*Article* **Clinical Spectrum and Functional Consequences Associated with Bi-Allelic Pathogenic** *PNPT1* **Variants**

**Rocio Rius 1,2, Nicole J. Van Bergen 1,2, Alison G. Compton 1,2, Lisa G. Riley 3,4, Maina P. Kava 5,6, Shanti Balasubramaniam 6,7,8, David J. Amor 1,2,9, Miriam Fanjul-Fernandez 2,9, Mark J. Cowley 10,11,12, Michael C. Fahey 13, Mary K. Koenig 14, Gregory M. Enns 15, Simon Sadedin 1,9, Meredith J. Wilson 16,17, Tiong Y. Tan 1,2,9, David R. Thorburn 1,2,9 and John Christodoulou 1,2,4,9,\***


Received: 9 October 2019; Accepted: 14 November 2019; Published: 19 November 2019

**Abstract:** *PNPT1* (PNPase—polynucleotide phosphorylase) is involved in multiple RNA processing functions in the mitochondria. Bi-allelic pathogenic *PNPT1* variants cause heterogeneous clinical phenotypes affecting multiple organs without any established genotype–phenotype correlations. Defects in PNPase can cause variable combined respiratory chain complex defects. Recently, it has been suggested that PNPase can lead to activation of an innate immune response. To better understand the clinical and molecular spectrum of patients with bi-allelic *PNPT1* variants, we captured detailed clinical and molecular phenotypes of all 17 patients reported in the literature, plus seven new patients, including a 78-year-old male with the longest reported survival. A functional follow-up of genomic

sequencing by cDNA studies confirmed a splicing defect in a novel, apparently synonymous, variant. Patient fibroblasts showed an accumulation of mitochondrial unprocessed *PNPT1* transcripts, while blood showed an increased interferon response. Our findings suggest that functional analyses of the RNA processing function of PNPase are more sensitive than testing downstream defects in oxidative phosphorylation (OXPHPOS) enzyme activities. This research extends our knowledge of the clinical and functional consequences of bi-allelic pathogenic *PNPT1* variants that may guide management and further efforts into understanding the pathophysiological mechanisms for therapeutic development.

**Keywords:** mitochondrial; *PNPT1*; PNPase; interferon; OXPHOS; respiratory chain; mutation; splice defect

#### **1. Introduction**

*PNPT1* encodes for polynucleotide phosphorylase (PNPase), a conserved homotrimeric 3- -to-5- exoribonuclease predominantly localized in the mitochondrial matrix and intermembrane space [1]. It is primarily involved in mitochondrial RNA (mtRNA) processing and degradation [2].

PNPase has been suggested to play a role in RNA import into mitochondria [3,4]; however, experimental data have been contradictory and, to date, there is no general agreement about an RNA import mechanism [5]. Recent reports suggest that disrupted PNPase RNA processing could lead to the accumulation of double-stranded mtRNAs, with the possibility of triggering an altered immune response [6,7].

Patients with bi-allelic *PNPT1* pathogenic variants have shown wide clinical heterogeneity ranging from non-syndromic hearing loss to multisystemic Leigh syndrome [8,9]. To date, no clear phenotype–genotype correlations have been drawn.

In a bid to better understand the clinical phenotype and functional consequences of patients with *PNPT1*-related diseases, we reported seven new patients with bi-allelic *PNPT1* variants and expanded upon the mutational spectrum. We also conducted functional studies and performed a thorough clinical review of previously published patients with *PNPT1* variants [6,8–13].

#### **2. Experimental Section**

#### *2.1. Patients*

We report seven new patients (P1, 2, 3, 3.2, 4, 7, 8) with bi-allelic variants in *PNPT1*, and provided updates for three previously published cases (P5, 6, 9). We also conducted a PubMed search using the terms PNPase (All Fields) OR (PNPT1 (All Fields) AND ("persons" (MeSH Terms) OR "persons" (All Fields) OR "individual" (All Fields))) up to September 2019 for publications limited to human subjects to describe the clinical features of all the patients with bi-allelic *PNPT1* pathogenic variants reported in the literature to date [6,8–13].

Functional studies were conducted in samples collected from four patients (P1, 2, 3, 4) in which the *PNPT1* variants were identified by whole genome sequencing (WGS; Garvan Institute, Sydney) or whole exome sequencing (WES; Victorian Clinical Genetics Services (VCGS), Melbourne; Broad Institute, Cambridge, MA, USA; and Baylor College of Medicine, Houston, TX, USA).

This study was performed in accordance with the Helsinki Declaration and ethical standards of the responsible ethics committees. The project was approved by the Human Research Ethics Committees of the Sydney Children's Hospitals Network (ID number HREC/10/CHW/114), Melbourne Health (ID number HREC/16/MH/251), and the Royal Children's Hospital (ID number HREC/16/RCHM/150).

#### *2.2. Next Generation Sequencing (NGS) and in Silico Tools*

The variants in P1 were identified through whole exome sequencing (WES) performed by the Genomics Platform at the Broad Institute of Harvard and MIT (Broad Institute, Cambridge, MA, USA). The variants in P2 were identified through trio whole genome sequencing (WGS) performed at the Kinghorn Centre for Clinical Genomics (Garvan Institute, Sydney) as previously described [14]. The variants in P3 and P4 were identified through WES performed at Victorian Clinical Genetics Services (VCGS), Melbourne. P7 and P8 variants were identified through WES performed at Baylor College of Medicine, Medical Genetics Laboratories, Whole Genome Laboratory (Houston, TX, USA).

In silico prediction analyses were performed using PolyPhen-2 [15], SIFT [16], Combined Annotation Dependent Depletion CADD [17], MutationTaster [18], and Human Splicing Finder v3.1 [19]. Visualization of variants in the Pfam [20] protein domains was conducted with MutationMapper [21]. Allele frequencies were determined using the Genome Aggregation Database [22].

#### *2.3. Western Blotting*

Fibroblast protein extraction and Western blotting were performed using total Abcam OXPHOS human WB antibody cocktail (ab11041) and PNPase (ab96176) as previously published [10].

#### *2.4. Mitochondrial Oxidative Phosphorylation (OXPHOS) Enzyme Activities*

Spectrophotometric analysis of OXPHOS enzyme activities in muscle and fibroblasts was performed as previously described [23]. The mitochondrial respiratory chain complex I (CI) and complex IV (CIV) dipstick activity assays (Abcam, Melbourne, VIC, Australia) were performed using 15 μg of whole-cell lysates fibroblasts as previously published [10].

#### *2.5. Fibroblast Culture, RNA Extraction, and Complementary DNA (cDNA) Studies*

Cycloheximide treatment of cultured fibroblasts from P2 and controls was performed as published [24]. Cultured fibroblasts from patients P1, P2, P3, and P4, and three control lines were incubated in the presence or absence of 100 U/mL interferon α-2a Roferon-A (Roche, Sydney, Australia) in HyClone Dulbecco's Modified Eagle Medium (GE Healthcare, Rydalmere, NSW, Australia) at 37 ◦C and 5% CO2 for 24 h.

