El Hout et al. Molecular Cancer (2020) 19:27 https://doi.org/10.1186/s12943-019-1126-8

REVIEW Open Access Crosstalk between autophagy and metabolic regulation of cancer stem cells Mouradi El Hout1,2, Emma Cosialls1,2, Maryam Mehrpour1,2* and Ahmed Hamaï1,2*

Abstract Cancer is now considered as a heterogeneous ecosystem in which tumor cells collaborate with each other and with host cells in their microenvironment. As circumstances change, the ecosystem evolves to ensure the survival and growth of the cancer cells. In this ecosystem, metabolism is not only a key player but also drives stemness. In this review, we first summarize our current understanding of how autophagy influences cancer stem cell phenotype. We emphasize metabolic pathways in cancer stem cells and discuss how autophagy-mediated regulation metabolism is involved in their maintenance and proliferation. We then provide an update on the role of metabolic reprogramming and plasticity in cancer stem cells. Finally, we discuss how metabolic pathways in cancer stem cells could be therapeutically targeted. Keywords: Cancer stem cells, Autophagy, Metabolic heterogeneity, Lipid metabolism, Therapeutic target

Background Autophagy in CSCs Cancer is a heterogeneous disease, and metabolic flexibility Autophagy is a process necessary for normal cellular func- of tumors contributes to this heterogeneity. As circum- tion involved in the tumor initiation, tumor interactions stances change, the tumor ecosystem evolves to ensure the with neighboring cells in the tumor microenvironment, and survival and growth of the cancer cells [1]. In this ecosys- cancer therapy. The role of autophagy in cancer is multifa- tem, metabolism is a driver of stemness [2]. Cancer stem ceted: Autophagy promotes tumor cell survival by supplying cells (CSCs) are a subset of cells within tumors that have recycled metabolites for growth, modulates mitochondrial the capacity to generate tumors and that exhibit self- function via mitophagy (the selective degradation of mito- renewal and differentiation properties. CSCs are resistant to chondria), and functions in tumor cell migration and inva- cancer therapies and are a distinct population associated sion via control of secretion of pro-migratory cytokines and with metastasis and relapse. focal adhesion turnover [3]. Also, several studies have dem- Only a few studies have directly examined the metab- onstrated that autophagy plays a central role in the tumor olism of CSCs in large part due to difficulties in isolating microenvironment [3, 4]. For example, autophagy is induced this population. Currently, there are more than 40 estab- in cancer-associated fibroblasts (CAFs) by their association lished CSC markers; however, most are also present in with tumor cells, and this results in increased fibroblast pro- human embryonic stem cells and/or adult stem cells duction of amino acids, which are provided in a paracrine from normal tissues and a consensus marker for the manner to tumor cells to sustain their growth [5]. Two im- identification of CSCs still a matter of debate. We begin portant elements that influence metabolic reprogramming this review with a brief discussion of autophagy and of tumors are their microenvironment and the distance to CSCs, and we review recent data on CSC metabolism. the vasculature [1, 4]. First, emerging evidence indicates the Finally, we discuss how CSC metabolism could be a unexpected ability of malignant cells (both CSCs and non- therapeutic target for treatment of cancer. CSCs) to supplement their metabolism with nutrients pro- vided by neighboring cells with complementary metabolic activities, enhancing tumor cell survival and proliferative capacity [6–8]. Second, cancer cells located closer to the * Correspondence: [email protected]; [email protected] 1Institut Necker-Enfants Malades (INEM), Inserm U1151-CNRS UMR 8253, blood supply generate ATP via oxidative stress, and this in- F-75993 Paris, France duces glycolysis and autophagy in the surrounding catabolic Full list of author information is available at the end of the article

© The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. El Hout et al. Molecular Cancer (2020) 19:27 Page 2 of 17

stromal/cancer cells (again, in both CSCs and non-CSCs), to autophagy induction [16]. Also, SOX2 and STAT3 which generate catabolites such as fatty acids, lactates, and have been shown to modulate autophagy genes and to ketones that in turn are taken up by anabolic cancer cells determine the stemness of CSCs. (both CSCs and non-CSCs) and used to fuel mitochondrial Mitophagy is emerging as a key in the control of normal metabolism and ATP production. This is known as the tissue stem cell homeostasis. Mitophagy functions to control reverse Warburg effect (Table 1). Parallel autophagic re- mitochondria quality and also regulates cellular metabolism. sponses activated in distal and poorly oxygenated tumor For example, removing damaged mitochondria, the main areas provide catabolic intermediates to sustain anabolic source of ROS, by mitophagy prevents senescence and limits demands and support cancer growth (Table 1). ROS-induced genome damage. Limiting ROS damage is es- Recent reviews have focused on the role of autophagy sential for maintenance of stemness. An essential role for in tumor metabolism [4], anti-tumor immunity [3], and mitophagy has reported during the glycolytic switch neces- cancer metastasis and cancer therapy [11]. Here, we sary for mouse developmental neurogenesis [12]. The turn- briefly discuss more recently reported roles for autoph- over of mitochondria through mitophagy helps to maintain agy in CSCs. Autophagy appears to be necessary for the the stem cell state by limiting the capacity of the stem cells maintenance of stemness in both normal tissue stem for oxidative phosphorylation (OXPHOS) and making the cells [12] and CSCs [13, 14] in diverse cancer types in- stem cells more dependent on glycolysis for energy demands. cluding breast, pancreatic, bladder, and colorectal can- Inhibition of mitophagy suppresses CD44 expression and cers, chronic myeloid leukemia, and glioblastoma (for also promotes translocation of p53 to the nucleus, where it review see [8]). The survival and quiescence of normal antagonizes expression of stem cell genes. tissue stem cells is dependent on autophagy, and au- The high levels of autophagy observed in CSCs are associ- tophagy has also been reported to promote pluripotency. ated with the maintenance of pluripotency, with resistance to In CSCs, autophagy promotes expression of stem cell chemotherapy, and with migration and invasion [3]. Autoph- markers such as CD44 as well as expression of mesen- agy allows CSCs to survive despite hypoxia and low levels of chymal markers such as vimentin [13]. Autophagy also nutrients in the tumor microenvironment [17]. Missing is a promotes spheroid formation in vivo tumorigenesis con- comprehensive view of how these processes drive CSC fate, sistent with a critical role in maintaining CSCs [14]. Fur- and few of the regulatory molecules involved have been iden- ther, the inhibition of autophagy limits tumor dormancy tified. Readers interested in detailed discussion of autophagy and promotes outgrowth of metastases [15]. Key tran- processes in CSCs should see a recent review [8]. scription factors have been linked to the induction of au- tophagy and the stem cell state including Forkhead CSCs are more glycolytic than other differentiated box 3A (FOXO3A), which induces expression of autoph- cancer cells agy genes in stem cells and is itself turned over by au- A growing body of evidence suggests that the metabolism tophagy. Other transcription factors, including the core of CSC also differs from that of the bulk tumors. The im- stemness factors sex determining region Y-box (SOX2) portance of glucose for the maintenance and propagation and Nanog Homeobox (NANOG), have also been linked of CSCs was first established in glioblastoma (GBM) [18] and then in other types of cancer including breast cancer, colon cancer, lung cancer, ovarian cancer, and osteosar- Table 1 Warburg and Reverse Warburg effects coma [2]. The side population cells with CSC characteris- Aerobic glycolysis, or the Warburg effect, is a phenomenon in cancer cells that results in reorientation of metabolism to the glycolytic tics avidly consume glucose and generate a significant pathway and to conversion of pyruvate resulting from glycolysis into amount of ATP and lactate [19]. In addition, in these cells lactate even in the presence of oxygen. This metabolic reprogramming the AKT Kinase pathway is activated by glucose and in- is a step in the process of tumorigenesis in many cancers. It is one of the best-described metabolic adaptations arising in cancer cells. It is hibition of glycolysis decreases the ability of these cells to + now established, however, that malignant transformation is not based form tumors in vivo. In ovarian cancer, the CD44 / solely on the Warburg effect. Indeed, tumor cells produce a significant MyD88+ CSCs depend only on glycolysis for their survival fraction of their ATP via oxidative phosphorylation (OXPHOS). Malignant cells adapt their energetic metabolism to the conditions of the microenviron- and are incapable of producing ATP by OXPHOS, result- ment, in particular to the oxygenation conditions of the tumor, which has ing in autophagic death in the absence of glucose [20]. the consequence of creating intra-tumor metabolic heterogeneity (for add- The metabolism of breast CSCs (BCSCs) grown as itional information see [1, 9, 10]). The reverse Warburg effect is observed when anabolic epithelial cancer cells induce aerobic glycolysis in neighbor- spheres is strongly associated with increased activities of ing stromal fibroblasts or neighboring catabolic cancer cells. These catabolic key enzymes of anaerobic glucose fate such as pyruvate kin- cells (epithelial cancer cells or cancer-associated fibroblasts) secrete catabo- ase isozyme M2 (PKM2), lactate dehydrogenase (LDH), and lites such as lactate, pyruvate (energy metabolites resulting from aerobic glycolysis), free fatty acids, and ketone bodies. Anabolic epithelial cancer cells glucose-6-phosphate dehydrogenase (G6PDH). Consistent take up these energy-rich metabolites and use them to fuel OXPHOS. This with this, BCSCs are highly sensitive to 2-deoxyglucose, a results in a higher proliferative capacity (see Fig. 1 and [1]). An absence of well-known inhibitor of glycolysis [21]. The overexpression stromal Cav-1 may be a biomarker for the reverse Warburg effect. of fructose-1,6- biphosphatase (FBP1) and the increase of El Hout et al. Molecular Cancer (2020) 19:27 Page 3 of 17

