Mitochondria on the move: Horizontal mitochondrial transfer in disease and health.

Mammalian genes were long thought to be constrained within somatic cells in most cell types. This concept was challenged recently when cellular organelles including mitochondria were shown to move between mammalian cells in culture via cytoplasmic bridges. Recent research in animals indicates transfer of mitochondria in cancer and during lung injury in vivo, with considerable functional consequences. Since these pioneering discoveries, many studies have confirmed horizontal mitochondrial transfer (HMT) in vivo, and its functional characteristics and consequences have been described. Additional support for this phenomenon has come from phylogenetic studies. Apparently, mitochondrial trafficking between cells occurs more frequently than previously thought and contributes to diverse processes including bioenergetic crosstalk and homeostasis, disease treatment and recovery, and development of resistance to cancer therapy. Here we highlight current knowledge of HMT between cells, focusing primarily on in vivo systems, and contend that this process is not only (patho)physiologically relevant, but also can be exploited for the design of novel therapeutic approaches.

Bioenergetic and Metabolic Adaptation in Tumor Progression and Metastasis.

The ability of cancer cells to adjust their metabolism in response to environmental changes is a well-recognized hallmark of cancer. Diverse cancer and non-cancer cells within tumors compete for metabolic resources. Metabolic demands change frequently during tumor initiation, progression and metastasis, challenging our quest to better understand tumor biology and develop novel therapeutics. Vascularization, physical constraints, immune responses and genetic instability promote tumor evolution resulting in immune evasion, opportunities to breach basement membrane barriers and spread through the circulation and lymphatics. In addition, the unfolded protein response linked to the ubiquitin proteasome system is a key player in addressing stoichiometric imbalances between nuclear and mitochondrially-encoded protein subunits of respiratory complexes, and nuclear-encoded mitochondrial ribosomal protein subunits. While progressive genetic changes, some of which affect metabolic adaptability, contribute to tumorigenesis and metastasis through clonal expansion, epigenetic changes are also important and more dynamic in nature. Understanding the role of stromal and immune cells in the tumor microenvironment in remodeling cancer cell energy metabolism has become an increasingly important area of research. In this perspective, we discuss the adaptations made by cancer cells to balance mitochondrial and glycolytic energy metabolism. We discuss how hypoxia and nutrient limitations affect reductive and oxidative stress through changes in mitochondrial electron transport activity. We propose that integrated responses to cellular stress in cancer cells are central to metabolic flexibility in general and bioenergetic adaptability in particular and are paramount in tumor progression and metastasis.

SMAD4 loss limits the vulnerability of pancreatic cancer cells to complex I inhibition via promotion of mitophagy.

Pancreatic cancer is one of the deadliest forms of cancer, which is attributed to lack of effective treatment options and drug resistance. Mitochondrial inhibitors have emerged as a promising class of anticancer drugs, and several inhibitors of the electron transport chain (ETC) are being clinically evaluated. We hypothesized that resistance to ETC inhibitors from the biguanide class could be induced by inactivation of SMAD4, an important tumor suppressor involved in transforming growth factor ß (TGFß) signaling, and associated with altered mitochondrial activity. Here we show that, paradoxically, both TGFß-treatment and the loss of SMAD4, a downstream member of TGFß signaling cascade, induce resistance to biguanides, decrease mitochondrial respiration, and fragment the mitochondrial network. Mechanistically, the resistance of SMAD4-deficient cells is mediated by increased mitophagic flux driven by MAPK/ERK signaling, whereas TGFß-induced resistance is autophagy-independent and linked to epithelial-to-mesenchymal transition (EMT). Interestingly, mitochondria-targeted tamoxifen, a complex I inhibitor under clinical trial, overcomes resistance mediated by SMAD4-deficiency or TGFß signaling. Our data point to differential mechanisms underlying the resistance to treatment in PDAC arising from TGFß signaling and SMAD4 loss, respectively. The findings will help the development of mitochondria-targeted therapy for pancreatic cancer patients with SMAD4 as a plausible predictive marker.

A simple indirect colorimetric assay for measuring mitochondrial energy metabolism based on uncoupling sensitivity.