RNA was isolated from fibroblasts using the RNeasy Plus kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Reverse transcription was performed using SuperScript III First-Strand synthesis kit (Thermo Fisher Scientific, Carlsbad, CA, USA) following the manufacturer's instructions.

#### *2.6. RNA Extraction from Blood*

PAXgene blood RNA tubes (PreAnalytix by Qiagen, Hombrechtikon, Switzerland) were used to collect peripheral blood samples. After collection, the tubes were left at room temperature between 2 h and 72 h before extracting RNA using the PAXgene blood RNA kit (PreAnalytix by Qiagen) following the manufacturer's instructions.

#### *2.7. PCR Quantification of Unprocessed Mitochondrial Transcripts*

qPCR for the quantification of unprocessed transcripts was performed with AccuPower 2X Greenstar qPCRMasterMix (Bioneer, Daejeon, Korea) using primers previously published byMatilainen and collaborators [9]. The relative accumulation of unprocessed mitochondrial transcripts was calculated using the 2(-ΔΔCt) method [25].

#### *2.8. Interferon Signature Analysis*

The relative expression of six interferon-stimulated genes was analyzed by qPCR using cDNA from 40 ng RNA, TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific), and Taq Man probes for *IFI27* (Hs01086370\_m1), *IFI44L* (Hs00199115\_m1), *IFIT1* (Hs00356631\_g1), *ISG15* (Hs00192713\_m1), *RSAD2* (Hs01057264\_m1), and *SIGLEC1* (Hs00988063\_m1). The relative abundance was normalized to *HPRT1* (Hs03929096\_g1) and *18S* (Hs999999001\_s1). Median expression was used to calculate an interferon score as described by Dhir, Rice, and collaborators [6,26].

#### **3. Results**

A total of 24 patients with bi-allelic variants in *PNPT1* were identified in 15 families, including seven new patients (P1, 2, 3, 3.1, 4, 7, 8), three previously published individuals with additional clinical information since the previous publication (P5, 6, 9), and fourteen other patients reported in the literature (Tables S1 and S2). A total of 22 different pathogenic variants were found, of which only three were reported in more than one family. The p.(Ala507Ser) variant was reported in 27% of the families (4/15), followed by the p.(Thr531Arg) variant at 13% (2/15), and p.(Arg136His) at 13% (2/15) (Figure 1).

**Figure 1.** Lollipop plot depicting the polynucleotide phosphorylase (PNPase) protein and location of pathogenic variants reported in 30 alleles (15 families). Missense variants are shown above the protein. Nonsense and splicing variants are shown below. Novel variants reported in this article are marked with #.

Most of the patients (75% *n* = 18) presented within the first year of life. The most common initial manifestations were tone abnormalities (42% *n* = 10), feeding difficulties (17% *n* = 4), and mild to severe sensorineural hearing loss (38% *n* = 9). Other reported initial features were regression, choreoathetosis, visual loss, and cataracts (Table S1).

The median age at the last follow up was 8.9 years (range 1–78). Three of the patients died due to acute encephalopathy (P15 at 4 years), infection (P16 at 2.4 years), and unstated reasons (P21 at 2 years) (Table S1).

The clinical symptoms of all the reported patients to date are summarized in Figure 2 and Table S1.

**Figure 2.** Clinical symptoms using Human Phenotype Ontology (HPO) terminology of patients with bi-allelic *PNPT1* variants (*n* = 24). Green indicates a present phenotype, red indicates an absent phenotype, and gray is unknown or not applicable. \* Respiratory chain deficiencies refer to reported reductions in oxidative phosphorylation (OXPHOS) enzyme activity measured by spectrophotometric analysis in any tissue. Neonatal (0–28 days), infant (28 days–1 year), and childhood (>1 year).

#### *3.1. cDNA Studies Identify a Splicing Defect in P2*

The NM\_033109.4:c.1818T>G heterozygous variant identified in P2 was originally believed to be synonymous p.(Val606=), however it was located 5 bp from the donor splice site and in silico studies suggested the possibility of a splicing defect (Table S2). cDNA studies performed from cultured fibroblasts grown with and without cycloheximide treatment to inhibit nonsense-mediated decay (NMD) confirmed a splicing defect resulting in a frameshift deletion and premature termination codon p.(Val607Lysfs\*21), leading the transcript to be subject to degradation by NMD (Figure 3).

**Figure 3.** cDNA studies from P2 fibroblast cells. (**a**) Schematic representation and partial electropherogram showing the creation of a new splice site generated by the NM\_033109.4:c.1818T>G variant in cells treated with cycloheximide. (**b**) Without cycloheximide treatment, the heterozygous NM\_033109.4:c.1818T>G variant was not detectable, suggesting that the mutant RNA is subject to nonsense-mediated decay.

#### *3.2. Mitochondrial OXPHOS Protein Expression and Activity*

In the fibroblasts, there was a reduction of the mitochondrial complex IV COX II subunit and the complex I NDUFB8 subunit in patients P1–4, although the levels of other OXPHOS subunits were normal (Figure 4a). The activities of complex I and complex IV were determined using a dipstick assay. Compared to the control means, there was a relatively mild reduction in complex I by 20% (P1), 45% (P2), 50% (P4), and a modest reduction in complex IV by 40% (P1), 57% (P2), 20% (P3), and 80% (P4) (Figure 4b). P5 was a previously reported patient with *PNPT1* variants [10] and was used as a positive control.

**Figure 4.** Protein expression and OXPHOS activity in fibroblasts. (**a**) Representative images of Western blot analysis performed in fibroblast proteins from controls (C) and patients (P). In the patients, there was a reduction in protein expression of PNPase (lower panel) and complex I and complex IV OXPHOS mitochondrial subunits (upper panel). (**b**) Complex I (upper panel) and complex IV (lower panel) enzyme activity was analyzed using dipstick activity assays. In the patients, there was a mild/moderate reduction in complex I and complex IV enzyme activities. The mean and variation (SEM) between three independent experiments are shown. (\*\*\* *p* < 0.001, \* *p* < 0.033).

However, spectrophotometric analysis of OXPHOS enzymes was normal in fibroblasts from P1, and was only borderline low for complex IV activity relative to protein and citrate synthase in muscle from P2 (Table 1).


**Table 1.** Spectrophotometric analysis of oxidative phosphorylation (OXPHOS) enzyme activities in fibroblasts from P1 and muscle from P2.

Activities of OXPHOS enzyme complexes I, II, III, IV, and citrate synthase (CS) are expressed as % relative to protein (residual activity), citrate synthase (CS ratio), and CII (CII ratio) of control samples.

