Increased mtDNA Copy Number Research Articles

The mitochondrial Ahi1/GR participates the regulation on mtDNA copy numbers and brain ATP levels and modulates depressive behaviors in mice

BackgroundPrevious studies have shown that depression is often accompanied by an increase in mtDNA copy number and a decrease in ATP levels; however, the exact regulatory mechanisms remain unclear.MethodsIn the present study, Western blot, cell knockdown, immunofluorescence, immunoprecipitation and ChIP-qPCR assays were used to detect changes in the Ahi1/GR-TFAM-mtDNA pathway in the brains of neuronal Abelson helper integration site-1 (Ahi1) KO mice and dexamethasone (Dex)-induced mice to elucidate the pathogenesis of depression. In addition, a rescue experiment was performed to determine the effects of regular exercise on the Ahi1/GR-TFAM-mtDNA-ATP pathway and depression-like behavior in Dex-induced mice and Ahi1 KO mice under stress.ResultsIn this study, we found that ATP levels decreased and mitochondrial DNA (mtDNA) copy numbers increased in depression-related brain regions in Dex-induced depressive mice and Ahi1 knockout (KO) mice. In addition, Ahi1 and glucocorticoid receptor (GR), two important proteins related to stress and depressive behaviors, were significantly decreased in the mitochondria under stress. Intriguingly, GR can bind to the D-loop control region of mitochondria and regulate mitochondrial replication and transcription. Importantly, regular exercise significantly increased mitochondrial Ahi1/GR levels and ATP levels and thus improved depression-like behaviors in Dex-induced depressive mice but not in Ahi1 KO mice under stress.ConclusionsIn summary, our findings demonstrated that the mitochondrial Ahi1/GR complex and TFAM coordinately regulate mtDNA copy numbers and brain ATP levels by binding to the D-loop region of mtDNA Regular exercise increases the levels of the mitochondrial Ahi1/GR complex and improves depressive behaviors.BE2neeR3CuVf7a9CPyYjGYVideo

Proteomic profiling reveals mitochondrial dysfunction in the cerebellum of transgenic mice overexpressing DYRK1A, a Down syndrome candidate gene.

DYRK1A is a dual-specificity kinase that is overexpressed in Down syndrome (DS) and plays a key role in neurogenesis, neuronal differentiation and function, cognitive phenotypes, and aging. Dyrk1A has also been implicated in cerebellar abnormalities observed in association with DS, and normalization of Dyrk1A dosage rescues granular and Purkinje cell densities in a trisomic DS mouse model. However, the underlying molecular mechanisms governing these processes are unknown. To shed light on the effects of Dyrk1A overexpression in the cerebellum, here we investigated the cerebellar proteome in transgenic Dyrk1A overexpressing mice in basal conditions and after treatment with green tea extract containing epigallocatechin-3-gallate (EGCG), a DYRK1A inhibitor. Our results showed that Dyrk1A overexpression alters oxidative phosphorylation and mitochondrial function in the cerebellum of transgenic mice. These alterations are significantly rescued upon EGCG-containing green tea extract treatment, suggesting that its effects in DS could depend in part on targeting mitochondria, as shown by the partially restoration by the treatment of the increased mtDNA copy number in TG non-treated mice.

NRG1/ErbB2 axis regulated mitochondrial function and antioxidant enzymes of neural stem cells in the cochlear nucleus partially through PGC-1α

Neuregulin-1 (NRG1)/erythroblastic leukaemia viral oncogene homologues 2 (ErbB2) pathway had been implicated in promoting differentiation and suppressing apoptosis of neuronal stem cells (NSCs) isolated from cochlear nucleus. In the current study, we aimed at determining the effects of NRG1/ErbB2 on mitochondrial (mt) function of NSCs. As expected, NRG1 increased the expression of mitofusin (Mfn) 1 and Mfn2 and decreased the expression of mitochondrial fission protein 1 (Fis1) and dynamin-related protein 1 (Drp1). However, after ErbB2 knockout, Mfn1 and Mfn2 expression decreased while Fis1 and Drp1 increased. Moreover, the increased mtDNA copy number and intracellular ATP level, elevated ATPase activities as well as decreased lactate production induced by NRG1 were partially reversed by ErbB2 knockout. Additionally, NRG1 treatment increased the activities of catalase, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and upregulated the protein expression of catalase, manganese superoxide dismutase (MnSOD), peroxisome proliferator-activated receptor-γ coactlvator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1) and transcription factor A, mitochondrial (TFAM), which were also reversed by ErbB2 knockout. Furthermore, PGC-1α overexpression partially reversed the above effects of ErbB2 knockout. In conclusion, these findings suggest that the promotion of mitochondrial function of NRG1/ErbB2 axis is at least in part mediated by PGC-1α in NSCs from cochlear nucleus.

