**3. Mitochondrial-nuclear crosstalk**

of mtDNA results in significant changes in methylation patterns of a number of nuclear-encoded genes, and these epigenetic modifications are reversed by the restoration of mtDNA content [55]. The molecular mechanism altering mtDNA copy number is still under investigation. In a study of 65 colorectal cancers, it has been suggested that hypomethylation of specific sites on CpG islands of the D-loop promoter may be involved in the regulation of mtDNA copy numbers [56]. Moreover, it has been reported that polymorphisms within the nuclear-encoded polymerase gamma gene (POLG), which codifies for a key component of the mitochondrial genome maintenance machinery, may lead to a decrease in mtDNA content and mitochondrial dysfunction [57]. Curiously, a homozygous polymorphic insertion (AluYb8MUTYH) in the 15th intron of the MUTYH base excision repair gene has been associated with a significant reduction of the type 1 MUTYH protein that localizes to mitochondria as well as lowered mtDNA content in age-related diseases [58]. Since biallelic mutations of MUTYH are associated with the MAP syndrome, it might be speculated that homozygous or compound heterozygous MUTYH variants may correlate with the mtDNA content in colorectal cancer [30].

The non-coding D-loop region contains essential transcription and replication elements and is formed by two hypervariable regions, namely HV-I (nt. 16,024–16,383) and HV-II (nt. 57–333) [59]. The latter includes the D310 sequence, a polycytidine repeat (nt. 303–309), which is essential for mtDNA replication in virtue of the H-strand replication origin. Replication of the leading strand initiates at the origin of H-strand synthesis and proceeds unidirectionally, displacing the parental H-strand as single-stranded DNA [60]. The D-loop is a well-known hotspot for somatic mutations in many types of cancer, with a mutation rate 100- to 200-fold higher than nuclear DNA. This finding may be partly explained by considering the direct relationship between mutational frequency and single-strandedness during mitochondrial replication [61]. mtDNA variants in the D-loop region have been repeatedly associated with risk and survival rates in cancer patients and, thus, they have been proposed as valuable prognostic markers. However, it has been argued that most of these studies could be biased due to artifacts related to genotyping errors or inadequate experimental design [62]. Mitochondrial microsatellite instability (mtMSI), that is a change in length in the repetitive sequences of the D-loop segment between normal and tumor tissues, has been described as a frequent molecular event in different cancers, but its prognostic value is still debated [63]. The variation of the homopolymeric tract length mainly arises through replication slippage of mitochondrial DNA polymerase and, importantly, this process may affect mtDNA replication and transcription. Intriguingly, the oxidative damage to mitochondrial polymerase γ may also contribute to the alteration in the length of the polycytidine repeat by impacting on mtDNA replication [64].

Instability of the D-loop hypervariable region-II (HV-II) has been associated with variants

early stages of MAP colorectal tumorigenesis [29]. Therefore, D-loop mutations probably do not directly drive carcinogenesis but are more likely an epiphenomenon, used as a universal clonal marker ("molecular clock") to estimate the relative mitotic history of tumors [65, 66].

instability might contribute to drive non-random accumulation of MT-CO2

gene in MAP patients, thus suggesting that genome

variants in the

**2.4. D-loop and mitochondrial instability**

200 Mitochondrial DNA - New Insights

specifically grouped inside the MT-CO<sup>2</sup>

Tight coordination between the nucleus and mitochondria is required for proper mitochondrial functioning and includes both anterograde (nucleus to mitochondria) and retrograde (mitochondria to nucleus) signals. This crosstalk is critical for the maintenance of cellular homeostasis, and accumulated mtDNA variants may perturb this subtle pathway [67]. It has been demonstrated that somatically acquired mitochondrial-nuclear genome fusion sequences are present in human cancer cells [68]. Although most of the genes encoding proteins of the OXPHOS machinery are transcribed in the nucleus (anterograde signaling), mitochondria may also exert retrograde regulatory control over the nucleus in terms of nuclear gene expression modulation [69]. This phenomenon suggests a strong association between nuclear and mitochondrial DNA alterations in driving tumor development and progression. Variants in nuclear-encoded mitochondrial genes, such as fumarate hydratase, iso-citrate dehydrogenase and succinate dehydrogenase) have been associated with a wide variety of human cancers, such as paragangliomas, uterine leiomyomas, renal carcinomas, breast cancers, gastrointestinal stromal cancers, leukemia, prostate cancer, glioblastomas and colorectal carcinomas [70–78]. Furthermore, it has been demonstrated that mtDNA changes and MAPK pathway alterations synergize to drive colorectal malignant transformation [79].