#### *3.3. Accumulation of Mitochondrial Unprocessed Transcripts*

To assess the PNPase mitochondrial RNA processing activity, we measured the accumulation of unprocessed polycistronic mitochondrial transcripts in fibroblasts.

Compared to controls, fibroblasts from patients P1–4 showed an accumulation of aberrantly processed mitochondrial RNA transcripts (Figure 5) in line with the PNPase function in mtRNA processing. The accumulation of unprocessed transcripts in myoblasts from a patient with bi-allelic pathogenic variants in *PNPT1* was previously described [9]. Notably, our patients presented different patterns of accumulation, which were not restricted to transcripts around *MT-ND6*. Further studies may help us to understand if the differences in the accumulation of unprocessed mitochondrial transcripts are related to specific *PNPT1* variants.

#### *3.4. Interferon Signature in Patients with PNPT1 Variants*

Increased expression of interferon-stimulated genes has been previously described in blood from P14, P22, and P23 with bi-allelic *PNPT1* variants [6]. To assess if the PNPase defects could elicit an increased interferon response in different tissues, we calculated the interferon score in fibroblasts (P1–4) and in blood (only available from P2 and P4). In fibroblasts from P1–4 stimulated with IFN-α2a (100 U/mL), the interferon response was not different to controls (interferon score below the 1.3 threshold in fibroblasts). On the other hand, in blood from P1 and P4, a positive interferon score was identified with a median of 2.53 and 4.65, respectively (above the 1.8 threshold in blood) (Figure 6).

**Figure 6.** Interferon (IFN) score in fibroblasts and in blood. (**a**) Median and individual values of the IFN score in fibroblasts of three controls and patients with *PNPT1* variants (P1–4) from two independent experiments. The IFN score threshold of 1.3 corresponds to the mean IFN score of controls + 2SD. Higher values are considered positive. (**b**) Median and individual values of the IFN score in blood of three controls and patients with *PNPT1* variants (P2 and P4) from two independent experiments. The IFN score threshold of 1.8 corresponds to the mean IFN score of controls + 2SD. Higher values are considered positive.

#### **4. Discussion**

This cohort illustrated the marked phenotypic heterogeneity associated with bi-allelic *PNPT1* variants. Severe developmental delay and regression were the most common neurodevelopmental abnormalities. Sensorineural hearing loss, optic atrophy, tone, and movement abnormalities—including choreoathetosis, dystonia, myoclonus, and ataxia—were also amongst the most prevalent clinical features. Most patients had a history of hypotonia (*n* = 19) and four patients presented a pattern of central hypotonia with peripheral hypertonia. Brain imaging was available in 17 patients, of which 16 patients had MRI abnormalities. The most common were white matter abnormalities (*n* = 8), followed by thin corpus callosum (*n* = 5), and basal ganglia abnormalities (*n* = 4) (Figure S1).

Interestingly, most of the patients in the cohort had normal to mildly deficient OXPHOS when measured by spectrophotometric enzyme assays. This finding has several implications. First, a normal OXPHOS enzyme analysis does not rule out the diagnosis of *PNPT1*-related disease. Second, reduced OXPHOS protein expression or activity measured by dipstick assays does not always correlate with spectrophotometric assays, as shown in P1 and P2. This discrepancy could be attributed to sample preparation and differences in the methodology, which has been discussed previously [10]. Finally, in order to confirm pathogenicity, functional analyses focusing on the primary role of *PNPT1* (mtRNA processing) and interferon signaling in blood rather than fibroblasts appear to be more sensitive, and potentially more specific than evaluating the downstream effects on the OXPHOS enzyme activities.

Most of the patients with *PNPT1*-related disease have been identified through NGS approaches. The identification of an apparently synonymous variant in P2 that was later demonstrated to result in abnormal mRNA splicing highlights the importance of careful interpretation of apparently "silent" variants in NGS data, the utility of in silico tools, and the need for cDNA studies when following up potential splicing variants.

The identification of an interferon signature in blood from patients with *PNPT1* variants raises the possibility that an immune response could contribute to disease pathogenesis [6]. Further longitudinal studies could elucidate if there is a correlation between the interferon signature and disease severity, or the possibility of being a useful clinical biomarker and/or therapeutic target.

So far, little is known about possible genotype–phenotype correlations, and it is not clear what factors could be contributing to the clinical spectrum of *PNPT1*-related diseases. Studies suggest that the rate of disease progression could be, in part, related to having two deleterious variants affecting the first core domain [9]. In line with this, three patients with rapidly progressive disease and early death (median age 2.4 years) were described (Table S1), in which the variants were located in the first core domain. P21 had the p.(Arg136His) variant in a homozygous state. P15 also had the p.(Arg136His) variant but in compound heterozygosity with the p.(Pro140Leu) variant, which was also located in the first core domain. P16, who had a p.(Gly76Asp) located in the first core domain and a p.(Arg192\*), was predicted to result in nonsense-mediated decay [6,9,12]. The novel variant p.(Arg136Cys) identified in P3 was an alternate amino acid change located in the same active site as described in P21 and P15, but her second pathogenic variant p.(Pro467His) was located in the second core domain. Interestingly, she is currently three years old and has a relatively slower progression. Her brother (P3.2) also seemed to be more mildly affected. Intrafamilial variability was also described in other families with *PNPT1* variants (P5, 6, 7, 8; Table S1), but bona fide genetic modifiers of disease severity have yet to be identified.

P4 had the longest survival reported to date (78 years), with a slowly progressive clinical course. He presented at 11 years old with hearing and visual loss. Subsequently, he developed blepharospasm and ptosis. Over the decades, he developed progressive ataxia, poor night vision, and external ophthalmoplegia. His hearing worsened during his 60s and, therefore, he received a cochlear implant. Eaton and collaborators [13] also described one family that presented with a slowly progressive pattern. Both affected individuals had congenital sensorineural hearing loss and developed neurological symptoms in adulthood. It is interesting to note that the pathogenic *PNPT1* variants in both these patients were distal variants located within the S1 domain. Further work and more patients are required to formally establish genotype–phenotype correlations.

Careful phenotypic analysis in rare monogenic diseases is valuable for identifying common clinical features [27,28]. In our cohort, this approach led us to describe the most common phenotypic findings associated with *PNPT1*-related disease that should increase the diagnostic suspicion of this genetic etiology, and could guide screening and management in patients with a molecular diagnosis. On the other hand, no single finding was present in 100% of the patients and, thus, the lack of any of these features does not rule out the diagnosis. Some of the clinical features have only been described in adult patients, such as obsessive–compulsive disorder (P19), depression (P4, P19), and paranoia (P20), or in single cases, such as renal artery stenosis in P9. The causal relationship of some of these findings is unknown and it remains to be seen whether some of these features are coincidental rather than linked to the pathophysiology of *PNPT1* dysfunction.

