaMKII) is a redox sensitive kinase upstream of AMPK(A) CaMKII oxidation was examined by Western blot using an antibody recognizing the oxidation of M281/M282 to methionine sulfoxide on CaMKII. Panel to the right shows quantification by densitometry of oxidized CaMKII detected in three independent experiments. (B) Impact of KN93 (a bona fide CaMKII inhibitor) on lactate production and steady ATP levels in neo, Mn44, Mn11. Water soluble KN93 (5 μM) was diluted into complete media. Cells were harvested at 36 h for assessments of lactate production and steady state levels of ATP as described in methods. (C) The impact of CaMKII inhibition with KN93 on AMPK phosphorylation and activity (pACC was used as a surrogate) was examined. Cells exposed to KN93 (5 μM) were harvested at the indicated time points after KN93 addition to full media, lysed and analyzed for AMPK(Thr172p) and pACC (Ser79) content by Western blot. *p<0.05; **p<0.05; ***p<0.01. Statistical analysis was performed using 6 independent replicates.
Mentions: Data presented above indicated that the constitutive elevation of cellular H2O2 maintained AMPK activation thereby promoting the irreversible conversion of the cellular metabolism to glycolytic. The mechanism of MnSOD-dependent AMPK constitutive activation was next investigated with a focus on CaMKII, a kinase responsive to H2O2-driven oxidative activation upstream of AMPK [43,44]. Oxidation of CaMKII at residues M281 and M282 had previously been shown to result in chronic activation, and this constitutive activation of CaMKII was apparent even in cells overexpressing low levels of MnSOD (2 – 3 fold over basal, Mn1), Figure 7A. Consistent with the hypothesis that AMPK is critical for maintaining increased glycolysis and steady state ATP levels in MnSOD overexpressing cells; the CaMKII inhibitor KN93 dampened glycolysis and reduced the steady state ATP levels in Mn44 and Mn11 cells (Figure 7B). Importantly, inhibition of CaMKII resulted in robust dephosphorylation of AMPK at the active site Thr172 (Figure 7C). Decreased AMPK phosphorylation was accompanied, as expected, by a reduction in AMPK activity as indicated by lower levels of phosphorylated ACC (Figure 7C), a downstream target of AMPK. This observation indicates that CaMKII oxidation by mtH2O2 leads to its constitutive activation and consequentially the activation of AMPK. Relevantly, inhibition of CaMKII markedly reduced the viability of cells expressing high MnSOD levels (Figure 8). The potential clinical importance of this finding was furthered by comparing the effect of CaMKII inhibitor KN93 with two commonly used breast cancer chemotherapeutic compounds, paclitaxel (a mitotic inhibitor) and 5-fluorouracil (a thymidylate synthase inhibitor). Exposure to KN93 resulted in 60–70% cell death of Mn44 and Mn11 cells while only minimally reducing the viability of MCF7/neo cells by 20% approximately, which was in clear contrast to the effect of paclitaxel and 5-flurouracil which were far more efficient in killing MCF7/neo (Figure 8). This observation indicated that cells expressing high MnSOD levelsare far more sensitive to CaMKII inhibition than to paclitaxel or 5-fluorouracil.

Bottom Line: Here we demonstrate that MnSOD upregulation in cancer cells establishes a steady flow of H2O2 originating from mitochondria that sustains AMP-activated kinase (AMPK) activation and the metabolic shift to glycolysis.Restricting MnSOD expression or inhibiting AMPK suppresses the metabolic switch and dampens the viability of transformed cells indicating that the MnSOD/AMPK axis is critical to support cancer cell bioenergetics.Recapitulating in vitro findings, clinical and epidemiologic analyses of MnSOD expression and AMPK activation indicated that the MnSOD/AMPK pathway is most active in advanced stage and aggressive breast cancer subtypes.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medicine, University of Illinois at Chicago, 909 South Wolcott Avenue, COMRB 1131, Chicago, Illinois 60612, USA [2] Department of Pathology, University of Illinois at Chicago, 909 South Wolcott Avenue, COMRB 1131, Chicago, Illinois 60612, USA.

ABSTRACT
Manganese superoxide dismutase (MnSOD/SOD2) is a mitochondria-resident enzyme that governs the types of reactive oxygen species egressing from the organelle to affect cellular signalling. Here we demonstrate that MnSOD upregulation in cancer cells establishes a steady flow of H2O2 originating from mitochondria that sustains AMP-activated kinase (AMPK) activation and the metabolic shift to glycolysis. Restricting MnSOD expression or inhibiting AMPK suppresses the metabolic switch and dampens the viability of transformed cells indicating that the MnSOD/AMPK axis is critical to support cancer cell bioenergetics. Recapitulating in vitro findings, clinical and epidemiologic analyses of MnSOD expression and AMPK activation indicated that the MnSOD/AMPK pathway is most active in advanced stage and aggressive breast cancer subtypes. Taken together, our results indicate that MnSOD serves as a biomarker of cancer progression and acts as critical regulator of tumour cell metabolism.

No MeSH data available.


Related in: MedlinePlus