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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 phosphorylation regulates mitochondrial morphology and stem cell marker expressiona. Immunofluorescent staining of mitochondria by TOM20 in 387 and 3565 non-brain tumor initiating cells (non-BTICs) transduced by lentiviral control vector or a DRP1S616E/S637A double mutant. b. Mitochondria morphology was assessed from 120 cells of three different experiments. Data are displayed as mean ± s.e.m. (387, fragmented: p = 0.0009, tubular: p = 0.0002; 3565, fragmented: p = 0.0030, tubular: p = 0.0016; statistical significance determined by Student’s t-test; n = 3). c. Immunoblot analysis of DRP1 protein in 387 and 3565 non-BTICs expressing control vector or a DRP1S616E/S637A double mutant. Images were cropped for presentation. Full-length blots are presented in Supplementary Fig. 10. d, e. 387 non-BTICs were transduced with vector encoding a DRP1S616E/S637A double mutant or control vector. Three days after infection, total RNA was isolated and cDNA was synthesized by reverse transcription. The mRNA levels of indicated genes were detected by real-time qPCR. Data are displayed as mean ± s.e.m. (387: Olig2, p = 0.0072; Oct4, p = 0.0097; Nanog, p = 0.0087; Nestin, p= 0.0309; Pou3f2, p= 0.0067; CD133, p = 0.0219; SSEA1, p = 0.0041; GFAP, p = 0.0095; MAP2, p = 0.0001. 3565: Olig2, p = 0.0334; Oct4, p = 0.0097; Nanog, p = 0.0074; Pou3f2, p = 0.0022; CD133, p = 0.0093; SSEA1, p = 0.0251; GFAP, p = 0.0164; MAP2, p = 0.0020. Statistical significance determined by Student’s t-test; n = 3).
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Figure 3: DRP1 phosphorylation regulates mitochondrial morphology and stem cell marker expressiona. Immunofluorescent staining of mitochondria by TOM20 in 387 and 3565 non-brain tumor initiating cells (non-BTICs) transduced by lentiviral control vector or a DRP1S616E/S637A double mutant. b. Mitochondria morphology was assessed from 120 cells of three different experiments. Data are displayed as mean ± s.e.m. (387, fragmented: p = 0.0009, tubular: p = 0.0002; 3565, fragmented: p = 0.0030, tubular: p = 0.0016; statistical significance determined by Student’s t-test; n = 3). c. Immunoblot analysis of DRP1 protein in 387 and 3565 non-BTICs expressing control vector or a DRP1S616E/S637A double mutant. Images were cropped for presentation. Full-length blots are presented in Supplementary Fig. 10. d, e. 387 non-BTICs were transduced with vector encoding a DRP1S616E/S637A double mutant or control vector. Three days after infection, total RNA was isolated and cDNA was synthesized by reverse transcription. The mRNA levels of indicated genes were detected by real-time qPCR. Data are displayed as mean ± s.e.m. (387: Olig2, p = 0.0072; Oct4, p = 0.0097; Nanog, p = 0.0087; Nestin, p= 0.0309; Pou3f2, p= 0.0067; CD133, p = 0.0219; SSEA1, p = 0.0041; GFAP, p = 0.0095; MAP2, p = 0.0001. 3565: Olig2, p = 0.0334; Oct4, p = 0.0097; Nanog, p = 0.0074; Pou3f2, p = 0.0022; CD133, p = 0.0093; SSEA1, p = 0.0251; GFAP, p = 0.0164; MAP2, p = 0.0020. Statistical significance determined by Student’s t-test; n = 3).

Mentions: To determine whether DRP1 phosphorylation is critical for the mitochondria morphological change between BTICs and non-BTICs, we constructed gain-of-function DRP1 encoding both S616E (to mimic activating phosphorylation) and S637A (to block inhibitory phosphorylation) mutations. Over-expression of DRP1S616E/S637A in non-BTICs potently induced remodeling of mitochondria morphology (Fig. 3a–c). Mitochondria in non-BTICs transduced by lentivirus expressing mutated DRP1S616E/S637A became more fragmented and less elongated than cells that expressed a control vector (Fig. 3a–c). Furthermore, forced expression of DRP1S616E/S637A induced expression of some, but not all, selected core stem cell regulators (Fig. 3d) and repression of differentiation markers (Fig. 3e) compared to vector control. Expression of DRP1S616E/S637A was not sufficient to induce sphere or in vivo tumor formation (data not shown), suggesting that DRP1 activity alone is not sufficient to fully reprogram non-BTICs into BTICs. Together, these results demonstrate that hyperactivated DRP1 plays an essential role in mitochondrial fission in the tumor hierarchy.


