Targeting mitochondria: Strategies,innovations and challenges: The future of medicine will come through mitochondria

Mitochondrial dysfunction has been associated with the aging process and a large variety of human disorders, such as cardiovascular and neurodegenerative diseases, cancer, migraine, infertility, kidney and liver diseases, toxicity of drugs and many more. It is well recognized that the physiological role of mitochondria widely exceeds that of solely being the biochemical power plant of our cells. Over the recent years, mitochondria have become an interesting target for drug therapy, and the research field aimed at “targeting mitochondria” is active and expanding as witnessed by this already third edition of the world congress on targeting mitochondria. It is becoming a necessity and an urge to know why and how to target mitochondria with bioactive molecules and drugs in order to treat and prevent mitochondria-based pathologies and chronic diseases. This special issue covers a variety of new strategies and innovations as well as clinical applications in mitochondrial medicine.     

Mitochondrial groups of germinal cells of teleost Cyprinidae. II. High resolution autoradiographic study of the incorporation of 3H phenylalanine and 3H uridine

A high resolution autoradiographic study of the incorporation of tritiated uridine and amino acids by the mitochondrial groups, which are typical of most of germ cells at the beginning of gametogenesis, has been made on tench spermatocytes. By this technique, we confirm the partially proteinacious composition of the intermitochondrial “cement”; and for the first time, the presence of RNA in the “cement” is demonstrated by autoradiography. Moreover, a study of the kinetics of the incorporation of both precursors makes likely the hypothesis that at least a part of the “cement” derived from nucleocytoplasmic transfer. Since the biogenesis of mitochondria results from the complementary functioning of the two protein synthesic systems, the cytoplasmic one, which is preponderant, and the mitochondrial one, the mitochondrial groups seem to be the direct visualization of the contribution of the nuclear genome to the edification of new mitochondria.     

Quality control of mitochondria during aging: Is there a good and a bad side of mitochondrial dynamics?

Maintenance of functional mitochondria is essential in order to prevent degenerative processes leading to disease and aging. Mitochondrial dynamics plays a crucial role in ensuring mitochondrial quality but may also generate and spread molecular damage through a population of mitochondria. Computational simulations suggest that this dynamics is advantageous when mitochondria are not or only marginally damaged. In contrast, at a higher degree of damage, mitochondrial dynamics may be disadvantageous. Deceleration of fusion‐fission cycles could be one way to adapt to this situation and to delay a further decline in mitochondrial quality. However, this adaptive response makes the mitochondrial network more vulnerable to additional molecular damage. The “mitochondrial infectious damage adaptation” (MIDA) model explains a number of inconsistent and counterintuitive data such as the “clonal expansion” of mutant mitochondrial DNA. We propose that mitochondrial dynamics is a double‐edged sword and suggest ways to test this experimentally.     

Biochemical studies on sub-fractions of rat liver mitochondria

Highly purified mitochondria from rat liver were separated into six sub-fractions by differential centrifugation. The sub-fractions represent a spectrum from “heavy” to “very light” mitochondria. Enzymes representative of mitochondrial compartments were assayed to see whether functional differences occurred among the various mitochondrial sub-fractions. Respiratory control and NADH oxidase activity, both of which are indicators of mitochondrial structural integrity, were also measured. An enzyme marker for endoplasmic reticulum (glucose-6-phosphatase, G-6-Pase) was also assayed. Specific activities for monoamine oxidase (outer membrane marker), cytochrome oxidase (inner membrane marker) and malate-cytochrome c reductase did not vary within experimental error in all sub-fractions; similarly, for respiratory control and NADH oxidase activity. Malate dehydrogenase, a component of malate-cytochrome c reductase is located within the matrix surrounded by the inner membrane. Specific activity of adenylate kinase (located between the outer and inner membrane) decreased markedly from the “heavy” mitochondria to the “very light” fractions. Specific activity for G-6-Pase, very low in the “heavy” fractions, increased markedly in the “light” to “very light” fractions. Isopycnic density centrifugation on a linear sucrose density gradient of each of the fractions indicated that the correlation coefficient for the sucrose concentrations at which cytochrome oxidase and G-6-Pase activities peaked was 0.995. Thus the “light” to “very light” mitochondria may represent mitochondria whose outer membrane is still contiguous with the endoplasmic reticulum. Microsomes containing the endoplasmic reticulum peaked on the gradient at a significantly lower sucrose concentration than any of the mitochondrial sub-fractions. A buoyant effect of endoplasmic reticulum still attached to any of the mitochondrial sub-fractions would be expected to lower the density of attached mitochondria and thus give rise to “light” and “very light” mitochondria.     