ROS are accompanied by a significant reduction in the De Luca et al. recently reported that mitochondrial bio- number of CD44high/CD24low/EpCAM+ CSCs and the for- genesis is required for maintenance of stem-like properties mation of spheres [22]. The increase of glucose uptake, [31]. The inhibition of mitochondrial biogenesis mediator glycolytic enzyme expression, lactate production, and ATP PGC1α decreases the stem-like properties of BCSCs [31]. content in CSCs compared with their differentiated coun- In pancreatic ductal adenocarcinoma (PDAC), the deadliest terparts seems to be linked to a concomitant reduction in cancer in western countries, it has been shown that CSCs mitochondrial activity [18, 21] and also to maintenance of are OXPHOS-dependent, unlike non-CSCs that are glyco- OXPHOS and beta-oxidation [23]. Mechanistic analysis lytic. In addition, suppression of MYC expression and in- demonstrated that decreased expression and activity of creased expression of PGC1α are key determinants for the pyruvate dehydrogenase (PDH), a key regulator of oxidative OXPHOS dependency of CSCs and their limited ability to phosphorylation, plays a critical role in promoting the pro- switch to glycolysis during mitochondrial inhibition [32]. glycolytic phenotype of CSCs. Metabolic reprogramming A recent study showed that Matcha green tea inhibits via forced activation of PDH preferentially eliminates CSCs the propagation of BCSCs. Interestingly, metabolic pheno- [24]. Tamada et al. showed that CD44, a marker of CSCs, typing revealed that treatment with this compound sup- acts as a metabolic modulator, activating glycolysis under presses both OXPHOS and glycolytic flux, shifting cancer hypoxic conditions and reducing glycolysis and antioxidant cells toward a more quiescent metabolic state [33]. responses and enhancing mitochondrial production with associated increases in ROS. CD44 interacts with PKM2 in Other metabolic pathways involved in CSC different cancer cell lines and inhibits its activity, correlating maintenance and proliferation with the glycolytic phenotypes of p53-deficient cells [25]. A Mevalonate metabolic pathway metabolic signature characteristic of colon cancer initiating A prominent role of the mevalonate metabolic pathway in cells has been associated with increased expression of genes regulating the self-renewal of basal/mesenchymal BCSCs has and metabolites of the glycolytic pathway and the tricarb- been demonstrated. Inhibition of this pathway with hydroxy- oxylic acid cycle (TCA) [26]. 3-methylglutaryl CoA reductase blockers results in a reduc- tion of BCSC proliferation independent of inhibition of chol- esterol biosynthesis and of protein farnesylation. Notably, CSCs rely on mitochondrial oxidative metabolism geranylgeranyl transferase I is crucial for BCSC maintenance. In contrast to data linking the stem phenotype of cancer cells The effect of geranylgeranyl transferase I on the CSC sub- to glycolytic metabolism, emerging evidence indicates that population is mediated by inactivation of Ras homolog family CSCs have a preference for OXPHOS metabolism (Fig. 1). member RHOA and increased accumulation of P27kip1 in In both cases, mitochondrial function is essential for stem- the nucleus [34]. Mesenchymal stem cells have been re- ness, migration, and drug resistance of CSCs [27]. Compared ported to shuttle mitochondria and/or mitochondrial DNA to differentiated progeny, BCSCs consume more glucose, in leukemia, lung, and breast tumors and to consume the produce less lactate, and have higher ATP content. BCSCs cysteine dimer cystine to provide leukemic cells with chemo- are heterogeneous in their metabolic phenotypes and have protective cysteine [7]. metabolic states distinct from their differentiated progeny. The increased mitochondrial mass in a distinct popu- Hypoxia and redox homeostasis lation of breast cancer cells is attributed to a stem-like Clinical data indicate that reduced oxygen availability, or phenotype and is associated with metastatic potential hypoxia, observed in intratumoral regions activates and chemotherapy resistance [28]. Despite a high rate of hypoxia-inducible factors (HIFs). These master regula- pentose phosphate pathway activity, which is not typical tors of oxygen homeostasis also play key roles in the of cells that prefer OXPHOS over glycolysis, the CSCs maintenance of BCSCs [35]. In response to intratumoral isolated from patients with epithelial cancer overexpress hypoxia or chemotherapy such as carboplatin or pacli- genes associated with glucose uptake, OXPHOS, and taxel, the increased expression of HIF-1α and HIF-2α in fatty acid beta oxidation, indicating that in these cells BCSCs leads to increased expression of pluripotency fac- pyruvate is preferentially directed toward the TCA cycle. tors such as Kruppel-like Factor 4 (KLF4), NANOG, Consistent with a metabolic OXPHOS profile, CSCs octamer-binding transcription factor 4 (OCT4), and have higher mitochondrial ROS production and elevated SOX2 [35]. HIF-1 coordinately regulates expression of membrane potential than normal cells and undergo genes encoding pyruvate dehydrogenase (PHGDH) and apoptosis upon inhibition of the mitochondrial respira- five downstream enzymes in the serine synthesis path- tory chain [29]. Consistent with previously reported data way and mitochondrial one-carbon (folate) cycle. Silen- [30], CSCs display enhanced antioxidant defenses com- cing of PHGDH expression leads to decreased NADPH pared to their non-tumorigenic counterparts, and this levels, disturbed mitochondrial redox homeostasis, and may contribute to tumor resistance to therapy. increased apoptosis, which abrogate BCSC enrichment El Hout et al. Molecular Cancer (2020) 19:27 Page 4 of 17

Fig. 1 The metabolic heterogeneity of cancer stem cells. Tumors are complex and dynamic structures encompassing populations of host cells (e.g., fibroblasts and immune cells) and cancer cells with different metabolic activities. These cells are affected in different ways by microenvironmental conditions and biological activities of other tumor cells. For example, cancer cells close to the vasculature show oxidative metabolism, whereas a shift toward a glycolytic metabolism is observed when glucose is present in cells residing in hypoxic areas. Despite metabolic heterogeneity, cancer cells cooperate to allow adaption to changes in conditions to ensure that metabolic requirements are met. Indeed, oxidative cancer cells, like proliferating cells, increase the consumption of glucose to produce ATP and generate biomass to support cell proliferation. The oxidative stress caused by rapidly proliferation of cancer cells induces glycolysis and autophagy/mitophagy in stromal cells and/or in glycolytic cancer cells leading to the release of high amounts of lactate, which fuels the metabolism of oxidative cancer cells. Key elements of lactate shuttles are the plasma membrane monocarboxylate transporters. MCT4 is involved in the export of lactate, and MCT1 and MCT2 are involved in the uptake of this catabolite. High levels of several factors including HIF-1α,NF-κB, TGF-β, and JNK/AP1 are associated with glycolytic phenotype. The metabolic status of a CSC depends on location. In actively growing regions of the tumor and in the presence of adequate levels of oxygen (normoxic conditions), CSCs rely on glycolytic and/or oxidative metabolism. Overexpression of HIF-1α in the hypoxic environment promotes upregulation of GLUT1, GLUT3, and glycolytic enzymes. In the metastatic niche, CSCs have increased utilization of extracellular catabolites. In nutrient-poor states, autophagy is activated to provide an alternative energy source. OXPHOS and the anabolic gluconeogenesis pathways control glucose homeostasis. Abbreviations: ATP, adenosine triphosphate; CSC, cancer stem cell; GLUT1/GLUT3, glucose transporter 1/3; HIF-1α, hypoxia-inducible factor 1α; HK2, hexokinase 2; JNK/AP1, c-Jun N-terminal kinases/activator protein 1; LDH, Lactate dehydrogenase; XMCT2/4, monocarboxylate transporter 2/4; NF-κB, nuclear factor-κB; OXPHOS, oxidative phosphorylation; PFKFB, phosphofructokinase/fructose bisphosphate; PKM2, pyruvate kinase isozyme M2; TGF-β, transforming growth factor β under hypoxic conditions. PHGDH-deficient cells are Genetic and pharmacological inhibition of mechanistic relatively weakly tumorigenic, and tumors that do form target of rapamycin (mTOR) abrogates low folate- are deficient in BCSCs and thus have no metastatic cap- activated AKT-mTOR-HIF1-FOXO3a signaling and acity [36]. Human non-small cell lung cancer cells cul- stemness-associated sonic hedgehog pathway activity, re- tured in low folate conditions have enhanced CSC-like verses the Warburg metabolic switch, and diminishes in- properties associated with elevated lactate release and vasiveness of non-small cell lung cancer cells. These medium acidification, suppressed expression of PDH, data suggest that lung CSCs may arise from a micro- and elevated redox status as shown by NADH/NAD+ environment low in folate through the activation of an and NADPH/NADP+ ratios. These data are indicative of AKT-mTOR-HIF1-FOXO3a signaling network, which the metabolic reprogramming to aerobic glycolysis. promotes bioenergetic reprogramming to enhance CSC- El Hout et al. Molecular Cancer (2020) 19:27 Page 5 of 17

like signatures and invasion and metastasis of lung can- availability and through downregulation of activities of DNA cers [37]. methyltransferases such as DNMT1 and DNMT3A [41].

NAD and nicotinamide phosphoribosyl transferase NAD+ and autophagy pathways are associated with tumorigenesis Decreased NAD+ availability compromises the PARP1- NAD is a cofactor essential for metabolism, energy associated base excision DNA repair pathway. Chemical production, DNA repair, maintenance of mitochondrial inhibition of PARP1 using the drug olaparib impairs base fitness, and signaling in many types of cancer cells. The excision DNA repair thereby enhancing temozolomide- biosynthesis of NAD occurs through both de novo and induced damage; this mechanism is responsible for syner- salvage pathways. NAD is primarily synthesized from gistic anti-tumor effects of the two drugs in GSC lines [42]. nicotinamide, a process known as the NAD salvage path- Mechanistic studies suggest that the activation of PARP1 way. Nicotinamide phosphoribosyl transferase (NAMPT) upregulates the AMP-activated protein kinase (AMPK) sig- catalyzes the conversion of nicotinamide to nicotinamide nal pathway and downregulates the mTOR signaling path- mononucleotide (NMN), which is the rate-limiting step in way, thereby promoting autophagy following ionizing the NAD salvage pathway. Thus, NAMPT is critical for radiation or starvation [43]. NAD biosynthesis. Inhibition of NAMPT leads to deple- NAD+ consumption by PARP1 generates a Ca2+ mo- tion of NAD+, which in turn inhibits ATP synthesis [38]. bilizing messenger and upregulates intracellular Ca2+ NAMPT is overexpressed in high-grade glioma and GBM signaling through transient receptor potential melastatin tumors, and its levels correlate with tumor grade and 2 channels, which can also enhance autophagy. However, prognosis. Ectopic overexpression of NAMPT in glioma further studies are required to confirm that NAD+ me- cell lines is associated with the enrichment of glioblastoma tabolism induced by PARP1 contributes to autophagy CSC population and inhibition of NAMPT blocks in vivo initiation in CSCs. Pharmacological or genetic manipula- tumorigenicity of glioblastoma CSCs. The self-renewal tion of NAD levels appears to modulate autophagy by al- properties of the glioblastoma CSC population and radi- tering SIRT1 activity. Inhibition of SIRT1 abolishes this ation resistance in GBM are orchestrated by a NAD- autophagy modulation, suggesting that SIRT1 is critical dependent transcriptional network [39]. Along the same for this process. The mechanisms underlying the NAD+- lines, Lucena-Cacace et al. also recently reported that dependent deacetylation by SIRT1 in the regulation of NAMPT plays an important role in regulation of the CSC autophagy involve the activation or inhibition of mul- survival and proliferation in colon cancer tumors [40]. tiple transcription factors, including FOXO3 and P53, This phenotype is mediated by poly (ADP-ribose) poly- and of ATG proteins such as ATG5, ATG12, ATG14, merases (PARPs) and sirtuins (SIRTs). Beclin-1, Bcl-2/adenovirus E1B interacting protein 3 Recently, Lucena-Cacace et al. raised the idea that (Bnip3), and Microtubule-associated Protein 1 Light NAMPT contributes to tumor dedifferentiation and, driven Chain 3 (LC3) [44]. However, further studies are re- by NAD supply, is responsible for the epigenetic reprogram- quired to confirm that NAD+ metabolism regulated by ming observed in tumors [37]. This idea is supported by SIRT1 contributes to autophagy initiation in CSCs. data reported by Jung et al. [41] who showed that mesen- chymal glioblastoma stem cells (GSCs) contain higher levels of NAD and lower levels of nicotinamide, methionine, and Glutaminolysis S-adenosyl methionine (SAM), a methyl donor generated Glutaminolysis is also essential for the proliferation and from methionine, compared to differentiated tumor cells. survival of epithelial CSCs largely because the ammonia Nicotinamide N-methyltransferase (NNMT), an enzyme molecules released from glutamine metabolism neutralize that catalyzes the transfer of a methyl group from the cofac- the excessive levels of protons (lactic acid) that result from tor SAM onto its various substrates such as nicotinamide the marked Warburg effect observed in these cells [45]. and other pyridines, is also overexpressed in GSCs. Increases Epithelial CSCs preferentially rely on aerobic glycolysis for in NNMT lead to a decrease in SAM. GSCs are hypomethy- bioenergetics, display an active serine-one-carbon-glycine lated in GBM, and this causes tumors to shift toward a mes- metabolism, and show an increased metabolic flexibility to enchymal phenotype with accelerated growth, a phenotype utilize different carbon sources (such as fatty acids and also associated with overexpression of NAMPT. NNMT glutamine) that offsets the decreased diversion of glucose- silencing decreases self-renewal and in vivo tumor growth of derived carbons into the TCA cycle. GSCs. Inhibition of NNMT expression or activity also di- Recent studies have shed light on the role of iron metab- minishes methyl donor availability, thus decreasing methio- olism in CSCs and suggest that specific targeting of iron nine and unmethylated cytosine levels. Available data metabolism in CSCs may improve the efficacy of cancer suggestthatNNMThasadualmechanism:Itpromotes therapy. Readers interested in detailed discussion of iron DNA hypomethylation through reduction of methyl donor metabolism in CSCs should see a recent review [46, 47]. El Hout et al. Molecular Cancer (2020) 19:27 Page 6 of 17