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 phosphorylation regulates mitochondrial morphology and stem cell marker expressiona. Immunofluorescent staining of mitochondria by TOM20 in 387 and 3565 non-brain tumor initiating cells (non-BTICs) transduced by lentiviral control vector or a DRP1S616E/S637A double mutant. b. Mitochondria morphology was assessed from 120 cells of three different experiments. Data are displayed as mean ± s.e.m. (387, fragmented: p = 0.0009, tubular: p = 0.0002; 3565, fragmented: p = 0.0030, tubular: p = 0.0016; statistical significance determined by Student’s t-test; n = 3). c. Immunoblot analysis of DRP1 protein in 387 and 3565 non-BTICs expressing control vector or a DRP1S616E/S637A double mutant. Images were cropped for presentation. Full-length blots are presented in Supplementary Fig. 10. d, e. 387 non-BTICs were transduced with vector encoding a DRP1S616E/S637A double mutant or control vector. Three days after infection, total RNA was isolated and cDNA was synthesized by reverse transcription. The mRNA levels of indicated genes were detected by real-time qPCR. Data are displayed as mean ± s.e.m. (387: Olig2, p = 0.0072; Oct4, p = 0.0097; Nanog, p = 0.0087; Nestin, p= 0.0309; Pou3f2, p= 0.0067; CD133, p = 0.0219; SSEA1, p = 0.0041; GFAP, p = 0.0095; MAP2, p = 0.0001. 3565: Olig2, p = 0.0334; Oct4, p = 0.0097; Nanog, p = 0.0074; Pou3f2, p = 0.0022; CD133, p = 0.0093; SSEA1, p = 0.0251; GFAP, p = 0.0164; MAP2, p = 0.0020. Statistical significance determined by Student’s t-test; n = 3).
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Figure 3: DRP1 phosphorylation regulates mitochondrial morphology and stem cell marker expressiona. Immunofluorescent staining of mitochondria by TOM20 in 387 and 3565 non-brain tumor initiating cells (non-BTICs) transduced by lentiviral control vector or a DRP1S616E/S637A double mutant. b. Mitochondria morphology was assessed from 120 cells of three different experiments. Data are displayed as mean ± s.e.m. (387, fragmented: p = 0.0009, tubular: p = 0.0002; 3565, fragmented: p = 0.0030, tubular: p = 0.0016; statistical significance determined by Student’s t-test; n = 3). c. Immunoblot analysis of DRP1 protein in 387 and 3565 non-BTICs expressing control vector or a DRP1S616E/S637A double mutant. Images were cropped for presentation. Full-length blots are presented in Supplementary Fig. 10. d, e. 387 non-BTICs were transduced with vector encoding a DRP1S616E/S637A double mutant or control vector. Three days after infection, total RNA was isolated and cDNA was synthesized by reverse transcription. The mRNA levels of indicated genes were detected by real-time qPCR. Data are displayed as mean ± s.e.m. (387: Olig2, p = 0.0072; Oct4, p = 0.0097; Nanog, p = 0.0087; Nestin, p= 0.0309; Pou3f2, p= 0.0067; CD133, p = 0.0219; SSEA1, p = 0.0041; GFAP, p = 0.0095; MAP2, p = 0.0001. 3565: Olig2, p = 0.0334; Oct4, p = 0.0097; Nanog, p = 0.0074; Pou3f2, p = 0.0022; CD133, p = 0.0093; SSEA1, p = 0.0251; GFAP, p = 0.0164; MAP2, p = 0.0020. Statistical significance determined by Student’s t-test; n = 3).
Mentions: To determine whether DRP1 phosphorylation is critical for the mitochondria morphological change between BTICs and non-BTICs, we constructed gain-of-function DRP1 encoding both S616E (to mimic activating phosphorylation) and S637A (to block inhibitory phosphorylation) mutations. Over-expression of DRP1S616E/S637A in non-BTICs potently induced remodeling of mitochondria morphology (Fig. 3a–c). Mitochondria in non-BTICs transduced by lentivirus expressing mutated DRP1S616E/S637A became more fragmented and less elongated than cells that expressed a control vector (Fig. 3a–c). Furthermore, forced expression of DRP1S616E/S637A induced expression of some, but not all, selected core stem cell regulators (Fig. 3d) and repression of differentiation markers (Fig. 3e) compared to vector control. Expression of DRP1S616E/S637A was not sufficient to induce sphere or in vivo tumor formation (data not shown), suggesting that DRP1 activity alone is not sufficient to fully reprogram non-BTICs into BTICs. Together, these results demonstrate that hyperactivated DRP1 plays an essential role in mitochondrial fission in the tumor hierarchy.

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