Role of Mitochondria in Cancer Stem Cell Resistance

cells Review Role of Mitochondria in Cancer Stem Cell Resistance JoséManuel García-Heredia 1,2,3,* and Amancio Carnero 1,3,* 1Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen del Rocío, Universidad de Sevilla, Consejo Superior de Investigaciones Científicas, Avda. Manuel Siurot s/n, 41013 Seville, Spain 2Departamento de Bioquímica Vegetal y Biología Molecular, Facultad de Biología, Universidad de Sevilla, Avda. de la Reina Mercedes 6, 41012 Seville, Spain 3Centro de Investigación Biomédica en Red de Cáncer, CIBERONC, Instituto de Salud Carlos III, 28029 Madrid, Spain * Correspondence: jmgher[email protected] (J.M.G.-H.); [email protected] (A.C.); Tel.: +34-955-923-115 (J.M.G.-H.); +34-955-923-110 (A.C.) Received: 4 June 2020; Accepted: 15 July 2020; Published: 15 July 2020   Abstract: Cancer stem cells (CSC) are associated with the mechanisms of chemoresistance to different cytotoxic drugs or radiotherapy, as well as with tumor relapse and a poor prognosis. Various studies have shown that mitochondria play a central role in these processes because of the ability of this organelle to modify cell metabolism, allowing survival and avoiding apoptosis clearance of cancer cells. Thus, the whole mitochondrial cycle, from its biogenesis to its death, either by mitophagy or by apoptosis, can be targeted by different drugs to reduce mitochondrial fitness, allowing for a restored or increased sensitivity to chemotherapeutic drugs. Once mitochondrial misbalance is induced by a specific drug in any of the processes of mitochondrial metabolism, two elements are commonly boosted: an increment in reactive nitrogen/oxygen species and, subsequently, activation of the intrinsic apoptotic pathway. Keywords: cancer stem cells; mitochondria; drug resistance; metabolic plasticity 1. Introduction Tumors and their microenvironment constitute a very heterogeneous structure, with multiple phenotypes that make them virtually metabolic ecosystems, different from structures in other parts of the body. These heterogeneous structures, in addition to different tumor cell types, comprise stromal cells such as infiltrated fibroblasts, endothelial cells and immune cells [ 1 – 3 ]. These non-tumor cells can greatly influence the plasticity and functionality of tumor cells [ 1 , 4 ]. Furthermore, within the tumor cell population, great heterogeneity exists, arising from both genetic and epigenetic differences [ 3 , 5 ]. Although tumors are largely clonal derivatives of a single cell, both the genomic instability of tumor cells and microenvironment variations make tumor cells a very heterogeneous population [ 5 ]. This heterogeneity allows cancer cells to adapt to different stresses that could appear in the tumor microenvironment, such as inflammation, hypoxia, low pH and nutrients. Thus, either different tumor cells or different cell metabolism properties can be found within a tumor with increasing variability, which increases the probability that a tumor can survive and invade other tissues [6,7]. Most tumor cells are considered differentiated—that is, unable to regenerate a tumor by themselves or metastasize—constituting the bulk of the tumor mass [ 8 , 9 ]. Infiltrated among them, a small percentage, generally less than 1–2% of the total, exhibits stem cell characteristics [ 9 , 10 ]. These cells were initially identified in leukemia (AML), but cancer stem cells (CSCs) have also been found in breast, lung, colon, brain, head and neck, prostate or liver tumors, among others [ 11 , 12 ]. These CSCs, also called stem-likecancer cells, tumor-initiating cells (TICs)or cancer-initiating cells, are considered tohave the highest associated risk due to their ability of both self-renewal and tumor initiation in vivo , as well Cells 2020,9, 1693; doi:10.3390/cells9071693 www.mdpi.com/journal/cells Cells 2020,9, 1693 2 of 28 as their ability to invade and migrate to other tissues, leading to metastasis [ 13 – 15 ]. These cells, which have a low proliferation rate, are the only cells within a tumor capable of generating tumor cells with different characteristics, an attribute shared with stem cells. Thus, tumors may arise due to mutations that occur in normal stem cells or from differentiated cells that acquire stem-like features [ 16 , 17 ]. Thus, an abnormal increase in gene transcription related to stem cell regulatory pathways, such as c-MYC, Bmi-1, Hedgehog, Notch and Wnt, has been observed in CSC populations [ 18 , 19 ]. Similarly, stress conditions in the tumor microenvironment, typically with low oxygen levels and a limited nutrient supply, promote the epithelial-to-mesenchymal transition (EMT), leading to increased self-renewal and cell migration [7,13,20]. CSCs have been shown to be resistant to commonly used