The diverse clinical heterogeneity of patients with bi-allelic *PNPT1* variants presents a diagnostic and management challenge to the clinician. As more patients are diagnosed, it is important to share detailed clinical information to understand the natural history, elucidate new insights into the mechanisms underlying the pathology, and aid with the identification of potential treatment strategies.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-0383/8/11/2020/s1, Table S1: Clinical phenotype of patients with bi-allelic PNPT1 variants; Table S2: In silico studies and population frequency of PNPT1 variants identified in new families; Figure S1: Brain MRI findings in patients with bi-allelic *PNPT1* variants.

**Author Contributions:** Conceptualization, J.C.; data curation, R.R., M.F.-F., M.K.K., and S.S.; formal analysis, R.R. and L.G.R.; funding acquisition, D.R.T., and J.C.; clinical evaluation and investigation, M.P.K., S.B., D.J.A., G.M.E., M.C.F., M.K.K., M.J.W., and T.Y.T.; methodology, R.R. and N.J.V.B.; software, M.J.C.; supervision, N.J.V.B., A.G.C., D.R.T., and J.C.; writing—Original draft, R.R.; writing—Review and editing, N.J.V.B., A.G.C., L.G.R., M.P.K., S.B., D.J.A., M.F.-F., M.J.C., M.C.F., M.K.K., G.M.E., S.S., M.J.W., T.Y.T., D.R.T., and J.C.

*J. Clin. Med.* **2019**, *8*, 2020

**Funding:** This research was supported by: A New South Wales Office of Health and Medical Research (OHMR) Council Sydney Genomics Collaborative grant (JC); the Australian Genomics Health Alliance (Australian Genomics) project, funded by an NHMRC Targeted Call for Research grant (GNT1113531; JC, DRT); NHMRC research fellowship 1155244 (DRT); OHMR Fellowship (MJC); and a CONACYT Postgraduate Research Scholarship (RR). Sequencing and analysis were provided by the Broad Institute of MIT and Harvard Center for Mendelian Genomics (Broad CMG) and were funded by the National Human Genome Research Institute, the National Eye Institute, the National Heart, Lung and Blood Institute grant UM1 HG008900, and, in part, by a National Human Genome Research Institute grant R01 HG009141.

**Acknowledgments:** We are grateful to the Crane and Perkins families for their generous financial support. The research conducted at the Murdoch Children's Research Institute was supported by the Victorian Government's Operational Infrastructure Support Program.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Mitochondrial Dysfunction in Aging and Cancer**

#### **Loredana Moro**

Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies, National Research Council, 70126 Bari, Italy; l.moro@ibiom.cnr.it; Tel.: +39-080-544-9807

Received: 10 October 2019; Accepted: 13 November 2019; Published: 15 November 2019

**Abstract:** Aging is a major risk factor for developing cancer, suggesting that these two events may represent two sides of the same coin. It is becoming clear that some mechanisms involved in the aging process are shared with tumorigenesis, through convergent or divergent pathways. Increasing evidence supports a role for mitochondrial dysfunction in promoting aging and in supporting tumorigenesis and cancer progression to a metastatic phenotype. Here, a summary of the current knowledge of three aspects of mitochondrial biology that link mitochondria to aging and cancer is presented. In particular, the focus is on mutations and changes in content of the mitochondrial genome, activation of mitochondria-to-nucleus signaling and the newly discovered mitochondria-telomere communication.

**Keywords:** mitochondrial DNA; mitochondria-to-nucleus signaling; aging; cancer

#### **1. Introduction**

Mitochondria are cellular organelles that play a pivotal role in maintaining cellular homeostasis by regulating energy metabolism, cell survival and proliferation. Production of adenosine triphosphate (ATP) and the generation of intermediate metabolites are the traditional functions ascribed to mitochondria. Within the mitochondria, the tricarboxylic acid cycle (TCA cycle) generates mitochondrial metabolites and reducing equivalents, in the form of reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2), which enter into the mitochondrial electron respiratory chain (ETC). ETC includes four membrane-bound protein complexes and catalyzes the oxidation of reducing equivalents using oxygen as the terminal electron acceptor. The electron transfer is coupled to the transfer of protons across the inner mitochondrial membrane to generate an electrochemical gradient (mitochondrial membrane potential) required both for ATP synthesis through the ATP synthase complex, and for efficient shuttling of proteins across the inner mitochondrial membrane. Electron leakage can arise along the ETC, leading to the formation of reactive oxygen species (ROS), important second messengers in aging, cancer and other physiopathological conditions. The intermediate metabolites generated in the TCA cycle are the precursors of lipids, carbohydrates, proteins and other macromolecules [1]. Mitochondria are able to migrate within the cell, fuse and divide through rapid fusion and fission processes and undergo turnover through mitophagy, a specialized form of autophagy. Mitochondrial dynamics is regulated by the large dynamin family of GTPases, enables mitochondrial recruitment to subcellular compartments that require more energy and is important for mitochondrial quality control and for communication with the cytosol and the nucleus (reviewed in [2]). Mitochondrial fusion begins with the joining of the mitochondrial outer membrane mediated by the mitofusin proteins Mfn1 and Mfn2, followed by fusion of the mitochondrial inner membrane catalyzed by OPA1. Fusion occurs similarly for the inner and outer membrane and involves a formation of interlocking coiled coils mediated by fusion proteins on the two membranes, followed by their fusion catalyzed by GTP hydrolysis [3].

The inner membrane fusion is dependent upon the mitochondrial membrane potential [4]. The mitochondrial fission is instead mediated by the GTPase DRP1, a protein that is recruited to the

outer membrane where it oligomerizes and forms a spiral around the outer and inner membrane that causes fragmentation of the mitochondrion [5,6]. Disruption of the normal fusion/fission balance results in mitochondrial dysfunction [6].

Recent evidences point to the mitochondrial content and functionality in the regulation of nuclear gene expression by affecting transcription and translation, as well as alternative splicing mechanisms [7]. Hence, mitochondrial dysfunction leading to changes in nuclear gene expression may affect the risk of degenerative diseases, cancer and aging [7–10].

Aging is the most important risk factor for cancer. According to the National Cancer Institute (NCI), the median age of a cancer diagnosis is 66 years (https://www.cancer.gov/about-cancer/causesprevention/risk/age). At first glance, aging and cancer may seem to be two opposite processes, the first implying a slow decline of cellular and organismal functions, the second providing fitness to the cells. In reality, these two processes are strictly interconnected and share common origins. Aging is associated with the progressive accumulation of genetic and cellular damage [11,12]. However, DNA damage may confer survival and growth advantages to certain cells, which can eventually thrive and evolve into a tumorigenic phenotype. Genetic damage affecting the functionality of mitochondria has been associated with both aging and cancer.

