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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 is hyperactivated in brain tumor initiating cellsa. Immunoblot analysis of DRP1 total protein levels and activating phosphorylation [phospho-DRP1S616] and repressive phosphorylation [phospho-DRP1S637] in BTICs and non-BTICs isolated from patient-derived xenografts (T387, T4302, T3565, and IN528). Images were cropped for presentation. Full-length blots are presented in Supplementary Fig. 9. b. Alternative enrichment of BTICs from patient-derived xenografts (387 and 3565) by SSEA1/CD15 confirmed differential activation on immunoblot of phospho-DRP1. c. Immunofluorescent staining of activating phosphorylation of DRP1 [phospho-DRP1S616] with several BTIC markers, including SOX2 and OLIG2 in two primary human GBM specimens (CW1617, CW1679). d. Immunoblot analysis of DRP1 protein and its activating phosphorylation [phospho-DRP1S616] and repressive phosphorylation [phopho-DRP1S637] in BTICs and non-BTICs directly derived from two primary human glioblastoma specimens (CCF3015 and CCF3038). e. Immunoblot analysis of phospho-DRP1S616 and phospho-DRP1S637 levels during BTIC (4302 and 387) differentiation induced by 10% serum over a time course.
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Figure 2: DRP1 is hyperactivated in brain tumor initiating cellsa. Immunoblot analysis of DRP1 total protein levels and activating phosphorylation [phospho-DRP1S616] and repressive phosphorylation [phospho-DRP1S637] in BTICs and non-BTICs isolated from patient-derived xenografts (T387, T4302, T3565, and IN528). Images were cropped for presentation. Full-length blots are presented in Supplementary Fig. 9. b. Alternative enrichment of BTICs from patient-derived xenografts (387 and 3565) by SSEA1/CD15 confirmed differential activation on immunoblot of phospho-DRP1. c. Immunofluorescent staining of activating phosphorylation of DRP1 [phospho-DRP1S616] with several BTIC markers, including SOX2 and OLIG2 in two primary human GBM specimens (CW1617, CW1679). d. Immunoblot analysis of DRP1 protein and its activating phosphorylation [phospho-DRP1S616] and repressive phosphorylation [phopho-DRP1S637] in BTICs and non-BTICs directly derived from two primary human glioblastoma specimens (CCF3015 and CCF3038). e. Immunoblot analysis of phospho-DRP1S616 and phospho-DRP1S637 levels during BTIC (4302 and 387) differentiation induced by 10% serum over a time course.

Mentions: Phosphorylation of DRP1S616 enhances DRP1 activity, whereas phosphorylation of DRP1S637 represses function33, 34. We quantified levels of phosphorylated DRP1 at DRP1S616 and DRP1S637 in matched cultures of BTICs and non-BTICs from short-term xenografts by immunoblot (Fig. 2a). In every model tested, BTICs displayed strikingly elevated activating DRP1 phosphorylation (S616) and significantly down-regulated inhibitory DRP1 phosphorylation (S637) levels compared to matched non-BTICs. To address the possible concern regarding CD133 as a marker, we used an alternative marker (CD15; stage-specific embryonic antigen 1, SSEA1), which has been suggested as marker of BTICs40. In confirmation of our results with CD133, SSEA1 positive cells displayed an identical pattern of activated DRP1 relative to non-BTICs (Fig. 2b). For yet an additional confirmation of specific activation of DRP1 in BTICs independent of the CD133 marker, we demonstrated co-expression of phosphorylated DRP1 (S616) and BTIC markers, SOX2 and OLIG2, by immunofluorescence staining of human primary glioblastoma tissue sections (Fig. 2c). To additionally rule out an effect of culture conditions underlying these observations, we confirmed these results using BTICs and non-BTICs directly isolated from primary glioblastoma patient specimens without culture (Fig. 2d). To determine the relationship between cellular differentiation and DRP1 regulation, we induced differentiation in BTICs and found a marked switch in DRP1 phosphorylation from the activating modification (S616) to an inhibitory state (S637) (Fig. 2e), indicating that dynamic regulation of DRP1 by phosphorylation is important for BTICs self-renewal and differentiation. Collectively, these findings support DRP1 is hyperactivated in BTICs.


