Mitochondrial DNA mutations and breast tumorigenesis

Breast cancer is a heterogeneous disease and genetic factors play an important role in its genesis. Although mutations in tumor suppressors and oncogenes encoded by the nuclear genome are known to play a critical role in breast tumorigenesis, the contribution of the mitochondrial genome to this process is unclear. Like the nuclear genome, the mitochondrial genome also encodes proteins critical for mitochondrion functions such as oxidative phosphorylation (OXPHOS), which is known to be defective in cancer including breast cancer. Mitochondrial DNA (mtDNA) is more susceptible to mutations due to limited repair mechanisms compared to nuclear DNA (nDNA). Thus changes in mitochondrial genes could also contribute to the development of breast cancer. In this review we discuss mtDNA mutations that affect OXPHOS. Continuous acquisition of mtDNA mutations and selection of advantageous mutations ultimately leads to generation of cells that propagate uncontrollably to form tumors. Since irreversible damage to OXPHOS leads to a shift in energy metabolism towards enhanced aerobic glycolysis in most cancers, mutations in mtDNA represent an early event during breast tumorigenesis, and thus may serve as potential biomarkers for early detection and prognosis of breast cancer. Because mtDNA mutations lead to defective OXPHOS, development of agents that target OXPHOS will provide specificity for preventative and therapeutic agents against breast cancer with minimal toxicity.     

Mitochondrial diseases

By convention, the term "mitochondrial diseases" refers to disorders of the mitochondrial respiratory chain, which is the only metabolic pathway in the cell that is under the dual control of the mitochondrial genome (mtDNA) and the nuclear genome (nDNA). Therefore, a genetic classification of the mitochondrial diseases distinguishes disorders due to mutations in mtDNA, which are governed by the relatively lax rules of mitochondrial genetics, and disorders due to mutations in nDNA, which are governed by the stricter rules of mendelian genetics. Mutations in mtDNA can be divided into those that impair mitochondrial protein synthesis in toto and those that affect any one of the 13 respiratory chain subunits encoded by mtDNA. Essential clinical features for each group of diseases are reviewed. Disorders due to mutations in nDNA are more abundant not only because most respiratory chain subunits are nucleus-encoded but also because correct assembly and functioning of the respiratory chain require numerous steps, all of which are under the control of nDNA. These steps (and related diseases) include: (i) synthesis of assembly proteins; (ii) intergenomic signaling; (iii) mitochondrial importation of nDNA-encoded proteins; (iv) synthesis of inner mitochondrial membrane phospholipids; (v) mitochondrial motility and fission.     

Mitochondrial genome changes and neurodegenerative diseases

Mitochondria are essential organelles within the cell where most of the energy production occurs by the oxidative phosphorylation system (OXPHOS). Critical components of the OXPHOS are encoded by the mitochondrial DNA (mtDNA) and therefore, mutations involving this genome can be deleterious to the cell. Post-mitotic tissues, such as muscle and brain, are most sensitive to mtDNA changes, due to their high energy requirements and non-proliferative status. It has been proposed that mtDNA biological features and location make it vulnerable to mutations, which accumulate over time. However, although the role of mtDNA damage has been conclusively connected to neuronal impairment in mitochondrial diseases, its role in age-related neurodegenerative diseases remains speculative. Here we review the pathophysiology of mtDNA mutations leading to neurodegeneration and discuss the insights obtained by studying mouse models of mtDNA dysfunction. This article is part of a Special Issue entitled: Misfolded Proteins, Mitochondrial Dysfunction, and Neurodegenerative Diseases.     

Molecular genetic and clinical aspects of mitochondrial disorders in childhood

Mitochondrial OXPHOS disorders are caused by mutations in mitochondrial or nuclear genes, which directly or indirectly affect mitochondrial oxidative phosphorylation (OXPHOS). Primary mtDNA abnormalities in children are due to rearrangements (deletions or duplications) and point mutations or insertions. Mutations in the nuclear-encoded polypeptide subunits of OXPHOS result in complex I and II deficiency, whereas mutations in the nuclear proteins involved in the assembly of OXPHOS subunits cause defects in complexes I, III, IV, and V. Here, we review recent progress in the identification of mitochondrial and nuclear gene defects and the associated clinical manifestations of these disorders in childhood.     

