Mitochondrial dysfunction in cancer

Nearly a century of scientific research has revealed a number of notable differences in the structure and function of mitochondria between normal and cancer cells, including differences in metabolic activity, molecular composition, and mtDNA sequence. This article reviews several of these differences and discusses their clinical implications, especially with regard to the use of mitochondria as biomarkers for early detection of cancer, or as unique cellular targets for novel and selective anti-cancer agents.     

Mitochondrial dysfunction impairs tumor suppressor p53 expression/function

Recently, mitochondria have been suggested to act in tumor suppression. However, the underlying mechanisms by which mitochondria suppress tumorigenesis are far from being clear. In this study, we have investigated the link between mitochondrial dysfunction and the tumor suppressor protein p53 using a set of respiration-deficient (Res(-)) mammalian cell mutants with impaired assembly of the oxidative phosphorylation machinery. Our data suggest that normal mitochondrial function is required for γ-irradiation (γIR)-induced cell death, which is mainly a p53-dependent process. The Res(-) cells are protected against γIR-induced cell death due to impaired p53 expression/function. We find that the loss of complex I biogenesis in the absence of the MWFE subunit reduces the steady-state level of the p53 protein, although there is no effect on the p53 protein level in the absence of the ESSS subunit that is also essential for complex I assembly. The p53 protein level was also reduced to undetectable levels in Res(-) cells with severely impaired mitochondrial protein synthesis. This suggests that p53 protein expression is differentially regulated depending upon the type of electron transport chain/respiratory chain deficiency. Moreover, irrespective of the differences in the p53 protein expression profile, γIR-induced p53 activity is compromised in all Res(-) cells. Using two different conditional systems for complex I assembly, we also show that the effect of mitochondrial dysfunction on p53 expression/function is a reversible phenomenon. We believe that these findings will have major implications in the understanding of cancer development and therapy.     

Mitochondrial DNA nucleoids determine mitochondrial genetics and dysfunction

Mitochondrial DNA plays a crucial role in cellular homeostasis; however, the molecular mechanisms underlying mitochondrial DNA inheritance and propagation are only beginning to be understood. To ensure the distribution and propagation of the mitochondrial genome, mitochondrial DNA is packaged into macromolecular assemblies called nucleoids, composed of one or more copies of mitochondrial DNA and associated proteins. We review current research on the mitochondrial nucleoid, including nucleoid-associated proteins, nucleoid dynamics within the cell, potential mechanisms to ensure proper distribution of nucleoids, and the impact of nucleoid organization on mitochondrial dysfunction. The nucleoid is the molecular organizing unit of mitochondrial genetics, and is the site of interactions that ultimately determine the bioenergetic state of the cell as a whole. Current and future research will provide essential insights into the molecular and cellular interactions that cause bioenergetic crisis, and yield clues for therapeutic rescue of mitochondrial dysfunction.     

Mitochondrial Dysfunction and Permeability Transition in Osteosarcoma Cells Showing the Warburg Effect

Metabolic reprogramming in cancer is manifested by persistent aerobic glycolysis and suppression of mitochondrial function and is known as the Warburg effect. The Warburg effect contributes to cancer progression and is considered to be a promising therapeutic target. Understanding the mechanisms used by cancer cells to suppress their mitochondria may lead to development of new approaches to reverse metabolic reprogramming. We have evaluated mitochondrial function and morphology in poorly respiring LM7 and 143B osteosarcoma (OS) cell lines showing the Warburg effect in comparison with actively respiring Saos2 and HOS OS cells and noncancerous osteoblastic hFOB cells. In LM7 and 143B cells, we detected markers of the mitochondrial permeability transition (MPT), such as mitochondrial swelling, depolarization, and membrane permeabilization. In addition, we detected mitochondrial swelling in human OS xenografts in mice and archival human OS specimens using electron microscopy. The MPT inhibitor sanglifehrin A reversed MPT markers and increased respiration in LM7 and 143B cells. Our data suggest that the MPT may play a role in suppression of mitochondrial function, contributing to the Warburg effect in cancer.     