Low mitochondrial DNA copy number induces chemotherapy resistance via epithelial-mesenchymal transition by DNA methylation in esophageal squamous cancer cells

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most severe cancers and is characterized by chemotherapy resistance and poor prognosis associated with epithelial-mesenchymal transition (EMT). In a previous study, a low mitochondrial DNA (mtDNA) copy number was associated with poorer prognosis and induced EMT in ESCC. However, the detailed mechanism related to mtDNA copy number and EMT is unclear. The aim of this study was to clarify the mechanism by which a change in mtDNA copy number contributes to EMT and to examine treatment of chemotherapy resistance in ESCC.MethodsThe association between low mtDNA copy number and chemotherapy resistance was investigated using specimens from 88 patients who underwent surgery after neoadjuvant chemotherapy. Then, the mtDNA content of human ESCC cell lines, TE8 and TE11, was depleted by knockdown of mitochondrial transcription factor A expression. The present study focused on modulation of mitochondrial membrane potential (MMP) and DNA methylation as the mechanisms by which mtDNA copy number affects EMT. mRNA and protein expression, chemotherapy sensitivity, proliferation, MMP and DNA methylation were evaluated, and in vitro and in vivo assays were conducted to clarify these mechanisms.ResultsESCC patients with decreased mtDNA copy number who underwent R0 resection after neoadjuvant chemotherapy had significantly worse pathological response and recurrence-free survival. Additionally, low mtDNA copy number was associated with resistance to chemotherapy in vitro and in vivo. mtDNA controlled MMP, and MMP depolarization induced EMT. Depletion of mtDNA and low MMP induced DNA methylation via a DNA methylation transcription factor (DNMT), and a DNMT inhibitor suppressed EMT and improved chemotherapy sensitivity in mtDNA-depleted ESCC cells, as shown by in vitro and in vivo assays.ConclusionThis study showed that decreased mtDNA copy number induced EMT via modulation of MMP and DNA methylation in ESCC. Therapeutic strategies increasing mtDNA copy number and DNMT inhibitors may be effective in preventing EMT and chemosensitivity resistance.

Oxygen as an important factor modulating in vitro MeHgCl toxicity associated with mitochondrial genes in hiPSCs

Mitochondria are energy factories of cells and important targets for methylmercury chloride (MgHgCl). Methylmercury (MeHg) is a well-known environmental toxicant that bioaccumulates in fish and shellfish. It readily crosses the placental barrier, making it a threat to correct fetal development. Despite being comprehensively investigated for years, this compound has not been assessed for its in vitro mitochondrial toxicity under different oxygen conditions. In this study, human induced pluripotent stem cells (hiPSCs) were used to evaluate the dependence of the expression of genes associated with pluripotency and mitochondria on atmospheric (21% O2) and low (5% O2) oxygen concentrations upon MeHgCl treatment. We showed that the toxicity of MeHgCl was strongly related to an increased mtDNA copy number and downregulation of the expression of an mtDNA replication and damage repair-associated gene POLG1 (Mitochondrial Polymerase Gamma Catalytic Subunit) in both tested oxygen conditions. In addition, the viability and mitochondrial membrane potential of hiPSCs were significantly lowered by MeHgCl regardless of the oxygen concentration. However, reactive oxygen species accumulation significantly increased only under atmospheric oxygen conditions; what was associated with increased expression of TFAM (Transcription Factor A, Mitochondrial) and NRF1 (Nuclear Respiratory Factor 1) and downregulation of PARK2 (Parkin RBR E3 Ubiquitin Protein Ligase). Taken together, our results demonstrated that MeHgCl could induce in vitro toxicity in hiPSCs through altering mitochondria-associated genes in an oxygen level-dependent manner. Thus, our work suggests that oxygen should be considered a factor was modulating the in vitro toxicity of environmental pollutants. Typical atmospheric conditions of in vitro culture significantly lower the predictive value of studies of such toxicity.

Mitochondrial supplementation of Sus scrofa metaphase II oocytes alters DNA methylation and gene expression profiles of blastocysts

