Thursday, 5 March 2015

Mitochondrial control by DRP1 in brain tumor initiating cells



Qi Xie, Qiulian Wu, Craig M Horbinski et al.

Brain tumor initiating cells (BTICs) are capable of self-renewal, they are highly proliferative and show chromosomal abnormalities. BTICs are known to take over the glucose transporter GLUT3 so that they can withstand metabolic stress more easily. All tumor cells have dysregulated metabolic pathways, but the highly proliferative nature of BTICs suggests that these tumor subpopulations have some metabolic features that distinguish them from the tumor bulk. In this paper they look at mitochondrial morphology in the most common primary intrinsic brain tumor, gliblastoma.

They compare mitochondrial morphology from BTICs with non-BTIC tumor cells and found out that mitochondria in BTICs are more fragmented and less tubular : in non-BTIC tumor cells, mitochondria show an elongated tubulated structure whereas in BTICs they are shorter and rounded. This suggest that BTICs have increased mitochondrial fragmentation or decreased fusion. It turns out that BTICs show increased phosphorylation of Drp1 at Ser-616 and decreased phosphorylation at Ser-637, both of these changes enhance fission activity of Drp1.

They then checked whether these changed in Drp1 phosphorylation levels were responsible for increased fission in BTICs. A Drp1 gain-of-function mutant, with increased Ser-616 phosphorylation activity and blocked Ser-637 phosphorylation, was expressed in non-BTIC tumor cells. Mitochondria in non-BTIC tumor cells expressing these mutants indeed became more fragmented and less elongated. It also induced expression of some stem cell regulators and repressed some differentiation markers. Expression of the Drp1 mutant was, however, not sufficient to reprogram non-BTIC tumor cells into BTICs.

They then tried to find out whether the change in Drp1 activity is crucial for BTIC maintenance, because it is observed that differentiation of BTICs reduces the hyperactivation of Drp1. Drp1 was knocked down in BTICs using small hairpin RNA lentiviral constructs (shDrp1) , which significantly decreased the growth of BTICs, whereas Drp1 knockdown had no effect on non-BTIC tumor cells or normal neuronal progenitor cells (which are, just like BTICs, capable of differentation and self-renewal). Targeting Drp1 lead to a fourfold decrease in tumorsphere size. They evaluated the potential anti-tumor effects of Drp1 knockdown in vivo in mice, and found that mice with BTICs expressing shDrp1 had reduced tumor formation and increased survival compared to mice with BTICs expressing non-targeted shRNA.

Mdivi-1 is an inhibitor of the GTPase activity of Drp1, and using Mdivi-1 to block Drp1 activity also lead to decreased growth of BTICs. BTICs were implanted into brains of mice, and the mice where then injected with Mdivi-1 which increased mice survival compared with control.

AMPK is a cellular stress sensor. They found that activation of AMPK decreased BTIC growth, and both Drp1 knockdown and Mdivi-1 treatment lead to an increase in AMPK activation. This suggests that the hyperactivity of Drp1 in BTICs may lead to a decreased AMPK activity. Downregulation of both Drp1 and AMPK activity did not reduce the growth of BTICs (as opposed to only downregulating Drp1) which indeed suggests that Drp1 is a critical node in the response of BTICs to metabolic stress through AMPK regulation.

They go on to find the molecular mechanisms that activate Drp1 in BTICs, it turns out that CDK5 and CAMK2 are important. CAMK2 inhibits Drp1 in non-BTIC tumor cells, and its expression is lower in BTICs. CDK5 activates Drp1 and is preferentially expressed in BTICs.

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