Metabolic reprogramming and plasticity AMPK-HIF1α pathway and is reversed by N-acetylcysteine. It has been shown that the epithelial-mesenchymal transi- Moreover, silencing of expression of the gene encoding nu- tion (EMT) can endow cancer cells with stem cell-like clear factor erythroid 2-like 2 (NRF2) or suppression of properties and can cause a switch from an epithelial pro- downstream thioredoxin and glutathione antioxidant path- gram to a motile mesenchymal phenotype [48]. However, ways result in ECSCs sensitive to ROS-induced differenti- in solid tumors, CSCs can arise independently of EMT. A ation and cytotoxicity. However, both MCSCs and ECSCs comparative analysis using metabolomic and fluxomic ap- are eliminated by co-inhibition of glycolysis and thioredoxin proaches identified metabolic profiles that differentiate and glutathione pathways. This co-inhibition abolishes metastatic prostate epithelial CSCs from non-CSCs tumor-initiating potential, tumor growth, and metastasis expressing a stable EMT signature. The epithelial CSCs [54]. The CSC phenotype is glycolyticininvitroexperi- (ECSCs) are distinguished by an enhanced Warburg effect ments, given the non-physiological concentrations of oxygen and a greater carbon and energy source flexibility resulting and glucose, whereas directly after cells are isolated from from amino acid and fatty acid metabolism. ECSCs are patients or after the first passage in culture CSCs depend on also characterized by a critical dependence on the proton OXPHOS [52]. The upregulation of the glucose transporter buffering capacity bestowed by glutamine metabolism. A Glut3 results in increased production of GSCs indicating metabolic gene signature for ECSCs has been correlated how the high-grade glioblastomas, which are endowed with with tumor progression and metastasis in several cancer a high metabolic plasticity, survive in glucose-poor environ- types [45]. ments [55]. The induction of EMT is associated with enhanced glycolysis and reduced mitochondrial activity. Mechanis- Metabolism as a therapeutic target for CSC tic analysis demonstrated that this process is the result Several pathways which regulate metabolism and autoph- of the activation of cytochrome c oxidase [49] or from agy of CSCs, are targeted for the treatment of cancer the suppression of fructose-1,6-bisphosphatase [22]. Fur- (Table 2 and Fig. 2). Anti-CSC therapies causing a defi- thermore, bioenergetic disorders resulting from the in- ciencies in energy and materials impairing CSC survival hibition of citrate synthase or succinate dehydrogenase and propagation establish the basis of the future therapies. subunit B can contribute to the acquisition of an EMT The following paragraphs provide a brief preview of these phenotype [50]. Luo et al. reported that EMT-driven therapeutic target and the compounds that influence me- CSCs can metabolize alternative high-energy metabo- tabolism and autophagy of CSCs. lites, the phenomenon known as reverse Warburg effect (Table 1)[51]. Mitochondrial metabolism The comparison between mesenchymal-like CSCs As mentioned above, mitochondria play a key role in the (MCSC) and ECSCs revealed distinct metabolic pathways. responses to oxidative stress, energy status changes, and MCSCs display enhanced glycolysis as well as reduced O2 apoptotic stimuli and are also involved in the regulation consumption, reduced ROS production, more antioxidant of stemness and differentiation of CSCs [59]. Several capacity, and reduced mitochondrial mass and membrane pathways that promote anaerobic and aerobic energy potential compared to ECSCs [30, 52]. Recent work has metabolism of CSCs have been evaluated as targets for divided GSCs into two subtypes with a mesenchymal GSC the treatment of cancer (Fig. 2). population as the more malignant subtype. Glycolytic and Aldehyde dehydrogenase 1A3 (ALDH1A3) activities are OXPHOS inhibitors remarkably elevated in mesenchymal GSCs but not in Various compounds that inhibit oxidative metabolism re- proneural GSCs. Moreover, irradiation of proneural GSCs sult in sensitization of CSCs to chemotherapies, leading to results in an up-regulation of mesenchymal-associated their eradication. This has been demonstrated in a model markers and a down-regulation of proneural-associated of PDAC. KRAS mutations are known to be a driver event markers, and this effect is attenuated by inhibition of of PDAC, but targeting mutant KRAS has proved challen- ALDH1A3 activity. For the high-grade glioma patients ging. Using a KRAS-inducible mouse model, Viale et al. with the mesenchymal signature, inhibition of ALDH1A3- demonstrated that a subpopulation of cells with CSC fea- mediated pathways is thus a promising therapeutic ap- tures survives KRAS-ablation therapy and induces tumor proach [53]. relapse [87]. Transcriptomic and metabolic analyses of Along the same line, Luo et al. also recently reported that surviving cells demonstrated a strong expression of genes proliferative ECSCs and quiescent MCSCs in breast cancer driving mitochondrial function and lysosomal and autoph- display different sensitivities to inhibitors of glycolysis and agic activity as well as a robust dependence on mitochon- redox metabolism. Metabolic or oxidative stress promotes drial respiration and a decreased dependence on glycolysis the transition of MCSCs with low levels of ROS to ECSCs for cellular energetics. Importantly, these cells depend on with high levels of ROS. This transition depends on the OXPHOS for survival. El Hout et al. Molecular Cancer (2020) 19:27 Page 7 of 17

Table 2 Drugs targeting CSC metabolism Metabolism-based strategies Compound Mechanism of action CSC or tumor type Reference Glycolysis inhibition 2-Deoxy-D-glucose Glycolysis inhibitor Breast CSCs [21] 3-BP Glycolysis inhibitor Glioblastoma CSCs, PDACs [56] DCA Metabolic shift from glycolysis GBM cells [57] to OXPHOS Inhibition of mitochondrial Metformin Complex I inhibitor Pancreatic CSCs, CSCs of HT29 [32, 58] respiration cell line derived from colorectal Phenformin cancer Rotenone Antimycin-A Complex III inhibitor Lung CSCs [59–62] Bedaquiline Complex V inhibitor Breast CSCs Oligomycin Complex V inhibitor Glioblastomas Salinomycin analogs Lysosomal iron sequestration Breast CSCs [63] Impairs autophagic flux Breast CSCs [64] Interference with Breast, AML, lung, gastric, [65] ABC transporters osteosarcoma, colorectal, pancreatic and prostate CSCs Activation of the Wnt/β-catenin signaling pathway 188Re-Liposome Mitophagy inhibitor Ovarian CSCs [66] Doxycycline Mitochondrial biogenesis Breast and cervical CSCs [67, 68] inhibitor and inducer of apoptosis XCT-790 ETC uncoupler, mitochondrial Breast CSCs [31] biogenesis inhibitor, inhibitor of ERRα-PGC-1 signaling pathway Mdivi-1 Fission inhibitor, inhibitor of Breast CSC, brain tumor [69, 70] assembly of Drp1 and its initiating cells GTPase activity Redox homeostasis and Zaprinast Inhibition of glutathione Non-small lung and glutamine- [30, 59, 71] antioxidant signaling biosynthesis, glutaminase addicted pancreatic cancer BPTES inhibitor cell lines 968 BSO Apigenin Neutralizer of ROS-induced NRF2 Ovarian and prostate CSCs, [72–76] activity, STAT3 signaling pathway triple-negative breast cancers, ATRA inhibitor, inhibitor of the NRF2 leukemia stem-like cells Brusatol and NF-ƙB pathways Disulfiram Trigonelline Lipid metabolism 2M14NQ Blocks CD36 activity and fatty Glioblastoma CSCs [77] acid uptake Soraphen A Inhibitor of ACC Breast CSCs [78–80] Chloroquine Inhibition of autophagy Breast CSCs [15, 64] TVB-2640 FASN inhibitor Breast CSCs [78] MF-438 SCD-1 inhibitor, inhibitor of FAO Lung CSCs, liver cancer, [81, 82] glioblastoma and AML cells SSI-4 A939572 Etomoxir ST1326 Inhibition of FAO AML cells [83, 84] Avocatin B Emodin ACLY inhibitor Lung CSCs [85] TVB-2640 Inhibitor of HMG-COAR, inhibitor Breast and brain CSCs [34, 86] of cholesterol synthesis through the mevalonate pathway El Hout et al. Molecular Cancer (2020) 19:27 Page 8 of 17

Fig. 2 Metabolic modulators with anti-CSC effects. Metabolic pathways such those involving glutamine, glycolysis, redox balance, lipids, and autophagy are potentially targetable in CSCs. Some of the metabolic enzymes that are currently being considered as therapeutic targets for CSC are indicated by blue rectangles in the figure. Transcription factor NRF2 plays a pivotal role in both intrinsic resistance and cellular adaptation to ROSand is shown in a yellow rectangle. The carnitine-dependent transporter, which inhibits the mitochondrial import of fatty acids is shown in a yellow ball. Inhibitors are indicated by red rectangles. Abbreviations: ACC, acetyl-CoA carboxylase; Ac-CoA, acetyl-coenzyme A; ACLY, ATP citrate lyase; ACSL,long- chain acyl-CoA synthetases; ATRA, all-trans retinoic acid; 3-BP, 3- bromopyruvate; BSO, L-buthionine-S,R-sulfoximine; CPT1, carnitine palmitoyltransferase; I/Q/II/III/IV/V, complexes of the electron transport chain; DCA, dichloroacetate; 2-DG, 2-deoxy-D-glucose; Doc, doxycycline; FASN, fatty acid synthetase; FAT/CD36, Fatty acid translocase; GCS, gamma glutamyl cysteine synthetase; GLS, glutaminase; GLUT1/4, glucose transporter 1/4; GSH, glutathione; HK2, hexokinase 2; HMG-CoAR, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase; 2M14NQ, 2-methylthio-1,4-naphthoquinone; Mito, mitochondrial; NRF2, nuclear factor erythroid 2-related factor 2; OAA, oxaloacetate; PDH, pyruvate dehydrogenase; ROS, reactive oxygen species; TC, tetracyclines; TCA, tricarboxylic acid cycle; SCD1, stearoyl-CoA desaturase-1; SLC1A5, solute carrier family 1 member 5;

These CSC have high sensitivity to OXPHOS inhibi- Interestingly, Kim et al. recently demonstrated that glu- tors and when OXPHOS inhibitors are combined with a tamine metabolism also plays an important role in regula- targeted inhibitor of the KRAS pathway tumor recur- tion of the sensitivity of colorectal CSCs to metformin rence is blocked [88]. However, metformin, which acts through a mechanism that depends on the AMPK/mTOR directly on the respiratory chain complex I in the mito- pathway. In the absence of glutamine, but not in low- chondria to inhibit OXPHOS and reduce mitochon- glucose medium, CSCs from SW620 cells were sensitive drial ATP production (Fig. 2), was not enough to to the CSC-suppressing effect of metformin with activa- eliminate the CSC subpopulation [32]. This is possibly tion of AMPK and suppression of mTOR. A combination due to their intermediate glycolytic/respiratory pheno- of metformin and glutaminase C inhibitor compound 968, type and also to the heterogeneity and plasticity of an inhibitor of glutamine metabolism, suppressed prolifer- PDAC cells. A previous study demonstrated that met- ation of CSCs in SW620 cells and enhanced the effect of formin increases ROS production in CSCs from metformin alone in HT29 cells (Fig. 2). Thus, the sensitiv- PDAC cells and reduces their mitochondrial trans- ity to metformin in this cell line is possibly due to activa- membrane potential. The AMPK/mTOR axis is not tion of AMPK pathway. involved in the subsequent induction of lethal energy Depletion of alanine serine cysteine transporter 2 crisis in CSCs. (ASCT2), glutaminase 1, and c-MYC induced significant El Hout et al. Molecular Cancer (2020) 19:27 Page 9 of 17