BackgroundMitochondrial DNA (mtDNA) copy number in oocytes correlates with oocyte quality and fertilisation outcome. The introduction of additional copies of mtDNA through mitochondrial supplementation of mtDNA-deficient Sus scrofa oocytes resulted in: (1) improved rates of fertilisation; (2) increased mtDNA copy number in the 2-cell stage embryo; and (3) improved development of the embryo to the blastocyst stage. Furthermore, a subset of genes showed changes in gene expression. However, it is still unknown if mitochondrial supplementation alters global and local DNA methylation patterns during early development.ResultsWe generated a series of embryos in a model animal, Sus scrofa, by intracytoplasmic sperm injection (ICSI) and mitochondrial supplementation in combination with ICSI (mICSI). The DNA methylation status of ICSI- and mICSI-derived blastocysts was analysed by whole genome bisulfite sequencing. At a global level, the additional copies of mtDNA did not affect nuclear DNA methylation profiles of blastocysts, though over 2000 local genomic regions exhibited differential levels of DNA methylation. In terms of the imprinted genes, DNA methylation patterns were conserved in putative imprint control regions; and the gene expression profile of these genes and genes involved in embryonic genome activation were not affected by mitochondrial supplementation. However, 52 genes showed significant differences in expression as demonstrated by RNAseq analysis. The affected gene networks involved haematological system development and function, tissue morphology and cell cycle. Furthermore, seven mtDNA-encoded t-RNAs were downregulated in mICSI-derived blastocysts suggesting that extra copies of mtDNA affected tRNA processing and/or turnover, hence protein synthesis in blastocysts. We also showed a potential association between differentially methylated regions and changes in expression for 55 genes due to mitochondrial supplementation.ConclusionsThe addition of just an extra ~ 800 copies of mtDNA into oocytes can have a significant impact on both gene expression and DNA methylation profiles in Sus scrofa blastocysts by altering the epigenetic programming established during oogenesis. Some of these changes may affect specific tissue-types later in life. Consequently, it is important to determine the longitudinal effect of these molecular changes on growth and development before considering human clinical practice.

Cancer/Testis Antigen 55 is required for cancer cell proliferation and mitochondrial DNA maintenance

Cancer/Testis Antigens (CTAs) represent a group of proteins whose expression under physiological conditions is restricted to testis but activated in many human cancers. Also, it was observed that co-expression of multiple CTAs worsens the patient prognosis. Five CTAs were reported acting in mitochondria and we recently reported 147 transcripts encoded by 67 CTAs encoding for proteins potentially targeted to mitochondria. Among them, we identified the two isoforms encoded by CT55 for whom the function is poorly understood. First, we found that patients with tumors expressing wild-type CT55 are associated with poor survival. Moreover, CT55 silencing decreases dramatically cell proliferation. Second, to investigate the role of CT55 on mitochondria, we first show that CT55 is localized to both mitochondria and endoplasmic reticulum (ER) due to the presence of an ambiguous N-terminal targeting signal. Then, we show that CT55 silencing decreases mtDNA copy number and delays mtDNA recovery after an acute depletion. Moreover, demethylation of CT55 promotor increases its expression, which in turn increases mtDNA copy number. Finally, we measured the mtDNA copy number in NCI-60 cell lines and screened for genes whose expression is strongly correlated to mtDNA amount. We identified CT55 as the second highest correlated hit. Also, we show that compared to siRNA scrambled control (siCtrl) treatment, CT55 specific siRNA (siCT55) treatment down-regulates aerobic respiration, indicating that CT55 sustains mitochondrial respiration. Altogether, these data show for first time that CT55 acts on mtDNA copy number, modulates mitochondrial activity to sustain cancer cell proliferation.

Mitochondrial DNA content reduction in the most fertile spermatozoa is accompanied by increased mitochondrial DNA rearrangement.

Is there an association between male fertility and spermatozoa mitochondrial DNA (mtDNA) copy number and genome rearrangements? Normal spermatozoa not only have a lower mtDNA copy number but also more DNA rearrangements than spermatozoa of men with severe oligoasthenospermia (SOA). While there is a consensus that mtDNA content is decreased in the most fertile spermatozoa, the role of mtDNA sequence alteration in male infertility is unclear. High-throughput sequencing, which allows an exhaustive analysis of mtDNA rearrangements and mutations, could be helpful in this context, but has yet to be used. This is an observational study of semen samples obtained from 44 men undergoing ART at an academic infertility centre in France, from October 2018 to November 2020. The men were classified into two groups: 20 men in the SOA group and 24 men with normal semen parameters in the control group. For each patient and control, mtDNA was isolated from sperm fractions from the 40% and 90% layers of the density gradient. The average mtDNA content of each sample was assessed using digital PCR. Deep sequencing was performed using next-generation sequencing. Signal processing and base calling were performed via the embedded pre-processing pipeline, the variants were analysed using an in-house workflow and a dedicated tool, based on soft-clipping, was used to study large mtDNA rearrangements. The distribution and the type of rearrangements and variants were compared between patients with SOA and controls on one hand, and between the 40% and 90% gradient layers, on the other hand. The mtDNA content of spermatozoa in the SOA group was significantly higher than in the control group (P < 0.0001). Moreover, mtDNA content was significantly higher in spermatozoa from the 40% layer (the most fertile spermatozoa) compared to the 90% layer, both in the SOA (P = 0.02) and the control group (P < 0.0001). The frequency of large mtDNA deletions and duplications was significantly higher in the control group (P = 0.002). Most of these rearrangements are potentially related to DNA breaks and their number was reduced by the removal of the linear mtDNA from the samples. Heteroplasmic variants were found more frequently in the SOA group (P = 0.05) and in the 40% layer (P = 0.03), but none had any obvious functional consequence. Our findings are novel and significant but should be verified in larger cohorts and other types of male infertility. Our findings suggest that sperm mtDNA rearrangements are not necessarily associated with mitochondrial dysfunction and male infertility. Instead, they seem to be concomitant with the process of mtDNA content reduction in the most potentially fertile spermatozoa. Furthermore, they refute the hypothesis that, in the case of mtDNA alteration, a compensatory mechanism allows an increase in mtDNA copy number to rectify the energy deficit. The increased frequency of mtDNA rearrangements in the most fertile spermatozoa is a novel result that offers new insight into the relation between sperm quality and mtDNA. This work was supported by Angers University Hospital (grant AOI CHU Angers 2018), Angers University and the French national research centres INSERM and CNRS. There are no competing interests. N/A.

