Targeting reduced mitochondrial DNA quantity as a therapeutic approach in pediatric high-grade gliomas

Abstract

Background: Despite increased understanding of the genetic events underlying pediatric high-grade gliomas (pHGGs), therapeutic progress is static, with poor understanding of nongenomic drivers. We therefore investigated the role of alterations in mitochondrial function and developed an effective combination therapy against pHGGs.

Methods: Mitochondrial DNA (mtDNA) copy number was measured in a cohort of 60 pHGGs. The implication of mtDNA alteration in pHGG tumorigenesis was studied and followed by an efficacy investigation using patient-derived cultures and orthotopic xenografts.

Results: Average mtDNA content was significantly lower in tumors versus normal brains. Decreasing mtDNA copy number in normal human astrocytes led to a markedly increased tumorigenicity in vivo. Depletion of mtDNA in pHGG cells promoted cell migration and invasion and therapeutic resistance. Shifting glucose metabolism from glycolysis to mitochondrial oxidation with the adenosine monophosphate-activated protein kinase activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) or the pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA) significantly inhibited pHGG viability. Using DCA to shift glucose metabolism to mitochondrial oxidation and then metformin to simultaneously target mitochondrial function disrupted energy homeostasis of tumor cells, increasing DNA damage and apoptosis. The triple combination with radiation therapy, DCA and metformin led to a more potent therapeutic effect in vitro and in vivo.

Conclusions: Our results suggest metabolic alterations as an onco-requisite factor of pHGG tumorigenesis. Targeting reduced mtDNA quantity represents a promising therapeutic strategy for pHGG.

Keywords: DIPG; gliomas; mitochondria; radiotherapy.

Fig. 1

Elimination of mtDNA leads to increased migration, invasion, and therapeutic resistance in pHGG. (A) MtDNA quantity was significantly lower in DIPG and supratentorial pHGG specimens than normal brain. (B) Generation of a mutant pediatric glioblastoma SF188 cell line (ρ ⁰) with complete removal of mtDNA. (C) Quantitative PCR assays demonstrating SF188 ρ ⁰ cells had markedly reduced mtDNA content compared with WT counterparts. (D, E) MtDNA depletion significantly reduced proliferative capacity. (F) JC-1 staining showed that SF188 ρ ⁰ cells had a notably decreased mitochondrial membrane potential compared with WT controls. (G) A significant reduction in total cellular ATP level was detected in ρ ⁰ cells in comparison with parental WT cells. (H) MtDNA-depleted SF188 cells exhibited a ~3-fold increase in L-lactate levels compared with controls. (I, J) Wound healing and transwell cell invasion assays show that lower mtDNA amount strongly enhanced cell migration and invasion. Scale bar: 50 μm. (K, L) Decreasing mtDNA copy number rendered SF188 cells more resistant to temozolomide and radiation. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2

Partial loss of mtDNA in iNHAs through shRNA-mediated silencing of TFAM promoted tumorigenesis. (A) Western blotting of TFAM and 2 key subunits of OXPHOS complexes, COXII and NDUFB2, in TFAM knockdown iNHAs and shRNA controls, with actin probed as a loading control. (B) Partial depletion of mtDNA greatly enhanced the production of L-lactate (~2-fold) in iNHAs relative to corresponding controls. (C) Stable knockdown of TFAM resulted in a ~30–40% decrease in relative mtDNA copy number in iNHAs compared with scrambled controls. (D) TFAM knockdown cells proliferated at a lower rate compared with controls. (E) NSG mice carrying TFAM-deficient iNHA orthotopic xenografts displayed enhanced tumorigenicity with a significantly worse survival time compared with the control group (n = 5/group). (F) Representative H&E staining of TFAM-deficient xenograft tumors showed some pathological characteristics of DIPG/pediatric high-grade astrocytoma compared with the control. Scale bar: 0.1 mm. (G) Quantitative PCR assays demonstrating a significantly lower mtDNA number in TFAM knockdown xenografts than iNHA non-targeting control (NTC) cells. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3

AICAR leads to cytotoxic effects on pHGG cells through AMPK activation, elevating mtDNA content and increasing levels of OXPHOS complex content via the PGC-1α/TFAM axis. (A) AICAR significantly reduced the viability of SU-DIPG4 and SF188 cells, while leaving nontransformed NHAs largely unaffected. (B) Exposure to AICAR triggered apoptosis in SU-DIPG4 and SF188 cells. (C) L-lactate concentration was significantly reduced by AICAR treatment in SU-DIPG4 and SF188 cells. (D) SU-DIPG4 and SF188 cells treated with 250 µM AICAR at various time points contained a significantly increased mtDNA content compared with controls. (E, F) AICAR treatment (250 µM) time-dependently increased expression of mitochondrial biogenesis modulators, PGC-1α and TFAM, induced activation of the AMPK signaling pathway, and markedly upregulated respiratory complex levels. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4

DCA induced cytotoxic and radiosensitizing effect on DIPG cells by shifting glucose metabolism from glycolysis to OXPHOS. (A) DCA treatment at 10 mM decreased the level of phosphorylated PDH-E1α in DIPG cells. (B) DCA treatment increased the PDH activity in HSJD-DIPG007. (C) DCA treatment induced an increase in oxygen consumption rate (OCR) and a decrease in extracellular acidification rate (ECAR) dose-dependently in HSJD-DIPG007. (D) DCA induced a dose-dependent decrease in mitochondrial reserve respiratory capacity in HSJD-DIPG007. (E) PDK1 is overexpressed in DIPG cultures compared with NHAs. (F) DCA selectively inhibited the proliferation of DIPG cells. (G) DCA treatment augmented RT-induced DNA DSBs. (H) DCA in combination with RT synergistically inhibited clonogenicity of DIPG cells. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5

Dual targeting of glucose metabolism with DCA and metformin leads to proliferative arrest and apoptosis in DIPG cells. (A) DCA/metformin combination synergistically inhibited cell proliferation. The combination index (CI) was calculated using CalcuSyn and CI < 1, = 1 and >1 was defined as synergistic, additive, and antagonistic effects, respectively. (B) DCA/metformin combination induced a decrease in the proportion of cells in G1 phase and an increase in G2-M phase. DCA/metformin combination induced higher levels of ROS production (C), H2AX (D), mitochondrial depolarization (E), and apoptosis (F), compared with each treatment alone. (G) DCA attenuated metformin-induced L-lactate production. (H) DCA/metformin combination further decreased cellular ATP level compared with monotherapy. (I) DCA/metformin combination activated the AMPK pathway. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6

DCA in combination with metformin enhances radiosensitivity of DIPG both in vitro and in vivo. (A) The clonogenicity of DIPG cells was maximally impaired by the triple combination. (B) The triple combination induced the highest level of DNA DSBs. (C) The triple combination treated orthotopic model of DIPG bearing HSJD-DIPG007 showed the longest median survival compared with other groups (n = 10/group). (D, E) The triple combination induced the lowest proliferation index (Ki-67) and the highest level of DNA DSBs (H2AX). Scale bar: 50 μm. *P < 0.05; **P < 0.01; ***P < 0.001.