Glucose Modulates Respiratory Complex I Activity in Response to Acute Mitochondrial Dysfunction

Proper coordination between glycolysis and respiration is essential, yet the regulatory mechanisms involved in sensing respiratory chain defects and modifying mitochondrial functions accordingly are unclear. To investigate the nature of this regulation, we introduced respiratory bypass enzymes into cultured human (HEK293T) cells and studied mitochondrial responses to respiratory chain inhibition. In the absence of respiratory chain inhibitors, the expression of alternative respiratory enzymes did not detectably alter cell physiology or mitochondrial function. However, in permeabilized cells NDI1 (alternative NADH dehydrogenase) bypassed complex I inhibition, whereas alternative oxidase (AOX) bypassed complex III or IV inhibition. In contrast, in intact cells the effects of the AOX bypass were suppressed by growth on glucose, whereas those produced by NDI1 were unaffected. Moreover, NDI1 abolished the glucose suppression of AOX-driven respiration, implicating complex I as the target of this regulation. Rapid Complex I down-regulation was partly released upon prolonged respiratory inhibition, suggesting that it provides an “emergency shutdown” system to regulate metabolism in response to dysfunctions of the oxidative phosphorylation. This system was independent of HIF1, mitochondrial superoxide, or ATP synthase regulation. Our findings reveal a novel pathway for adaptation to mitochondrial dysfunction and could provide new opportunities for combatting diseases.     

Mitochondrial and metabolic alterations in cancer cells

Metabolic alterations have been observed in many cancer types. The deregulated metabolism has thus become an emerging hallmark of the disease, where the metabolism is frequently rewired to aerobic glycolysis. This has led to the concept of “metabolic reprogramming”, which has therefore been extensively studied. Over the years, it has been characterized the enhancement of aerobic glycolysis, where key mutations in some of the enzymes of the TCA cycle, and the increased glucose uptake, are used by cancer cells to achieve a “metabolic phenotype” useful to gain a proliferation advantage. Many studies have highlighted in detail the signaling pathways and the molecular mechanisms responsible for the glycolytic switch. However, glycolysis is not the only metabolic process that cancer cells rely on. Oxidative Phosphorylation (OXPHOS), gluconeogenesis or the beta-oxidation of fatty acids (FAO) may be involved in the development and progression of several tumors. In some cases, these metabolisms are even more crucial than aerobic glycolysis for the tumor survival. This review will focus on the contribution of these alterations of metabolism to the development and survival of cancers. We will also analyze the molecular mechanisms by which the balance between these metabolic processes may be regulated, as well as some of the therapeutical approaches that can derive from their study.     

Replication of mitochondrial DNA occurs throughout the mitochondria of cultured human cells

Replication of mitochondrial DNA (mtDNA) is dependent on nuclear-encoded factors. It has been proposed that this reliance may exert spatial restrictions on the sites of mtDNA replication within the cytoplasm, as a previous study only detected mtDNA synthesis in perinuclear mitochondria. We have studied mtDNA replication in situ in a variety of human cell cultures labeled with 5-bromo-2'-deoxyuridine. In contrast to what has been reported, mtDNA synthesis was detected at multiple sites throughout the mitochondrial network following short pulses with bromodeoxyuridine. Although no bromodeoxyuridine incorporation was observed in anuclear platelets, incorporation into mtDNA of fibroblasts that had been enucleated 2 h prior to labeling was readily detectable. Blotting experiments indicated that the bromodeoxyuridine incorporation into mtDNA observed in situ represents replication of the entire mtDNA molecule. The studies also showed that replication of mtDNA occurred at any stage of the cell cycle in proliferating cells and continued in postmitotic cells, although at a lower level. These results demonstrate that mtDNA replication is not restricted to mitochondria in the proximity of the nucleus and imply that all components of the replication machinery are available at sufficient levels throughout the mitochondrial network to permit mtDNA replication throughout the cytoplasm.     