PURPOSE: Cancer cells rapidly adjust their balance between glycolytic and mitochondrial ATP production in response to changes in their microenvironment and to treatments like radiation and chemotherapy. Reliable, simple, high throughput assays that measure the levels of mitochondrial energy metabolism in cells are useful determinants of treatment effects. Mitochondrial metabolism is routinely determined by measuring the rate of oxygen consumption (OCR). We have previously shown that indirect inhibition of plasma membrane electron transport (PMET) by the mitochondrial uncoupler, FCCP, may also be a reliable measure of mitochondrial energy metabolism. Here, we aimed to validate these earlier findings by exploring the relationship between stimulation of oxygen consumption by FCCP and inhibition of PMET. METHODS: We measured PMET by reduction of the cell impermeable tetrazolium salt WST-1/PMS. We characterised the effect of different growth conditions on the extent of PMET inhibition by FCCP. Next, we compared FCCP-mediated PMET inhibition with FCCP-mediated stimulation of OCR using the Seahorse XF96e flux analyser, in a panel of cancer cell lines. RESULTS: We found a strong inverse correlation between stimulation of OCR and PMET inhibition by FCCP. PMET and OCR were much more severely affected by FCCP in cells that rely on mitochondrial energy production than in cells with a more glycolytic phenotype. CONCLUSION: Indirect inhibition of PMET by FCCP is a reliable, simple and inexpensive high throughput assay to determine the level of mitochondrial energy metabolism in cancer cells.

Mitochondrial DNA Affects the Expression of Nuclear Genes Involved in Immune and Stress Responses in a Breast Cancer Model.

Tumor cells without mitochondrial (mt) DNA (ρ0 cells) are auxotrophic for uridine, and their growth is supported by pyruvate. While ATP synthesis in ρ0 cells relies on glycolysis, they fail to form tumors unless they acquire mitochondria from stromal cells. Mitochondrial acquisition restores respiration that is essential for de novo pyrimidine biosynthesis and for mitochondrial ATP production. The physiological processes that underpin intercellular mitochondrial transfer to tumor cells lacking mtDNA and the metabolic remodeling and restored tumorigenic properties of cells that acquire mitochondria are not well understood. Here, we investigated the changes in mitochondrial and nuclear gene expression that accompany mtDNA deletion and acquisition in metastatic murine 4T1 breast cancer cells. Loss of mitochondrial gene expression in 4T1ρ0 cells was restored in cells recovered from subcutaneous tumors that grew from 4T1ρ0 cells following acquisition of mtDNA from host cells. In contrast, the expression of most nuclear genes that encode respiratory complex subunits and mitochondrial ribosomal subunits was not greatly affected by loss of mtDNA, indicating ineffective mitochondria-to-nucleus communication systems for these nuclear genes. Further, analysis of nuclear genes whose expression was compromised in 4T1ρ0 cells showed that immune- and stress-related genes were the most highly differentially expressed, representing over 70% of those with greater than 16-fold higher expression in 4T1 compared with 4T1ρ0 cells. The monocyte recruiting chemokine, Ccl2, and Psmb8, a subunit of the immunoproteasome that generates MHCI-binding peptides, were the most highly differentially expressed. Early monocyte/macrophage recruitment into the tumor mass was compromised in 4T1ρ0 cells but recovered before mtDNA could be detected. Taken together, our results show that mitochondrial acquisition by tumor cells without mtDNA results in bioenergetic remodeling and re-expression of genes involved in immune function and stress adaptation.

Vaccines adjuvanted with an NKT cell agonist induce effective T-cell responses in models of CNS lymphoma.