Glia Maturation Factor Beta as a Novel Biomarker and Therapeutic Target for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common types of cancer. The novel sensitive biomarkers and therapeutic targets are urgently needed for the early diagnosis of HCC and improvement of clinical outcomes. Glia maturation factor-β (GMFB) is a growth and differentiation factor for both glia and neurons and has been found to be tightly involved in inflammation and neurodegeneration conditions. In our study, the expression level of GMFB was significantly up-regulated in patients with HCC and positively co-expression with tumor node metastases (TNM) stage and histopathological grade of HCC. The high expression level of GMFB was remarkably associated with poor overall survival, which mainly occurred in males rather than females. Multivariate analysis revealed GMFB to be an independent prognostic factor for overall survival in patients with HCC. Results of Gene Ontology (GO) and KEGG pathways analysis showed that down-regulation of pathways related to protein translation and mitochondria function were enriched. Protein-protein interaction analysis revealed the central role of mitochondria protein in HCC. The downregulation of genes involved in glycolysis and gluconeogenesis was observed among the co-expression genes of GMFB. Knockdown of GMFB in Hep3B significantly inhibited proliferation, migration, and invasion of Hep3B cells, and also downregulated the expression levels of some of metal matrix proteinase (MMP), increased mtDNA copy number and loss of mitochondrial transmembrane potential. GMFB influences the malignancy rate of HCC possibly through regulation of the expression of MMPs, mtDNA function and glycolysis. We proposed that GMFB was a promising HCC diagnostic and prognostic biomarker and therapeutic target in HCC.

Mitochondrial DNA Copy Number in Rett Syndrome Caused by Methyl-CpG-Binding Protein-2 Variants

To determine changes in mitochondrial DNA (mtDNA) copy number in peripheral blood in Rett syndrome caused by methyl-CpG-binding protein-2 (MECP2) variants and explore the mechanism of mitochondrial dysfunction in Rett syndrome. Female patients who were diagnosed with Rett syndrome and had an MECP2 variant (n=142) were recruited in this study, along with the same number of age- and sex-matched healthy controls. MtDNA copy number was quantified by real-time quantitative polymerase chain reaction with TaqMan probes. The differences in mtDNA copy number between the Rett syndrome group and the control group were analyzed using the independent-samples t test. Linear regression, biserial correlation analysis, and one-way ANOVA were applied for the correlations between mtDNA copy number and age, clinical severity, variant types, functional domains, and hot-spot variants. MtDNA copy number was found to be significantly increased in the patients with Rett syndrome with MECP2 gene variants compared with the control subjects. Age, clinical severity, variant types, functional domains, and hot-spot variants were not related to mtDNA copy number in patients with Rett syndrome. MtDNA copy number is increased significantly in patients with Rett syndrome, suggesting that changes in mitochondrial function in Rett syndrome trigger a compensatory increase in mtDNA copy number and providing new possibilities for treating Rett syndrome, such as mitochondria-targeted therapies.

Mitonuclear Interactions Produce Diverging Responses to Mild Stress in Drosophila Larvae.