CSC suppression. The compounds 968 and metformin a cofactor of peroxisome proliferator-activated receptor also induced CSC elimination, and the activities were gamma co-activator (PGC-1α). PGC-1α is the master regu- enhanced by silencing of ASCT2 and c-MYC. Thus, the lator of mitochondrial biogenesis and is essential for the effect of metformin on CSCs varies depending on the activation of numerous nuclear transcription factors that extent of activation of the AMPK/mTOR pathway and control the transcription of many mitochondrial genes [94]. glutamine metabolism [58]. Consistent with a metabolic Independent of its inhibition of ERRα and mitochondrial profile dominated by OXPHOS, ovarian CSCs undergo biogenesis, XCT-790 is a potent mitochondrial electron apoptosis upon inhibition of the mitochondrial respiratory transport chain uncoupler [95](Fig.2). chain by oligomycin, antimycin, rotenone, and metformin Numerous classes of FDA-approved antibiotics also in- (Fig. 2). hibit mitochondrial biogenesis to eliminate CSCs [96]. CSCs have higher mitochondrial ROS production and These include the erythromycins, the tetracyclines, the elevated membrane potential as well as enhanced pen- glycylcyclines, an anti-parasitic drug, and chlorampheni- tose phosphate pathway activity compared to normal col. Efficacy was observed across eight different tumor counterparts. This metabolic characteristic is not repre- types (breast, ductal carcinoma in situ, ovarian, prostate, sentative of cells that privilege OXPHOS over glycolysis lung, pancreatic, melanoma, and glioblastoma), suggest- and may instead reflect the role of the pentose phosphate ing that cancer can be treated as an infectious disease. pathway in reloading scavenging enzymes [29]. In agree- Indeed, simultaneous inhibition of autophagy and treat- ment with this, combined treatment with 5-fluorouracil, ment with antibiotics significantly reduces tumorigenic an inhibitor of thymidine synthesis, and a pharmacological properties of cancer cells suggesting that this should be inhibitor of OXPHOS abolishes drug resistance of colon tested as a potential strategy for anticancer therapy [97]. cancer cells in culture and diminishes the expression of However, continuous treatment with antibiotics for can- stem-like markers [89]. cer therapy may not succeed due to induction of autoph- The efficacy of metformin has prompted efforts to re- agy or a glycolytic shift. purpose available drugs to target CSCs (for review see [59]). Various FDA-approved antibiotics known to target the mitochondrial respiratory chain have been shown to Mitochondrial dynamics inhibitors selectively decrease CSC survival or proliferation (Fig. 2 Mitochondria are dynamic organelles that often undergo and Table 2). Examples are antimycin A, a powerful fusion and fission events to sustain mitochondrial integ- complex III inhibitor that decreases lung spheroids; the rity and appropriate bioenergetics and spatial distribu- anti-tuberculosis agent bedaquiline (a complex V inhibi- tion. High levels of mitochondrial fission activity are tor) that inhibits mammosphere formation; oligomycin associated with high proliferation and invasiveness in (another complex V inhibitor) that synergistically sup- some cancer cells and with self-renewal and resistance presses growth and motility of glioblastoma cell lines when to differentiation in some stem cells [98]. A specific in- combined with 2-deoxy-D-glucose (2-DG); and niclosa- hibitor of the fission events, mDIVI1, induced apoptosis mide, an anti-helminthic with OXPHOS uncoupling prop- in brain tumor initiating cells and inhibited tumor erties [90], that inhibits proliferation of CSCs from ovarian growth. mDIVI1 is an inhibitor of dynamin-related pro- and breast cancers. Niclosamide also prevents the conver- tein 1 (DRP1), a mitochondrial fission protein, induces sion of breast non-CSCs into CSCs induced by IL-6 [91]. mitochondrial oxidative stress and reduces mitochon- Salinomycin also inhibits CSC formation in diverse cancer drial metabolism. CDK5-dependent DRP1 activation in types [65]; OXPHOS is known to be inhibited by salinomy- brain tumor initiating cells stimulates mitochondrial cin [92]. Depletion of ATP levels and induction of mito- fission preventing cell death and sustaining self-renewal phagy and mitoptosis are observed in cancer cells treated and growth. DRP1 activation in brain tumor initiating with salinomycin [93].Asapleotropiccompoundthatalso cells correlates with poor glioblastoma patient survival interferes with Wnt signaling and ABC transporters, the [69]. Recently, Peiris-Pages et al. showed that mDIVI1 antitumoral effect of salinomycin likely results from a com- prevents breast CSC survival and propagation [70]. bination of factors [65]. We recently demonstrated that Mitochondrial fission can produce an impaired daugh- salinomycin impairs autophagic flux [64] and kills CSCs by ter unit that is targeted by the autophagic machinery. sequestering iron in lysosomes by ferroptosis [63](Fig.2). Mitochondrial fusion, on the other hand, may serve to The compound known as XCT-790 also prevents breast dilute impaired respiratory components and thereby pre- CSC survival and propagation. The rescue of the effect of vent their removal. The inverse dependency of fusion XCT-790 by acetyl-l-carnitine (a mitochondrial fuel) indi- and mitophagy on membrane potential allows these two cates that mitochondria are the target of XCT-790 in CSCs processes to act in a complementary rather than com- [31]. XCT-790 is a strong and selective inverse agonist lig- petitive fashion on the daughter mitochondrion after a and of the estrogen-related receptor alpha (ERRα), which is fission event [99]. Intraperitoneal delivery of the El Hout et al. Molecular Cancer (2020) 19:27 Page 10 of 17

nanomedicine 188Re-Liposome killed the CSCs-like cells development. This suggests that high levels of ROS may in tumors with a degree of selectivity and switched me- not eradicate CSCs. tabolism from glycolysis to OXPHOS in an animal CSCs are characterized by a finely regulated redox me- model of ovarian cancer [66] (Fig. 2). A study showed tabolism [103]. Glutathione plays an essential role in that dynamin 1-like -mediated mitochondrial fission in- maintenance of stemness characteristics [104]. Glutathione duced by liensinine, a novel mitophagy inhibitor, sensi- synthesis may be inhibited either directly or indirectly by tizes breast cancer cells to chemotherapy [100]. blocking glutamine synthetase (GS) or glutaminase en- Recently, Chang et al. showed that mitophagy inhibitors zymes (GLS). Several glutaminase inhibitors have been eval- such as liensinine and 188Re-Liposome abolish drug re- uated preclinically [30], including buthionine sulfoximine sistance in ovarian CSC-like cells [101]. (BSO), 968, and bis-2-(5-phenylacetamido-1,3,4-thiadiazol- 2-yl) ethyl sulfide (BPTES) [59](Fig.2). Recently, zaprinast, Glycolysis inhibitors an asthma medication was identified as a glutaminase in- Previous findings suggest that CSCs may be specifically hibitor [71]. Depletion of glutathione and inhibition of dependent on a high glucose turnover; therefore, target- thioredoxin reductase activity could also enhance radiation ing the glycolytic pathway is a promising therapeutic ap- responses in human breast and pancreatic CSCs by a mech- proach. Zhou et al. demonstrated that the combination anism involving thiol-dependent oxidative stress. The fol- of a glycolysis inhibitor such as 3-bromopyruvate (3-BP) lowing pharmacologically agents inhibit glutathione and with standard therapeutic agents such as doxorubicin thioredoxin metabolism: BSO; sulfasalazine (SSZ), an in- killed glioblastoma CSCs and inhibited tumor formation. hibitor of xc (−) cysteine/glutamate antiporter; auranofin, a This study suggests that stem-like cancer cells prefer a thioredoxin reductase inhibitor; and 2-AAPA, a GSH- low oxygen microenvironment and actively utilize the reductase inhibitor. Combined inhibition of glutathione- glycolytic pathway [18]. Recently, Isayev et al. showed and thioredoxin-dependent thiol metabolism can enhance that treatment with 3-bromopyruvate almost completely responses of CSC to conventional therapies [105]. blocked cell viability, self-renewal potential, NF-κB bind- As mentioned above, NRF2 is a transcription factor ing activity, and stem cell-related signaling and reverted that mediates the cytoprotective response to oxidative gemcitabine resistance of CSCs from PDAC [56]. and electrophilic stress. Under the oxidative stress, The switch from mitochondrial OXPHOS to cytoplasmic NRF2 dissociates from its molecular inhibitor Kelch-like glycolysis is accompanied by development of the resistance ECH-associating protein 1 (KEAP1) and translocates to cell death in glioblastoma multiforme. This metabolic into the nucleus. There NRF2 binds to the antioxidant switch is accompanied by mitochondrial hyperpolarization. response element (ARE) of its target genes to induce Michelakis et al. demonstrated that dichloroacetate (DCA), their expression. NRF2 target genes include NADPH a small-molecule drug, induced a metabolic shift from gly- quinone oxidoreductase-1 (NQO-1) and aldo-keto reduc- colysis to OXPHOS, resulting in increased ROS, and in- tase 1C1 and genes encoding glutathione generating en- duced apoptosis in CSC glioblastoma [57]. By inhibiting zymes and drug efflux transporters such as breast cancer pyruvate dehydrogenase kinase (PDK), DCA activates PDH, resistance protein (BCRP). Recently, Kim et al. reported increasing the ratio of glucose oxidation to glycolysis. On that all-trans retinoic acid (ATRA) inhibits NRF2 activa- activation of PDH, however, pyruvate can be decarboxy- tion, suppresses ALDH1 expression, and leads to the at- lated to acetyl-coenzyme A, enter the TCA cycle, and tenuation of ovarian CSC-like properties [72] (Fig. 2). complete glucose oxidation in the mitochondrial matrix, In the presence of copper, administration of disulfiram, an generating up to 36 mol of ATP per glucose molecule in approved anti-alcoholism drug, significantly downregulates thepresenceofoxygen. ALDH1A1, CD44, and phospho-STAT3 levels. Disulfiram suppresses stem-like properties in triple-negative breast Targeting redox homeostasis and antioxidant cancer by targeting the STAT3 signaling pathway [73]. signaling Moreover, in leukemia stem-like cells, disulfiram and copper Growing evidence supports a model for redox homeosta- selectively activate the stress-related ROS-JNK pathway and sis in which the ROS-antioxidant interaction acts as a simultaneously inactivate the NRF2 and NF-κB pathways metabolic interface for signals derived from metabolism [74]. Although disulfiram is now begin tested in phase III and from the environment. This interface regulates pro- clinical trials, it is extremely unstable in blood. To increase cesses that allow cells to acclimate or, alternatively, to disulfiram blood levels, a nanocarrier system of mPEG- die. The efficacy of clinically used classical chemo and PLGA/PCL has been used for the delivery [106]. radiotherapy is due to high levels of intracellular ROS- Many other NRF2 inhibitors, including brusatol, api- induced cancer cell death. However, Yuan et al. recently genin, and trigonelline, have been identified (Fig. 2). Bru- reported that ROS generated from OXPHOS is essential satol treatment suppresses NRF2 at the protein level, in CSC activation [102], which promotes tumor which results in enhanced intracellular ROS, sensitization El Hout et al. Molecular Cancer (2020) 19:27 Page 11 of 17