Inhibition of alanine aminotransferase in silico and in vivo promotes mitochondrial metabolism to impair malignant growth

Cancer cells commonly exhibit increased nonoxidative D-glucose metabolism whereas induction of mitochondrial metabolism may impair malignant growth. We have first used an in silico method called elementary mode analysis to identify inhibition of ALAT (L-alanine aminotransferase) as a putative target to promote mitochondrial metabolism. We then experimentally show that two competitive inhibitors of ALAT, L-cycloserine and β-chloro-L-alanine, inhibit L-alanine production and impair D-glucose uptake of LLC1 Lewis lung carcinoma cells. The latter inhibition is linked to an initial energy deficit, as quantified by decreased ATP content, which is then followed by an activation of AMP-activated protein kinase and subsequently increased respiration rates and mitochondrial production of reactive oxygen species, culminating in ATP replenishment in ALAT-inhibited LLC1 cells. Moreover, we observe altered phosphorylation of p38 MAPK (mitogen-activated protein kinase 14), ERK (extracellular signal-regulated kinase 1/2), and Rb1 (retinoblastoma 1) proteins, as well as decreased expression of Cdc25a (cell decision cycle 25 homolog A) and Cdk4 (cyclin-dependent kinase 4). Importantly, these sequelae of ALAT inhibition culminate in similarly reduced anchorage-dependent and anchorage-independent growth rates of LLC1 cells, together suggesting that inhibition of ALAT efficiently impairs cancer growth by counteracting the Warburg effect due to compensatory activation of mitochondrial metabolism.     

Essential Roles of Neutral Ceramidase and Sphingosine in Mitochondrial Dysfunction Due to Traumatic Brain Injury

In addition to immediate brain damage, traumatic brain injury (TBI) initiates a cascade of pathophysiological events producing secondary injury. The biochemical and cellular mechanisms that comprise secondary injury are not entirely understood. Herein, we report a substantial deregulation of cerebral sphingolipid metabolism in a mouse model of TBI. Sphingolipid profile analysis demonstrated increases in sphingomyelin species and sphingosine concurrently with up-regulation of intermediates of de novo sphingolipid biosynthesis in the brain. Investigation of intracellular sites of sphingosine accumulation revealed an elevation of sphingosine in mitochondria due to the activation of neutral ceramidase (NCDase) and the reduced activity of sphingosine kinase 2 (SphK2). The lack of change in gene expression suggested that post-translational mechanisms are responsible for the shift in the activities of both enzymes. Immunoprecipitation studies revealed that SphK2 is complexed with NCDase and cytochrome oxidase (COX) subunit 1 in mitochondria and that brain injury hindered SphK2 association with the complex. Functional studies showed that sphingosine accumulation resulted in a decreased activity of COX, a rate-limiting enzyme of the mitochondrial electron transport chain. Knocking down NCDase reduced sphingosine accumulation in mitochondria and preserved COX activity after the brain injury. Also, NCDase knockdown improved brain function recovery and lessened brain contusion volume after trauma. These studies highlight a novel mechanism of secondary TBI involving a disturbance of sphingolipid-metabolizing enzymes in mitochondria and suggest a critical role for mitochondrial sphingosine in promoting brain injury after trauma.     

Long-term depletion of cereblon induces mitochondrial dysfunction in cancer cells

Cereblon (CRBN) is a multi-functional protein that acts as a sub-strate receptor of the E3 ligase complex and a molecular chaperone. While CRBN is proposed to function in mitochondria, its specific roles are yet to be established. Here, we showed that knockdown of CRBN triggers oxidative stress and calcium overload in mitochondria, leading to disruption of mitochondrial membrane potential. Notably, long-term CRBN depletion using PROteolysis TArgeting Chimera (PROTAC) induced irreversible mitochondrial dysfunction, resulting in cell death. Our collective findings indicate that CRBN is required for mitochondrial homeostasis in cells.     

The role of compartmentalized signaling pathways in the control of mitochondrial activities in cancer cells