Genetic insights into OXPHOS defect and its role in cancer

Warburg proposed that cancer originates from irreversible injury to mitochondrial oxidative phosphorylation (mtOXPHOS), which leads to an increase rate of aerobic glycolysis in most cancers. However, despite several decades of research related to Warburg effect, very little is known about the underlying genetic cause(s) of mtOXPHOS impairment in cancers. Proteins that participate in mtOXPHOS are encoded by both mitochondrial DNA (mtDNA) as well as nuclear DNA. This review describes mutations in mtDNA and reduced mtDNA copy number, which contribute to OXPHOS defects in cancer cells. Maternally inherited mtDNA renders susceptibility to cancer, and mutation in the nuclear encoded genes causes defects in mtOXPHOS system. Mitochondria damage checkpoint (mitocheckpoint) induces epigenomic changes in the nucleus, which can reverse injury to OXPHOS. However, irreversible injury to OXPHOS can lead to persistent mitochondrial dysfunction inducing genetic instability in the nuclear genome. Together, we propose that "mitocheckpoint" led epigenomic and genomic changes must play a key role in reversible and irreversible injury to OXPHOS described by Warburg. These epigenetic and genetic changes underlie the Warburg phenotype, which contributes to the development of cancer.     

Pharmacological targeting of mitochondrial complex I deficiency: the cellular level and beyond

Complex I (CI) represents a major entry point of electrons in the mitochondrial electron transport chain (ETC). It consists of 45 different subunits, encoded by the mitochondrial (mtDNA) and nuclear DNA (nDNA). In humans, mutations in nDNA-encoded subunits cause severe neurodegenerative disorders like Leigh Syndrome with onset in early childhood. The pathophysiological mechanism of these disorders is still poorly understood. Here we summarize the current knowledge concerning the consequences of nDNA-encoded CI mutations in patient-derived cells, present mouse models for human CI deficiency, and discuss potential treatment strategies for CI deficiency.     

Molecular research technologies in mitochondrial diseases: the microarray approach

Mitochondria are ubiquitous in eukaryotic cells where they generate much of the cellular energy by the process of oxidative phosphorylation (OXPHOS). The approximately 1500 genes of the mitochondrial genome are distributed between the cytoplasmic, maternally-inherited, mitochondrial DNA (mtDNA) which encodes 37 genes and the nuclear DNA (nDNA) which encompasses the remaining mitochondrial genes. The interplay between the mtDNA and nDNA encoded mitochondrial genes and their role in mitochondrial disorders is still largely unclear. One approach for elucidating the pathophysiology of mitochondrial diseases has been to look at changes in the expression of mtDNA and nDNA-encoded genes in response to specific mitochondrial genetic defects. Initial studies of gene expression changes in response to mtDNA defect employed blot technologies to analyze changes in the expression of individual genes one at a time. While Southern/Northern blot experiments confirmed the importance of nDNA-mtDNA interactions in the pathophysiology of mitochondrial myopathy, the methodology used limited the number of genes that could be analyzed from each patient. This barrier has been overcome, in part by the advent of DNA microarray technology. In DNA microarrays gene sequences or oligonucleotides homologous to gene sequences are arrayed on a solid support. The RNA from the subject is then isolated, the mRNA converted to cDNA and the cDNA labeled with a fluorescent probe. The labeled cDNA is hybridized on the microarray and the fluorescence bound to each array is then quantified. Recently, these technologies have been applied to mitochondrial disease patient tissues and the presence of coordinate changes in mitochondrial gene expression confirmed.     

Mitochondrial involvement in Alzheimer's disease

The causes of most neurodegenerative diseases, including sporadic Alzheimer's disease (AD), remain enigmatic. There is, however, increasing evidence implicating mitochondrial dysfunction resulting from deafferentiation of disconnected neural circuits in the pathogenesis of energy deficit in AD. The patterns of reduced expression of both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) encoded genes is consistent with a physiological down-regulation of the mitochondrial respiratory chain in response to reduced neuronal activity. On the other hand, the role(s) of somatic cell or maternally inherited mtDNA mutations in the pathogenesis of mitochondrial dysfunction in AD are still controversial.     