chemotherapeutic agents in leukemia, malignant melanoma, and brain, head and neck, breast, pancreas and colorectal tumors [ 21 – 27 ]. Similarly, brain and breast tumors have also shown resistance to radiation therapy [ 28 , 29 ]. Thus, CSCs are responsible for tumor relapse in many tumor types because they can resist cancer therapies, either intrinsically or because of changes arising from treatment. The relative abundance of CSCs is associated with the clinical outcome [ 30 ]. Thus, although traditional chemotherapy and radiotherapy can remove most tumor cells, CSCs are thought to survive, developing resistance during treatment [ 18 , 20 , 26 , 31 , 32 ]. Consequently, these cells remain hidden in the body of the patients until its reactivation [ 33 ], regenerating the tumor or migrating to other organs to metastasize. This makes them an attractive target to design new cancer therapies, because conventional treatments can kill, by autophagy and apoptosis, most of the tumor, but do not affect CSCs [ 13 , 14 , 28 , 34 , 35 ]. This fact increases the CSC population and causes a more aggressive tumor relapse [ 14 , 15 , 26 ]. The cellular mechanisms underlying this phenotype of drug resistance remain largely unknown, although there are some common elements, including the following: —Cell quiescence, that is, a state in which CSCs do not divide, or do so very slowly [ 13 , 15 , 27 , 36 , 37 ]. Quiescence properties, such as maintenance in the G0/G1 stage in stem cells, protect them from cytotoxic stress [ 38 ]. Most chemotherapeutic drugs are based on tumor cells dividing faster than normal cells, producing little or no effect on CSCs [39]; —Overexpression of drug transporters that allow an active outflow of the drug [ 40 – 42 ]. Thus, the ABC genes constitute a broad family of ATP-dependent transporters in which at least 16 of them have been linked to resistance to anticancer drugs [ 43 ]. Moreover, a similar phenotype is caused by the loss of cell surface receptors or transporters that allow the drug to enter the cell [14,44]; —An increase in the levels of expression of the molecular targets of chemotherapeutic drugs, which causes, consequently, a decrease in effectiveness [ 44 , 45 ], that can be achieved, in a similar way, by the accumulation of chemotherapeutic drugs in intracellular vesicles, reducing its biological effect [ 46 ]; —Increased ability to repair DNA damage [ 28 , 42 ]. Treatment with chemotherapeutic drugs, such as cisplatin or carboplatin, can induce apoptotic death in sensitive cancer cells through the accumulation of DNA damage, reaching a level that makes repair impossible; —Overexpression of antiapoptotic proteins or reduced expression of proteins related to apoptosis [ 47 , 48 ]. Certain CSCs exhibited higher expression levels of prosurvival proteins from the BCL-2 family [49]; —Resistance to reactive nitrogen and oxygen species (RNOS) [ 14 , 15 , 35 , 36 ]. Many drugs produce RNOS in excess, usually generating DNA damage and modifying protein functionality, triggering apoptosis; —Deregulation of autophagy [ 13 , 34 , 42 ], the process in which double-membrane vesicles (autophagosomes) encompass different parts of the cell, including organelles, allowing the recycling of its components after fusion with the lysosome. This process is highly related to the maintenance of CSCs but is not restricted to them. In cancer-associated fibroblasts (CAFs), an increase in autophagy has been observed, increasing the nutrients received by tumor cells [ 3 , 4 ]. CAFs also provide cytokines that stimulate mitochondrial biogenesis in cancer cells [ 50 ]. Likewise, an increment in mitochondrial Cells 2020,9, 1693 3 of 28 autophagy (mitophagy) reduces oxidative metabolism, making tumor cells dependent on glycolysis and less efficient in generating ATP and favoring a slow cell cycle, typical of some stem cells [ 13 , 14 , 34 , 35 , 51 ]. —Metabolicchanges. MostCSCs exhibithighmetabolic plasticity, whichwould facilitatetheability of these cells to thrive in adverse microenvironmental conditions, such as those of hypoxia that usually exist in tumors [ 6 , 18 , 35 , 36 ]. This metabolic plasticity would allow them to modify their metabolism from oxidative respiration to aerobic glycolysis. In this way, and even within the same tumor, CSCs with glycolytic and oxidative metabolism can coexist, in addition to differentiated cells with different metabolic phenotypes, because of different genetic or microenvironmental factors [ 52 , 53 ]. Additionally, other metabolites, such as fatty acids, ketones or amino acids, can be used by cancer cells to support metabolism [ 6 , 35 , 54 ]. Due to the crucial contribution of metabolism in malignant transformation and tumor progression, metabolic reprogramming/plasticity has become one of the characteristics