Mitochondria have been the focus of the aging research for decades, when Harman proposed the free radical theory of aging [13,14], then revisited by Alexeyev as the mitochondrial theory of aging [15]. Both theories hypothesize that ROS are the main determinants of the loss of cellular performance observed with increasing age, with the mitochondrial theory implying mitochondria as the main producers of ROS, and consequently, mitochondria as key drivers of aging. In the last decade, knowledge of these organelles has expanded, providing more connections between mitochondrial biology and the aging process [16]. Similarly to aging, changes in mitochondrial functionality have been strictly linked to cancer [17]. Indeed, several studies have shown that mutations in mitochondrial proteins encoded by either the mitochondrial or the nuclear genome may favor cancer development and progression [8,9,18,19]. This review provides an overview of the convergent and divergent roles that mitochondrial dysfunction may play in cellular processes linked to aging and cancer, with a specific focus on mitochondrial DNA, mitochondria-to-nucleus signaling and telomere shortening.

#### **2. The Mitochondrial Genome**

Mitochondria are equipped with their own genome, the mitochondrial genome (mtDNA), a circular, double-stranded DNA molecule of ~16.5 kb in humans, present in one to several thousand copies per cell, depending on the tissue and cell type. The two mtDNA strands have different resolution on a cesium chloride gradient and can be separated in a heavy strand (H-strand) rich in G and a light strand (L-strand) rich in C [20]. In addition, the mtDNA has a unique regulatory region, the displacement loop (D-loop), a triple-stranded region that includes a short single-strand DNA molecule, the 7S DNA [21]. The mitochondrial genome encodes for 13 proteins belonging to the respiratory complexes I, III, IV and V, and for two rRNAs and 22 tRNAs required for mitochondrial protein synthesis. Most of the mtDNA information is encoded by the H-strand (12 proteins, 2 rRNAs, 14 tRNAs). The mtDNA is maternally inherited and depends upon nuclear-encoded proteins for its replication and transcription. Replication of mtDNA is performed by DNA polymerase γ (POLγ), twinkle helicase and mitochondrial single-stranded binding protein (mtSSB), and proceeds throughout the entire life of an organism in both dividing and terminally differentiating cells. The exact molecular mechanism involved in the regulation of mtDNA replication in response to extracellular stimuli/stress remains a poorly investigated area [22]. The mitochondrial proteome is dynamic; i.e., the protein composition and the levels of a given protein may vary depending on the tissue and cell type. Even within the same cell type the mitochondrial proteome may vary over time depending on specific external stimuli or stress conditions [23].

Though the majority of the mammalian mitochondrial proteome (about 1200 proteins) is encoded by the nuclear genome and then imported into mitochondria, the 13 proteins encoded by the mtDNA (1% of the mitochondrial proteome) play an essential role for the mitochondrial function. Mutations or

depletion of the mtDNA can indeed impair the energy metabolism and alter the intracellular signaling, resulting in mitochondrial dysfunction that may affect the cell performance at various degrees, leading to "mitochondrial diseases" in extreme cases [8,9,24,25]. MtDNA accumulates mutations at a considerably higher rate than nuclear DNA [26]. Traditionally, the increased mutational load was ascribed to a lack of efficient repair systems, lack of protective histones and to the close proximity of mtDNA to the respiratory chain complexes, the main site of production of ROS. Though mtDNA lacks traditional histones, the mitochondrial transcriptional factor A (TFAM) is tightly associated to the mtDNA molecules and plays an histone-like protective role [27]. However, mitochondria lack nucleotide excision repair (NER), efficient homologous recombination, microhomology-mediated end joining (MMEJ) and non-homologous end joining (NHEJ). The short and long patch base excision repair (BER) and single strand break repair pathways are instead preserved [28]. Hundreds of mtDNA mutations have been reported leading to a variety of human disorders (www.mitomap.org). In most cases, wild-type and mutated mtDNA coexist within the same cell, a condition known as heteroplasmy, as opposed to the term homoplasmy, when all the copies of the mtDNA are identical. The severity of the mtDNA-associated disorders is dependent on the level of heteroplasmy. For example, the mitochondrial dysfunction observed in the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) syndrome, usually caused by a mutation within the mtDNA-encoded tRNA Leu (UUR), can be rescued by levels of wild-type mtDNA above 6% [29]. In general, a pathogenic mtDNA mutation would need to reach a threshold level of more than 60% in a given tissue or cell type to produce a measurable bioenergetic defect [30].

#### *2.1. MtDNA and Aging*

MtDNA point mutations and deletions are known to accumulate with age in human tissues, including brain, heart and skeletal muscles [31–34]. A debate is still ongoing to define whether accumulation of mtDNA mutations is causal or just a correlation with aging. So far, the strongest evidence favoring the hypothesis of a causal role of mtDNA changes in aging comes from the generation of homozygous knock-in mice for the catalytic subunit of POLγ that lacks proofreading activity. These mice exhibit a mtDNA mutator phenotype, with accumulation of extremely high levels of mtDNA mutations, a shortened lifespan and early onset of age-associated phenotypes, including hair graying, weight loss, reduced subcutaneous fat, osteoporosis, alopecia and kyphosis [35,36]. Of note, while an increased load of mtDNA point mutations has not been linked to the aging phenotype [37], the presence of large mtDNA deletions has been reported as a driver of premature aging in mitochondrial mutator mice [38]. These mice apparently did not exhibit a net increase in ROS levels [35], and in general, in oxidative stress markers [36]. However, Kolesar et al. [39] have recently shown that the mitochondrial protein content in the muscle tissues of Polγ-knock-out mice is significantly reduced compared with wild-type mice, which results in a net increase in ROS levels per mitochondrial proteins, and a burst in mtDNA and mitochondrial protein oxidative stress. Increased oxidative stress in the muscle of Polγ-knock-out mice has been confirmed by a subsequent study [40]. These findings support previous experimental evidences showing an anti-aging role of mitochondria-targeted catalase, an anti-oxidant enzyme [41–43]. Further experiments in mice have shown that maternally inherited mtDNA mutations may cause premature aging signs, such as alopecia, kyphosis and premature death [44,45]. In humans, HIV patients treated with nucleoside analog reverse transcriptase inhibitor antiretroviral drugs have shown signs of premature aging, including increased cardiovascular disease and bone fractures [46]. Subsequent studies have shown that these antiretroviral drugs have mitochondria as off-target sites, and in particular, they inhibit POLγ, causing mtDNA mutations and depletion that result in mitochondrial dysfunction [47]. Another example linking mtDNA to the aging phenotype comes from human subjects carrying mutations in POLγ. More than 300 pathogenic mutations in human POLγ have been identified so far (https://tools.niehs.nih.gov/polg/), and they have been linked to several disorders, including age-related pathologies such as Parkinson's disease [10,48,49].