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 is hyperactivated in brain tumor initiating cellsa. Immunoblot analysis of DRP1 total protein levels and activating phosphorylation [phospho-DRP1S616] and repressive phosphorylation [phospho-DRP1S637] in BTICs and non-BTICs isolated from patient-derived xenografts (T387, T4302, T3565, and IN528). Images were cropped for presentation. Full-length blots are presented in Supplementary Fig. 9. b. Alternative enrichment of BTICs from patient-derived xenografts (387 and 3565) by SSEA1/CD15 confirmed differential activation on immunoblot of phospho-DRP1. c. Immunofluorescent staining of activating phosphorylation of DRP1 [phospho-DRP1S616] with several BTIC markers, including SOX2 and OLIG2 in two primary human GBM specimens (CW1617, CW1679). d. Immunoblot analysis of DRP1 protein and its activating phosphorylation [phospho-DRP1S616] and repressive phosphorylation [phopho-DRP1S637] in BTICs and non-BTICs directly derived from two primary human glioblastoma specimens (CCF3015 and CCF3038). e. Immunoblot analysis of phospho-DRP1S616 and phospho-DRP1S637 levels during BTIC (4302 and 387) differentiation induced by 10% serum over a time course.
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Figure 2: DRP1 is hyperactivated in brain tumor initiating cellsa. Immunoblot analysis of DRP1 total protein levels and activating phosphorylation [phospho-DRP1S616] and repressive phosphorylation [phospho-DRP1S637] in BTICs and non-BTICs isolated from patient-derived xenografts (T387, T4302, T3565, and IN528). Images were cropped for presentation. Full-length blots are presented in Supplementary Fig. 9. b. Alternative enrichment of BTICs from patient-derived xenografts (387 and 3565) by SSEA1/CD15 confirmed differential activation on immunoblot of phospho-DRP1. c. Immunofluorescent staining of activating phosphorylation of DRP1 [phospho-DRP1S616] with several BTIC markers, including SOX2 and OLIG2 in two primary human GBM specimens (CW1617, CW1679). d. Immunoblot analysis of DRP1 protein and its activating phosphorylation [phospho-DRP1S616] and repressive phosphorylation [phopho-DRP1S637] in BTICs and non-BTICs directly derived from two primary human glioblastoma specimens (CCF3015 and CCF3038). e. Immunoblot analysis of phospho-DRP1S616 and phospho-DRP1S637 levels during BTIC (4302 and 387) differentiation induced by 10% serum over a time course.
Mentions: Phosphorylation of DRP1S616 enhances DRP1 activity, whereas phosphorylation of DRP1S637 represses function33, 34. We quantified levels of phosphorylated DRP1 at DRP1S616 and DRP1S637 in matched cultures of BTICs and non-BTICs from short-term xenografts by immunoblot (Fig. 2a). In every model tested, BTICs displayed strikingly elevated activating DRP1 phosphorylation (S616) and significantly down-regulated inhibitory DRP1 phosphorylation (S637) levels compared to matched non-BTICs. To address the possible concern regarding CD133 as a marker, we used an alternative marker (CD15; stage-specific embryonic antigen 1, SSEA1), which has been suggested as marker of BTICs40. In confirmation of our results with CD133, SSEA1 positive cells displayed an identical pattern of activated DRP1 relative to non-BTICs (Fig. 2b). For yet an additional confirmation of specific activation of DRP1 in BTICs independent of the CD133 marker, we demonstrated co-expression of phosphorylated DRP1 (S616) and BTIC markers, SOX2 and OLIG2, by immunofluorescence staining of human primary glioblastoma tissue sections (Fig. 2c). To additionally rule out an effect of culture conditions underlying these observations, we confirmed these results using BTICs and non-BTICs directly isolated from primary glioblastoma patient specimens without culture (Fig. 2d). To determine the relationship between cellular differentiation and DRP1 regulation, we induced differentiation in BTICs and found a marked switch in DRP1 phosphorylation from the activating modification (S616) to an inhibitory state (S637) (Fig. 2e), indicating that dynamic regulation of DRP1 by phosphorylation is important for BTICs self-renewal and differentiation. Collectively, these findings support DRP1 is hyperactivated in BTICs.

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