of cancer. Many of these pathways are mediated by redox imbalance and the involvement of ROS detoxification systems, which usually show CSCs involved in the upregulation of ROS metabolism [ 14 , 55 ]. In this way, tumor cells can modulate different metabolic pathways to obtain energy and different metabolites, while also maintaining redox balance. In many of these processes, the mitochondria play an essential role. Thus, they are responsible for the synthesis of many metabolites, participating actively in apoptotic mechanisms, in addition to being the bioenergetic power station of the cell and where most intracellular ROS are produced and eliminated [ 52 , 53 ]. Thus, in some CSCs, an increase was observed in the transcription of nuclear genes that encode mitochondrial proteins [ 56 , 57 ]. These results suggest that certain CSCs depend on both metabolism and mitochondrial biogenesis for their survival and spread. Throughout this review, we will focus on the role of mitochondria in the mechanisms of CSC resistance to different drugs, as well as in possible strategies that enable the circumvention of these mechanisms. 2. Mitochondria as a Crucial Element in Cancer Mitochondria perform an essential function in cells by coordinating both the production and distribution of energy through oxidative phosphorylation (OXPHOS) based on oxygen and substrate availability. It also carries out other important metabolic reactions, such as the citric acid or tricarboxylic acid (TCA) cycle, fatty acid oxidation (FAO) or glutaminolysis. Due to its role in essential cellular function, this organelle has been linked to multiple aspects of tumorigenesis and tumor progression [ 58 ]. In many metabolic pathways in which mitochondria are involved, alterations have been found in tumor cells and, specifically, in CSCs [ 35 , 53 , 54 , 59 , 60 ]. Consequently, multiple chemotherapeutic drugs target mitochondria, either directly or through pathways regulating mitochondrial activity. This turns drugs that target mitochondria into promising agents to interfere with tumor adaptations, allowing the elimination of CSCs [ 61 ]. It is important to consider that mitochondria do not play a unique role in tumorigenesis and tumor progression or the response of tumor cells to treatments. Depending on the tissue in which the tumor appears, there will be epigenetic or tumor microenvironment differences that can modify mitochondrial functionality [ 5 , 54 , 59 ]. Even within the same tumor, differences between cells can mean differences in mitochondrial functionality or metabolism, thus promoting tumor adaptation to its microenvironment and the resistance capacity of the cells. We must consider the cell as a whole so that modifications in an organelle will have side effects in other parts of the cell. To illustrate this, we can consider the direct communication between mitochondria and the nucleus, called the retrograde response, which suggests that changes in mitochondrial physiology or metabolism may induce changes in gene expression [ 51 , 52 , 54 , 57 ]. Thus, the NAD + /NADH ratio, levels of acetyl-CoA, ATP, ROS or specific mitochondrial metabolites are involved in this process [ 62 ]. Furthermore, because most of the proteins that play a role in the mitochondria are encoded in the nucleus, there is a required nucleo-mitochondria communication to allow not only the proper mitochondrial function but also the replication of mitochondrial DNA (mtDNA) through the import into mitochondria of proteins such as TFAM, POLG and POLGY2 [ 52 , 63 ]. Cells 2020,9, 1693 4 of 28 Multiple signaling pathways can modify mitochondrial function, such as the PI3K/AKT/mTOR pathway, with multiple roles in tumorigenesis [ 64 ]. This fact makes it challenging to describe in a review all the processes implied in CSCs’ drug resistance mediated by mitochondria. Thus, herein, we will mainly focus on mitochondrial processes. If we analyze the cycle of a mitochondrion, three key steps are involved: the generation of new mitochondria (biogenesis), mitochondrial metabolism, and the autophagy of aging or non-functional mitochondria (mitophagy) [ 13 , 34 , 54 , 60 , 65 ]. For each step, CSCs exhibit resistance mechanisms to different drugs. To consider the mitochondrial mechanisms of CSC resistance, the crosstalk between different processes inside a cell must be discussed. Multiple drugs generate oxidative stress, inducing increased ROS, against which there are multiple defense mechanisms in mitochondria [ 14 , 55 ]. Although some drugs cause DNA damage that will produce increased ROS, other drugs produce increased ROS that will damage DNA [ 66 , 67 ]. Additionally, mitochondria are crucial in cell death/survival due to the role of some of their related proteins, such as cytochrome c, in triggering the intrinsic apoptotic pathway [ 68 ]. Throughout this review, we will discuss how alterations in the regulation of each of the three key steps (biogenesis, metabolism and mitophagy) modify the resistance of CSCs to different chemotherapeutic drugs. In addition, we will discuss how that resistance can be overcome, inducing apoptotic pathway for tumor cell clearance. 