Loss of mitochondrial fusion in the skeletal muscle of *Mfn1*- and *Mfn2*-knockout mice has been linked to increased mtDNA mutations and deletions, as well as mtDNA depletion. These molecular events precede the phenotypic changes observed in these mice, such as impaired mitochondrial functionality, abnormal mitochondria proliferation and muscle loss [50]. Overall, increased mitochondrial fusion may support longevity, as highlighted by a recent study in *C. elegans*[51]. In addition, a decline in mtDNA content has been observed in peripheral mononuclear blood cells (PMBC) during aging, and it has been linked to reduced immune response in the elderly [52,53]. Recently, a novel and very precise technique has allowed researchers to ascertain that healthy centenarians retain more mtDNA copies and less mtDNA deletions during immune cell stimulation than old people and frail centenarians [54], implying that the retention of a high amount of mtDNA is a hallmark of healthy aging.

#### *2.2. MtDNA and Cancer*

MtDNA mutations and changes in mtDNA content have been associated with cancer progression in a variety of cancer types (reviewed in [8,9]). A large-scale analysis performed on several cancer types of the TCGA dataset has recently confirmed that many, but not all cancers, display a significant depletion of the mtDNA content, which is directly associated with reduction in the expression of genes of the mitochondrial respiratory chain, and inversely correlated with the expression of genes of the immune response and cell cycle [55]. Some somatic mtDNA mutations were reported to favor tumorigenesis [56] and others to promote cancer metastasis [57] through an increase in ROS production [56,57]. However, a definitive link between mtDNA depletion/somatic mutations and tumorigenesis or cancer progression is still missing. Experiments in cell culture have shown that induced mtDNA depletion may restrain tumorigenesis [58,59] but support cancer cell invasion and metastasis [9,60–64]. Partial mtDNA depletion results in decreased mitochondrial membrane potential, increased DRP1 mitochondrial localization and decreased OPA1 levels, accompanied by increased mitochondrial fission, as demonstrated by enrichment in fragmented mitochondria [65]. In turn, increased mitochondrial fission alters the cytoskeleton and promotes the formation of pseudopodia-like structures, typical of invasive cells [65]. Consistently, increased mitochondrial fission has been frequently detected in carcinoma samples (reviewed in [66]). Analysis of the TCGA dataset reported a direct correlation between mtDNA depletion and reduced patients' survival [55]. An independent study performed on 8161 normal and cancer samples in the TCGA dataset showed that decreased oxidative phosphorylation is associated with poor survival across multiple cancer types and with an epithelia-to-mesenchymal (EMT) gene expression profile [67], typical of cells with acquired invasive abilities [9]. Furthermore, analysis of the Skin Cutaneous Melanoma dataset containing 367 metastatic lesions and 103 primary cancer samples demonstrated that: (i) Reduced oxidative phosphorylation was the most significant metabolic signature in the metastatic lesions compared with the primary cancers; and (ii) EMT was strongly upregulated in the metastatic cancer samples [67], supporting a role of mitochondrial dysfunction in the metastatic process. A previous study performed on 49 patients with advanced metastatic melanoma has shown that stage IV melanomas may be dependent either on glycolysis or a combination of glycolysis and oxidative phosphorylation, which may suggest a metabolic symbiosis within the same tumor [68]. In agreement with this study, Najjar et al. [69] reported that metabolic adaptation can vary in melanoma samples, with some cells exhibiting only deregulated glycolysis and others only deregulated oxidative phosphorylation. Patient-derived melanoma cell lines with a prevalent oxidative metabolism showed resistance to immunotherapy as well [69]. Recent evidences have pointed out to the horizontal transfer of whole mitochondria from host cells present in the tumor microenvironment to respiratory-deficient tumor cells, depleted of mtDNA [70]. This transfer would recover mitochondrial respiratory activity and promote tumor growth [70]. Based on these observations, it is tempting to speculate that mtDNA depletion/deletions and pathogenic mtDNA mutations may confer a selective advantage to certain cancer cells for invading and thriving in hostile microenvironments, such as the circulating system, while abating cell proliferation. Partial

reconstitution of the wild-type mtDNA pool by horizontal transfer from stromal cells would allow cancer cells to recover their proliferative capacity, and thus, their full tumorigenic potential.

#### **3. Retrograde Signaling in Aging and Cancer**

Besides being considered as the cell's "energy hubs", mitochondria have emerged as critical "signaling hubs" in regulating cell homeostasis [71]. Communication from mitochondria to the nucleus is known as "retrograde signaling", opposed to the traditional anterograde signaling, i.e., the communication pathways from the nucleus to the mitochondria. It has been first described by Butow in yeast. That is, in yeast cells depleted of mtDNA, Butow described changes in the transcription of nuclear-encoded genes, and an increase in the transcription of genes associated with a metabolic shift from aerobic to anaerobic respiration (reviewed in [72]). Screenings performed in *C. elegans* have revealed that impairing the mitochondrial respiratory chain unexpectedly extends the lifespan [73]. This observation seems a paradox given that aging tissues are characterized by loss of mitochondrial fitness. Similarly, in *C. elegans* a transient increase of mitochondrial ROS triggers changes in gene expression that promote longevity instead of aging, sharply in contrast with the mitochondrial theory of aging [74]. These findings have been corroborated in mammals using mouse models. For example, mice heterozygous for Mclk1, a mitochondrial protein necessary for the biosynthesis of ubiquinone, show increased lifespan correlated with early impairment of the mitochondrial functionality, significant reduction of mitochondrial electron transport, ATP and NAD+ levels [75]. The reason why mitochondrial stress may counteract the aging process is still not completely understood. Some experimental evidences suggest that the retrograde signaling activated by the mitochondrial stress may play a critical role. In the context of aging, activation of the mitochondrial unfolded protein response (UPRmt) seems to be important in signaling a mitochondrial stress to the nucleus. This retrograde response is elicited by mtDNA depletion or by protein misfolding occurring in these mitochondria [76]. It was described for the first time in mammalian cells depleted of mtDNA through ethidium bromide treatment, which exhibited induction of mRNAs for mitochondrial proteases and chaperons [77]. A similar transcriptional profile was observed upon overexpression in the mitochondrial matrix of a dominant negative ornithine transcarbamylase, an enzyme essential for protein processing and folding, suggesting a link between mitochondrial dysfunction, alteration of proteostasis mechanisms and the activation of UPRmt [78]. Work in *C. elegans* and other organisms has identified several components of this retrograde response (for a comprehensive review, see [79]). In *C. elegans*, mitochondrial dysfunction reduces the mitochondrial import efficiency of the transcription factor ATFS-1, allowing it to accumulate in the nucleus where it induces the transcription of hundreds of genes, including antioxidant proteins, proteases and enzymes involved in cellular metabolism that would promote survival and recovery of the mitochondrial functionality, thus supporting longevity and lifespan [78]. However, prolonged UPRmt activation occurring in the context of a heteroplasmic mtDNA pool seems to exacerbate mitochondrial dysfunction because it would result in an accumulation of damaged mtDNA (for a detailed review see [76]).