Microbiota and mitobiota. Putting an equal sign between mitochondria and bacteria

The recent revival of old theories and setting them on modern scientific rails to a large extent are also relevant to mitochondrial science. Given the widespread belief that mitochondria are symbionts of ancient bacterial origin, the processes inherent to mitochondrial physiology can be revised based on their comparative analysis with possible involvement of bacteria. Such comparison combined with discussion of the role of microbiota in pathogenesis allows discussion of the role of “mitobiota” (we introduce this term) as the combination of different phenotypic manifestations of mitochondria in the organism reflecting pathological changes in the mitochondrial genome. When putting an equal sign between mitochondria and bacteria, we find similarity between the mitochondrial and bacterial theories of cancer. The presence of the term “bacterial infection” suggests “mitochondrial infection”, and mitochondrial (oxidative) theory of aging can in some way be transformed into a “bacterial theory of aging”. The possible existence of such processes and the data confirming their presence are discussed in this review. If such a comparison has the right to exist, the homeostasis of “mitobiota” is of not lesser physiological importance than homeostasis of microbiota, which has been so intensively discussed recently.     

Musical chairs during mitophagy

Mitophagy, or the autophagic degradation of mitochondria, is thought to be important in mitochondrial quality control, and hence in cellular physiology. Defects in mitophagy correlate with late onset pathologies and aging. Here, we discuss recent results that shed light on the interrelationship between mitophagy and mitochondrial dynamics, based on proteomic analyses of protein dynamics in wild-type and mutant cells. These studies show that different mitochondrial matrix proteins undergo mitophagy at different rates, and that the rate differences are affected by mitochondrial dynamics. These results are consistent with models in which phase separation within the mitochondrial matrix leads to unequal segregation of proteins during mitochondrial fission. Repeated fusion and fission cycles may thus lead to “distillation” of components that are destined for degradation.     

Identification of two discrete states of energized mitochondria: Experiments on single mitochondria

Dynamic phase microscopy was used to measure the refractivity of a single mitochondrion. Our previous studies showed that application of an electric potential to artificial and natural mitochondrial membranes sharply increases their refractivity. Under the conditions of proton pump activity, the refractivity of a single mitochondrion is 2 to 4 times higher than an average refractivity of deenergized mitochondria. This study demonstrates that the membrane potential of energized mitochondria varies depending on environmental conditions and is controlled by the mitochondrial osmoregulation system. The refractivity of energized mitochondria, i.e., the difference between the refraction indexes of a single mitochondrion and the medium, is 0.02 ± 0.01, i.e., several times lower than that of the energized mitochondria whose membranes bear an electric charge. Earlier it was shown that refractivity of model multilayer systems formed from purified natural lecithin depends linearly on the electric field strength. These data point to a relationship between the refractivity of a single mitochondrion and the membrane potential generated during operation of the proton pump. Under normal conditions (250 mOsm), the mitochondrion behaves as a dynamic system oscillating on a minute scale between two functional states with different values of the refractivity index and different membrane potentials. The transition time is 10–30 s; the lifetime of both states is several minutes. The histograms reflecting the distribution of refractivities of single energized mitochondria within a population (n = 20–30) revealed the presence of two independent peaks (fractions II and III) with average refractivity values of 0.05 ± 0.01 and 0.09 ± 0.01, respectively; these fractions correspond to two long-lived states of mitochondria. However, under hypotonic conditions (120 mOsm), only one (“static”) state was identified, in which oscillations were absent and the refractivity of the overall mitochondrial population does not exceed 0.05 ± 0.01 (fraction II). Studies on mitoplast showed that values of refractivity are related to the inner mitochondrial membrane. It is inferred from these data that there exist two discrete states of mitochondria. Analysis of low-amplitude fluctuations of the refractivity of single mitochondria revealed the presence of frequency components at 1–3 Hz, presumably generated in response to non-uniform functioning of mitochondrial proton pumps. It is suggested that frequency components at 1.8-2.6 Hz are more characteristic of the ATPase pump, while the 1–1.3 Hz frequencies predominate during the functioning of respiratory proton pumps.     