of mammospheres to taxol, and reduced anchorage- from ALDH+/CD133+ ovarian cancer cells [111] and independent growth. However, further studies are needed colorectal cancer cells [112]. Thus, increased lipid stor- to establish its in vivo action. Apigenin [75] and trigonel- age in lipid droplets appears as a CSCs marker [112]. line [76] are transcriptional and translational NRF2 inhibi- Lipids from extracellular sources can also be stored in tors, respectively, that were developed as adjuvants to lipid droplets. Thus, lipid metabolism is controlled by chemotherapeutic drugs. Mechanistic analyses demon- fatty acid synthesis and fatty acid oxidation (FAO) as strated that NRF2 silencing or treatment with trigonelline well as by NADH, which is necessary for the production abolishes the ferroptosis resistance of KEAP1-deficient of ATP [113]. Lipolysis of lipids stored in lipid droplets and cisplatin-resistant cancer cells to artesunate [76]. was earlier considered to be solely carried out by cyto- Paradoxically, some natural antioxidants, including sul- solic lipases. However, recent studies demonstrate that foraphane, curcumin, resveratrol, oleanane triterpenoid, lipophagy (autophagic degradation of lipids by acidic li- and carnosol, which all increase NRF2 expression pases) serves as an alternate pathway for the degradation levels, also have therapeutic potential. Readers inter- of lipid droplets [113]. Among target tested for CSC ested in detailed discussion of this paradox should see elimination, lipid metabolism is promising [59]. Several recent reviews [59, 107]. compounds that influence lipid metabolism have been tested preclinically (Fig. 2 and Table 2). Lipid metabolism The role of lipid metabolism as a major source of energy Lipid uptake inhibition and metabolic intermediates was recently demonstrated for Lipid uptake can be inhibited by pharmacological inhib- processes implicated in transformation of normal cells into ition or by antibody-mediated inhibition of the transporter malignant cells and tumor progression [59]. Lipid metabol- fatty acid translocase CD36. Dietary lipid dependence of ism is necessary for synthesis of membrane components. metastasis-initiating cells has recently been reported in mel- Fatty acids are broken down through mitochondria beta anoma and breast cancer. Inhibition of CD36, by specific oxidation, which produces acetyl-coenzyme A (Ac-CoA) antibodies, impairs metastasis [114]. Conversely, over- for anaplerosis. Citrate, a TCA cycle intermediate, can be expression of CD36 significantly increases lymph node me- used as a precursor for fatty acid synthesis and for NADPH tastasis of oral squamous cell carcinoma cell lines with low production through the ATP citrate lyase (ACLY) (Fig. 2). metastatic potential [114]. Metastatic-initiating cells are also Citrate is subsequently converted to acetyl-CoA and oxalo- characterized by a distinct lipid metabolic signature related acetate in the cytoplasm. ACLY links glycolytic and lipidic to fatty acid degradation, de novo lipogenesis, and lipid stor- metabolism. ACLY is overexpressed in cancer cells, and age. CD36 is enriched in CSCs from glioblastoma and func- siRNA-mediated silencing of ACLY limits cancer cell prolif- tionally distinguishes self-renewing cells. CD36 is co- eration and reduces the capacity of A549 lung cancer cells expressed with integrin alpha 6 and CD133, previously de- to form spheres [108]. ACLY inhibitors, previously devel- scribed CSC markers, and CD36 reduction results in con- oped for metabolic disorders, have recently attracted inter- comitant loss of integrin alpha 6 expression, self-renewal, est as promising anti-cancer agents [109]. Koerner et al. and tumor initiation capacity. 2-Methylthio-1,4-naphtoqui- recently synthesized an emodin derivative and demon- none (2M14NQ), a unique sulfur-containing quinone, strated that this novel ACLY inhibitor prevents prolifera- which inhibits CD36 activity, decreases self-renewal ability tion of lung CSCs in vitro [85](Fig.2 and Table 2). and induces apoptosis in glioblastoma CD133+ CSCs [77] The committed step of fatty acid synthesis is the acti- (Fig. 2). CD36+ leukemia CSCs are enriched in gonadal adi- vation of acetyl-CoA to malonyl-CoA. This is an energy- pose tissue and have elevated fatty acid uptake and beta oxi- consuming process catalyzed by acetyl-CoA carboxylase dation [115]. Sulfosuccinimidyl oleate, another CD36 (ACC). The acetyl and malonyl groups are then coupled inhibitory compound, decreases proliferation of chemoresis- to the acyl-carrier protein domain of the multifunctional tant leukemic CD36+/CD34+ stem cells [115]. The conver- fatty acid synthase (FASN). Repeated condensations of sion of long chain fatty acids to fatty acyl-CoAs is catalyzed acetyl groups generate the basic 16-carbon saturated by the long-chain acyl-CoA synthetases (ACSL), which have palmitic acid. FASN activity is higher in adult murine been linked to carcinogenesis [116]. The ACSL inhibitor neuronal stem and progenitor cells than in differentiated triacsin C is under investigation for the treatment of acyl- progeny [110]. Fatty acids are required for the produc- CoA synthetase-dependent tumors. However, there is no tion of phosphoglycerides, which, together with choles- data available on the ACSL as a therapeutic target for CSC. terol, can be used for building cell membranes. Lipid droplets store triacyl glycerides and cholesteryl esters. Inhibition of lipogenesis and acyl-CoA synthetase lipid Emerging data indicate that stored cholesteryl ester and desaturation: FASN and stearoyl-CoA desaturase-1 accumulation of lipid droplets are correlated with Results of a recent study support the theory of re- tumorigenicity of CSCs derived from neurospheres and activation of de novo lipogenesis in solid tumors as part El Hout et al. Molecular Cancer (2020) 19:27 Page 12 of 17

of cancer metabolic reprogramming [113]. In invasive CAY 10566 and SC26196, SCD1 was identified as the en- ductal carcinoma, acetyl CoA synthetase 2 (ACSS2), an zyme responsible for the increased desaturation in stem enzyme that catalyzes the conversion of acetate to acetyl cells. Mechanistically, UFAs increase NF-κBactivity, CoA, is overexpressed under hypoxic and lipid-depleted which upregulates expression of ALDHA1 and SCD-1 conditions. ACSS2 increases acetate consumption and mRNAs. Increased SCD1 in turn promotes UFA synthesis thereby fatty acid biosynthesis in the harsh tumor micro- from saturated fatty acids, forming a positive feedback environment where there is a scarcity of alternate carbon loop [118]. sources for lipogenesis. Thus, targeting de novo lipogen- Two studies support the use of combination therapy esis and lipid desaturation could provide a selective with SCD1 inhibitors to achieve better control of cancer mechanism to interfere with tumor growth. [81]. The first study reported that SCD1-mediated endo- A specific inhibitor of the first committed step of lipid plasmic reticulum stress regulates liver tumor-initiating biosynthesis catalyzed by ACC, the antifungal polyketide cells and sorafenib sensitivity. SCD1 inhibitors A939572 soraphen A, suppresses growth of breast CSCs [78](Fig.2 or SSI-4 alone or in combination with sorafenib thus and Table 2). Numerous classes of ACC inhibitors have have potential for treatment liver cancer [81]. In parallel, been evaluated in clinical trials for metabolic diseases (i.e., Pisnau et al. reported that co-treatment with cisplatin obesity and metabolic syndrome). Mechanistic analysis and the SCD1 inhibitor MF-438 decreases expression of suggests that the biotin carboxylase domain of the ACC, lung CSCs markers, strongly synergizes in the inhibition which is the soraphen A binding subunit dimerization site, of sphere formation, and induces apoptosis of lung CSCs may be an ideal target for ACC inhibitors with potential [82] (Fig. 2 and Table 2). However, clinical utilization of for use in cancer therapy. SCD1 inhibitors for anti-cancer therapy should proceed with extreme caution. SCD1 is also involved in the regu- FASN inhibitors lation of inflammation and stress in various cell types, Given the involvement of the enzyme FASN in numerous including β-cells, adipocytes, macrophages, endothelial tumor types, FASN inhibitors including C75, C93, epigallo- cells, and myocytes. catechin gallate, G28UCM, orlistat, Fasnall, GSK2194069, Considering the established link between obesity and risk and GSK837149A have been evaluated in a mouse model for many types of cancer, the observation that SCD1 defi- of breast cancer [79]. Inhibition of FASN by cerulenin and ciency protects mice against high-fat diet-induced obesity of mevalonate pathways by atorvastatin prevents prolifera- and hepatic steatosis [119] suggests that SCD1 inhibitors tion of CSCs in vitro [80](Fig.2 and Table 2). C75 at non- could serve the dual purpose of blunting tumor growth and cytotoxic concentrations significantly reduces the capacity preventing obesity and associated metabolic conditions. On of MCF-7/HER2 cells to form mammospheres, an in vitro the other hand, loss of SCD1 function is associated with the indicator of cancer stem-like cells [117]. Despite these ef- development of inflammatory diseases such as dermatitis, forts, however, the majority of FASN inhibitors have failed atherosclerosis, intestinal colitis, pancreatic β-cell dysfunc- to advance into clinical trials due to unexpected toxicities. tion, and liver dysfunction [119]. Furthermore, SCD1 is Currently, TVB-2640 is the only selective FASN inhibitor highly expressed in the brain. Small-molecule inhibitors of in clinical trials for the treatment of advanced solid tumors, SCD1 could cross the blood-brain barrier and interfere with including HER2+ advanced breast cancer, high-grade astro- the axon myelination process. Therefore, therapeutic strat- cytoma, colon cancer, and non-small cell lung carcinoma egies that target the re-activation of de novo lipogenesis of with mutations in KRAS. tumor tissues should take into consideration the risks of interference with active de novo lipogenesis in normal SCD1 inhibitors tissues. A recent report using hyperspectral-stimulated Raman spectroscopic imaging and mass spectrometry analysis of Inhibition of fatty acid oxidation extracted lipids showed that ovarian CSCs contain un- FAO is a promising target for elimination of CSCs. Etomoxir, usually high levels of unsaturated fatty acids (UFAs) and an inhibitor of the carnitine-dependent transporter CPT1 that UFAs are essential for the cells to retain stemness. (also known as CPT1A), which inhibits the mitochondrial These data suggest that increases in lipid unsaturation import of fatty acids mediated by the carnitine shuttle, de- might be a general marker for CSCs in ovarian cancer and creases intracellular ATP levels as well as the viability and re- a new target for CSC-specific therapy [118]. Stearoyl-CoA sistance to chemotherapy of glioblastoma and acute myeloid desaturase-1 (SCD1), the most abundant desaturase, is leukemia cells [120]. Silencing of Nanog or overexpression of expressed in lipogenic tissues and catalyzes the formation cytochrome c oxidase subunit 6A and/or inhibition of FAO of double bonds at the ninth carbon atom of saturated by etomoxir, sensitizes CSCs to sorafenib treatment. These fatty acids, leading to mono-unsaturated fatty acids. Using data suggest that FAO inhibition or OXPHOS reestablish- molecular approaches and chemical inhibitors such as ment to induce metabolic reprogramming of CSCs should El Hout et al. Molecular Cancer (2020) 19:27 Page 13 of 17