Aim: The efficacy of anti-lymphoma vaccines that exploit the cellular adjuvant properties of activated natural killer T (NKT) cells were examined in mouse models of CNS lymphoma. Materials & methods: Vaccines were prepared by either loading the NKT cell agonist, α-galactosylceramide onto irradiated and heat-shocked B- and T-lymphoma cells, or chemically conjugating α-galactosylceramide to MHC-binding peptides from a lymphoma-associated antigen. Vaccine efficacy was analyzed in mice bearing intracranial tumors. Results: Both forms of vaccine proved to be effective in preventing lymphoma engraftment through activity of T cells that accessed the CNS. Established lymphoma was harder to treat with responses constrained by Tregs, but this could be overcome by depleting Tregs prior to vaccination. Conclusion: Simply designed NKT cell-activating vaccines enhance T-cell responses and have the potential to protect against CNS lymphoma development or prevent CNS relapse. To be effective against established CNS lymphoma, vaccines need to be combined with Treg suppression.

High-intensity interval exercise increases humanin, a mitochondrial encoded peptide, in the plasma and muscle of men.

Humanin is a small regulatory peptide encoded within the 16S ribosomal RNA gene (MT-RNR2) of the mitochondrial genome that has cellular cyto- and metabolo-protective properties similar to that of aerobic exercise training. Here we investigated whether acute high-intensity interval exercise or short-term high-intensity interval training (HIIT) impacted skeletal muscle and plasma humanin levels. Vastus lateralis muscle biopsies and plasma samples were collected from young healthy untrained men (n = 10, 24.5 ± 3.7 yr) before, immediately following, and 4 h following the completion of 10 × 60 s cycle ergometer bouts at V̇o2peak power output (untrained). Resting and postexercise sampling was also performed after six HIIT sessions (trained) completed over 2 wk. Humanin protein abundance in muscle and plasma were increased following an acute high-intensity exercise bout. HIIT trended (P = 0.063) to lower absolute humanin plasma levels, without effecting the response in muscle or plasma to acute exercise. A similar response in the plasma was observed for the small humanin-like peptide 6 (SHLP6), but not SHLP2, indicating selective regulation of peptides encoded by MT-RNR2 gene. There was a weak positive correlation between muscle and plasma humanin levels, and contraction of isolated mouse EDL muscle increased humanin levels ~4-fold. The increase in muscle humanin levels with acute exercise was not associated with MT-RNR2 mRNA or humanin mRNA levels (which decreased following acute exercise). Overall, these results suggest that humanin is an exercise-sensitive mitochondrial peptide and acute exercise-induced humanin responses in muscle are nontranscriptionally regulated and may partially contribute to the observed increase in plasma concentrations.NEW & NOTEWORTHY Small regulatory peptides encoded within the mitochondrial genome (mitochondrial derived peptides) have been shown to have cellular cyto- and metabolo-protective roles that parallel those of exercise. Here we provide evidence that humanin and SHLP6 are exercise-sensitive mitochondrial derived peptides. Studies to determine whether mitochondrial derived peptides play a role in regulating exercise-induced adaptations are warranted.

Sodium sulfide selectively induces oxidative stress, DNA damage, and mitochondrial dysfunction and radiosensitizes glioblastoma (GBM) cells.

Glioblastoma (GBM) has a poor prognosis despite intensive treatment with surgery and chemoradiotherapy. Previous studies using dose-escalated radiotherapy have demonstrated improved survival; however, increased rates of radionecrosis have limited its use. Development of radiosensitizers could improve patient outcome. In the present study, we report the use of sodium sulfide (Na2S), a hydrogen sulfide (H2S) donor, to selectively kill GBM cells (T98G and U87) while sparing normal human cerebral microvascular endothelial cells (hCMEC/D3). Na2S also decreased mitochondrial respiration, increased oxidative stress and induced γH2AX foci and oxidative base damage in GBM cells. Since Na2S did not significantly alter T98G capacity to perform non-homologous end-joining or base excision repair, it is possible that GBM cell killing could be attributed to increased damage induction due to enhanced reactive oxygen species production. Interestingly, Na2S enhanced mitochondrial respiration, produced a more reducing environment and did not induce high levels of DNA damage in hCMEC/D3. Taken together, this data suggests involvement of mitochondrial respiration in Na2S toxicity in GBM cells. The fact that survival of LN-18 GBM cells lacking mitochondrial DNA (ρ0) was not altered by Na2S whereas the survival of LN-18 ρ+ cells was compromised supports this conclusion. When cells were treated with Na2S and photon or proton radiation, GBM cell killing was enhanced, which opens the possibility of H2S being a radiosensitizer. Therefore, this study provides the first evidence that H2S donors could be used in GBM therapy to potentiate radiation-induced killing.

Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells.

Cancer cells without mitochondrial DNA (mtDNA) do not form tumors unless they reconstitute oxidative phosphorylation (OXPHOS) by mitochondria acquired from host stroma. To understand why functional respiration is crucial for tumorigenesis, we used time-resolved analysis of tumor formation by mtDNA-depleted cells and genetic manipulations of OXPHOS. We show that pyrimidine biosynthesis dependent on respiration-linked dihydroorotate dehydrogenase (DHODH) is required to overcome cell-cycle arrest, while mitochondrial ATP generation is dispensable for tumorigenesis. Latent DHODH in mtDNA-deficient cells is fully activated with restoration of complex III/IV activity and coenzyme Q redox-cycling after mitochondrial transfer, or by introduction of an alternative oxidase. Further, deletion of DHODH interferes with tumor formation in cells with fully functional OXPHOS, while disruption of mitochondrial ATP synthase has little effect. Our results show that DHODH-driven pyrimidine biosynthesis is an essential pathway linking respiration to tumorigenesis, pointing to inhibitors of DHODH as potential anti-cancer agents.

Metabolic reprogramming of mitochondrial respiration in metastatic cancer.

Tumor initiation, progression, and metastasis are tissue context-dependent processes. Cellular and non-cellular factors provide the selective microenvironment that determines the fate of the evolving tumor through mechanisms that include metabolic reprogramming. Genetic and epigenetic changes contribute to this reprogramming process, which is orchestrated through ongoing communication between the mitochondrial and nuclear genomes. Metabolic flexibility, in particular the ability to rapidly adjust the balance between glycolytic and mitochondrial energy production, is a hallmark of aggressive, invasive, and metastatic cancers. Tumor cells sustain damage to both nuclear and mitochondrial DNA during tumorigenesis and as a consequence of anticancer treatments. Nuclear and mitochondrial DNA mutations and polymorphisms are increasingly recognized as factors that influence metabolic reprogramming, tumorigenesis, and tumor progression. Severe mitochondrial DNA damage compromises mitochondrial respiration. When mitochondrial respiration drops below a cell-specific threshold, metabolic reprogramming and plasticity fail to compensate and tumor formation is compromised. In these scenarios, tumorigenesis can be restored by acquisition of respiring mitochondria from surrounding stromal cells. Thus, intercellular mitochondrial transfer has the potential to confer treatment resistance and to promote tumor progression and metastasis. Understanding the constraints of metabolic, and in particular bioenergetic reprogramming, and the role of intercellular mitochondrial transfer in tumorigenesis provides new insights into addressing tumor progression and treatment resistance in highly aggressive cancers.

Intercellular Communication in Tumor Biology: A Role for Mitochondrial Transfer.

Intercellular communication between cancer cells and other cells in the tumor microenvironment plays a defining role in tumor development. Tumors contain infiltrates of stromal cells and immune cells that can either promote or inhibit tumor growth, depending on the cytokine/chemokine milieu of the tumor microenvironment and their effect on cell activation status. Recent research has shown that stromal cells can also affect tumor growth through the donation of mitochondria to respiration-deficient tumor cells, restoring normal respiration. Nuclear and mitochondrial DNA mutations affecting mitochondrial respiration lead to some level of respiratory incompetence, forcing cells to generate more energy by glycolysis. Highly glycolytic cancer cells tend to be very aggressive and invasive with poor patient prognosis. However, purely glycolytic cancer cells devoid of mitochondrial DNA cannot form tumors unless they acquire mitochondrial DNA from adjacent cells. This perspective article will address this apparent conundrum of highly glycolytic cells and cover aspects of intercellular communication between tumor cells and cells of the microenvironment with particular emphasis on intercellular mitochondrial transfer.

Mitochondrial Genome Transfer to Tumor Cells Breaks The Rules and Establishes a New Precedent in Cancer Biology.