Mitochondrial function depends on direct interactions between respiratory proteins encoded by genes in two genomes, mitochondrial and nuclear, which evolve in very different ways. Serious incompatibilities between these genomes can have severe effects on development, fitness and viability. The effect of subtle mitonuclear mismatches has received less attention, especially when subject to mild physiological stress. Here, we investigate how two distinct physiological stresses, metabolic stress (high-protein diet) and redox stress [the glutathione precursor N-acetyl cysteine (NAC)], affect development time, egg-to-adult viability, and the mitochondrial physiology of Drosophila larvae with an isogenic nuclear background set against three mitochondrial DNA (mtDNA) haplotypes: one coevolved (WT) and two slightly mismatched (COX and BAR). Larvae fed the high-protein diet developed faster and had greater viability in all haplotypes. The opposite was true of NAC-fed flies, especially those with the COX haplotype. Unexpectedly, the slightly mismatched BAR larvae developed fastest and were the most viable on both treatments, as well as control diets. These changes in larval development were linked to a shift to complex I-driven mitochondrial respiration in all haplotypes on the high-protein diet. In contrast, NAC increased respiration in COX larvae but drove a shift toward oxidation of proline and succinate. The flux of reactive oxygen species was increased in COX larvae treated with NAC and was associated with an increase in mtDNA copy number. Our results support the notion that subtle mitonuclear mismatches can lead to diverging responses to mild physiological stress, undermining fitness in some cases, but surprisingly improving outcomes in other ostensibly mismatched fly lines.

MITOQUINONE RESCUES STRESS INDUCED MITOPHAGY IN AGED OOCYTES

It is well established that decreased oocyte quality from advanced maternal age (AMA) women are associated with dysfunctional mitochondria. Mitophagy is a critical pathway to regulate mitochondrial quality. Our previous research demonstrated reduction in key mitophagy protein Parkin (PRKN) and its activity in oocytes from AMA women. The objective of this study was to investigate if maturing aged mouse oocytes with Mitoquinone (MQ) would improve oocyte mitochondrial quality by rescuing mitophagy. First, oocytes from young (2 months) and aged (13.5 months) mice were matured with or without MQ and the levels of p-PRKN and PRKN were analyzed by Western blot (Jess, ProteinSimple). In the second experiment, young oocytes with or without MQ were matured under normal (6.5%) or high oxygen (20%) environment, and the expression of p-PRKN and PRKN, mitochondria DNA (mtDNA) copy number (analyzed by qPCR) and blastocyst development after in vitro fertilization, blastocyst hatching, and inner cell mass (ICM) ratio were examined. We observed a significant decrease in p-PRKN (0.32±0.09, p≤0.01) and PRKN (0.30±0.1, p≤0.05) in aged compared to young mouse oocytes (normalized to 1). Treatment of aged oocytes with MQ rescued p-PRKN (1.11±0.19 p≤0.01) and PRKN (0.67±0.04, p≤0.05) protein expression. Oocytes matured under high oxygen environment exhibited significantly reduced p-PRKN (0.5±0.1, p≤0.05) compared to oocytes matured under normal oxygen conditions. This coincided with an increase in mtDNA copy number (Young: 56957, High oxygen: 72758 copies). MQ treatment significantly increased p-PRKN protein expression (1.2±0.2, p≤0.05) as well as decreased mtDNA copy number (50312 copies) in oxidatively stressed oocytes. Addition of MQ significantly improved blastocyst development (High O2: 39.5%, High O2+MQ: 70%, p≤0.05), hatching (High O2: 59%, High O2+MQ: 93%, p≤0.05), and ICM ratio (High O2: 14.61%, High O2+MQ: 22.5%, p≤0.0001) of oxidatively stressed oocytes that showed decreased trends in blastocyst development potential. We have demonstrated previously that oocytes from AMA women (>38 years old) exhibited significantly less p-PRKN and PRKN than oocytes from young women (<32 years old). Similar PRKN expression pattern was obtained in young and aged mice and we also observed decreased p-PRKN in oxidatively stressed mouse oocytes, indicating that mitophagy via Parkin pathway is altered in aged and oxidatively stressed oocytes. We have shown for the first time that treating aged and oxidatively stressed oocytes with MQ rescues p-PRKN and PRKN protein expression to ameliorate mitophagy. MQ also improved blastocyst development potential of oxidatively stressed oocytes.

High levels of TFAM repress mammalian mitochondrial DNA transcription in vivo.

Mitochondrial transcription factor A (TFAM) is compacting mitochondrial DNA (dmtDNA) into nucleoids and directly controls mtDNA copy number. Here, we show that the TFAM-to-mtDNA ratio is critical for maintaining normal mtDNA expression in different mouse tissues. Moderately increased TFAM protein levels increase mtDNA copy number but a normal TFAM-to-mtDNA ratio is maintained resulting in unaltered mtDNA expression and normal whole animal metabolism. Mice ubiquitously expressing very high TFAM levels develop pathology leading to deficient oxidative phosphorylation (OXPHOS) and early postnatal lethality. The TFAM-to-mtDNA ratio varies widely between tissues in these mice and is very high in skeletal muscle leading to strong repression of mtDNA expression and OXPHOS deficiency. In the heart, increased mtDNA copy number results in a near normal TFAM-to-mtDNA ratio and maintained OXPHOS capacity. In liver, induction of LONP1 protease and mitochondrial RNA polymerase expression counteracts the silencing effect of high TFAM levels. TFAM thus acts as a general repressor of mtDNA expression and this effect can be counterbalanced by tissue-specific expression of regulatory factors.