Mitochondria are the powerhouse organelles present in all eukaryotic cells. They play a fundamental role in cell respiration, survival and metabolism. Stimulation of G-protein coupled receptors (GPCRs) by dedicated ligands and consequent activation of the cAMP·PKA pathway finely couple energy production and metabolism to cell growth and survival. Compartmentalization of PKA signaling at mitochondria by A-Kinase Anchor Proteins (AKAPs) ensures efficient transduction of signals generated at the cell membrane to the organelles, controlling important aspects of mitochondrial biology. Emerging evidence implicates mitochondria as essential bioenergetic elements of cancer cells that promote and support tumor growth and metastasis. In this context, mitochondria provide the building blocks for cellular organelles, cytoskeleton and membranes, and supply all the metabolic needs for the expansion and dissemination of actively replicating cancer cells. Functional interference with mitochondrial activity deeply impacts on cancer cell survival and proliferation. Therefore, mitochondria represent valuable targets of novel therapeutic approaches for the treatment of cancer patients. Understanding the biology of mitochondria, uncovering the molecular mechanisms regulating mitochondrial activity andmapping the relevant metabolic and signaling networks operating in cancer cells will undoubtly contribute to create a molecular platform to be used for the treatment of proliferative disorders.Here, we will highlight the emerging roles of signaling pathways acting downstream to GPCRs and their intersection with the ubiquitin proteasome system in the control of mitochondrial activity in different aspects of cancer cell biology.     

Electron microscopy morphology of the mitochondrial network in human cancer

Mitochondria have been implicated in the process of carcinogenesis, which includes alterations of cellular metabolism and cell death pathways. The aim of this review is to describe and analyze the electron microscopy morphology of the mitochondrial network in human cancer. The structural mitochondrial alterations in human tumors are heterogeneous and not specific for any neoplasm. These findings could be representing an altered structural and functional mitochondrial network. The mitochondria in cancer cells, independently of histogenesis, predominantly are seen with lucent-swelling matrix associated with disarrangement and distortion of cristae and partial or total cristolysis and with condensed configuration in minor scale. Mitochondrial changes are associated with mitochondrial-DNA mutations, tumoral microenvironment conditions and mitochondrial fusion–fission disequilibrium. Functionally, the structural alterations suppose the presence of hypoxia-tolerant and hypoxia-sensitive cancer cells. Possibly, hypoxia-tolerant cells are related with mitochondrial condensed appearance and are competent to produce adequate amount of ATP by mitochondrial respiration. Hypoxia-sensitive cells are linked with lucent-swelling and cristolysis mitochondria profile and have an inefficient or null oxidative phosphorylation, which consequently use the glycolytic pathway to generate energy. Additionally, mitochondrial fragmentation is associated with apoptosis; however, alterations in the mitochondrial network are linked with the reduction in sensitivity to apoptosis induces and/or pro-apoptotic conditions. Pharmacological approaches designed to act on both glycolysis and oxidative phosphorylation can be considered as a new approach to selectively kill cancer cells.     

The human mitochondrial replication fork in health and disease

Mitochondria are organelles whose main function is to generate power by oxidative phosphorylation. Some of the essential genes required for this energy production are encoded by the mitochondrial genome, a small circular double stranded DNA molecule. Human mtDNA is replicated by a specialized machinery distinct from the nuclear replisome. Defects in the mitochondrial replication machinery can lead to loss of genetic information by deletion and/or depletion of the mtDNA, which subsequently may cause disturbed oxidative phosphorylation and neuromuscular symptoms in patients. We discuss here the different components of the mitochondrial replication machinery and their role in disease. We also review the mode of mammalian mtDNA replication.     

Mammalian MTHFD2L encodes a mitochondrial methylenetetrahydrofolate dehydrogenase isozyme expressed in adult tissues

Previous studies in our laboratory showed that isolated, intact adult rat liver mitochondria are able to oxidize the 3-carbon of serine and the N-methyl carbon of sarcosine to formate without the addition of any other cofactors or substrates. Conversion of these 1-carbon units to formate requires several folate-interconverting enzymes in mitochondria. The enzyme(s) responsible for conversion of 5,10-methylene-tetrahydrofolate (CH(2)-THF) to 10-formyl-THF in adult mammalian mitochondria are currently unknown. A new mitochondrial CH(2)-THF dehydrogenase isozyme, encoded by the MTHFD2L gene, has now been identified. The recombinant protein exhibits robust NADP(+)-dependent CH(2)-THF dehydrogenase activity when expressed in yeast. The enzyme is localized to mitochondria when expressed in CHO cells and behaves as a peripheral membrane protein, tightly associated with the matrix side of the mitochondrial inner membrane. The MTHFD2L gene is subject to alternative splicing and is expressed in adult tissues in humans and rodents. This CH(2)-THF dehydrogenase isozyme thus fills the remaining gap in the pathway from CH(2)-THF to formate in adult mammalian mitochondria.     