be a powerful therapy in hepatocellular carcinoma [121]. Un- Abbreviations fortunately, the clinical development of etomoxir was termi- 2-DG: 2-deoxy-D-glucose; 2M14NQ: 2-methylthio-1,4-naphtoquinone; 3-BP: 3- bromopyruvate; ACC: Acetyl-CoA carboxylase; Ac-CoA: Acetyl-coenzyme A; nated because of severe hepatotoxicity and hematopoietic ACLY: ATP citrate lyase; ACSL: Long-chain acyl-CoA synthetases; stem cell exhaustion associated with treatment [120]. How- ACSS2: Acetyl CoA synthetase 2; ALDH1A3: Aldehyde dehydrogenase 1A3; ever, alternative FAO inhibitors are under investigation. For AMPK: AMP-activated protein kinase; ARE: Antioxidant response element; ASCT2: Alanine serine cysteine transporter 2; ATP: Adenosine triphosphate; instance, the compound ST1326 strongly inhibits chemore- ATRA: All-trans retinoic acid; BCRP: Breast cancer resistance protein; sistance of leukemia cells with no effect on normal stem cells BCSCs: Breast cancer stem cells; Bnip3: Bcl-2/adenovirus E1B interacting [83]. Additionally, another FAO inhibitor, avocatin B, which protein 3; BPTES: Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide; BSO: L-buthionine-S,R-sulfoximine; CAFs: Cancer-associated fibroblasts; acts as a lipid that accumulates in mitochondria, eliminates CPT1: Carnitine palmitoyltransferase; CS: Citrate synthase; CSC: Cancer stem CSCs from acute myeloid leukemia with no effect on normal cell; DCA: Dichloroacetate; DNMT: DNA methyltransferases; Doc: Doxycycline; blood stem cells [84](Fig.2 and Table 2). DRP1: Dynamin-related protein 1; ECSCs: Epithelial CSCs; EMT: Epithelial- mesenchymal transition; ERRα: Estrogen-related receptor alpha; F6P: Fructose- 6-phosphate; FAO: Fatty acid oxidation; FASN: Fatty acid synthetase; FAT/ CD36: Fatty acid translocase; FBP1: Fructose-1,6-biphosphatase; Cholesterol synthesis through the mevalonate pathway FOXO3A: Forkhead box 3A; G6PDH: Glucose-6-phosphate dehydrogenase; GBM: Glioblastoma; GCS: Gamma-glutamylcysteine synthetase; GDH: Glutamate Cholesterol synthesis from acetyl-CoA proceeds through dehydrogenase; GFAT: Glutamine-fructose-6-phosphate transaminase 1; the mevalonate pathway. Analysis of a large cohort of GLS: Glutaminase; GLS1: Mitochondrial glutaminase; GLS2: Cytosolic isoform breast cancer patients provided evidence of reduced mor- glutaminase; GLUT1, 2, 3, 4: Glucose transporter 1,2, 3,4; GS: Glutamine synthetase; GSC: Glioblastoma stem cells; GSH: Glutathione; HIF-1α:Hypoxia- tality in statin users. Statins are inhibitors of 3-hydroxy-3 inducible factor 1α; HK2: Hexokinase 2; HMG-CoAR: 3-hydroxy-3-methyl-glutaryl- methylglutaryl-CoA reductase (HMG-COAR), the limiting coenzyme A reductase; I/Q/II/III/IV/V: Complexes of the electron transport chain; step of the mevalonate pathway. However, these associa- JNK/AP1: c-Jun N-terminal kinases/activator protein 1; KEAP1: Kelch-like ECH- associating protein 1; KLF4: Kruppel-like Factor 4; LC3: Microtubule-associated tions are weak in magnitude and attenuated in some Protein 1 Light Chain 3; LDH: Lactate dehydrogenase; MCSC: Mesenchymal-like sensitivity analyses [122]. As mentioned above, treatment CSCs; MCT2/4: Monocarboxylate transporter 2/4; Mito: Mitochontrial; with various statins targeting CSC self-renewal resulted in NADH: Nicotinamide adenine dinucleotide (reduced); NADPH: Nicotinamide adenine dinucleotide phosphate (reduced); NAMPT: Nicotinamide elimination of CSCs in breast [34] and brain [86]cancers. phosphoribosyl transferase; NANOG: Nanog Homeobox; NF-κB: Nuclear factor- Moreover, a mixture of brutieridin and melitidin, which κB; NMN: Nicotinamide mononucleotide; NNMT: Nicotinamide N- has statin-like properties, eradicates CSCs by targeting methyltransferase; NQO-1: NADPH quinone oxidoreductase-1; NRF2: Nuclear factor erythroid 2–related factor 2; OAA: Oxaloacetate; OCT4: Octamer-binding mevalonate, Rho-GDI-signaling, and mitochondrial me- transcription factor 4; OXPHOS: Oxidative phosphorylation; P: Phosphate (or tabolism [123]. In addition, bergamot metabolically in- phospho); PARPs: Poly (ADP-ribose) polymerases; PDAC: Pancreatic ductal hibits OXPHOS and FAO [123]. adenocarcinoma; PDH: Pyruvate dehydrogenase; PDK1: Pyruvate dehydrogenase kinase 1; PFKFB: Phosphofructokinase/fructose bisphosphate; PGC-1α: Peroxisome proliferator-activated receptor gamma co-activator; PHGDH: Phosphoglycerate dehydrogenase; PKM2: Pyruvate kinase isozyme M2; Concluding remarks RHOA: Homolog family member; ROS: Reactive oxygen species; SAM: S- It is now clear that the cancer is a heterogeneous disease adenosyl methionine; SCD1: Stearoyl-CoA desaturase-1; SFA: Saturated fatty acids; SIRTs: Sirtuins; SLC1A5: Solute carrier family 1 member 5; SOX2: Sex and that metabolic heterogeneity and flexibility of tumor determining region Y-box 2 S; SZ: Sulfasalazine; TC: Tetracyclines; cells contributes to this heterogeneity. Location influences TCA: Tricarboxylic acid cycle; TGF-β: Transforming growth factor β; CSC metabolic status. In actively growing regions of the UFA: Unsaturated fatty acids. tumorandinthepresenceofadequatelevelsofoxygen, Acknowledgments CSCs rely on glycolytic and/or oxidative metabolism. In We apologize for the omission of any primary citations. nutrient-poor states, autophagy is activated as an alternative energy source. The catabolic glycolysis/oxidative phosphor- Authors’ contributions AH and MM have contributed equally to the writing process of this ylation and the anabolic gluconeogenesis pathway control manuscript. MEH, AH, and MM drafted the manuscript. MEH and EC glucose homeostasis. The metabolic adaptation of CSCs to discussed, helped to design all figures and contributed to writing the review. the tumor microenvironment may provide an explanation All authors read and approved the final manuscript. for the metabolic differences observed in CSCs. However, Funding further investigation are necessary to demonstrate the role Supported in part by grants from INSERM, Université Paris Descartes- of autophagy in plasticity and metabolic reprogramming. Sorbonne Paris Cité, and Comité de Paris de la ligue contre le cancer. Current studies have revealed details of CSC metabolism in Mouradi EL Hout thanks LASER association for funding. terms of redox state, lipid metabolism, and use of alterna- Availability of data and materials tive fuels, such as amino acids or ketone bodies, identifying Not applicable. important vulnerabilities that could provide new thera- peutic opportunities. However, to interrogate the metabolic Ethics approval and consent to participate traits of CSCs, metabolism must be analyzed directly after Not applicable. isolation from patients or after very few passages in culture Consent for publication to avoid artifactual switches in metabolic characteristics. The authors declare that they consent for publication. El Hout et al. Molecular Cancer (2020) 19:27 Page 14 of 17

Competing interests 2. Snyder V, Reed-Newman TC, Arnold L, Thomas SM, Anant S. Cancer stem The authors declare that they have no competing interests. cell metabolism and potential therapeutic targets. Front Oncol. 2018;8:203. 3. Mowers EE, Sharifi MN, Macleod KF. Functions of autophagy in the tumor Glossary microenvironment and cancer metastasis. FEBS J. 2018;285(10):1751–66. Electron transport chain 4. Poillet-Perez L, White E. Role of tumor and host autophagy in cancer A series of transmembrane protein complexes, present on the inner metabolism. Genes Dev. 2019;33(11–12):610–9. membrane of mitochondria, that transfer electrons via redox 5. Poillet-Perez L, Xie X, Zhan L, Yang Y, Sharp DW, Hu ZS, et al. Autophagy maintains reactions to the terminal electron acceptor oxygen, which is tumour growth through circulating arginine. Nature. 2018;563(7732):569–73. reduced by binding of protons to a water molecule. This generates 6. Palm W, Thompson CB. Nutrient acquisition strategies of mammalian cells. a proton gradient that powers ATP synthase to produce ATP. Nature. 2017;546(7657):234–42. Premature leakage of electrons to oxygen can lead to production 7. Lyssiotis CA, Kimmelman AC. Metabolic interactions in the tumor of reactive oxygen species. microenvironment. Trends Cell Biol. 2017;27(11):863–75. Anaplerosis 8. Smith AG, Macleod KF. Autophagy, cancer stem cells and drug resistance. J The act of replenishing TCA cycle intermediates used for Pathol. 2018. macromolecule biosynthesis. 9. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg Cataplerosis effect: the metabolic requirements of cell proliferation. Science. 2009; The removal of intermediate metabolites, especially those resulting 324(5930):1029–33. from the TCA cycle, to prevent their accumulation in the mitochondrial 10. Cordier-Bussat M, Thibert C, Sujobert P, Genestier L, Fontaine E, Billaud M. matrix. Even the Warburg effect can be oxidized: metabolic cooperation and tumor Glutathione development. Med Sci (Paris). 2018;34(8–9):701–8. A tripeptide (glutamate-cysteine-glycine) that acts as an important 11. Galluzzi L, Baehrecke EH, Ballabio A, Boya P, Bravo-San Pedro JM, Cecconi F, antioxidant. The reduced form of glutathione can react with H2O2 et al. Molecular definitions of autophagy and related processes. EMBO J. to form the oxidized form (GSSG). 2017;36(13):1811–36. Glutaminolysis 12. Boya P, Codogno P, Rodriguez-Muela N. Autophagy in stem cells: repair, A series of biochemical reactions by which the glutamine is lysed remodelling and metabolic reprogramming. Development. 2018;145(4). to generate molecules such as CO2,alanine,aspartate,citrate, 13. Cufi S, Vazquez-Martin A, Oliveras-Ferraros C, Martin-Castillo B, Vellon L, glutamate, lactate, and pyruvate. When glycolytic energy production Menendez JA. Autophagy positively regulates the CD44(+) CD24(−/low) is low, glutamine, the most common amino acid in the plasma, breast cancer stem-like phenotype. Cell Cycle. 2011;10(22):3871–85. serves as an additional energy source for tumor cells. Glutamine is 14. Gong C, Bauvy C, Tonelli G, Yue W, Delomenie C, Nicolas V, et al. Beclin 1 also a nitrogen source for amino acids, hexosamines (amino sugars and autophagy are required for the tumorigenicity of breast cancer stem- involved in the synthesis of glycosylated molecules), and like/progenitor cells. Oncogene. 2013;32(18):2261–72 72e 1–11. nucleotides. In cancer cells, glutamine metabolism fuels the TCA 15. Vera-Ramirez L, Vodnala SK, Nini R, Hunter KW, Green JE. Autophagy cycle, fatty acid and nucleotide biosynthesis, and redox balance. promotes the survival of dormant breast cancer cells and metastatic tumour Most cancer cells consume glutamine, and many tumors are recurrence. Nat Commun. 2018;9(1):1944. thought to be addicted to glutamine [124]. This addiction results 16. Sharif T, Martell E, Dai C, Kennedy BE, Murphy P, Clements DR, et al. largely from the contribution of glutamine to anaplerosis [125]. Autophagic homeostasis is required for the pluripotency of cancer stem Macroautophagy cells. Autophagy. 2017;13(2):264–84. Referred to here as autophagy, allows the orderly degradation of 17. Nazio F, Bordi M, Cianfanelli V, Locatelli F, Cecconi F. Autophagy and cancer cellular components and recycling of amino acids, lipids, nucleic stem cells: molecular mechanisms and therapeutic applications. Cell Death acids, and saccharides from intracellular nutrient stores. In this Differ. 2019;26(4):690–702. catabolic process, double-membraned vesicles called 18. Zhou Y, Zhou Y, Shingu T, Feng L, Chen Z, Ogasawara M, et al. Metabolic autophagosomes form around cellular cargo, including organelles, alterations in highly tumorigenic glioblastoma cells: preference for hypoxia protein aggregates, and intracellular pathogens. After fusion of and high dependency on glycolysis. J Biol Chem. 2011;286(37):32843–53. autophagosome with a lysosome, the cargo is degraded. 19. Liu PP, Liao J, Tang ZJ, Wu WJ, Yang J, Zeng ZL, et al. Metabolic regulation Nicotinamide adenine dinucleotide (NAD) metabolism of cancer cell side population by glucose through activation of the Akt NAD acts as an electron and hydrogen acceptor during glycolysis, fatty pathway. Cell Death Differ. 2014;21(1):124–35. acid oxidation, and the TCA cycle. NAD also serves as a coenzyme for 20. Alvero AB, Montagna MK, Sumi NJ, Joo WD, Graham E, Mor G. Multiple oxidoreductases and dehydrogenases that generate ATP during nutrient blocks in the engagement of oxidative phosphorylation in putative ovarian degradation, playing a key role in cellular respiration. NAD and its cancer stem cells: implication for maintenance therapy with glycolysis phosphorylated and reduced forms, including NADP, NADH, and NADPH, inhibitors. Oncotarget. 2014;5(18):8703–15. are essential for cell metabolism activities. NAD most often functions in 21. Ciavardelli D, Rossi C, Barcaroli D, Volpe S, Consalvo A, Zucchelli M, et al. catabolic energy-generating reactions where it is reduced to NADH. The Breast cancer stem cells rely on fermentative glycolysis and are sensitive to phosphorylated form, NADPH, participates in anabolic reactions, such as 2-deoxyglucose treatment. Cell Death Dis. 2014;5:e1336. the synthesis of fatty acids and cholesterol. In addition to its function in 22. Dong C, Yuan T, Wu Y, Wang Y, Fan TW, Miriyala S, et al. Loss of FBP1 by oxidative phosphorylation and redox reactions, NAD is a substrate for snail-mediated repression provides metabolic advantages in basal-like evolutionarily conserved NAD cleavage enzymes such as poly (ADP- breast cancer. Cancer Cell. 2013;23(3):316–31. ribose) polymerases (PARPs), sirtuins (SIRTs), and cADP-ribose synthases 23. Viale A, Pettazzoni P, Lyssiotis CA, Ying H, Sanchez N, Marchesini M, et al. such as CD38 and CD157. Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function. Nature. 2014;514(7524):628–32. Author details 24. Feng W, Gentles A, Nair RV, Huang M, Lin Y, Lee CY, et al. Targeting 1 Institut Necker-Enfants Malades (INEM), Inserm U1151-CNRS UMR 8253, unique metabolic properties of breast tumor initiating cells. Stem Cells. 2 F-75993 Paris, France. Université Paris Descartes-Sorbonne Paris Cité, F-75993 2014;32(7):1734–45. Paris, France. 25. Tamada M, Nagano O, Tateyama S, Ohmura M, Yae T, Ishimoto T, et al. Modulation of glucose metabolism by CD44 contributes to Received: 20 August 2019 Accepted: 26 December 2019 antioxidant status and drug resistance in cancer cells. Cancer Res. 2012;72(6):1438–48. 26. Chen KY, Liu X, Bu P, Lin CS, Rakhilin N, Locasale JW, et al. A metabolic References signature of colon cancer initiating cells. Conf Proc IEEE Eng Med Biol Soc. 1. Martinez-Outschoorn UE, Peiris-Pages M, Pestell RG, Sotgia F, Lisanti MP. 2014;2014:4759–62. Cancer metabolism: a therapeutic perspective. Nat Rev Clin Oncol. 2017; 27. Chae YC, Kim JH. Cancer stem cell metabolism: target for cancer therapy. 14(2):113. BMB Rep. 2018;51(7):319–26. El Hout et al. Molecular Cancer (2020) 19:27 Page 15 of 17