Sodium—A Functional Plant Nutrient

Plant scientists usually classify plant mineral nutrients based on the concept of “essentiality” defined by Arnon and Stout as those elements necessary to complete the life cycle of a plant. Certain other elements such as Na have a ubiquitous presence in soils and waters and are widely taken up and utilized by plants, but are not considered as plant nutrients because they do not meet the strict definition of “essentiality.” Sodium has a very specific function in the concentration of carbon dioxide in a limited number of C₄ plants and thus is essential to these plants, but this in itself is insufficient to generalize that Na is essential for higher plants. The unique set of roles that Na can play in plant metabolism suggests that the basic concept of what comprises a plant nutrient should be reexamined. We contend that the class of plant mineral nutrients should be comprised not only of those elements necessary for completing the life cycle, but also those elements which promote maximal biomass yield and/or which reduce the requirement (critical level) of an essential element. We suggest that nutrients functioning in this latter manner should be termed “functional nutrients.” Thus plant mineral nutrients would be comprised of two major groups, “essential nutrients” and “functional nutrients.” We present an array of evidence and arguments to support the classification of Na as a “functional nutrient,” including its requirement for maximal biomass growth for many plants and its demonstrated ability to replace K in a number of ways, such as being an osmoticium for cell enlargement and as an accompanying cation for long-distance transport. Although in this paper we have only attempted to make the case for Na being a “functional nutrient,” other elements such as Si and Se may also confirm to the proposed category of “functional nutrients.”     

Regulation of mitochondrial matrix volume

Mitochondrial volume homeostasis is a housekeeping cellular function essential for maintaining the structural integrity of the organelle. Changes in mitochondrial volume have been associated with a wide range of important biological functions and pathologies. Mitochondrial matrix volume is controlled by osmotic balance between cytosol and mitochondria. Any dysbalance in the fluxes of the main intracellular ion, potassium, will thus affect the osmotic balance between cytosol and the matrix and promote the water movement between these two compartments. It has been hypothesized that activity of potassium efflux pathways exceeds the potassium influx in functioning mitochondria and that potassium concentration in matrix could be actually lower than in cytoplasm. This hypothesis provides a clear-cut explanation for the mitochondrial swelling observed after mitochondrial depolarization, mitochondrial calcium overload, or opening of permeability transition pore. It should also be noted that the rate of water flux into or out of the mitochondrion is determined not only by the osmotic gradient that acts as the driving force for water transport but also by the water permeability of the inner membrane. Recent data suggest that the mitochondrial inner membrane has also specific water channels, aquaporins, which facilitate water movement between cytoplasm and matrix. This review discusses different phases of mitochondrial swelling and summarizes the potential effects of mitochondrial swelling on cell function. potassium homeostasis; depolarization; mitochondrial swelling     

Energy transfer in "parasitic" cancer metabolism

It is now widely recognized that the tumor microenvironment promotes cancer cell growth and metastasis via changes in cytokine secretion and extracellular matrix remodeling. However, the role of tumor stromal cells in providing energy for epithelial cancer cell growth is a newly emerging paradigm. For example, we and others have recently proposed that tumor growth and metastasis is related to an energy imbalance. Host cells produce energy-rich nutrients via catabolism (through autophagy, mitophagy, and aerobic glycolysis), which are then transferred to cancer cells to fuel anabolic tumor growth. Stromal cell-derived L-lactate is taken up by cancer cells and is used for mitochondrial oxidative phosphorylation (OXPHOS) to produce ATP efficiently. However, “parasitic” energy transfer may be a more generalized mechanism in cancer biology than previously appreciated. Two recent papers in Science and Nature Medicine now show that lipolysis in host tissues also fuels tumor growth. These studies demonstrate that free fatty acids produced by host cell lipolysis are re-used via beta-oxidation (beta-OX) in cancer cell mitochondria. Thus, stromal catabolites (such as lactate, ketones, glutamine and free fatty acids) promote tumor growth by acting as high-energy onco-metabolites. As such, host catabolism, via autophagy, mitophagy and lipolysis, may explain the pathogenesis of cancer-associated cachexia and provides exciting new druggable targets for novel therapeutic interventions. Taken together, these findings also suggest that tumor cells promote their own growth and survival by behaving as a “parasitic organism.” Hence, we propose the term “Parasitic Cancer Metabolism” to describe this type of metabolic coupling in tumors. Targeting tumor cell mitochondria (OXPHOS and beta-OX) would effectively uncouple tumor cells from their hosts, leading to their acute starvation. In this context, we discuss new evidence that high-energy onco-metabolites (produced by the stroma) can confer drug resistance. Importantly, this metabolic chemo-resistance is reversed by blocking OXPHOS in cancer cell mitochondria with drugs like Metformin, a mitochondrial “poison.” In summary, parasitic cancer metabolism is achieved architecturally by dividing tumor tissue into at least two well-defined opposing “metabolic compartments:” catabolic and anabolic.     