Cellular responses to replication stress: Implications in cancer biology and therapy

DNA replication is essential for cell proliferation. Any obstacles during replication cause replication stress, which may lead to genomic instability and cancer formation. In this review, we summarize the physiological DNA replication process and the normal cellular response to replication stress. We also outline specialized therapies in clinical trials based on current knowledge and future perspectives in the field.     

Metformin Increases Mitochondrial Energy Formation in L6 Muscle Cell Cultures

A popular hypothesis for the action of metformin, the widely used anti-diabetes drug, is the inhibition of mitochondrial respiration, specifically at complex I. This is consistent with metformin stimulation of glucose uptake by muscle and inhibition of gluconeogenesis by liver. Yet, mitochondrial inhibition is inconsistent with metformin stimulation of fatty acid oxidation in both tissues. In this study, we measured mitochondrial energy production in intact cells adapting an in vivo technique of phosphocreatine (PCr) formation following energy interruption (“PCr recovery”) to cell cultures. Metformin increased PCr recovery from either dinitrophenol (DNP) or azide in L6 cells. We found that metformin alone had no effect on cell viability as measured by total ATP concentration, trypan blue exclusion, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction. However, treatments with low concentrations of DNP or azide reversibly decreased ATP concentration. Metformin increased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction during recovery from either agent. Viability measured by trypan blue exclusion indicated that cells were intact under these conditions. We also found that metformin increased free AMP and, to a smaller extent, free ADP concentrations in cells, an action that was duplicated by a structurally unrelated AMP deaminase inhibitor. We conclude that, in intact cells, metformin can lead to a stimulation of energy formation, rather than an inhibition.     

Complexities and pitfalls in analyzing and interpreting mitochondrial DNA content in human cancer

Mutations in the human mitochondrial genome have been observed in all types of human cancer, indicating that mutations might contribute to tumorigenesis, metastasis, recurrence, or drug response. This possibility is appealing because of the known shift from oxidative metabolism to glycolysis, known as the Warburg effect, that occurs in malignancy. Mitochondrial DNA (mtDNA) mutations could either be maternally inherited and predispose to cancer (germ line mutations) or occur sporadically in the mtDNA of specific tissues (tissue- or tumor-specific somatic mutations) and contribute to the tumor initiation and progression process. High-throughput sequencing technologies now enable comprehensive detection of mtDNA variation in tissues and bodily fluids, with the potential to be used as an early detection tool that may impact the treatment of cancer. Here, we discuss insights into the roles of mtDNA mutations in carcinogenesis, highlighting the complexities involved in the analysis and interpretation of mitochondrial genomic content, technical challenges in studying their contribution to pathogenesis, and the value of mtDNA mutations in developing early detection, diagnosis, prognosis, and therapeutic strategies for cancer.     

Mne1 is a novel component of the mitochondrial splicing apparatus responsible for processing of a COX1 group I intron in yeast

Saccharomyces cerevisiae cells lacking Mne1 are deficient in intron splicing in the gene encoding the Cox1 subunit of cytochrome oxidase but contain wild-type levels of the bc(1) complex. Thus, Mne1 has no role in splicing of COB introns or expression of the COB gene. Northern experiments suggest that splicing of the COX1 aI5β intron is dependent on Mne1 in addition to the previously known Mrs1, Mss116, Pet54, and Suv3 factors. Processing of the aI5β intron is similarly impaired in mne1Δ and mrs1Δ cells and overexpression of Mrs1 partially restores the respiratory function of mne1Δ cells. Mrs1 is known to function in the initial transesterification reaction of splicing. Mne1 is a mitochondrial matrix protein loosely associated with the inner membrane and is found in a high mass ribonucleoprotein complex specifically associated with the COX1 mRNA even within an intronless strain. Mne1 does not appear to have a secondary function in COX1 processing or translation, because disruption of MNE1 in cells containing intronless mtDNA does not lead to a respiratory growth defect. Thus, the primary defect in mne1Δ cells is splicing of the aI5β intron in COX1.