Spectrum of mitochondrial genomic variation in parathyroid neoplasms.

Mitochondrial DNA (mtDNA) variations have been implicated in various cancer types. Several attempts have been made in benign parathyroid adenoma (PA); however, mtDNA variants and copy number alterations have not been investigated in parathyroid carcinoma (PC). The present study aimed to explore the variations in the mitochondrial genome in parathyroid neoplasms. In this study, ultradeep targeted sequencing of mtDNA was performed in 12 cases with PC and 25 cases with PA. Germline and somatic variants in the mitochondrial genome were analysed according to clinical features. The mtDNA copy number relative to nuclear DNA was measured. A total of 821 germline variants and 23 somatic mutations were identified. All 160 germline nonsynonymous variants in the coding sequence (CDS) were predicted to be likely benign, and germline variants frequently occurred (26.3%) in the displacement loop region. Tumour somatic mutation in a CDS with heteroplasmic factor (HF) >0.8 showed a higher mtDNA copy number (p = 0.039). Using Spearman's rank test, tumour mtDNA copy number revealed a positive correlation with serum levels of intact parathyroid hormone and calcium. Our results demonstrate that null recurrent mtDNA mutational hotspots were PC specific. An increased mtDNA copy number in parathyroid neoplasms was associated with somatic CDS mutations with a high HF and major clinical features. Larger cohort investigations should be performed in future analyses.

Abstract 2404: Increased mitochondrial DNA copy number occurs during prostate cancer progression and in cancer precursor lesions across multiple organs

Abstract Accurately measuring mitochondrial DNA copy number (mtDNAcn) is important for improving our understanding of cancer biology. Prior studies have revealed differences in mtDNAcn between matched bulk tumor-normal pairs from a range of different organs. However, there is little consistency across tumor types; compared to their normal tissue counterparts, some cancers appear to have increased mtDNAcn, others appear to have decreased mtDNAcn, and in others, such as prostate cancer, studies have shown contradictory results. The lack of consistency of mtDNAcn changes in tumor-normal pairs may reflect a failure to address the marked heterogeneity in mtDNAcn across different cell types within normal tissues. In addition, tumor cell heterogeneity in mtDNAcn has also not been taken into account in most prior studies, which can further confound tumor-normal comparisons. Finally, little is known about mtDNAcn alterations in cancer precursor lesions. To address these technical limitations, we combined a recently introduced quantitative in situ hybridization method with immunohistochemistry in a multiplex assay. This enabled us to measure mtDNAcn using digital image analysis in specific cell populations of interest on formalin fixed paraffin embedded (FFPE) and frozen tissue samples. We determined that normal prostate basal cells have approximately 3-fold higher mtDNA levels than normal prostate luminal cells. Compared to the adjacent normal prostate glands, we found markedly increased levels of mtDNA in high-grade prostatic intraepithelial neoplasia (HGPIN), the presumptive precursor lesions to most prostate cancers. We also found increased levels as well as heterogeneity of mtDNAcn in invasive prostate cancer lesions, and more consistently elevated levels in castration-resistant metastatic prostate cancer (CRPC). Increased mtDNA copy number was also identified by whole genome sequencing and quantitative PCR from laser capture micro-dissected human prostate tumor-normal pairs. We also observed higher mtDNAcn in two other precancer lesion types, pancreatic intraepithelial neoplasia and colorectal adenomas. Our approach represents a technological advance as it facilitates mtDNAcn measurements in a manner that preserves morphology, allowing for the evaluation of specific cell populations of interest. These findings raise the hypothesis that increased mitochondrial mass and function may drive the development of precancerous lesions in the prostate, pancreas and colon, as well as invasive prostate cancer development and progression. Citation Format: Jiayu Chen, Qizhi Zheng, Jessica L. Hicks, Levent Trabzonlu, Emmanuel S. Antonarakis, Mark C. Markowski, Samuel R. Denmeade, Busra Ozbek, Anuj Gupta, Tatianna C. Larman, Ralph H. Hruban, Sarah Wheelan, Srinivasan Yegnasubramanian, Angelo M. De Marzo. Increased mitochondrial DNA copy number occurs during prostate cancer progression and in cancer precursor lesions across multiple organs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2404.

