Mitochondrial DNA Alterations in Glioblastoma (GBM)

Glioblastoma (GBM) is an extremely aggressive tumor originating from neural stem cells of the central nervous system, which has high histopathological and genomic diversity. Mitochondria are cellular organelles associated with the regulation of cellular metabolism, redox signaling, energy generation, regulation of cell proliferation, and apoptosis. Accumulation of mutations in mitochondrial DNA (mtDNA) leads to mitochondrial dysfunction that plays an important role in GBM pathogenesis, favoring abnormal energy and reactive oxygen species production and resistance to apoptosis and to chemotherapeutic agents. The present review summarizes the known mitochondrial DNA alterations related to GBM, their cellular and metabolic consequences, and their association with diagnosis, prognosis, and treatment.


Introduction
Grade IV glioma or glioblastoma (GBM) is the most common and aggressive malignant central nervous system (CNS) cancer in adults [1], accounting for 55% of all gliomas. It is associated with high rates of morbidity, relapse, and mortality [2][3][4] and can arise via a de novo pathway without clinical or histologic evidence of a less malignant precursor lesion (primary GBM), or via a progressive pathway through development from a low-grade astrocytoma (secondary GBM) [5,6].
Although age-related prognosis has no significant difference between the two histological types, primary GBM is frequently diagnosed at around 60 years of age and a more advanced stage, while secondary GBM is diagnosed at an early age, around 45 years [6]. One of the challenging features of GBM is its chemoresistance due to high biological complexity observed during tumorigenesis and progression [3,7].
Several GBM changes in mitochondrial function are observed at different levels, such as structural and functional changes, affecting mitogenic, hemodynamic, bioenergetic, and apoptotic signaling [8,9]. Altogether, these changes may indicate a decreased function of the oxidative phosphorylation system (OXPHOS) and energy linkage in glioma cells [10]. Thus, understanding the role of mitochondrial DNA (mtDNA) alterations in the development of glioblastoma carcinogenesis is extremely important [11]; hence, we provide a review of such phenomena. resulting in its up-regulated aberrant expression instead of its wild-type isoform hexokinase 1 (HK1), which helps the maintenance of the Warburg effect [35].
In this sense, it is known that mitochondria play a role in reprogramming the cellular metabolism [36], with metabolic flexibility serving to balance tumor cell energy needs with requirements for metabolites and precursors [37,38].

Mitochondria: Genome and Activity
Mitochondria are essential cellular organelles delimited by a double membrane system, external and internal (mitochondrial ridges), involved in numerous complex physiological processes, including cellular metabolism regulation, redox signaling, energy generation, regulation of cellular proliferation, and apoptosis [39]. As general characteristics, mitochondria have their own genetic material, and their biogenesis does not depend on the cell cycle [40].
Mitochondria are, besides other processes, responsible for cell energy generation due to the presence of a series of multi-protein complexes (I-IV, forming the MRC) within the mitochondrial inner membrane, which generates reflux of protons used by complex V (ATP synthase) for the OXPHOS process to produce ATP and inorganic phosphate [14,45].
Electron transfers and proton translocations from the MRC and ATP generation from OXPHOS are related to several cellular processes, such as tricarboxylic acid cycle flow, regulation of nucleotide pools, generation of NADPH, carbon metabolism, ROS signaling, exchange ATP/ADP, calcium transport, protein import, apoptosis, inorganic phosphate transport, and mitochondrial membrane potential. Thus, this system has an important role in cell regulation as well as in molecular alterations in mitochondrial and/or nuclear genes, and thus, it could be related to the development and/or maintenance of diseases [10,38,39].
Most solid tumors, including glioma cells, behave according to the Warburg effect [14]. Therefore, tumor cells depend mainly on cytosolic ATP produced from glycolysis, rather than ATP derived from mitochondria [3,46].

Mutations and Polymorphisms
Considering the substantial evidence supporting an involvement of mitochondria in the process of tumorigenesis, some studies have focused on the role of mtDNA changes in cancer development and maintenance. However, the precise underlying mechanism remains unknown [32,41,42].
Mutations in mtDNA can be found in about 60% of solid tumors, according to the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas Program (TCGA), and could result in the coexistence of mixed mtDNA molecules in a single cell, a phenomenon known as heteroplasmy. These mutations occur in MRC subunits, the F o F 1 -ATPase subunit, or the protein biosynthesis on mitochondrial ribosomes, affecting ATP production and increasing oxidative stress [39]. Hence, understanding the mtDNA role in GBM development, progression, and drug resistance [32] is highly desirable because deregulation of mitochondrial function is considered a GBM marker [47].
Besides D-LOOP, codifying regions of mtDNA, especially those involved in MRC and OXPHOS, are also subject to mutations. Protein-coding genes of the MRC Complex I were the most affected, especially NADH dehydrogenase 4 (ND4) and NADH dehydrogenase 6 (ND6) genes. This suggests that such variants of the mitochondrial genome could offer advantage to the cells for promoting the tumorigenesis of glioblastoma [55]. One should notice that Complex I is involved in the cellular capacity to respond to oxygen deficiency and the establishment of hypoxia mechanisms; thus, genetic changes leading to its dysfunction can cause resistance to chemotherapeutic agents which need the redox cycle to be activated. For example, the T14634C mutation in ND6 causes an amino acid change, resulting in a modification of the structure and orientation of the transmembrane helix of the ND6 protein [50].
The A10398G polymorphism in the NADH dehydrogenase 3 (ND3) gene results in a decrease of complex I function and, consequently, an increase of ROS cell production and oxidative stress, resulting in mtDNA damage, which can favor the onset and promotion of carcinogenesis [49].
Apart from being considered a hotspot for substitution mutations, genes involved in Complex I are also hotspots for other mutation types. As an example, it was observed that around 65% of the truncating somatic missense mutations of the mtDNA in GBM occurred in the NADH dehydrogenase 5 (NAD5) gene [57].
Alterations on Complexes III and IV coding genes are also frequent in GBM, some resulting in protein structural and functional alterations, considered as independent predictors of poor prognosis [58,61].
Recently, it was described that the T14798C mutation, which results in an amino acid change (F18L) in the central subunit of Cytochrome b (Cyt b), part of the MRC Complex III, can alter the activity and sensitivity to complex III-targeting drugs, altering ROS production, cell behavior, and the patient's response to treatment, resulting in a poor prognosis. Thus, as a germline alteration, the T14798C mutation could be used as a non-invasive biomarker for prognostic and treatment response prediction [32]. Other mutations on Cyt b are related to Complex III activity or drug sensitivity. For example, G15257A and T14798C mutations increase drug sensitivity to atovaquone and clomipramine, respectively [60].
Even though most tumor-and non-tumor specific mtDNA mutations are not considered as deleterious events correlated with pathogenic processes, a small but important number of mutations on protein-coding genes may lead to alterations on mitochondrial function and might explain the metabolic plasticity observed on GBM [3].