Mitochondrial models of pathologies with oxidative stress. Efficiency of alkalization to reduce mitochondrial damage

Previously, we developed a method to monitor the development of oxidative stress in isolated liver mitochondria. The method is based on recording of membrane potential changes in response to sequential introduction of low concentrations (5–20 μM) of tert-butyl hydroperoxide (tBHP). It allows monitoring of the extent of amplification or attenuation of oxidative stress caused by external influences (changes in incubation conditions, additions of biologically active substances). Based on this method, we created a mitochondrial model for the study and improvement of treatment of pathologies associated with oxidative stress. The following two processes were simulated in the experiments: 1) introduction of desferal for treatment of serious diseases caused by cell overload with iron (high desferal concentrations were shown to suppress mitochondrial energetics); 2) efficiency of alkalization to reduce mitochondrial damage induced by oxidative stress. The experiments have shown that even a small increase in pH (alkalization) increases the amount of tBHP that can be added to mitochondria before the MPTP (“mitochondrial permeability transition pore”) is induced. The effect of alkalization was shown to be close to the effect of cyclosporin A in the pH range 7.2–7.8. The mechanism of the similarities of these effects in the organism and in mitochondrial suspensions is explained by the increase in toxic reactive oxygen species in both systems under oxidative stress.     

Nuclear mitochondrial pseudogenes

As has been demonstrated recently, the transfer of genetic material from mitochondria to the nucleus and its integration into the nuclear genome is a continuous and dynamic process. Fragments of mitochondrial DNA (mtDNA) are incorporated in the nuclear genome as noncoding sequences, which are called nuclear mitochondrial pseudogenes (NUMT pseudogenes or NUMT inserts). In various eukaryotes, NUMT pseudogenes are distributed through different chromosomes to form a “library” of mtDNA fragments, providing important information on genome evolution. The escape of mtDNA from mitochondria is mostly associated with mitochondrial damage and mitophagy. Fragments of mtDNA may be integrated into nuclear DNA (nDNA) during repair of double-strand breaks (DSBs), which are caused by endogenous or exogenous agents. DSB repair of nDNA with a capture of mtDNA fragments may occur via nonhomologous end joining or a similar mechanism that involves microhomologous terminal sequences. An analysis of the available data makes it possible to suppose that the NUMT pseudogene formation rate depends on the DSB rate in nDNA, the activity of the repair systems, and the number of mtDNA fragments leaving organelles and migrating into the nucleus. Such situations are likely after exposure to damaging agents, first and foremost, ionizing radiation. Not only do new NUMT pseudogenes change the genome structure in the regions of their integration, but they may also have a significant impact on the actualization of genetic information. The de novo integration of NUMT pseudogenes in the nuclear genome may play a role in various pathologies and aging. NUMT pseudogenes may cause errors in PCR-based analyses of free mtDNA as a component of total cell DNA because of their coamplification.     

Mitochondrial functional complementation in mitochondrial DNA-based diseases

Mitochondria exist in networks that are continuously remodeled through fusion and fission. Why do individual mitochondria in living cells fuse and divide continuously? Protein machinery and molecular mechanism for the dynamic nature of mitochondria have been almost clarified. However, the biological significance of the mitochondrial fusion and fission events has been poorly understood, although there is a possibility that mitochondrial fusion and fission are concerned with quality controls of mitochondria. trans-mitochondrial cell and mouse models possessing heteroplasmic populations of mitochondrial DNA (mtDNA) haplotypes are quite efficient for answering this question, and one of the answers is “mitochondrial functional complementation” that is able to regulate respiratory function of individual mitochondria according to “one for all, all for one” principle. In this review, we summarize the observations about mitochondrial functional complementation in mammals and discuss its biological significance in pathogeneses of mtDNA-based diseases.     