28. Farnie G, Sotgia F, Lisanti MP. High mitochondrial mass identifies a sub- 49. Lee SY, Jeon HM, Ju MK, Kim CH, Yoon G, Han SI, et al. Wnt/snail signaling population of stem-like cancer cells that are chemo-resistant. Oncotarget. regulates cytochrome C oxidase and glucose metabolism. Cancer Res. 2012; 2015;6(31):30472–86. 72(14):3607–17. 29. Pasto A, Bellio C, Pilotto G, Ciminale V, Silic-Benussi M, Guzzo G, et al. 50. Aspuria PP, Lunt SY, Varemo L, Vergnes L, Gozo M, Beach JA, et al. Succinate Cancer stem cells from epithelial ovarian cancer patients privilege oxidative dehydrogenase inhibition leads to epithelial-mesenchymal transition and phosphorylation, and resist glucose deprivation. Oncotarget. 2014;5(12): reprogrammed carbon metabolism. Cancer Metab. 2014;2:21. 4305–19. 51. Luo M, Brooks M, Wicha MS. Epithelial-mesenchymal plasticity of breast 30. Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN, et al. Association cancer stem cells: implications for metastasis and therapeutic resistance. of reactive oxygen species levels and radioresistance in cancer stem cells. Curr Pharm Des. 2015;21(10):1301–10. Nature. 2009;458(7239):780–3. 52. Peiris-Pages M, Martinez-Outschoorn UE, Pestell RG, Sotgia F, Lisanti MP. 31. De Luca A, Fiorillo M, Peiris-Pages M, Ozsvari B, Smith DL, Sanchez-Alvarez R, Cancer stem cell metabolism. Breast Cancer Res. 2016;18(1):55. et al. Mitochondrial biogenesis is required for the anchorage-independent 53. Mao P, Joshi K, Li J, Kim SH, Li P, Santana-Santos L, et al. Mesenchymal survival and propagation of stem-like cancer cells. Oncotarget. 2015;6(17): glioma stem cells are maintained by activated glycolytic metabolism 14777–95. involving aldehyde dehydrogenase 1A3. Proc Natl Acad Sci U S A. 2013; 32. Sancho P, Burgos-Ramos E, Tavera A, Bou Kheir T, Jagust P, Schoenhals M, 110(21):8644–9. et al. MYC/PGC-1alpha balance determines the metabolic phenotype and 54. Luo M, Shang L, Brooks MD, Jiagge E, Zhu Y, Buschhaus JM, et al. Targeting plasticity of pancreatic Cancer stem cells. Cell Metab. 2015;22(4):590–605. breast Cancer stem cell state equilibrium through modulation of redox 33. Bonuccelli G, Sotgia F, Lisanti MP. Matcha green tea (MGT) inhibits the signaling. Cell Metab. 2018;28(1):69–86 e6. propagation of cancer stem cells (CSCs), by targeting mitochondrial 55. Flavahan WA, Wu Q, Hitomi M, Rahim N, Kim Y, Sloan AE, et al. Brain tumor metabolism, glycolysis and multiple cell signalling pathways. Aging (Albany initiating cells adapt to restricted nutrition through preferential glucose NY). 2018;10(8):1867–83. uptake. Nat Neurosci. 2013;16(10):1373–82. 34. Ginestier C, Monville F, Wicinski J, Cabaud O, Cervera N, Josselin E, et al. 56. Isayev O, Rausch V, Bauer N, Liu L, Fan P, Zhang Y, et al. Inhibition of Mevalonate metabolism regulates basal breast cancer stem cells and is a glucose turnover by 3-bromopyruvate counteracts pancreatic cancer stem potential therapeutic target. Stem Cells. 2012;30(7):1327–37. cell features and sensitizes cells to gemcitabine. Oncotarget. 2014;5(13): 35. Xiang L, Semenza GL. Hypoxia-inducible factors promote breast cancer 5177–89. stem cell specification and maintenance in response to hypoxia or cytotoxic 57. Michelakis ED, Sutendra G, Dromparis P, Webster L, Haromy A, Niven E, et al. chemotherapy. Adv Cancer Res. 2019;141:175–212. Metabolic modulation of glioblastoma with dichloroacetate. Sci Transl Med. 36. SamantaD,ParkY,AndrabiSA,SheltonLM,GilkesDM,SemenzaGL. 2010;2(31):31ra4. PHGDH expression is required for mitochondrial redox homeostasis, 58. Kim JH, Lee KJ, Seo Y, Kwon JH, Yoon JP, Kang JY, et al. Effects of metformin breast Cancer stem cell maintenance, and lung metastasis. Cancer Res. on colorectal cancer stem cells depend on alterations in glutamine 2016;76(15):4430–42. metabolism. Sci Rep. 2018;8(1):409. 37. Lucena-Cacace A, Umeda M, Navas LE, Carnero A. NAMPT as a 59. Jagust P, de Luxan-Delgado B, Parejo-Alonso B, Sancho P. Metabolism- dedifferentiation-inducer gene: NAD(+) as Core Axis for Glioma Cancer based therapeutic strategies targeting Cancer stem cells. Front Pharmacol. stem-like cells maintenance. Front Oncol. 2019;9:292. 2019;10:203. 38. Garten A, Schuster S, Penke M, Gorski T, de Giorgis T, Kiess W. Physiological 60. Yeh CT, Su CL, Huang CY, Lin JK, Lee WH, Chang PM, et al. A preclinical and pathophysiological roles of NAMPT and NAD metabolism. Nat Rev evaluation of antimycin a as a potential antilung cancer stem cell agent. Endocrinol. 2015;11(9):535–46. Evid Based Complement Alternat Med. 2013;2013:910451. 39. Gujar AD, Le S, Mao DD, Dadey DY, Turski A, Sasaki Y, et al. An NAD+- 61. Fiorillo M, Lamb R, Tanowitz HB, Cappello AR, Martinez-Outschoorn UE, dependent transcriptional program governs self-renewal and radiation Sotgia F, et al. Bedaquiline, an FDA-approved antibiotic, inhibits resistance in glioblastoma. Proc Natl Acad Sci U S A. 2016;113(51): mitochondrial function and potently blocks the proliferative expansion of E8247–E56. stem-like cancer cells (CSCs). Aging (Albany NY). 2016;8(8):1593–607. 40. Lucena-Cacace A, Otero-Albiol D, Jimenez-Garcia MP, Munoz-Galvan S, 62. Kennedy CR, Tilkens SB, Guan H, Garner JA, Or PM, Chan AM. Differential Carnero A. NAMPT is a potent oncogene in Colon Cancer progression that sensitivities of glioblastoma cell lines towards metabolic and signaling modulates Cancer stem cell properties and resistance to therapy through pathway inhibitions. Cancer Lett. 2013;336(2):299–306. Sirt1 and PARP. Clin Cancer Res. 2018;24(5):1202–15. 63. Mai TT, Hamai A, Hienzsch A, Caneque T, Muller S, Wicinski J, et al. 41. Jung J, Kim LJ, Wang X, Wu Q, Sanvoranart T, Hubert CG, et al. Salinomycin kills cancer stem cells by sequestering iron in lysosomes. Nat Nicotinamide metabolism regulates glioblastoma stem cell maintenance. JCI Chem. 2017;9(10):1025–33. Insight. 2017;2(10). 64. Yue W, Hamai A, Tonelli G, Bauvy C, Nicolas V, Tharinger H, et al. 42. Lu Y, Kwintkiewicz J, Liu Y, Tech K, Frady LN, Su YT, et al. Chemosensitivity Inhibition of the autophagic flux by salinomycin in breast cancer stem- of IDH1-mutated Gliomas due to an impairment in PARP1-mediated DNA like/progenitor cells interferes with their maintenance. Autophagy. 2013; repair. Cancer Res. 2017;77(7):1709–18. 9(5):714–29. 43. ChenZT,ZhaoW,QuS,LiL,LuXD,SuF,etal.PARP-1promotes 65. Naujokat C, Steinhart R. Salinomycin as a drug for targeting human cancer autophagy via the AMPK/mTOR pathway in CNE-2 human stem cells. J Biomed Biotechnol. 2012;2012:950658. nasopharyngeal carcinoma cells following ionizing radiation, while 66. Shen YA, Lan KL, Chang CH, Lin LT, He CL, Chen PH, et al. Intraperitoneal inhibition of autophagy contributes to the radiation sensitization of (188)re-liposome delivery switches ovarian cancer metabolism from CNE-2 cells. Mol Med Rep. 2015;12(2):1868–76. glycolysis to oxidative phosphorylation and effectively controls ovarian 44. Zhang DX, Zhang JP, Hu JY, Huang YS. The potential regulatory roles tumour growth in mice. Radiother Oncol. 2016;119(2):282–90. of NAD(+) and its metabolism in autophagy. Metabolism. 2016;65(4): 67. Scatena C, Roncella M, Di Paolo A, Aretini P, Menicagli M, Fanelli G, et al. 454–62. Doxycycline, an inhibitor of mitochondrial biogenesis, effectively reduces 45. Aguilar E. Marin de mas I, Zodda E, Marin S, Morrish F, Selivanov V, et al. Cancer stem cells (CSCs) in early breast Cancer patients: a clinical pilot metabolic reprogramming and dependencies associated with epithelial study. Front Oncol. 2018;8:452. Cancer stem cells independent of the epithelial-Mesenchymal transition 68. Yang B, Lu Y, Zhang A, Zhou A, Zhang L, Zhang L, et al. Doxycycline program. Stem Cells. 2016;34(5):1163–76. induces apoptosis and inhibits proliferation and invasion of human cervical 46. El Hout M, Dos Santos L, Hamai A, Mehrpour M. A promising new approach carcinoma stem cells. PLoS One. 2015;10(6):e0129138. to cancer therapy: targeting iron metabolism in cancer stem cells. Semin 69. Xie Q, Wu Q, Horbinski CM, Flavahan WA, Yang K, Zhou W, et al. Cancer Biol. 2018;53:125–38. Mitochondrial control by DRP1 in brain tumor initiating cells. Nat Neurosci. 47. Recalcati S, Gammella E, Cairo G. Dysregulation of iron metabolism in 2015;18(4):501–10. cancer stem cells. Free Radic Biol Med. 2018. 70. Peiris-Pages M, Bonuccelli G, Sotgia F, Lisanti MP. Mitochondrial fission as a 48. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al. The driver of stemness in tumor cells: mDIVI1 inhibits mitochondrial function, epithelial-mesenchymal transition generates cells with properties of stem cell migration and cancer stem cell (CSC) signalling. Oncotarget. 2018;9(17): cells. Cell. 2008;133(4):704–15. 13254–75. El Hout et al. Molecular Cancer (2020) 19:27 Page 16 of 17