3. Mitochondrial Biogenesis and CSC Resistance Mitochondrial biogenesis is a crucial step in the maintenance of the cell cycle and for correct metabolism. In all cells, mitochondria must be synthesized from a pre-existing one, an essential step for correct cell division [ 65 ]. Each cell contains a variable number of copies of mitochondrial DNA (mtDNA), encoding 13 polypeptides essential for OXPHOS, as well as rRNAs and tRNAs, with the remaining mitochondrial proteins encoded in the nuclear genome [ 57 ]. Thus, mitochondrial proteins encoded by nuclear and mitochondrial genes must be transcribed in a coordinated way to form some of the mitochondrial complexes associated with OXPHOS [ 69 ]. Increased mitochondrial biogenesis is usually connected with a higher tumorigenic rate [ 70 , 71 ], which supports the maintenance of stem-like properties in a certain population of CSCs, independently of their metabolism [ 72 – 74 ]. Thus, an increased mitochondrial mass is usually detected in CSCs, reflecting increased mitochondrial biogenesis, which is also related to increased chemoresistance [ 18 , 72 , 73 , 75 , 76 ] (Figure 1). However, this is not a universal event because it has been also reported that cells with increased mitochondrial biogenesis are less glycolytic and exhibit reduced invasive properties [77]. According to endosymbiotic theory and the bacterial origin of mitochondria, antibiotics designed for bacteria also target mitochondria. This means that some antibiotics can be used to inhibit the spread of CSCs. Among these are doxycycline (tetracycline), tigecycline (glycylcycline), azithromycin (erythromycin), antiparasitic drugs (pyrvinium pamoate, atovaquone), and antimycobacterial drugs, such as bedaquiline [ 75 , 78 ]. These drugs have acceptable side effects, which would allow their use as therapeutic agents to eradicate CSCs [ 75 ] (Figure 1). Thus, doxycycline, tigecycline and azithromycin inhibit protein translation, which blocks mitochondrial biogenesis [ 73 , 75 , 79 , 80 ]. Due to the high number of mitochondria in many tissues, such as the skeletal muscle, brain, and heart, normal cells would be slightly affected by antibiotic treatment. However, mitochondrial biogenesis is higher in tumor cells, with a higher mitochondrial mass, making them more sensitive to these antibiotics. Clinical trials with doxycycline and azithromycin showed positive effects, such as increased patient survival and a reduction in the percentage of CSCs in multiple cancer types [ 81 , 82 ]. Additionally, doxycycline was shown to overcome paclitaxel resistance in CSCs [ 79 ] and reduce the CSC population in early breast cancer patients [ 81 ]; additionally, it is a strong radiosensitizer [ 75 ]. Furthermore, this drug could target paclitaxel-resistant CSCs [ 79 ]. However, continuous treatment of cancer with antibiotics may prove ineffective in the long term due to the appearance of resistance in cultured cells caused by alterations in mtDNA, such as its reduction (Figure 1). In this way, doxycycline-resistant CSCs, with lower mtDNA content, exhibited an inflexible metabolism [ 83 ]. Thus, the cells, according to glycolysis, Cells 2020,9, 1693 5 of 28 are more sensitive to other metabolic inhibitors, such as Vitamin C, which has been described to block aerobic glycolysis by targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [84]. Figure 1. Mitochondrial biogenesis misbalance in cancer stem cells (CSCs). In normal cells, mitochondrial mass is usually maintained. However, both increased mitochondrial biogenesis, on the one hand, and alterations on mtDNA, on the other hand, are connected with an increased resistance in CSCs. Black arrows refer to increased chemoresistance, from a normal situation where mitochondrial biogenesis allows the maintenance of mitochondrial population. Treatment with antibiotics of CSCs with high mitochondrial biogenesis can overcome chemoresistance (red arrow), but prolonged treatments can force the appearance of a CSC population with reduced mtDNA (dotted red arrow). To complete correct mitochondrial biogenesis, mtDNA must be correctly maintained. The mutational rate of the mitochondrial genome is higher than that exhibited by the nuclear genome [ 85 ], with frequently detected mutations being associated with the appearance of tumors [ 86 ] (Figure 1). Thus, mtDNA damage can induce tumor progression to an advanced phenotype in different tumor types [ 