TOPIARY PRUNING AND WEIGHTING REINFORCE AN AFRICAN ORIGIN FOR THE HUMAN MITOCHONDRIAL DNA TREE

A method is presented for removing recent homoplastic events from a phylogenetic tree. This “topiary pruning” method produces a series of progressively modified duplicates of the original set of data, from which more and more of the most recent substitutions have been removed. The edited sets of data have increased amounts of information per remaining taxon, while similar but randomized data sets subjected to topiary pruning do not. The ability of topiary pruning to “unscramble” artificial data sets that have high levels of homoplasy is demonstrated, and is shown to be similar in its effects to the weighting method of Kluge and Farris (1969), although with the additional advantage of reducing the number of taxa to the point where bootstrapping is feasible. Pruning and weighting used together produce closer approximations to the “true” tree than either method used separately. It is further shown that in these artificial data sets midpoint rooting is more likely to be accurate than outgroup rooting. When pruning and weighting are applied to the extensive sets of mitochondrial DNA data of Cann et al. (1987) and Vigilant et al. (1991), trees result that have deep branch points, some of which lead to entirely African branches. In the case of the Vigilant et al. data, the three African branches have bootstrap values between 0.94 and 1.0, and the consensus and bootstrap midpoint roots also have high bootstrap values and occur on these African branches near their junction. An African origin of the human mitochondrial tree is not proved by this approach, particularly since sequences from non-African groups are underrepresented in current data sets, but it is rendered more likely.     

The role of mitochondria in the oncogenic signal transduction

Mitochondria are intracellular organelles thought to have evolved from an alphaproteobacterium engulfed by the ancestor of the eukaryotic cell, an archeon, two billion years ago. Although mitochondria are frequently recognised as the “power plant” of the cell, the function of these organelles go beyond the simple generation of ATP. In fact, mounting evidence suggests that mitochondria are involved in several cellular processes, from regulation of cell death to signal transduction. Given this important role in cell physiology, mitochondrial dysfunction has been frequently associated with human diseases including cancer. Importantly, recent evidence suggests that mitochondrial function is directly regulated by oncogenes and tumour suppressors. However, the consequences of deregulation of mitochondrial function in tumour formation are still unclear. In this review, I propose that mitochondria play a pivotal role in shaping the oncogenic signalling cascade and that mitochondrial dysfunction, in some circumstances, is a required step for cancer transformation.     

Epidemiology of trace elements deficiencies in Belgian beef and dairy cattle herds

Selenium (Se), iodine (I), zinc (Zn) and copper (Cu) deficiencies in cattle have been reported in Europe. These deficiencies are often associated with diseases. The aim of the study was to assess trace element status in Belgian cattle herds showing pathologies and to compare them to healthy cattle herds. Eighty-two beef herds with pathologies, 11 healthy beef herds, 65 dairy herds with pathologies and 20 healthy dairy herds were studied during barn period. Blood and/or milk samples were taken in healthy animals. Plasma Zn, Cu, inorganic I (PII) and activity of glutathione peroxidase in erythrocytes (GPX) were assayed. In milk, I concentration was measured. Data about pathologies and nutrition in the herds were collected. According to defined thresholds, it appeared that a large proportion of deficient herds belonged to “sick” group of herds. This conclusion was supported by the mean value of trace elements and by the fact that a majority of individual values of trace elements was below the threshold. Dairy herds had mean values of trace elements higher than beef herds. More concentrates and minerals were used in healthy herds versus “sick” herds. These feed supplements were also used more often in dairy herds, compared to beef herds. Trace elements deficiencies are present in cattle herds in Belgium and are linked to diseases. Nutrition plays a major role in the trace elements status.     

New Data on Effects of SkQ1 and SkQT1 on Rat Liver Mitochondria and Yeast Cells

Mitochondria are involved in many processes in eukaryotic cells. They play a central role in energy conservation and participate in cell metabolism and signaling pathways. Mitochondria are the main source of reactive oxygen species, excessive generation of which provokes numerous pathologies and cell death. One of the most promising approaches to the attenuation of oxidative stress in mitochondria is the use of targeted (i.e., transported exclusively into mitochondria) lipophilic cationic antioxidants. These compounds offer advantages over conventional water-soluble antioxidants because they induce the so-called “mild uncoupling” and can prevent collapse of the membrane potential in low, nontoxic concentrations. A novel mitochondria-targeted antioxidant, SkQT1, was synthesized and tested within the framework of the research project guided by V. P. Skulachev. The results of these experiments were initially reported in 2013; however, one publication was not able to accommodate all the data on the SkQT1 interactions with isolated mitochondria and cells. Here, we examined comparative effects of SkQT1 and SkQ1 on rat liver mitochondria (with broader spectrum of energy parame- ters being studied) and yeast cells. SkQT1 was found to be less effective uncoupler, depolarizing agent, inhibitor of respiration and ATP synthesis, and “opener” of a nonspecific pore compared to SkQ1. At the same time SkQ1 exhibited higher antioxidant activity. Both SkQT1 and SkQ1 prevented oxidative stress and mitochondria fragmentation in yeast cells exposed to t-butyl hydroperoxide and promoted cell survival, with SkQT1 being more efficient than SkQ1. Together with the results presented in 2013, our data suggest that SkQT1 is the most promising mitochondria-targeted antioxidant that can be used for preventing various pathologies associated with the oxidative stress in mitochondria.     

