The regulation of mitochondrial DNA copy number in glioblastoma cells

Abstract

As stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions. It is not understood whether tumor-initiating cells regulate their mtDNA in a similar manner or whether mtDNA is essential for tumorigenesis. We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity. Differentiating multipotent glioblastoma cells failed to match the expansion in mtDNA copy number, patterns of gene expression and increased respiratory capacity observed in hNSCs. Partial depletion of glioblastoma cell mtDNA rescued mtDNA replication events and enhanced cell differentiation. However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including POU class 5 homeobox 1 (OCT4) and sonic hedgehog (SHH). The transfer of glioblastoma cells depleted to varying degrees of their mtDNA content into immunocompromised mice resulted in tumors requiring significantly longer to form compared with non-depleted cells. The number of tumors formed and the time to tumor formation was relative to the degree of mtDNA depletion. The tumors derived from mtDNA depleted glioblastoma cells recovered their mtDNA copy number as part of the tumor formation process. These outcomes demonstrate the importance of mtDNA to the initiation and maintenance of tumorigenesis in glioblastoma multiforme.

Figure 1

MtDNA copy number and gene expression in differentiating GBM cells and hNSCs. Mean mtDNA copy number in differentiating hNSCs, HSR-GBM1, GBM-L1 and GBM-L2 cells (a). Gene expression analysis of the nuclear-encoded mtDNA replication and transcription factors in differentiating HSR-GBM1 cells and hNSCs (b–f). Fold change in expression relative to undifferentiated cells, weighted to β-ACTIN, for TFAM (b), POLGA (c), POLGB (d), TWINKLE (e) and MTSSB (f). Bars represent mean values±S.E.M. *P<0.05, **P<0.01 and ***P<0.001

Figure 2

MtDNA depletion of GBM cells. Mean mtDNA copy number was assessed over 50 days of mtDNA depletion (a). Gene expression analysis of undifferentiated and mtDNA depleted HSR-GBM1 cells was determined relative to β-ACTIN and as fold changes relative to non-depleted cells for NESTIN (b), MUSASHI1 (c), CD133 (d) and GFAP (e). Population-doubling times of undifferentiated and mtDNA depleted HSR-GBM1 cells (f). Columns represent mean values±S.E.M. *P<0.05, **P<0.01 and ***P<0.001

Figure 3

Gene expression analysis of markers of pluripotency and self-renewal in undifferentiated and mtDNA depleted GBM cells. Gene expression for undifferentiated and mtDNA depleted HSR-GBM1 cells was determined relative to β-ACTIN and as fold changes relative to non-depleted cells for OCT4 (a), NANOG (b), SOX2 (c), c-MYC (d) and hTERT (e). Bars represent mean values±S.E.M. *P<0.05, **P<0.01 and ***P<0.001

Figure 4

Recovery of mtDNA copy number and gene expression in depleted GBM cells. Mean (±S.E.M.) mtDNA copy number following 14 days of recovery for undifferentiated HSR-GBM1 cells depleted for up to 50 days (a). Gene expression relative to β-ACTIN for HSR-GBM1 cells following 14 days of recovery after depletion for up to 25 days. Fold change in expression of NESTIN (b), MUSASHI1 (c), CD133 (d) and GFAP (e) is relative to non-depleted GBM cells. Bars represent mean values±S.E.M. *P<0.05, **P<0.01 and ***P<0.001

Figure 5

MtDNA copy number in mtDNA depleted, differentiating GBM cells. MtDNA copy for HSR-GBM1 cells depleted for 7 days (a), 14 days (b) and 21 days (c) and differentiated for 14 days are shown as fold changes relative to non-depleted HSR-GBM1 cells. Columns represent mean values±S.E.M. ***P<0.001

Figure 6

HSR-GBM1 tumor formation assay and assessment of mtDNA copy number. Tumor growth curve analysis of mtDNA depleted (mtDNA⁵⁰, mtDNA²⁰, mtDNA³ and mtDNA^0.2) and non-depleted (mtDNA¹⁰⁰) HSR-GBM1 cells (a). Kaplan–Meier survival plot for non-depleted and depleted HSR-GBM1 cells (b). Immunohistochemical labeling of proliferating cell nuclear antigen. in depleted (c) and non-depleted (d–f) HSR-GBM1 tumors. Negative control (g) and positive control (h). Quantification of proliferating cell nuclear antigen positive cells in depleted and non-depleted HSR-GBM1 tumors (i). Mean mtDNA copy number analysis of depleted and non-depleted HSR-GBM1 tumors (j). Columns represent mean values±S.E.M. *P<0.05 and **P<0.01. Scale bars=25 μm