57 ]. The presence of mutations in the mitochondrial genome is common in tumors, with frequent resistance to chemotherapeutic agents such as 5-fluorouracil (5-FU) and cisplatin facilitating these mutations’ resistance to other drugs, such as carboplatin, based on a similar principle of drug action [ 87 ]. Damage to mtDNA, or even its loss, causes a reduction in the rate of cell proliferation, characteristic of CSCs [ 39 , 52 ]. Thus, large mtDNA deletions and a reduced copy number of mtDNA by the cell are usually associated with advanced tumors, increased metastasis and a poor prognosis [ 52 , 57 ]. Additionally, a low mtDNA content was detected in up to 80% of breast tumors [ 88 ]. These tumors with low mtDNA content also induced EMT and an increase in invasive and metastatic properties, constituting a more malignant phenotype [ 89 ]. Furthermore, despite poor metabolism, anti-apoptotic and survival pathways are activated by retrograde signaling from the mitochondria to the nucleus [ 52 ]. Therefore, these traits produce an increase in the population of CSCs resistant to chemotherapeutic drugs, such as vincristine, doxorubicin and paclitaxel [ 52 , 57 , 90 ]. Thus, mitochondrial-deficient cells have shown resistance to increased ROS levels, such as direct treatment with hydrogen peroxide or ROS-inducing agents, such as doxorubicin, paraquat and menadione, possibly due to increased expression levels of antioxidant enzymes [ 91 ]. Similarly, mtDNA-depleted cells also showed resistance to cisplatin, hydroxytamoxifen, paclitaxel or TNF-induced apoptosis in different cancer cells [ 90 , 92 , 93 ]. However, despite the damage to mtDNA, or even its absence, the mitochondrial structure, mainly encoded in the nuclear genome, is necessary to allow the CSC phenotype. Indeed, non-tumor cells with mtDNA-deficient mitochondria can resist staurosporine-mediated apoptosis by increased expression Cells 2020,9, 1693 6 of 28 of the antiapoptotic proteins BCL-2 and BCL-XL, sequestration of proapoptotic factors (BID, BAX, BAD) in the internal mitochondrial membrane and reduced activation of caspases 3, 8 and 9, among others properties [ 94 ]. These results show that, in general, the reduction in mtDNA content is related to increased resistance to different drugs. Additionally, mtDNA-depleted mitochondria usually show defective mitochondrial function, with a common metabolic switch towards the Warburg effect [ 52 , 86 ]. Interestingly, cells with low mtDNA levels exhibited lower levels of the tumor suppressor BRCA2, becoming more sensitive to PARP inhibitors, probably due to synthetic lethality [95,96]. Mitochondrial biogenesis is finely tuned by cells, with multiple pathways or regulators, such as PGC-1 α , MYBBP1a or the MAPK/ERK pathway, which can modify their activity [ 63 , 97 – 100 ]. PGC-1 α (peroxisome proliferator-activated receptor gamma coactivator 1α ) is an important regulator of the transcription of nuclear-encoded genes implicated in mitochondrial biogenesis [ 97 ]. Through its role as a transcriptional coactivator, PGC-1 α also regulates both OXPHOS and ROS detoxifying enzymes [ 101 ]. Thus, in breast cancer, higher PGC-1 α expression is correlated with a poor prognosis [ 102 ]. The use of XCT790, a compound that reduces colony formation in soft agar by regulating PGC-1 α , showed a reduction in CSCs [ 73 ]. Additionally, pancreatic CSCs exhibited high levels of PGC-1 α , making these cells more sensitive to metformin [ 18 ]. The MAPK/ERK pathway is also implied in mitochondrial biogenesis due to its role in both intrinsic and acquired resistance to MAPK inhibitors (MAPKi). MAPKi treatment showed the presence of a resistant population, with high mitochondrial biogenesis and an active OXPHOS metabolism [63]. All these results show that mitochondrial population should be finely tuned in normal cells to avoid an imbalance, either due to excessive biogenesis or due to defects (mutations or deletions) in mtDNA, which will probably cause changes in metabolism. 4. Mitochondrial Metabolism in CSC Resistance Importantly, each CSC subpopulation inside a tumor may exhibit a different metabolic profile to other CSCs or the tumor mass, with this heterogeneity associated with resistance to treatment. Thus, contradictory results have been obtained regarding the metabolic profile of CSCs. In some cases, they have been described as mainly glycolytic [ 61 , 103 – 106 ]; other articles have indicated that CSCs are primarily dependent on OXPHOS [18,61,76,107]. The metabolic status of CSCs is very important in drug resistance mechanisms because, when glycolytic CSCs differentiate and proliferate, a change from anaerobic to aerobic metabolism can be observed [14]. However, CSCs from different tumor types have also been described as depending on OXPHOS for most