Horizontal gene transfer is known to occur in bacteria and archaea whereas higher organisms including mammals undergo vertical transfer. Our recent results demonstrate horizontal transfer of mitochondrial DNA (mtDNA) from normal host cells to tumor cells lacking mitochondrial DNA (mtDNA). This mtDNA migration results in recovery of respiration, restored tumor initiation, and metastasis.

Alternative assembly of respiratory complex II connects energy stress to metabolic checkpoints.

Cell growth and survival depend on a delicate balance between energy production and synthesis of metabolites. Here, we provide evidence that an alternative mitochondrial complex II (CII) assembly, designated as CIIlow, serves as a checkpoint for metabolite biosynthesis under bioenergetic stress, with cells suppressing their energy utilization by modulating DNA synthesis and cell cycle progression. Depletion of CIIlow leads to an imbalance in energy utilization and metabolite synthesis, as evidenced by recovery of the de novo pyrimidine pathway and unlocking cell cycle arrest from the S-phase. In vitro experiments are further corroborated by analysis of paraganglioma tissues from patients with sporadic, SDHA and SDHB mutations. These findings suggest that CIIlow is a core complex inside mitochondria that provides homeostatic control of cellular metabolism depending on the availability of energy.

Functional Mitochondria in Health and Disease.

The ability to rapidly adapt cellular bioenergetic capabilities to meet rapidly changing environmental conditions is mandatory for normal cellular function and for cancer progression. Any loss of this adaptive response has the potential to compromise cellular function and render the cell more susceptible to external stressors such as oxidative stress, radiation, chemotherapeutic drugs, and hypoxia. Mitochondria play a vital role in bioenergetic and biosynthetic pathways and can rapidly adjust to meet the metabolic needs of the cell. Increased demand is met by mitochondrial biogenesis and fusion of individual mitochondria into dynamic networks, whereas a decrease in demand results in the removal of superfluous mitochondria through fission and mitophagy. Effective communication between nucleus and mitochondria (mito-nuclear cross talk), involving the generation of different mitochondrial stress signals as well as the nuclear stress response pathways to deal with these stressors, maintains bioenergetic homeostasis under most conditions. However, when mitochondrial DNA (mtDNA) mutations accumulate and mito-nuclear cross talk falters, mitochondria fail to deliver critical functional outputs. Mutations in mtDNA have been implicated in neuromuscular and neurodegenerative mitochondriopathies and complex diseases such as diabetes, cardiovascular diseases, gastrointestinal disorders, skin disorders, aging, and cancer. In some cases, drastic measures such as acquisition of new mitochondria from donor cells occurs to ensure cell survival. This review starts with a brief discussion of the evolutionary origin of mitochondria and summarizes how mutations in mtDNA lead to mitochondriopathies and other degenerative diseases. Mito-nuclear cross talk, including various stress signals generated by mitochondria and corresponding stress response pathways activated by the nucleus are summarized. We also introduce and discuss a small family of recently discovered hormone-like mitopeptides that modulate body metabolism. Under conditions of severe mitochondrial stress, mitochondria have been shown to traffic between cells, replacing mitochondria in cells with damaged and malfunctional mtDNA. Understanding the processes involved in cellular bioenergetics and metabolic adaptation has the potential to generate new knowledge that will lead to improved treatment of many of the metabolic, degenerative, and age-related inflammatory diseases that characterize modern societies.

The mobility of mitochondria: Intercellular trafficking in health and disease.

The view that genes are constrained within somatic cells is challenged by in vitro evidence, and more recently by in vivo studies which demonstrate that mitochondria with their mitochondrial DNA (mtDNA) payload not only can, but do move between cells in tumour models and in mouse models of tissue damage. Using mouse tumour cell models without mtDNA to reflect mtDNA damage, we have shown that these cells grow tumours only after acquiring mtDNA from cells in the local microenvironment resulting in respiration recovery, tumorigenesis and metastasis. Mitochondrial transfer between cells has also been demonstrated following ischaemia-induced injury in the heart and brain and in lung epithelium, and following lung inflammation. In vitro investigations suggest that stem cells may be mitochondrial donors. The ability of mitochondria to move between cells appears to be an evolutionarily-conserved phenomenon, relevant to diseases with compromised mitochondrial function including neurodegenerative, neuromuscular and cardiovascular diseases as well as cancer and ageing.