Variation on MtDNA Content
Unlike the nuclear genome, mtDNA has several copies per cell as mitochondria undergo different dynamic processes, such as fusion and fission [62]. Even so, mtDNA content in each cell type is kept within a constant range to maintain cellular energy levels and, thus, cell function [59]. However, changes in mtDNA content are reportedly occurring at early stages of carcinogenesis [62,63], as a reflection of essential mutations for neoplastic transformation [62], leading to alterations in OXPHOS and, consequently, ATP production [64,65].
Currently, there are few studies about the influence of mtDNA copy number variation in GBM tumorigenesis, some with contradictory results. While mtDNA content maintenance during the differentiation process has been suggested [59,66], a higher mtDNA content was observed in patients with gliomas (GBM included) when compared to the con-trol samples. Besides, a negative correlation between the increased mtDNA copy number and the histopathological grade of the glioma and tumor recurrence was also observed [47].
On the other hand, several studies have suggested a relationship between mtDNA content, GBM prognosis, and treatment response. However, in GBM cell lines (U87 and LN229), a low mtDNA copy number was associated with resistance to radiation and Temozolomide (TMZ) therapies [67] and with changes in nuclear genes methylation and expression patterns, leading to the expression of stemness markers [44,66]; studies using case-control samples observed a correlation between a high mtDNA copy number and the increase in overall survival [41,59,62], suggesting a better prognosis.

MtDNA Alterations and Liquid Biopsies
Currently, the diagnosis and monitoring of GBM patients consist, basically, of the use of neuroimaging approaches, such as magnetic resonance imaging (MRI) and computed tomography (CT) [68,69]. However, some limitations are related to those approaches, as imaging exams may not differentiate a true tumor progression from a pseudoprogression, leading to a misinterpretation of the patient's response to therapy and delaying possible clinical interventions; additionally, tissue biopsies are extremely invasive and can cause cerebral edema and neurological disorders, which brings considerable risks to the patient [69,70]. Thus, the use of a more precise, less invasive strategies for the diagnosis, prognosis, molecular classification, response to treatment prediction, and assessment of GBM progression [68,71] are desirable. In recent years, the practice of liquid biopsy, fluid biopsy, or fluid phase biopsy have been proposed to overcome the limitations in the diagnosis and monitoring of GBM patients [70].
Regarding cfDNA, which can be found freely, bound to proteins, or inside extracellular vesicles, it can be divided into two main classes: cell-free nuclear (cfnDNA) and mitochondrial (cfmtDNA) DNA, with the latter composed by generally short fragments, with 30 to 80 base pairs [74].
Body fluids such as blood, urine, saliva, and CSF can be used to detect cfmtDNA that are linked to GBM prognosis, response to treatment, and tumor recurrence [63,65,67]. In that sense, as tumoral mtDNA content variation is correlated with the patient's prognosis, variations in mtDNA copy number in peripheral blood may be used as a biomarker for diagnosis [12,59] as well as prognosis [75].

Conclusions and Perspectives
Molecular alterations in mtDNA can deregulate mitochondria function. In that sense, despite several mtDNA mutations that have been described in GBM, only a few, located in protein-coding genes related with OXPHOS, have been considered deleterious, resulting in alteration of the energy-production process and in abnormal cell function, which can lead to resistance to some chemotherapeutic agents. However, due to a lack of characterization of their functional consequences, their real role in GBM tumorigenesis has not yet been determined. Considering this, the utilization of mtDNA mutations and polymorphisms in clinical routine as GBM prognosis biomarkers, although promising, should be carefully considered.
On the other hand, alterations on mtDNA content, as a result or not from mutations on the D-LOOP region, support their use as a diagnostic, prognostic, and responseto-treatment marker, making them a promising biomarker mainly for non-invasive approaches, such as liquid biopsy. In this respect, the mtDNA content quantification may be useful, especially in monitoring GBM recurrences in a less invasive and less stressful way for the patients.