of their energy. The increased efficiency of OXPHOS-based metabolism would, in theory, allow cancer cells to use the nutrients better, allowing them to survive in nutritionally poor environments [ 35 ]. Studies have suggested that treatment-resistant CSCs are less glycolytic, indicating that specific OXPHOS inhibition could attack these cells, helping them to escape from damage in hypoxic areas of the tumor. Thus, cells under severe hypoxia become more resistant to irradiation than cells in normoxia [108]. At the molecular level, the typical metabolic flexibility of cancer is based on the reconnection among the different metabolic pathways, achieved through the synthesis or degradation of key proteins that allow metabolites to change pathways according to cells’ needs. The first of the described mechanisms of this metabolic remodeling is the so-called Warburg effect, in which cells use large amounts of glucose in the presence of oxygen [ 109 ]. This leads to the formation and secretion of lactate in a mechanism called aerobic glycolysis [ 86 , 109 ]. Although this mechanism is inefficient in energy production in the form of ATP, glucose absorption from tumor cells is usually higher, leading to a net ATP production similar to the level achieved with OXPHOS, caused by different mutations in respiratory complexes [48]. The low ROS levels present in CSCs could be due to events leading to metabolic reprogramming, which is essential for maintaining self-renewal and improving the antioxidant defense mechanism [ 14 ]. Thus, glycolytic CSCs are usually adapted to the typical hypoxic environment of many tumors, in Cells 2020,9, 1693 7 of 28 which the glycolytic metabolism prevails to counteract a low mitochondrial level. Specific physiological metabolites, such as pyruvate, tetrahydrofolate, and glutamine, can function as cytotoxic agents in CSCs when administered at doses that disrupt the redox NADP + /NADPH ratio [ 110 ]. Thus, pyruvate accumulation mimics the blockade of the respiratory chain performed by the binding of antimycin A to cytochrome c reductase in mitochondrial complex III [110,111]. High ALDH1 (aldehyde dehydrogenase-1) expression levels are characteristic of CSCs, which are related to increased resistance to chemotherapeutic agents in sarcoma, breast and lung CSCs [ 112 – 114 ]. High ALDH expression protects against the toxic effects of RNOS production derived from chemotherapeutic treatment, so that its inhibition further raises the RNOS levels, triggering apoptosis [ 115 ]. High ALDH activity is associated with high levels in the mitochondrial mass, both in cell lines and samples derived from patients, suggesting higher mitochondrial biogenesis or retarded degradation of mitochondria. Thus, the mitochondrial biogenesis inhibitor doxycycline targets ALDH + breast CSCs [ 116 ]. These results could be related to higher expression of mitochondrial ALDH isoforms [117]. The regulation among the enzymes of the glycolytic pathway, OXPHOS and TCA cycle, all involved in the synthesis of ATP and maintenance of the NAD + /NADH ratio, results in high metabolic plasticity in a large proportion of CSCs. The mitochondria take small molecules, such as pyruvate, fatty acids and amino acids, from catabolic reactions to obtain reduced power in the form of NADH and/or FADH 2 . These molecules allow, through their oxidation, the electron transfer in the mitochondrial respiratory chain from water to molecular oxygen. Importantly, design strategies that target a type of metabolism can lead to tumor cells, especially CSCs, with higher metabolic plasticity, changing their metabolism to adapt to the new situation. Thus, considering combined treatment is important to short-circuit mitochondrial metabolic plasticity. 4.1. Role of the Mitochondrial Respiratory Chain in CSC Resistance The mitochondrial respiratory chain, or electron transport chain (ETC), comprises four enzymatic complexes, among which complex II is completely encoded in the nuclear genome, while complexes I, III and IV are still encoded in mtDNA [ 86 ]. The higher mutation rate of mtDNA than nuclear DNA allows the appearance of certain mutations that can affect the assembly of the mitochondrial complexes but allowing its partial functioning. Thus, due to the heteroplasmy existing in cells [ 86 ], mutations in mtND1 allow the functioning of Complex I but, when the mutated population reached a threshold, they behave as tumor suppressors, preventing the assembly of complex I and also reducing its metastatic ability [ 118 ]. Thus, mtDNA mutations comprising both the activity and assembly of OXPHOS complexes may decrease or even inhibit tumor growth in xenograft models [ 118 ]. However, a partial ETC inhibition caused by mutations in mtDNA, that mimics the effect of mtDNA depletion, promoted