Abnormalities in Mitochondrial DNA Copy Number Have Pathogenetic and Prognostic Implications in Multiple Myeloma

Mitochondria are bioenergetic and biosynthetic organelles which control crucial biological pathways such as cell growth, proliferation and apoptosis among others. Over the last two decades, many studies have examined the role of mitochondrial DNA (mtDNA) alterations in carcinogenesis, via inactivating genetic mutations or dysregulation of the mtDNA copy number in the cell. However, the status of the mtDNA copy number and its implication in the pathogenesis of Multiple Myeloma (MM) remains unknown. Here, we have analyzed the mtDNA copy number in asymptomatic and symptomatic stages of MM and evaluated its clinical impact.We have estimated the mtDNA copy number in purified plasma cells from 139 patients across different stages of the disease (4 healthy donor, 19 MGUS, 14 Smoldering MM (SMM), 78 newly diagnosed MM (ndMM) and 24 relapse (rMM). The mtDNA copy number quantification was performed by RT-PCR using 2 Taqman probes which targeted the mitochondrial gene MT-RNR1 and the nuclear gene RPPH1. We have also used whole-genome or whole-exome sequencing data to examine changes in mtDNA copy number in newly-diagnosed or relapsed MM (n=64 samples) including paired sample from SMM patients progressing to ndMM (n=10). FastMitocalc and Samtools were used to extract reads aligning to mitochondrial genome from bam files and calculate de mtDNA copy number by comparing to the number of reads aligning to nuclear genome. Differential gene expression analysis was done by using canEvolve.org ocogenomics web portal (Multiple Myeloma, GSE6477 study).We observed a significant increase in the average mtDNA copy number in myeloma cells compared to healthy plasma cells (39 vs 228 copies; p-value = 0.02). Plasma cells from MGUS cells showed lower mtDNA content than more advanced stages of the disease (129 vs 242 mtDNA copies). Interestingly, amongst 10 paired samples from SMM progressing to MM an increase in the mtDNA copy number was observed in 8 of the 10 patients, being more evident when the time to progression was longer. Additionally, we also observed an increase in the mitochondrial content in 17 of the 21 patient samples examined pre and post relapse, including those patients with three consecutive relapsed samples available. We have evaluated the impact on survival of the mtDNA copy number in 69 newly diagnosed patients and we have found that patients with greater than 400 mtDNA copy number had significantly shorter progression free survival compared to the rest (p-value = 0.024). The differential expression analysis of plasma cells from MGUS, SMM, ndMM and rMM patients compared to normal plasma cells confirmed an overexpression of the mitochondrial biogenesis regulators (TFAM, TUFM, MYC and HNRNPK).We describe for the first time that mtDNA copy number increase across different stages of MM. Our data provides evidence that the malignant transformation of the myeloma cells from asymptomatic stages implies activation of mitochondrial biogenesis resulting in increased mtDNA levels. Functional experiments are ongoing to confirm the metabolic activation of myeloma cells over the time. These results highlight mitochondria as an important component of pathogenesis and progression of MM and as a potential target for therapy. DisclosuresNo relevant conflicts of interest to declare.

1152-P: Elucidating Protective Mechanisms of Targeting of hOGG1 to Mitochondria on the HFD-Induced Insulin Resistance in Mice Lacking OGG1

Ogg1 (8-oxoguanine DNA glycosylase-1) is a DNA glycosylase mediating the first step in the base excision repair which removes 8-oxoguanine and repairs oxidized nuclear and mitochondrial (mtDNA). Obesity-induced mtDNA damage heightens mitochondrial oxidative stress, which leads to the development of insulin resistance. Thus, it is important to understand molecular mechanisms by which mtDNA damage induces insulin resistance and to develop therapies including preventative and treatment strategies. Previously we found that mitochondrial targeting of human OGG1 (mt-hOGG1) ameliorated high fat diet (HFD)-induced insulin resistance in Ogg1 deficient (KO) mice in both genetic model (KO/TG mice) and via TAT-protein delivery using intraperitoneal injections (Diabetes 68, Suppl 1, 2019). As we previously demonstrated that Ogg1 contributed to HFD-induced insulin resistance via hepatic insulin resistance (Diabetes 67, Suppl 1, 2018), the goal of the current study was to elucidate the underlying mechanisms for the protective effect of mt-hOGG1 delivery specifically in liver. In this study we found that targeting of mt-hOGG1 prevented hepatic mtDNA from oxidative damage in both genetic model (KO/TG) and via TAT-protein delivery (TAT-mt-hOGG1) to KO mice. Furthermore, delivery of mt-hOGG1 via TAT-protein transduction significantly increased copy number of mtDNA and expression of VDAC1, a mitochondrial marker in liver isolated from TAT-mt-hOGG1 compared to the vehicle treated mice. Studies are underway which will explore the mechanisms by which TAT-mt-hOGG1 increased mtDNA copy number, including evaluation of mitophagy and mitochondrial biogenesis. Collectively, our findings suggested that mt-hOGG1 is critical to preserve mtDNA integrity in liver and provides strong rationale to use TAT-mediated delivery of DNA repair enzymes into mitochondria as a new translational approach for treatment of obesity-induced insulin resistance. Disclosure L. Yuzefovych: None. V. M. Pastukh: None. L. Rachek: None.

