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Mitochondrial control by DRP1 in brain tumor initiating cells.

Xie Q, Wu Q, Horbinski CM, Flavahan WA, Yang K, Zhou W, Dombrowski SM, Huang Z, Fang X, Shi Y, Ferguson AN, Kashatus DF, Bao S, Rich JN - Nat. Neurosci. (2015)

Bottom Line: Targeting DRP1 using RNA interference or pharmacologic inhibition induced BTIC apoptosis and inhibited tumor growth.Downstream, DRP1 activity regulated the essential metabolic stress sensor, AMP-activated protein kinase (AMPK), and targeting AMPK rescued the effects of DRP1 disruption.DRP1 activation correlated with poor prognosis in glioblastoma, suggesting that mitochondrial dynamics may represent a therapeutic target for BTICs.

View Article: PubMed Central - PubMed

Affiliation: Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland, Ohio, USA.

ABSTRACT
Brain tumor initiating cells (BTICs) co-opt the neuronal high affinity glucose transporter, GLUT3, to withstand metabolic stress. We investigated another mechanism critical to brain metabolism, mitochondrial morphology, in BTICs. BTIC mitochondria were fragmented relative to non-BTIC tumor cell mitochondria, suggesting that BTICs increase mitochondrial fission. The essential mediator of mitochondrial fission, dynamin-related protein 1 (DRP1), showed activating phosphorylation in BTICs and inhibitory phosphorylation in non-BTIC tumor cells. Targeting DRP1 using RNA interference or pharmacologic inhibition induced BTIC apoptosis and inhibited tumor growth. Downstream, DRP1 activity regulated the essential metabolic stress sensor, AMP-activated protein kinase (AMPK), and targeting AMPK rescued the effects of DRP1 disruption. Cyclin-dependent kinase 5 (CDK5) phosphorylated DRP1 to increase its activity in BTICs, whereas Ca(2+)-calmodulin-dependent protein kinase 2 (CAMK2) inhibited DRP1 in non-BTIC tumor cells, suggesting that tumor cell differentiation induces a regulatory switch in mitochondrial morphology. DRP1 activation correlated with poor prognosis in glioblastoma, suggesting that mitochondrial dynamics may represent a therapeutic target for BTICs.

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DRP1 inhibition induces AMPK activity in BTICsa. Lysates of 387 and 3565 BTICs expressing NT control shRNA, shDrp1#1, or shDrp1#2 were immunoblotted with the indicated antibodies. shRNA mediated knockdown of DRP1 induced AMPK activity. Images were cropped for presentation. Full-length blots are presented in Supplementary Fig. 10. b. Lysates of 387 and 3565 BTICs treated with Midivi-1 or DMSO were immunoblotted with the indicated antibodies. Inhibition DRP1 by Midivi-1 induced AMPK activity. c. Growth curves of 387 BTICs expressing NT control shRNA, shDrp1, shAMPKα, or shDrp1 and shAMPKα together. Data are plotted as mean ± s.e.m. p < 0.0001 by repeated measures ANOVA. d. In vitro extreme limiting dilution assays (ELDA) of 387 BTICs expressing NT control shRNA, shDrp1, shAMPKα, or shDrp1 and shAMPKα together. p = 1.7 × 10−34 by ANOVA.
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Figure 6: DRP1 inhibition induces AMPK activity in BTICsa. Lysates of 387 and 3565 BTICs expressing NT control shRNA, shDrp1#1, or shDrp1#2 were immunoblotted with the indicated antibodies. shRNA mediated knockdown of DRP1 induced AMPK activity. Images were cropped for presentation. Full-length blots are presented in Supplementary Fig. 10. b. Lysates of 387 and 3565 BTICs treated with Midivi-1 or DMSO were immunoblotted with the indicated antibodies. Inhibition DRP1 by Midivi-1 induced AMPK activity. c. Growth curves of 387 BTICs expressing NT control shRNA, shDrp1, shAMPKα, or shDrp1 and shAMPKα together. Data are plotted as mean ± s.e.m. p < 0.0001 by repeated measures ANOVA. d. In vitro extreme limiting dilution assays (ELDA) of 387 BTICs expressing NT control shRNA, shDrp1, shAMPKα, or shDrp1 and shAMPKα together. p = 1.7 × 10−34 by ANOVA.

Mentions: AMP-activated protein kinase (AMPK) is a central cellular sensor of energy stress, suggesting a potentially critical role in determining the survival of cells under metabolic stress. Using a pharmacologic activator of AMPK, we found that AMPK activation decreased BTIC growth and induced caspase activation (Supplementary Fig. 4b, c), concordant with studies in glioma cell lines41, phenocopying DRP1 targeting. We therefore interrogated a potential link to AMPK activation under DRP1 inhibition in BTICs. DRP1 inhibition by either shRNA knockdown (Fig. 6a) or treatment with Mdivi-1 (Fig. 6b) led to upregulation of AMPK activation, measured by increased phosphorylation of AMPKα. To determine if AMPK regulation may serve as a critical downstream mediator of DRP1, we interrogated the potential for rescuing the phenotype caused by DRP1 targeting. While knocking down AMPKα by itself had minimal effect on BTIC growth or neurosphere formation, targeting AMPKα expression in the context of DRP1 inhibition rescued both the BTIC growth defect (Fig. 6c) and compromised neurosphere formation capacity (Fig. 6d), which strongly supports AMPKα as a critical downstream mediator of BTIC response following DRP1 inhibition. Collectively, our results suggest that DRP1 serves as a critical node in the response of BTICs to metabolic stress through AMPK regulation.