Molecular Evolution of Cytochrome c Oxidase in High-Performance Fish (Teleostei: Scombroidei)

The 13 peptides encoded by vertebrate mitochondrial DNA (mtDNA) are essential subunits of oxidative phosphorylation (OXPHOS) enzymes. These genes normally experience purifying selection and also coevolve with nuclear-encoded subunits of OXPHOS complexes. However, the role of positive selection on mtDNA evolution is still unclear, as most examples of intergenomic coevolution appear to be the result of compensation by nuclear-encoded genes for mildly deleterious mtDNA mutations, and not simultaneous positive selection in both genomes. Organisms that have experienced strong selective pressures to increase aerobic capacity or adapt to changes in thermal environment may be better candidates in which to examine the impact of positively selected changes on mtDNA evolution. The tuna (suborder Scombroidei, family Scombridae) and billfish (suborder Scombroidei, families Xiphiidae and Istiophoridae) are highly aerobic fish with multiple specializations in muscle energetics, including a high mitochondrial content and regional endothermy. We examined the role of positively selected mtDNA substitutions in the production of these unique phenotypes. Focusing on a catalytic subunit of cytochrome c oxidase (COX II), we found that the rate ratio of nonsynonymous (d(N); amino acid changing)-to-synonymous (d(S); silent) substitutions was not increased in lineages leading to the tuna but was significantly increased in the lineage preceding the billfish. Furthermore, there are a number of individual positively selected sites that, when mapped onto the COX crystal structure, appear to interact with other COX subunits and may affect OXPHOS function and regulation in billfish.     

Multifactorial nature of high frequency of mitochondrial DNA mutations in somatic mammalian cells

The high frequency of mitochondrial DNA (mtDNA) mutations in somatic mammalian cells, which is more than two orders of magnitude higher than the mutation frequency of nuclear DNA (nDNA), significantly correlates with development of a variety of mitochondrial diseases (neurodegenerative diseases, cardiomyopathies, type II diabetes mellitus, cancer, etc.). A direct cause—consequence relationship has been established between mtDNA mutations and aging phenotypes in mammals. However, the unclear nature of the high frequency of mtDNA mutations requires a comprehensive consideration of factors that contribute to this phenomenon: oxidative stress, features of structural organization and repair of the mitochondrial genome, ribonucleotide reductase activity, replication errors, mutations of nuclear genes encoding mitochondrial proteins.     

Molecular basis of mitochondrial DNA disease

Mitochondrial ATP production via oxidative phosphorylation (OXPHOS) is essential for normal function and maintenance of human organ systems. Since OXPHOS biogenesis depends on both nuclear- and mitochondrial-encoded gene products, mutations in both genomes can result in impaired electron transport and ATP synthesis, thus causing tissue dysfunction and, ultimately, human disease. Over 30 mitochondrial DNA (mtDNA) point mutations and over 100mtDNA rearrangements have now been identified as etiological factors in human disease. Because of the unique characteristics of mtDNA genetics, genotype/phenotype associations are often complex and disease expression can be influenced by a number of factors, including the presence of nuclear modifying or susceptibility alleles. Accordingly, these mutations result in an extraordinarily broad spectrum of clinical phenotypes ranging from systemic, lethal pediatric disease to late-onset, tissue-specific neurodegenerative disorders. In spite of its complexity, an understanding of the molecular basis of mitochondrial DNA disease will be essential as the first step toward rationale and permanent curative therapy.     

Mitogroup: continent-specific clusters of mitochondrial OXPHOS complexes based on nuclear non-synonymous polymorphisms

OXPHOS polymorphisms are known to be population specific and to influence disease. Previous studies have focused on mtDNA polymorphisms. Based on a world sampling of 629 unrelated individuals, we have now studied the polymorphisms of the 80 genes encoding OXPHOS nuclear subunits. We have shown that (i) amino-acid replacement frequencies are significantly correlated with their pathogenicity probability, and (ii) populations can be distinguished based only on amino-acid replacements in nuclear encoded OXPHOS subunits. These results are congruent with the major mtDNA haplogroups, which suggests that OXPHOS complexes are different across the populations in both nuclear and in mitochondrial encoded subunits.     