Liposomes and Liposome-like Vesicles for Drug and DNA Delivery to Mitochondria

Mitochondrial research is presently one of the fastest growing disciplines in biomedicine. Since the early 1990s, it has become increasingly evident that mitochondrial dysfunction contributes to a large variety of human disorders, ranging from neurodegenerative and neuromuscular diseases, obesity, and diabetes to ischemia-reperfusion injury and cancer. Most remarkably, mitochondria, the “power house” of the cell, have also become accepted as the “motor of cell death” reflecting their recognized key role during apoptosis. Based on these recent exciting developments in mitochondrial research, increasing pharmacological efforts have been made leading to the emergence of “Mitochondrial Medicine” as a whole new field of biomedical research. The identification of molecular mitochondrial drug targets in combination with the development of methods for selectively delivering biologically active molecules to the site of mitochondria will eventually launch a multitude of new therapies for the treatment of mitochondria-related diseases, which are based either on the selective protection, repair, or eradication of cells. Yet, while tremendous efforts are being undertaken to identify new mitochondrial drugs and drug targets, the development of mitochondria-specific drug carrier systems is lagging behind. To ensure a high efficiency of current and future mitochondrial therapeutics, colloidal vectors, i.e., delivery systems, need to be developed able to selectively transport biologically active molecules to and into mitochondria within living human cells. Here we review ongoing efforts in our laboratory directed toward the development of different phospholipid- and non-phospholipid-based mitochondriotropic drug carrier systems.     

Mitochondrial content is central to nuclear gene expression: Profound implications for human health

We review a recent paper in Genome Research by Guantes et al. showing that nuclear gene expression is influenced by the bioenergetic status of the mitochondria. The amount of energy that mitochondria make available for gene expression varies considerably. It depends on: the energetic demands of the tissue; the mitochondrial DNA (mtDNA) mutant load; the number of mitochondria; stressors present in the cell. Hence, when failing mitochondria place the cell in energy crisis there are major effects on gene expression affecting the risk of degenerative diseases, cancer and ageing. In 2015 the UK parliament approved a change in the regulation of IVF techniques, allowing “Mitochondrial replacement therapy” to become a reproductive choice for women at risk of transmitting mitochondrial disease to their children. This is the first time that this technique will be available. Therefore understanding the interaction between mitochondria and the nucleus has never been more important.     

Mitochondrial dysfunction in breast cancer cells prevents tumor growth

Metformin is a well-established diabetes drug that prevents the onset of most types of human cancers in diabetic patients, especially by targeting cancer stem cells. Metformin exerts its protective effects by functioning as a weak “mitochondrial poison,” as it acts as a complex I inhibitor and prevents oxidative mitochondrial metabolism (OXPHOS). Thus, mitochondrial metabolism must play an essential role in promoting tumor growth. To determine the functional role of “mitochondrial health” in breast cancer pathogenesis, here we used mitochondrial uncoupling proteins (UCPs) to genetically induce mitochondrial dysfunction in either human breast cancer cells (MDA-MB-231) or cancer-associated fibroblasts (hTERT-BJ1 cells). Our results directly show that all three UCP family members (UCP-1/2/3) induce autophagy and mitochondrial dysfunction in human breast cancer cells, which results in significant reductions in tumor growth. Conversely, induction of mitochondrial dysfunction in cancer-associated fibroblasts has just the opposite effect. More specifically, overexpression of UCP-1 in stromal fibroblasts increases β-oxidation, ketone body production and the release of ATP-rich vesicles, which “fuels” tumor growth by providing high-energy nutrients in a paracrine fashion to epithelial cancer cells. Hence, the effects of mitochondrial dysfunction are truly compartment-specific. Thus, we conclude that the beneficial anticancer effects of mitochondrial inhibitors (such as metformin) may be attributed to the induction of mitochondrial dysfunction in the epithelial cancer cell compartment. Our studies identify cancer cell mitochondria as a clear target for drug discovery and for novel therapeutic interventions.