a migratory phenotype in cultured cells, behaving as oncogenic mutations [ 119 ]. In this way, certain mutations behave as oncogenic when appearing at low levels in the mtDNA population but became tumor suppressors in homoplasmy. Due to the role of the four complexes involved in OXPHOS in electron flux, inhibiting a specific complex is similar to that obtained by inhibiting others (Figure 2A). Drugs inhibiting mitochondrial respiration include metformin, a complex I inhibitor that reduces tumorigenesis [ 120 ]. In non-CSCs from pancreatic cancer cells, metformin induced cell cycle arrest, while apoptosis was induced in CSCs [ 18 , 107 ]. This molecule can reverse the resistance to chemotherapy drugs in breast cancer cells [ 121 ]. In fact, metformin treatment specifically eliminates CD44 + /CD24 − CSCs, showing positive synergy with doxorubicin and resulting in delayed tumor recurrence [ 122 ]. Additionally, metformin, also described as a PI3K/Akt/mTOR signaling inhibitor, reduced CSC resistance to temozolomide, a chemotherapy drug that causes DNA damage [ 123 ]. However, metformin resistance has also been detected in certain tumors in vivo [ 18 ], making it necessary to use combined treatments or more powerful derivatives. Thus, the combination of metformin with 2-deoxy-D-glucose (2-DG), a glycolysis inhibitor, increased the cell death percentage and reduced tumor growth in xenograft Cells 2020,9, 1693 8 of 28 models [ 124 ]. The use of phenformin, a biguanide derivative similar to metformin and a complex I inhibitor, but with higher antineoplastic efficiency, can be used alone or combined with other drugs against CSCs [ 125 ]. Combined treatment of phenformin with gossypol, an inhibitor of ALDH and various anti-apoptotic components of the BCL-2 family, suppresses stemness and produces a significant reduction in invasion capacity and cell viability [ 126 ]. Another drug, menadione, exerts a double effect through complex I inhibition and the induction of ROS, and prevents the appearance of resistance [ 127 ]. Pirvinium pamoate, a complex II inhibitor, reduces tumorsphere formation in different cancer cell lines [ 75 ]. Similarly, antimycin A and atovaquone, both complex III inhibitors, significantly reduce CSCs [ 78 , 128 ]. Atovaquone target CSCs preferentially, producing an increased glycolytic rate, with no effect on normal fibroblasts [ 78 ]. ATP synthase, also called Complex V, can be inhibited by oligomycin, but its high toxicity prevents its use as a chemotherapeutic drug [ 76 ]. However, it could be used at lower concentrations combined with other drugs, such as 2-DG or niclosamide, reducing the CSC percentage in glioblastoma and ovarian and breast cancer cells [129–131]. Another Complex V inhibitor, bedaquiline, is a drug approved for multidrug-resistant tuberculosis [ 132 ] that targets CSCs preferentially [ 133 ]. Other drugs, such as resveratrol, can correct defects in complexes I and IV, decreasing the growth rate and invasive potential of different cancer cell lines [ 77 , 134 ]. This drug has been described to induce mitochondrial dysfunction, cytochrome crelease and caspase activation in pancreatic cancer [ 135 ]. In other cases, OXPHOS must be inhibited to bypass acquired resistance to certain drugs. Thus, OXPHOS promotes resistance to cytarabine, an antimetabolite, in animal AML models, and combined treatment with OXPHOS inhibitors restores sensitivity [136]. OXPHOS can also be inhibited by changes in the mitochondrial membrane potential. Salinomycin is a K + ionophore that, acting as an OXPHOS inhibitor, seems to reduce the CSC percentage both in vitro and in vivo in different types of cancer [ 137 – 139 ]. Salinomycin can kill cells resistant to different chemotherapeutic drugs, such as doxorubicin, cisplatin, gemcitabine, temozolomide, verapamil or imatinib and sensitizes radioresistant cells [ 139 ]. The toxicity of salinomycin is amplified under low oxygen and/or glucose levels, increasing RNOS levels [ 140 ]. In this situation, AMP-activated protein kinase (AMPK) is activated, triggering autophagy and allowing this drug to act as an anti-CSC molecule. Thus, the combination of AMPK agonists, such as metformin and 2-DG, with salinomycin could be used to overcome CSC resistance [ 139 ]. Salinomycin also causes mitochondrial hyperpolarization, inducing mitophagy of dysfunctional mitochondria [ 141 ]. Salinomycin can target and kill cancer cells preferentially, but not primary cells [ 141 ]. A similar effect, the preference for cancer cells, has been found for another OXPHOS inhibitor, VLX600, increasing the effect of irinotecan in xenograft models [ 142 ]. This compound also induces AMPK phosphorylation in tumor cell lines [ 142 ] and sensitizes cells to glucose starvation [143]. In most of these cases, OXPHOS