Mitochondrial control by DRP1 in brain tumor initiating cells.

Xie Q, Wu Q, Horbinski CM, Flavahan WA, Yang K, Zhou W, Dombrowski SM, Huang Z, Fang X, Shi Y, Ferguson AN, Kashatus DF, Bao S, Rich JN - Nat. Neurosci. (2015)

DRP1 inhibition induces AMPK activity in BTICsa. Lysates of 387 and 3565 BTICs expressing NT control shRNA, shDrp1#1, or shDrp1#2 were immunoblotted with the indicated antibodies. shRNA mediated knockdown of DRP1 induced AMPK activity. Images were cropped for presentation. Full-length blots are presented in Supplementary Fig. 10. b. Lysates of 387 and 3565 BTICs treated with Midivi-1 or DMSO were immunoblotted with the indicated antibodies. Inhibition DRP1 by Midivi-1 induced AMPK activity. c. Growth curves of 387 BTICs expressing NT control shRNA, shDrp1, shAMPKα, or shDrp1 and shAMPKα together. Data are plotted as mean ± s.e.m. p < 0.0001 by repeated measures ANOVA. d. In vitro extreme limiting dilution assays (ELDA) of 387 BTICs expressing NT control shRNA, shDrp1, shAMPKα, or shDrp1 and shAMPKα together. p = 1.7 × 10−34 by ANOVA.
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Figure 6: DRP1 inhibition induces AMPK activity in BTICsa. Lysates of 387 and 3565 BTICs expressing NT control shRNA, shDrp1#1, or shDrp1#2 were immunoblotted with the indicated antibodies. shRNA mediated knockdown of DRP1 induced AMPK activity. Images were cropped for presentation. Full-length blots are presented in Supplementary Fig. 10. b. Lysates of 387 and 3565 BTICs treated with Midivi-1 or DMSO were immunoblotted with the indicated antibodies. Inhibition DRP1 by Midivi-1 induced AMPK activity. c. Growth curves of 387 BTICs expressing NT control shRNA, shDrp1, shAMPKα, or shDrp1 and shAMPKα together. Data are plotted as mean ± s.e.m. p < 0.0001 by repeated measures ANOVA. d. In vitro extreme limiting dilution assays (ELDA) of 387 BTICs expressing NT control shRNA, shDrp1, shAMPKα, or shDrp1 and shAMPKα together. p = 1.7 × 10−34 by ANOVA.
Mentions: AMP-activated protein kinase (AMPK) is a central cellular sensor of energy stress, suggesting a potentially critical role in determining the survival of cells under metabolic stress. Using a pharmacologic activator of AMPK, we found that AMPK activation decreased BTIC growth and induced caspase activation (Supplementary Fig. 4b, c), concordant with studies in glioma cell lines41, phenocopying DRP1 targeting. We therefore interrogated a potential link to AMPK activation under DRP1 inhibition in BTICs. DRP1 inhibition by either shRNA knockdown (Fig. 6a) or treatment with Mdivi-1 (Fig. 6b) led to upregulation of AMPK activation, measured by increased phosphorylation of AMPKα. To determine if AMPK regulation may serve as a critical downstream mediator of DRP1, we interrogated the potential for rescuing the phenotype caused by DRP1 targeting. While knocking down AMPKα by itself had minimal effect on BTIC growth or neurosphere formation, targeting AMPKα expression in the context of DRP1 inhibition rescued both the BTIC growth defect (Fig. 6c) and compromised neurosphere formation capacity (Fig. 6d), which strongly supports AMPKα as a critical downstream mediator of BTIC response following DRP1 inhibition. Collectively, our results suggest that DRP1 serves as a critical node in the response of BTICs to metabolic stress through AMPK regulation.

Bottom Line: Targeting DRP1 using RNA interference or pharmacologic inhibition induced BTIC apoptosis and inhibited tumor growth.Downstream, DRP1 activity regulated the essential metabolic stress sensor, AMP-activated protein kinase (AMPK), and targeting AMPK rescued the effects of DRP1 disruption.DRP1 activation correlated with poor prognosis in glioblastoma, suggesting that mitochondrial dynamics may represent a therapeutic target for BTICs.

View Article: PubMed Central - PubMed

Affiliation: Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland, Ohio, USA.

ABSTRACT
Brain tumor initiating cells (BTICs) co-opt the neuronal high affinity glucose transporter, GLUT3, to withstand metabolic stress. We investigated another mechanism critical to brain metabolism, mitochondrial morphology, in BTICs. BTIC mitochondria were fragmented relative to non-BTIC tumor cell mitochondria, suggesting that BTICs increase mitochondrial fission. The essential mediator of mitochondrial fission, dynamin-related protein 1 (DRP1), showed activating phosphorylation in BTICs and inhibitory phosphorylation in non-BTIC tumor cells. Targeting DRP1 using RNA interference or pharmacologic inhibition induced BTIC apoptosis and inhibited tumor growth. Downstream, DRP1 activity regulated the essential metabolic stress sensor, AMP-activated protein kinase (AMPK), and targeting AMPK rescued the effects of DRP1 disruption. Cyclin-dependent kinase 5 (CDK5) phosphorylated DRP1 to increase its activity in BTICs, whereas Ca(2+)-calmodulin-dependent protein kinase 2 (CAMK2) inhibited DRP1 in non-BTIC tumor cells, suggesting that tumor cell differentiation induces a regulatory switch in mitochondrial morphology. DRP1 activation correlated with poor prognosis in glioblastoma, suggesting that mitochondrial dynamics may represent a therapeutic target for BTICs.

Show MeSH
Related in: MedlinePlus