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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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CDK5 activates DRP1 in brain tumor initiating cellsa. Lysates of 387 and 3565 brain tumor initiating cells (BTICs) treated with the CDK1/2/5 inhibitor Roscovitine or DMSO vehicle control were subjected to immunoblot analysis with the indicated antibodies. Images were cropped for presentation. Full-length blots are presented in Supplementary Fig. 11. b. Lysates of 387 and 3565 BTICs treated the CDK1/2 inhibitor BMS-265246 or DMSO were immunoblotted with the indicated antibodies. c. Immunoblot analysis of CDK1, CDK5, stem and differentiation markers in BTICs and non-BTICs isolated from multiple patient-derived glioma xenografts (4302, 387, and 3565). d. In vitro kinase assays were performed with or without CDK5/p25 and either wild type or mutant (S616A) GST-C-terminal fragment of DRP1 (AA 518–736), GST-DRP1CT. e. Lysates of 387 BTICs expressing NT control shRNA or three independent shRNA constructs targeting CDK1 or CDK5 were immunoblotted with the indicated antibodies. Knockdown CDK5 but not CDK1 decreased phospho-DRP1S616 levels. f. Immunofluorescent staining of the mitochondrial marker TOM20 in 387 and 3565 BTICs expressing NT control shRNA or shCDK5. Data are represented as mean ± s.e.m. (387: fragmented, p = 0.0088; tubular, p = 0.0011. 3565: fragmented, p = 0.0098; tubular, p = 0.0018; Student’s t-test; n = 3).
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Figure 7: CDK5 activates DRP1 in brain tumor initiating cellsa. Lysates of 387 and 3565 brain tumor initiating cells (BTICs) treated with the CDK1/2/5 inhibitor Roscovitine or DMSO vehicle control were subjected to immunoblot analysis with the indicated antibodies. Images were cropped for presentation. Full-length blots are presented in Supplementary Fig. 11. b. Lysates of 387 and 3565 BTICs treated the CDK1/2 inhibitor BMS-265246 or DMSO were immunoblotted with the indicated antibodies. c. Immunoblot analysis of CDK1, CDK5, stem and differentiation markers in BTICs and non-BTICs isolated from multiple patient-derived glioma xenografts (4302, 387, and 3565). d. In vitro kinase assays were performed with or without CDK5/p25 and either wild type or mutant (S616A) GST-C-terminal fragment of DRP1 (AA 518–736), GST-DRP1CT. e. Lysates of 387 BTICs expressing NT control shRNA or three independent shRNA constructs targeting CDK1 or CDK5 were immunoblotted with the indicated antibodies. Knockdown CDK5 but not CDK1 decreased phospho-DRP1S616 levels. f. Immunofluorescent staining of the mitochondrial marker TOM20 in 387 and 3565 BTICs expressing NT control shRNA or shCDK5. Data are represented as mean ± s.e.m. (387: fragmented, p = 0.0088; tubular, p = 0.0011. 3565: fragmented, p = 0.0098; tubular, p = 0.0018; Student’s t-test; n = 3).

Mentions: To determine the molecular mechanism activating DRP1 in BTICs, we investigated potential kinases regulating the phosphorylation status of DRP1. Cyclin-dependent kinase 1 (CDK1) has been reported to phosphorylate DRP1 at Ser61633. CDK family kinases -- especially CDK1, CDK2 and CDK5 -- often share substrates. To determine if the CDKs regulate BTIC DRP1S616 phosphorylation, we treated BTICs with Roscovitine, a pan-CDK1/2/5 inhibitor, and determined that DRP1S616 phosphorylation was significantly reduced (Fig. 7a) with associated loss of fragmented mitochondrial morphology (Supplementary Fig. 5). In contrast, treatment with BMS-265246, a CDK inhibitor more specific for CDK1/2, did not alter DRP1S616 phosphorylation (Fig. 7b) or mitochondrial morphology (Supplementary Fig. 5), indicating that CDK5 might be the dominant regulating kinase responsible for DRP1S616 phosphorylation in BTICs. We screened the expression of the CDKs in matched BTICs and non-BTICs and found that both CDK1 and CDK5 were preferentially expressed by BTICs in multiple tumors (Fig. 7c).