Evidence is accumulating on the important role that mitochondrial metabolites may play as second messengers eliciting epigenetic changes in the nucleus (for comprehensive reviews see [80,81]). In this context, acetyl-CoA is one of the most studied signaling molecules. This metabolite is present in mitochondria, cytosol and the nucleus. In mitochondria, it can be produced through different metabolic pathways, including conversion of pyruvate to acetyl-CoA by the pyruvate dehydrogenase complex (PDH), fatty acid β-oxidation, amino acid metabolism, direct synthesis by a reaction of ligation of acetate with CoA catalyzed by mitochondrial acyl-CoA synthetase short-chain family member 1 (ACSS1). Mitochondrial acetyl-CoA enters into the TCA cycle together with oxaloacetate to produce citrate, which is oxidized, allowing production of ATP through the oxidative phosphorylation.

Once inside mitochondria, acetyl-CoA is unable to cross the inner mitochondrial membrane. To replenish its cytosolic and nuclear pool, citrate exported from mitochondria via the tricarboxylate carrier is converted to acetyl-CoA and oxaloacetate by the ATP-citrate lyase (ACLY) [82]. Cytosolic acetyl-CoA can participate to biosynthetic pathways, such as to fatty acids synthesis, and provides acetyls for lysine acetylation of proteins, thereby regulating their activity and localization [83]. In the nucleus, protein lysine acetylation modulates histone acetylation. It has been recently reported that the PDH complex can translocate from mitochondria to the nucleus during active cell proliferation, generating a nuclear pool of acetyl-CoA for fine-tuning histone acetylation [84]. Acetyl-CoA may thus function as a "sentinel metabolite", where under stress conditions, acetyl-CoA would be diverted to mitochondria to provide energy and mitochondria-generated products, such as ketone bodies. Instead, elevated nucleo-cytosolic levels of acetyl-CoA would signal permissive conditions for growth, biosynthesis of lipids and histone acetylation. Indeed, a recent work has reported that high cytosolic/nuclear levels of acetyl-CoA due to overexpression of ACLY promote pancreatic cancer development [85]. In aging cells, elevated nucleo-cytosolic acetyl-CoA levels function as the metabolic repressor of autophagy [86]. Consistently, brain-specific knock-down of acetyl-CoA synthetase increases lifespan in *Drosophila* [86], suggesting that mitochondrial metabolites may play opposite roles in aging and cancer.

Chronic inflammation has been linked to aging and age-related pathologies, as well as to cancer (for detailed reviews see [87,88]). The inflammation process represents an essential immunological defense system that promotes survival. It has been extensively reported that mitochondrial dysfunction can promote the release of factors, known as damage-associated molecular patterns (DAMPs), into the cytosol, which can trigger an inflammatory response by engaging pattern recognition receptors. Mitochondrial DAMPs (mtDAMPs) include several molecules/effectors, such as mtDNA and mitochondria-produced ROS, which can activate the inflammasomes, multi-protein cytosolic complexes consisting of the adaptor apoptosis-associated speck-like protein (ASC), the inflammatory cysteine protease caspase-1, NOD-, LRR- and pyrin domain-containing protein 1 (NLRP1), NLRP3, NOD-, LRR- and CARD-containing protein 4 (NLRC4), and absent in melanoma 2 (AIM2). Among different inflammasomes, mitochondrial stress has been implicated in the regulation of NLRP3 activity, with mtDNA released into the cytosol as a triggering DAMP of NLRP3 inflammasome. In macrophages, cytosolic accumulation of mtDNA upon stress activates caspase-1 and promotes the secretion of IL-1β and IL-18 through activation of the NLRP3 inflammasome [89]. This retrograde response requires mitochondria-generated ROS that would cause a release of mtDNA into the cytosol [89]. Degraded mtDNA has been shown to induce the release of pro-inflammatory cytokines in brain cells and represents a possible trigger of neurodegenerative processes [90]. Recent reports further support the role of mitochondrial ROS and mtDNA in the activation of NLRP3 in age-dependent diseases, including atherosclerosis [91–96], as well as in cancer, though the activation of inflammasomes during tumor development and progression still remains controversial and with conflicting outcomes (for a detailed review see [97]). Recent evidences point to a role of DAMPs released by dying cancer cells upon chemotherapy and inflammasome's activation in bursting an immune response that could help the removal of the remaining live cancer cells. In this context, it has been proposed that a particular class of mitocans (mitochondrial-targeted anti-cancer drugs), like vitamin E analogs, that selectively induce cell death by triggering ROS production in cancer cell mitochondria, may be promising candidates for anti-cancer therapy (reviewed in [98]).

Work from Avadhani's group has shown that mammalian cells can activate another mitochondriato-nucleus signaling pathway upon mitochondrial respiratory stress [72,99]. This signaling pathway considers Ca2<sup>+</sup> as the main second messenger involved in the transmission of stress signals from mitochondria to nucleus and has been related to the response to a mitochondrial stress in the context of cancer cells. Mitochondria store large amounts of Ca2<sup>+</sup>. Upon mitochondrial stress/dysfunction leading to reduced mitochondrial membrane potential, Ca2<sup>+</sup> is released into the cytosol where it can activate Ca2+-dependent signaling. An early event of this signaling is activation of the phosphatase Calcineurin. This Ca<sup>2</sup>+/calcineurin-mediated pathway can be ROS-dependent or -independent, in relation to the cell type. For example, it is ROS-independent in skeletal myocytes, but ROS-dependent in macrophages [60,100,101]. In turn, the Ca2+/calcineurin signaling would activate the insulin-like growth factor receptor I (IGF1R) in a growth factor-independent manner that, by promoting the PI3-kinase/AKT

pathway, would culminate in the induction of the transcription of nuclear genes (reviewed in [65,99]). Ca2<sup>+</sup> may also activate other pathways independently on calcineurin. From a "phenotypic" point of view, Ca2<sup>+</sup>-mediated signaling upon mitochondrial stress results in survival, resistance to pro-apoptotic drugs, increased glycolysis and invasion of otherwise not invasive cells [8,9,61,63,99,102,103]. Taken together, these experimental evidences are consistent with a role of mild mitochondrial dysfunction in supporting health and fitness of the cells, both in aging and in cancer, through the activation of a protective retrograde signaling.