Effect of exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs) on mitochondrial DNA (mtDNA) copy number in rats

Mitochondria are intracellular organelles responsible for biological oxidation and energy production. These organelles are susceptible to damage from oxidative stress and compensate for damage by increasing the number of copies of their own genome, mitochondrial DNA (mtDNA). Cancer and environmental exposure to some pollutants have also been associated with altered mtDNA copy number. Since exposures to polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been shown to increase oxidative stress, we hypothesize that mtDNA copy number will be altered with exposure to these compounds. mtDNA copy number was measured in DNA from archived frozen liver and lung specimens from the National Toxicology Program (NTP) study of female Harlan Sprague Dawley rats exposed to TCDD (3, 10, or 100 ng/kg/day), dioxin-like (DL) PCB 126 (10, 100, or 1000 ng/kg/day), non-DL PCB 153 (10, 100, or 1000 μg/kg/day), and PCB 126 + PCB 153 (10 ng/kg/day + 10 μg/kg/day, 100 ng/kg/day + 100 μg/kg/day, or 1000 ng/kg/day + 1000 μg/kg/day, respectively) for 13 and 52 weeks. An increase in mtDNA copy number was observed in the liver and lung of rats exposed to TCDD and the lung of rats exposed to the mixture of PCB 126 and PCB 153. A statistically significant positive dose-dependent trend was also observed in the lung of rats exposed to PCB 126 and a mixture of PCB 153 and PCB 126, although in neither case was the control copy number significantly exceeded at any dose level. These exposures produced a range of pathological responses in these organs in the two-year NTP studies. Conversely, there was a significant decrease or no change in mtDNA copy number in the liver and lung of rats exposed to non-DL PCB 153. This is consistent with a general lack of PCB 153 mediated liver or lung injury in the NTP study, with the exception of liver hypertrophy. Together, the results suggest that an increase in mtDNA copy number may serve as a sensitive, early biomarker of mitochondrial injury and oxidative stress that contributes to the development of the toxicity of dioxin-like compounds.

Mitochondrial DNA Copy Number in Human Blastocyst: A Novel Biomarker for the Prediction of Implantation Potential

The relationship between mitochondrial DNA (mtDNA) copy number and the outcome of embryo transfer is under debate. Our aim was to explore the relationship between mtDNA copy number in human blastocysts and embryonic development to determine whether mtDNA represents a novel biomarker for the prediction of implantation potential. A total of 246 blastocysts were analyzed by next-generation sequencing. There was no correlation between mtDNA copy number and maternal age in all blastocyst groups and euploid blastocyst groups. Additionally, the mtDNA copy number was not significantly higher in aneuploid blastocysts. Subsequently, no relationship was observed between mtDNA copy number and blastocyst quality. The assessment of clinical pregnancy outcome after the transfer of euploid blastocysts to the uterus indicated that the mtDNA copy number was significantly lower in the clinical pregnancy group than in those who failed implantation. The cut-off value of mtDNA copy number was 320.5, which was a highly predictive value. Blastocysts with an increased mtDNA copy number had lower implantation potential, and mtDNA copy number was largely equal in terms of maternal age, chromosome ploidy, and quality of blastocysts.

Association Between Mitochondrial DNA Copy Number and Head and Neck Squamous Cell Carcinoma: A Systematic Review and Dose-Response Meta-Analysis

BackgroundThe association between mitochondrial DNA (mtDNA) copy number and head and neck squamous cell carcinoma (HNSCC) risk remains unclear. Therefore, we aimed to evaluate the relationship between mtDNA copy number and HNSCC risk.Material/MethodsWe searched PubMed, Web of Science, and EMBASE until August 2020. Studies that assessed the association between mtDNA copy number and HNSCC as the outcome of interest were included. We performed a 2-class and dose-response meta-analysis to assess the association between cancer risk and mtDNA.ResultsEight articles (2 cohort studies and 6 case-control studies) with a total of 3913 patients were included in our meta-analysis. The overall results showed that mean mtDNA copy number level from 9 studies was 0.71 higher in patients with cancer than in non-cancer controls (the standardized mean differences (SMD) 0.71, 95% CI: 0.28–1.15, P<0.001). However, when 4 studies were pooled by dichotomizing mtDNA copy number at the median value into high- and low-content groups, no significant association between mtDNA content and overall cancer risk was found (odds ratio (OR)=0.87, 95% CI: 0.52–1.44, P=0.584). Furthermore, we observed a non-linear association from 3 studies between increased mtDNA copy number levels (P for nonlinearity <0.001).ConclusionsThe elevated mtDNA copy number could predict the risk of HNSCC as a biomarker. Moreover, there was non-linear relationship of risk between HNSCC and mtDNA copy number.