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)

CDK5 activates DRP1 in brain tumor initiating cellsa. Lysates of 387 and 3565 brain tumor initiating cells (BTICs) treated with the CDK1/2/5 inhibitor Roscovitine or DMSO vehicle control were subjected to immunoblot analysis with the indicated antibodies. Images were cropped for presentation. Full-length blots are presented in Supplementary Fig. 11. b. Lysates of 387 and 3565 BTICs treated the CDK1/2 inhibitor BMS-265246 or DMSO were immunoblotted with the indicated antibodies. c. Immunoblot analysis of CDK1, CDK5, stem and differentiation markers in BTICs and non-BTICs isolated from multiple patient-derived glioma xenografts (4302, 387, and 3565). d. In vitro kinase assays were performed with or without CDK5/p25 and either wild type or mutant (S616A) GST-C-terminal fragment of DRP1 (AA 518–736), GST-DRP1CT. e. Lysates of 387 BTICs expressing NT control shRNA or three independent shRNA constructs targeting CDK1 or CDK5 were immunoblotted with the indicated antibodies. Knockdown CDK5 but not CDK1 decreased phospho-DRP1S616 levels. f. Immunofluorescent staining of the mitochondrial marker TOM20 in 387 and 3565 BTICs expressing NT control shRNA or shCDK5. Data are represented as mean ± s.e.m. (387: fragmented, p = 0.0088; tubular, p = 0.0011. 3565: fragmented, p = 0.0098; tubular, p = 0.0018; Student’s t-test; n = 3).
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Figure 7: CDK5 activates DRP1 in brain tumor initiating cellsa. Lysates of 387 and 3565 brain tumor initiating cells (BTICs) treated with the CDK1/2/5 inhibitor Roscovitine or DMSO vehicle control were subjected to immunoblot analysis with the indicated antibodies. Images were cropped for presentation. Full-length blots are presented in Supplementary Fig. 11. b. Lysates of 387 and 3565 BTICs treated the CDK1/2 inhibitor BMS-265246 or DMSO were immunoblotted with the indicated antibodies. c. Immunoblot analysis of CDK1, CDK5, stem and differentiation markers in BTICs and non-BTICs isolated from multiple patient-derived glioma xenografts (4302, 387, and 3565). d. In vitro kinase assays were performed with or without CDK5/p25 and either wild type or mutant (S616A) GST-C-terminal fragment of DRP1 (AA 518–736), GST-DRP1CT. e. Lysates of 387 BTICs expressing NT control shRNA or three independent shRNA constructs targeting CDK1 or CDK5 were immunoblotted with the indicated antibodies. Knockdown CDK5 but not CDK1 decreased phospho-DRP1S616 levels. f. Immunofluorescent staining of the mitochondrial marker TOM20 in 387 and 3565 BTICs expressing NT control shRNA or shCDK5. Data are represented as mean ± s.e.m. (387: fragmented, p = 0.0088; tubular, p = 0.0011. 3565: fragmented, p = 0.0098; tubular, p = 0.0018; Student’s t-test; n = 3).
Mentions: To determine the molecular mechanism activating DRP1 in BTICs, we investigated potential kinases regulating the phosphorylation status of DRP1. Cyclin-dependent kinase 1 (CDK1) has been reported to phosphorylate DRP1 at Ser61633. CDK family kinases -- especially CDK1, CDK2 and CDK5 -- often share substrates. To determine if the CDKs regulate BTIC DRP1S616 phosphorylation, we treated BTICs with Roscovitine, a pan-CDK1/2/5 inhibitor, and determined that DRP1S616 phosphorylation was significantly reduced (Fig. 7a) with associated loss of fragmented mitochondrial morphology (Supplementary Fig. 5). In contrast, treatment with BMS-265246, a CDK inhibitor more specific for CDK1/2, did not alter DRP1S616 phosphorylation (Fig. 7b) or mitochondrial morphology (Supplementary Fig. 5), indicating that CDK5 might be the dominant regulating kinase responsible for DRP1S616 phosphorylation in BTICs. We screened the expression of the CDKs in matched BTICs and non-BTICs and found that both CDK1 and CDK5 were preferentially expressed by BTICs in multiple tumors (Fig. 7c).

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