inhibition causes mitochondrial imbalance, causing increased RNOS levels and/or metabolic switch to glycolysis. In solid tumors, due to the poor blood supply, both the oxygen and glucose low levels will compromise metabolic rewiring from OXPHOS to glycolysis if mitochondrial OXPHOS is inhibited. Thus, as a consequence of OXPHOS imbalance, oxidative stress due to RNOS accumulation can promote the triggering of apoptosis in CSCs based on OXPHOS metabolism. Cells 2020,9, 1693 9 of 28 Figure 2. Mitochondrial metabolism targeting can overcome CSC resistance. ( A ) Mitochondrial eltron transport chain (ETC), core of oxidative metabolism, showing some of the described inhibitors for each complex (blue lines) used for CSC treatment. CI: complex I; CII: complex II; CIII: Complex III, CIV: Complex IV; CV: Complex V; Q: ubiquinone; Cc: cytochrome c. Outer mitochondrial membrane appears in red, while inner mitochondrial membrane is colored in grey. Salinomycin causes mitochondrial membrane potential ( ∆Ψ m) disruption. ( B ) Reactive oxygen species produced as a consequence of ETC. Inhibitors of mitochondrial detoxifying enzymes are labelled with a blue line. Red arrow indicates a RNOS enhancer. ATO: Arsenic trioxide; GSH: glutathione; GSSG: glutathione disulfide; GR: glutathione reductase; GPX: gluthathione peroxidase. ( C ) Glycolytic metabolism, showing inhibitors of GLUT1 and hexokinase, blocking glycolysis at its beginning, and dichloroacetate (DCA), that inhibits pyruvate dehydrogenase kinase (PDK) and allow the incorporation of pyruvate into the tricarboxylic acid (TCA) cycle. Blue lines correspond to glycolytic inhibitors, while red arrows belong to HIF-1 α , a glycolytic enhancer. 2-DG: 2-deoxyglucose; GPI: glucose-6-phosphate isomerase; G3PDH: glyceraldehyde-3-phosphate dehydrogenase; PGK1: phosphoglycerate kinase; PGM: phosphoglycerate mutase. ( D ) Summary of fatty acid oxidation (FAO) metabolism. Etomoxir and perhexiline, inhibitors of CPT1 (blue line) diminished CSC population, by reducing mitochondrial incorporation of fatty acids. ( E ) Pathways related to the TCA cycle frequently modified in CSCs. Blue arrows correspond to aconitase (ACO) knockdown. SDH: succinate dehydrogenase; FH: fumarate hydratase; IDH: isocitrate dehydrogenase; mIDH: mutated IDH. GLS: glutaminase; GDH: glutamate dehydrogenase; IDH: isocitrate dehydrogenase; ACLY: ATP citrate lyase. Cells 2020,9, 1693 16 of 28 induce apoptosis in paclitaxel-resistant cells [ 167 ]. BCL-2 inhibition also impairs OXPHOS, reducing the survival of OXPHOS-dependent CSCs in AML [238]. Due to apoptosis-acquired resistance of CSCs, the use of inhibitors of BCL2 family would reduce the CSC population. Figure 4. Summary of intrinsic apoptotic pathway. BCL-2, as antiapoptotic protein, inhibits the formation of pores by BAX/BAK that allow cytochrome crelease to the cytoplasm, where it interacts with Apaf-1 to constitute apoptosome, allowing caspase cascade. Drugs targeting apoptosis comprise dissipaters of mitochondrial membrane potential ( ∆Ψ m) or BCL-2 inhibitors (blue lines) or molecules enhancing formation of BAX/BAK pores (red arrow). 8. Conclusions Mitochondria behave as an organelle of extraordinary metabolic plasticity, capable of modifying their different pathways to support (cancer) cell survival. Additionally, the combination of different chemotherapeutic agents or radiation is a good strategy to overcome the resistance of CSCs. Thus, blocking at least two metabolic pathways simultaneously would reduce the possibility of a relapse, as well as the possible development of resistance. However, and due to metabolic plasticity, it would be important to know which pathway(s) is prevalent in a specific CSC population, in order to use drugs that could target them, increasing its effectiveness. Additionally, it would be interesting to study the design of synergistic treatments focused on CSCs mitochondrial metabolism, to reduce relapse probability. Funding: This research was funded by grants from the Ministerio de Ciencia, Innovaci ó n y Universidades (MCIU) Plan Estatal de I+D+I 2018, a la Agencia Estatal de Investigaci ó n (AEI) y al Fondo Europeo de Desarrollo Regional (MCIU/AEI/FEDER, UE): RTI2018-097455-B-I00; grant from AEI-MICIU/FEDER (RED2018-102723-T); from CIBER de C á ncer (CB16/12/00275), co-funded by FEDER from Regional Development European Funds (European Union); from Consejer í a de Salud (PI-0397-2017) and Consejer í a of Econom í a, Conocimiento, Empresas y Universidad of the Junta de Andaluc í a (P18-RT-2501). Also especial thanks to the Fundaci ó n AECC and Fundaci ó n Eugenio Rodriguez Pascual for supporting this work. Cells 2020,9, 1693 17 of 28 Conflicts of Interest: The authors declare no conflict of interest. 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