#### **4. Telomeres in Aging and Cancer**

Telomeres are DNA-protein structures present at both ends of each chromosome that protect the genome from interchromosomal fusion and rapid nucleolytic degradation. These specialized structures function as biological clocks: most normal cells have a limited lifespan and at each cell division, telomeres lose a portion of DNA. After a certain number of cell divisions, telomeres reach a critical minimal length and cells undergo apoptosis [104]. Telomere length decreases with the aging process and the rate of telomere shortening may indicate the pace of aging [104]. Stem cells and cancer cells express high levels of telomerase, an enzyme known as hTERT in humans (human TElomerase Reverse Transcriptase), which is able to prevent telomeres' shortening by elongating the telomeres' DNA. Most somatic cells instead do not express telomerase. In addition to its established role in regulating telomere length in the nucleus, TERT exerts a protective role within mitochondria by shuttling from the nucleus to the mitochondria upon exogenous stress [105–109]. Within mitochondria, TERT binds mtDNA and protects it from damage, thus preserving the mitochondrial function and promoting resistance to apoptosis in cancer cells [108–110]. Consistently, expression of a mutant telomerase lacking a nuclear export signal inhibits cancer cell proliferation, prevents immortalization, promotes mitochondrial dysfunction and sensitivity to ionizing radiation and hydrogen peroxide exposure, two genotoxic stresses [111–113].

Inherited mutations in genes encoding for protein components of the telomeres cause accelerated aging symptoms (cardiovascular disease, hair graying, diabetes, poor immune response) [114]. Interestingly, in a yeast model system, lack of telomerase accelerated aging independently on de-protected telomeres [115]. Overexpression of telomerase can delay aging but would increase the risk of tumor formation [116]. Consistently, 80%–90% of malignant tumors show overexpression of hTERT, which may occur via multiple mechanisms, including epigenetic modifications (h*TERT* gene promoter methylation), h*TERT* amplification or structural variants (reviewed in [117]). Overall, up-regulation of hTERT confers unlimited replicative potential to cancer cells, and is one of the distinctive hallmarks of cancer [118].

Despite overexpression of hTERT in cancer cells, increasing evidence demonstrates that telomere shortening correlates with cancer aggressiveness (reviewed in [119]), suggesting a threshold of telomere shortening in cancer. In this context, hTERT may guarantee an optimal telomere length that would prevent replicative senescence. In addition, expression of hTERT splice variants in cancer cells has been shown to confer a growth advantage and resistance to chemotherapeutic drugs independently upon telomere protection [120], supporting the notion that hTERT plays multiple roles in different pathophysiological conditions.

A recent work has shown a direct connection between mitochondrial dysfunction and telomere attrition [121]. Qian et al. have used a chemoptogenetic approach to produce short-lived singlet oxygen (a highly reactive ROS) in the mitochondrial matrix, whose destiny in the whole cells may be easily monitored. One pulse of singlet oxygen produced secondary ROS (superoxide radical anion and hydrogen peroxide) that promoted mtDNA damage, decrease in mitochondrial respiration and cell cycle arrest. Intriguingly, these secondary ROS were detected in the nucleus where they induced telomere fragility and loss, without causing general nuclear DNA strand breaks. DNA double-strand breaks were exclusively present in telomeres. In turn, telomere dysfunction promoted Ataxia-Telangiectasia Mutated (ATM)-dependent repair of DNA damage, preventing cells from undergoing apoptosis [121]. These findings highlight a novel mitochondria-telomere axis activated by mitochondrial ROS and associated with mitochondrial dysfunction that may be important both in aging and in cancer. Future studies are needed to assess whether this new retrograde signaling pathway may also activate hTERT, thus supporting cell survival in the long term, and tumorigenesis. Alternatively, since in progeroid syndrome this telomere dysfunction has been shown to impair the mitochondrial function [122], it is possible that an initial, transient mitochondrial dysfunction may cause progressive loss of cell fitness and apoptosis via induction of telomere shortening in a vicious cycle.

#### **5. Conclusions**

Mitochondrial function is critical for cell homeostasis, and alteration of mitochondrial performance is linked both to aging and cancer (Figure 1), two opposite processes, being aging associated with "loss of function" and cancer with "gain of fitness".

**Figure 1.** Mitochondrial dysfunction in aging and cancer. Aging and cancer share common mechanisms that include an alteration of the mitochondrial genome (mtDNA), activation of mitochondria-to-nucleus signaling pathways (retrograde signaling; see text for details) and telomere shortening. The latter is the biological clock of the cells, and beyond a limit, would cause cell cycle arrest and apoptosis, preventing the unlimited replication of normal cells. In cancer cells, expression of the telomerase hTERT would prevent excessive shortening of telomeres. Recent evidence points to mitochondrial dysfunction as a regulator of telomere shortening.

MtDNA mutations and/or decreased mtDNA content have been described in aging tissues and cancer cells. In aging, they have been associated with reduced respiration and available energy, thus decreasing the rate of anabolic reactions for biosynthesis of the cell's bricks (lipids, proteins, carbohydrates) and leading to physiological decline. Genetic studies in mouse models have provided evidence for a causal link between mtDNA depletion and the aging symptoms [35,44,45]. In cancer, some mtDNA point mutations seem to favor tumorigenesis, and other mutations would promote cancer metastasis, while mtDNA depletion correlates with poor patients' survival in certain cancers. Though there are several evidences in support of the causal role of mtDNA changes in cancer, a definitive link between mtDNA depletion/somatic mutations and tumorigenesis/cancer progression is still missing.

Intriguingly, mouse models with a mtDNA mutator phenotype that display progeroid syndrome do not exhibit increased production of mitochondrial ROS [35], in contradiction with the core hypothesis of the mitochondrial theory of aging. In cancer, increase in ROS elicited by certain mtDNA mutations favors tumorigenesis and metastasis [56,57]. Recent studies have highlighted the role of alternative retrograde signaling transmitted by dysfunctional mitochondria to the nucleus that may modulate aging and cancer.

These signals include the UPRmt, relevant to the aging process, and Ca2<sup>+</sup>-mediated signaling pathways, involved in the acquisition of some hallmarks of cancer cells. Mitochondrial dysfunction can also trigger telomere fragility and shortening, a common trait of aging and cancer. However, at variance with the aging process, cancer cells would not undergo cell cycle arrest because of the genetic/epigenetic upregulation of telomerase. These pathways are not mutually exclusive; rather they may intersect and contribute to the phenotype. Future studies, aimed at understanding the relative contribution of different mitochondrial retrograde signaling to aging and cancer, may open the road to the development of biologically active molecules targeting specific retrograde signaling pathways, in order to delay progression of age-related symptoms and/or to inhibit tumor growth and metastasis.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**


*J. Clin. Med.* **2019**, *8*, 1983


© 2019 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel. +41 61 683 77 34 Fax +41 61 302 89 18 www.mdpi.com

*Journal of Clinical Medicine* Editorial Office E-mail: jcm@mdpi.com www.mdpi.com/journal/jcm

MDPI St. Alban-Anlage 66 4052 Basel Switzerland

Tel: +41 61 683 77 34 Fax: +41 61 302 89 18