In a study on colorectal adenoma and adenocarcinoma samples, an increased number of mutations in nuclear genes encoding proteins involved in critical mitochondrial processes, such as fusion, fission and localization were found [80]. It has also been suggested that mtDNA depletion may disrupt crucial nuclear processes, leading to centrosome amplification and mitotic spindle multipolarity, both participating in cancer cell transformation [81, 82]. mtDNA variants have the potential to induce molecular signals through the mitochondrial-nuclear crosstalk mechanism, thereby promoting nuclear compensation in response to mitochondrial malfunction [67]. Interestingly, some typical nuclear transcription factors, such as the tumor-suppressor p53 and estrogen receptor (ER), are localized within mitochondria, where they exert various transcriptionindependent functions [83]. By using transmitochondrial cybrid systems ("cybrids"), Kaipparettu et al. [69] elegantly demonstrated that mitochondria derived from the non-transformed breast epithelial cell line MCF10A reverse the tumorigenic properties of osteosarcoma metastatic cells (e.g. cell proliferation and viability under hypoxic conditions, anchorage-independent cell growth, resistance to anticancer drugs) by suppressing several oncogenic pathways involving HER2, SRC, RAS and TP53; on the other hand, some of the tumor-suppressor genes including VHL, PTEN and RB1 were overexpressed in cytoplasmic hybrids (cybrids) with non-cancerous mitochondria.

Other studies suggested that mitochondrial dysfunction may induce epigenetic modifications within the nuclear genome, such as aberrant methylation patterns in CpG-rich regions [84, 85]. These epigenetic alterations, including DNA and chromatin modifications and signaling through small RNAs, may contribute to the maintenance of mitochondria-mediated oncogenic transformation. However, the mitochondrial signals that potentially might trigger these epigenetic changes in the nucleus remain still largely unknown [30].

ROS-induced mitochondrial deregulation has been reported to trigger a survival response by inducing the nuclear factor NF-κB pathway and stimulating the synthesis of anti-apoptotic molecules (such as Bcl-xL/Bcl-2), which in turn promote cell survival and proliferation [86]. Moreover, oxidative stress may also affect the expression of nuclear genes involved in tumorigenic and invasive phenotypes [87]. Altogether these findings suggest that targeting the retrograde signaling could be a successful therapeutic strategy for cancer.

case of diphenyleneiodonium chloride (DPI), a strong inhibitor of mitochondrial complex I and II flavin-containing enzymes, which effectively depletes cancer stem-like cells (CSCs), one of the main drivers of poor clinical outcome in a wide variety of tumor types and especially in advanced disease states [103]. Interestingly, mitochondrial inhibition with VLX600 has also been proposed in combination with imatinib in the treatment of drug-resistant gas-

Mitochondrial DNA Variations in Tumors: Drivers or Passengers?

http://dx.doi.org/10.5772/intechopen.75188

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Recently, morphological and ultrastructural changes in the mitochondrial cristae structure (cristae remodeling), for example, through the optic atrophy 1 (OPA1) pathway, represent an important step in apoptosis and autophagy, and a potential target for future pharmacological

Chromosomal translocations generating in-frame oncogenic gene fusions also represent successful examples of targeted cancer therapies, and recently it has been shown that the FGFR3- TACC3 (F3–T3) gene fusion—initially discovered in human glioblastoma and then reported in many other cancers—promotes oxidative phosphorylation, mitochondrial biogenesis and tumor

Whole mitochondrial genome analysis by high-throughput next-generation sequencing (NGS) techniques enables the detection of low-level heteroplasmic mtDNA variants and completely revolutionized mitogenomics in the last few years [109]. This approach has been extensively applied to different mitochondrial disorders to carefully investigate the transmission dynamics of low-level maternal germline mtDNA variants across generations [110–112]. In a comparative analysis, it has been demonstrated that Sanger sequencing is valid for quantification of heteroplasmies with more than 10% of cells/mitochondria carrying the mutation, whereas NGS is capable of reliably detecting and quantifying heteroplasmic variants down to the 1% level [113]. Recently, a massive parallel sequencing (MPS) protocol reliably quantified low frequency, large mtDNA deletions in single cells with a lower detection limit of 0.5% [114]. mtDNA NGS has been also suggested as a useful quality check of pluripotent stem cells

Conventionally, DNA variants detected in a tumor sample but not in the germline counterpart (such as peripheral blood, buccal swab or saliva) are scored as somatic (likely pathogenic) mtDNA variants, otherwise they are considered as germinal variants (likely polymorphic/ benign). High-throughput NGS approaches may unveil low-level germinal heteroplasmies having a tumoral tissue counterpart with higher heteroplasmy simply because of increased cell replication rate or random genetic drift phenomena and, therefore, without any deleterious oncogenic effect. The ultra-sensitive detection rate of NGS methods may be used to monitor even subtle shifts in the heteroplasmy levels of the tumor during time and potentially correlate them with tumor evolution [116]. Moreover, the possibility to easily analyze the circulating cell-free mtDNA isolated from plasma/serum ("liquid biopsy") or urine [117–119],

**5. Ultra-sensitive next-generation sequencing techniques and** 

for drug discovery and regenerative medicine purposes [115].

may allow non-invasive serial sampling from the same patient.

trointestinal stromal tumors (GISTs) [104].

modulation in cancer [105].

growth [106–108].

**mitogenomics**