Horizontal transfer of whole mitochondria restores tumorigenic potential in mitochondrial DNA-deficient cancer cells.

Recently, we showed that generation of tumours in syngeneic mice by cells devoid of mitochondrial (mt) DNA (ρ0 cells) is linked to the acquisition of the host mtDNA. However, the mechanism of mtDNA movement between cells remains unresolved. To determine whether the transfer of mtDNA involves whole mitochondria, we injected B16ρ0 mouse melanoma cells into syngeneic C57BL/6Nsu9-DsRed2 mice that express red fluorescent protein in their mitochondria. We document that mtDNA is acquired by transfer of whole mitochondria from the host animal, leading to normalisation of mitochondrial respiration. Additionally, knockdown of key mitochondrial complex I (NDUFV1) and complex II (SDHC) subunits by shRNA in B16ρ0 cells abolished or significantly retarded their ability to form tumours. Collectively, these results show that intact mitochondria with their mtDNA payload are transferred in the developing tumour, and provide functional evidence for an essential role of oxidative phosphorylation in cancer.

Mitochondrial Transfer from Astrocytes to Neurons following Ischemic Insult: Guilt by Association?

Intercellular mitochondrial transfer has been shown in tumor models, following lung injury and in xenotransplants of leukemic cells, but trafficking between cells in the brain remains unexplored. A suggestion that mitochondria move from astrocytes to neurons in a model of ischemia in a recent article in Nature by Hayakawa et al. (2016) should be interpreted with caution.

Iterative sorting reveals CD133+ and CD133- melanoma cells as phenotypically distinct populations.

BACKGROUND: The heterogeneity and tumourigenicity of metastatic melanoma is attributed to a cancer stem cell model, with CD133 considered to be a cancer stem cell marker in melanoma as well as other tumours, but its role has remained controversial. METHODS: We iteratively sorted CD133+ and CD133- cells from 3 metastatic melanoma cell lines, and observed tumourigenicity and phenotypic characteristics over 7 generations of serial xeno-transplantation in NOD/SCID mice. RESULTS: We demonstrate that iterative sorting is required to make highly pure populations of CD133+ and CD133- cells from metastatic melanoma, and that these two populations have distinct characteristics not related to the cancer stem cell phenotype. In vitro, gene set enrichment analysis indicated CD133+ cells were related to a proliferative phenotype, whereas CD133- cells were of an invasive phenotype. However, in vivo, serial transplantation of CD133+ and CD133- tumours over 7 generations showed that both populations were equally able to initiate and propagate tumours. Despite this, both populations remained phenotypically distinct, with CD133- cells only able to express CD133 in vivo and not in vitro. Loss of CD133 from the surface of a CD133+ cell was observed in vitro and in vivo, however CD133- cells derived from CD133+ retained the CD133+ phenotype, even in the presence of signals from the tumour microenvironment. CONCLUSION: We show for the first time the necessity of iterative sorting to isolate pure marker-positive and marker-negative populations for comparative studies, and present evidence that despite CD133+ and CD133- cells being equally tumourigenic, they display distinct phenotypic differences, suggesting CD133 may define a distinct lineage in melanoma.

Horizontal transfer of mitochondria between mammalian cells: beyond co-culture approaches.

Current dogma holds that genes are the property of individual mammalian cells and partition between daughter cells during cell division. However, and rather unexpectedly, recent research has demonstrated horizontal cell-to-cell transfer of mitochondria and mitochondrial DNA in several mammalian cell culture systems. Furthermore, unequivocal evidence that mitochondrial DNA transfer occurs in vivo has now been published. While these studies show horizontal transfer of mitochondrial DNA in pathological settings, it is also possible that intercellular mitochondrial transfer is a fundamental physiological process with a role in development and tissue homeostasis.