The expanding spectrum of nuclear gene mutations in mitochondrial disorders

Our understanding of the molecular basis of mitochondrial disorders has come primarily from the discovery of an expanding number of mutations of mtDNA. However, a variety of recent observations indicate that many syndromes are due to abnormalities in nuclear genes related to oxidative-phosphorylation (OXPHOS). Nuclear genes encode hundreds of proteins involved in mitochondrial OXPHOS. Nevertheless, the identification of these genes has proceeded at a much slower pace, compared with the discovery and characterization of mtDNA mutations. This scenario is rapidly changing, thanks to the discovery of several OXPHOS-related human genes, and to the identification of mutations responsible for different clinical syndromes.     

Electrophoresis techniques to investigate defects in oxidative phosphorylation

Defects in mitochondrial oxidative phosphorylation (OXPHOS) are a frequent cause of severe inherited metabolic disorders and also contribute to aging. The OXPHOS system constitutes five multi-subunit complexes embedded in the mitochondrial inner membrane. Correct function of this system requires proper assembly of the 80 proteins in the complexes, as well as numerous assembly factors. Blue native electrophoresis has become a crucial tool to investigate OXPHOS-related defects in mitochondrial disease patients. In addition, OXPHOS-assembly profiles can be obtained by two dimensional blue native/SDS gel electrophoresis, which provides additional information for identifying disease-causing mutations and insight in the role of specific proteins in the biogenesis of the OXPHOS system. Here we provide a practical guide on how to set-up the basic technique to study OXPHOS defects in patient-derived cells and tissues.     

Mitochondrial medicine

After reviewing the history of mitochondrial diseases, I follow a genetic classification to discuss new developments and old conundrums. In the field of mitochondrial DNA (mtDNA) mutations, I argue that we are not yet scraping the bottom of the barrel because: (i) new mtDNA mutations are still being discovered, especially in protein-coding genes; (ii) the pathogenicity of homoplasmic mutations is being revisited; (iii) some genetic dogmas are chipped but not broken; (iv) mtDNA haplotypes are gaining interest in human pathology; (v) pathogenesis is still largely enigmatic. In the field of nuclear DNA (nDNA) mutations, there has been good progress in our understanding of disorders due to faulty intergenomic communication. Of the genes responsible for multiple deletions and depletion of mtDNA, mutations in POLG have been associated with a great variety of clinical phenotypes in humans and to precocious aging in mice. Novel pathogenetic mechanisms include alterations in the lipid milieu of the inner mitochondrial membrane and mutations in genes controlling mitochondrial motility, fission, and fusion.     

Mitonuclear Coevolution,but not Nuclear Compensation,Drives Evolution of OXPHOS Complexes in Bivalves

In Metazoa, four out of five complexes involved in oxidative phosphorylation (OXPHOS) are formed by subunits encoded by both the mitochondrial (mtDNA) and nuclear (nuDNA) genomes, leading to the expectation of mitonuclear coevolution. Previous studies have supported coadaptation of mitochondria-encoded (mtOXPHOS) and nuclear-encoded OXPHOS (nuOXPHOS) subunits, often specifically interpreted with regard to the “nuclear compensation hypothesis,” a specific form of mitonuclear coevolution where nuclear genes compensate for deleterious mitochondrial mutations due to less efficient mitochondrial selection. In this study, we analyzed patterns of sequence evolution of 79 OXPHOS subunits in 31 bivalve species, a taxon showing extraordinary mtDNA variability and including species with “doubly uniparental” mtDNA inheritance. Our data showed strong and clear signals of mitonuclear coevolution. NuOXPHOS subunits had concordant topologies with mtOXPHOS subunits, contrary to previous phylogenies based on nuclear genes lacking mt interactions. Evolutionary rates between mt and nuOXPHOS subunits were also highly correlated compared with non-OXPHO-interacting nuclear genes. Nuclear subunits of chimeric OXPHOS complexes (I, III, IV, and V) also had higher dN/dS ratios than Complex II, which is formed exclusively by nuDNA-encoded subunits. However, we did not find evidence of nuclear compensation: mitochondria-encoded subunits showed similar dN/dS ratios compared with nuclear-encoded subunits, contrary to most previously studied bilaterian animals. Moreover, no site-specific signals of compensatory positive selection were detected in nuOXPHOS genes. Our analyses extend the evidence for mitonuclear coevolution to a new taxonomic group, but we propose a reconsideration of the nuclear compensation hypothesis.