71. Elhammali A, Ippolito JE, Collins L, Crowley J, Marasa J, Piwnica-Worms D. A 95. Eskiocak B, Ali A, White MA. The estrogen-related receptor alpha inverse high-throughput fluorimetric assay for 2-hydroxyglutarate identifies agonist XCT 790 is a nanomolar mitochondrial uncoupler. Biochemistry. Zaprinast as a glutaminase inhibitor. Cancer Discov. 2014;4(7):828–39. 2014;53(29):4839–46. 72. Kim D, Choi BH, Ryoo IG, Kwak MK. High NRF2 level mediates cancer stem 96. Lamb R, Ozsvari B, Lisanti CL, Tanowitz HB, Howell A, Martinez-Outschoorn cell-like properties of aldehyde dehydrogenase (ALDH)-high ovarian cancer UE, et al. Antibiotics that target mitochondria effectively eradicate cancer cells: inhibitory role of all-trans retinoic acid in ALDH/NRF2 signaling. Cell stem cells, across multiple tumor types: treating cancer like an infectious Death Dis. 2018;9(9):896. disease. Oncotarget. 2015;6(7):4569–84. 73. Kim YJ, Kim JY, Lee N, Oh E, Sung D, Cho TM, et al. Disulfiram suppresses 97. Esner M, Graifer D, Lleonart ME, Lyakhovich A. Targeting cancer cells cancer stem-like properties and STAT3 signaling in triple-negative breast through antibiotics-induced mitochondrial dysfunction requires autophagy cancer cells. Biochem Biophys Res Commun. 2017;486(4):1069–76. inhibition. Cancer Lett. 2017;384:60–9. 74. Xu B, Wang S, Li R, Chen K, He L, Deng M, et al. Disulfiram/copper selectively 98. Chen H, Chan DC. Mitochondrial dynamics in regulating the unique eradicates AML leukemia stem cells in vitro and in vivo by simultaneous induction phenotypes of Cancer and stem cells. Cell Metab. 2017;26(1):39–48. of ROS-JNK and inhibition of NF-kappaB and Nrf2. Cell Death Dis. 2017;8(5):e2797. 99. Bordi M, Nazio F, Campello S. The close interconnection between 75. Erdogan S, Turkekul K, Serttas R, Erdogan Z. The natural flavonoid apigenin mitochondrial dynamics and Mitophagy in Cancer. Front Oncol. 2017;7:81. sensitizes human CD44(+) prostate cancer stem cells to cisplatin therapy. 100. Zhou J, Li G, Zheng Y, Shen HM, Hu X, Ming QL, et al. A novel autophagy/ Biomed Pharmacother. 2017;88:210–7. mitophagy inhibitor liensinine sensitizes breast cancer cells to 76. Roh JL, Kim EH, Jang H, Shin D. Nrf2 inhibition reverses the resistance of chemotherapy through DNM1L-mediated mitochondrial fission. Autophagy. cisplatin-resistant head and neck cancer cells to artesunate-induced 2015;11(8):1259–79. ferroptosis. Redox Biol. 2017;11:254–62. 101. Chang CM, Lan KL, Huang WS, Lee YJ, Lee TW, Chang CH, et al. (188)Re- 77. Hale JS, Otvos B, Sinyuk M, Alvarado AG, Hitomi M, Stoltz K, et al. Cancer Liposome Can Induce Mitochondrial Autophagy and Reverse Drug stem cell-specific scavenger receptor CD36 drives glioblastoma progression. Resistance for Ovarian Cancer: From Bench Evidence to Preliminary Clinical Stem Cells. 2014;32(7):1746–58. Proof-of-Concept. Int J Mol Sci. 2017;18(5). 78. Corominas-Faja B, Cuyas E, Gumuzio J, Bosch-Barrera J, Leis O, Martin AG, 102. Yuan S, Lu Y, Yang J, Chen G, Kim S, Feng L, et al. Metabolic activation et al. Chemical inhibition of acetyl-CoA carboxylase suppresses self-renewal of mitochondria in glioma stem cells promotes cancer development growth of cancer stem cells. Oncotarget. 2014;5(18):8306–16. through a reactive oxygen species-mediated mechanism. Stem Cell Res 79. Alwarawrah Y, Hughes P, Loiselle D, Carlson DA, Darr DB, Jordan JL, Ther. 2015;6:198. et al. Fasnall, a selective FASN inhibitor, shows potent anti-tumor 103. Chang CW, Chen YS, Tsay YG, Han CL, Chen YJ, Yang CC, et al. ROS- activity in the MMTV-Neu model of HER2(+) breast Cancer. Cell Chem independent ER stress-mediated NRF2 activation promotes Warburg effect Biol. 2016;23(6):678–88. to maintain stemness-associated properties of cancer-initiating cells. Cell 80. Yasumoto Y, Miyazaki H, Vaidyan LK, Kagawa Y, Ebrahimi M, Yamamoto Y, Death Dis. 2018;9(2):194. et al. Inhibition of fatty acid synthase decreases expression of Stemness 104. Ishimoto T, Nagano O, Yae T, Tamada M, Motohara T, Oshima H, et al. CD44 markers in Glioma stem cells. PLoS One. 2016;11(1):e0147717. variant regulates redox status in cancer cells by stabilizing the xCT subunit 81. Ma MKF, Lau EYT, Leung DHW, Lo J, Ho NPY, Cheng LKW, et al. Stearoyl- of system xc(−) and thereby promotes tumor growth. Cancer Cell. 2011; CoA desaturase regulates sorafenib resistance via modulation of ER stress- 19(3):387–400. induced differentiation. J Hepatol. 2017;67(5):979–90. 105. Onodera T, Momose I, Kawada M. Potential anticancer activity of Auranofin. 82. Pisanu ME, Noto A, De Vitis C, Morrone S, Scognamiglio G, Botti G, et al. Chem Pharm Bull (Tokyo). 2019;67(3):186–91. Blockade of Stearoyl-CoA-desaturase 1 activity reverts resistance to cisplatin 106. Song W, Tang Z, Lei T, Wen X, Wang G, Zhang D, et al. Stable loading and in lung cancer stem cells. Cancer Lett. 2017;406:93–104. delivery of disulfiram with mPEG-PLGA/PCL mixed nanoparticles for tumor 83. Ricciardi MR, Mirabilii S, Allegretti M, Licchetta R, Calarco A, Torrisi MR, et al. therapy. Nanomedicine. 2016;12(2):377–86. Targeting the leukemia cell metabolism by the CPT1a inhibition: functional 107. Milkovic L, Zarkovic N, Saso L. Controversy about pharmacological preclinical effects in leukemias. Blood. 2015;126(16):1925–9. modulation of Nrf2 for cancer therapy. Redox Biol. 2017;12:727–32. 84. Lee EA, Angka L, Rota SG, Hanlon T, Mitchell A, Hurren R, et al. Targeting 108. Hanai JI, Doro N, Seth P, Sukhatme VP. ATP citrate lyase knockdown impacts mitochondria with Avocatin B induces selective leukemia cell death. Cancer cancer stem cells in vitro. Cell Death Dis. 2013;4:e696. Res. 2015;75(12):2478–88. 109. Granchi C. ATP citrate lyase (ACLY) inhibitors: an anti-cancer strategy at the 85. Koerner SK, Hanai JI, Bai S, Jernigan FE, Oki M, Komaba C, et al. Design and crossroads of glucose and lipid metabolism. Eur J Med Chem. 2018;157: synthesis of emodin derivatives as novel inhibitors of ATP-citrate lyase. Eur J 1276–91. Med Chem. 2017;126:920–8. 110. Knobloch M, Braun SM, Zurkirchen L, von Schoultz C, Zamboni N, Arauzo- 86. Wang L, Li P, Hu W, Xia Y, Hu C, Liu L, et al. CD44(+)CD24(+) subset of Bravo MJ, et al. Metabolic control of adult neural stem cell activity by Fasn- PANC-1 cells exhibits radiation resistance via decreased levels of reactive dependent lipogenesis. Nature. 2013;493(7431):226–30. oxygen species. Oncol Lett. 2017;14(2):1341–6. 111. Sun P, Xia S, Lal B, Shi X, Yang KS, Watkins PA, et al. Lipid metabolism 87. Peixoto J, Lima J. Metabolic traits of cancer stem cells. Dis Model Mech. enzyme ACSVL3 supports glioblastoma stem cell maintenance and 2018;11(8). tumorigenicity. BMC Cancer. 2014;14:401. 88. Vitale I, Manic G, Dandrea V, De Maria R. Role of autophagy in the maintenance 112. Tirinato L, Liberale C, Di Franco S, Candeloro P, Benfante A, La Rocca R, et al. and function of cancer stem cells. Int J Dev Biol. 2015;59(1–3):95–108. Lipid droplets: a new player in colorectal cancer stem cells unveiled by 89. Denise C, Paoli P, Calvani M, Taddei ML, Giannoni E, Kopetz S, et al. 5- spectroscopic imaging. Stem Cells. 2015;33(1):35–44. fluorouracil resistant colon cancer cells are addicted to OXPHOS to survive 113. Maan M, Peters JM, Dutta M, Patterson AD. Lipid metabolism and lipophagy and enhance stem-like traits. Oncotarget. 2015;6(39):41706–21. in cancer. Biochem Biophys Res Commun. 2018;504(3):582–9. 90. Chen W, Mook RA Jr, Premont RT, Wang J. Niclosamide: beyond an 114. Pascual G, Avgustinova A, Mejetta S, Martin M, Castellanos A, Attolini CS, antihelminthic drug. Cell Signal. 2018;41:89–96. et al. Targeting metastasis-initiating cells through the fatty acid receptor 91. Kim SY, Kang JW, Song X, Kim BK, Yoo YD, Kwon YT, et al. Role of the IL-6- CD36. Nature. 2017;541(7635):41–5. JAK1-STAT3-Oct-4 pathway in the conversion of non-stem cancer cells into 115. Ye H, Adane B, Khan N, Sullivan T, Minhajuddin M, Gasparetto M, et al. cancer stem-like cells. Cell Signal. 2013;25(4):961–9. Leukemic stem cells evade chemotherapy by metabolic adaptation to an 92. Mitani M, Yamanishi T, Miyazaki Y, Otake N. Salinomycin effects on adipose tissue niche. Cell Stem Cell. 2016;19(1):23–37. mitochondrial ion translocation and respiration. Antimicrob Agents 116. Young PA, Senkal CE, Suchanek AL, Grevengoed TJ, Lin DD, Zhao L, et al. Chemother. 1976;9(4):655–60. Long-chain acyl-CoA synthetase 1 interacts with key proteins that activate 93. Jangamreddy JR, Ghavami S, Grabarek J, Kratz G, Wiechec E, Fredriksson BA, and direct fatty acids into niche hepatic pathways. J Biol Chem. 2018; et al. Salinomycin induces activation of autophagy, mitophagy and affects 293(43):16724–40. mitochondrial polarity: differences between primary and cancer cells. 117. Corominas-Faja B, Vellon L, Cuyas E, Buxo M, Martin-Castillo B, Serra D, Biochim Biophys Acta. 2013;1833(9):2057–69. et al. Clinical and therapeutic relevance of the metabolic oncogene 94. Deblois G, St-Pierre J, Giguere V. The PGC-1/ERR signaling axis in cancer. fatty acid synthase in HER2+ breast cancer. Histol Histopathol. 2017; Oncogene. 2013;32(30):3483–90. 32(7):687–98. El Hout et al. Molecular Cancer (2020) 19:27 Page 17 of 17

118. Li J, Condello S, Thomes-Pepin J, Ma X, Xia Y, Hurley TD, et al. Lipid desaturation is a metabolic marker and therapeutic target of ovarian Cancer stem cells. Cell Stem Cell. 2017;20(3):303–14 e5. 119. Liu X, Strable MS, Ntambi JM. Stearoyl CoA desaturase 1: role in cellular inflammation and stress. Adv Nutr. 2011;2(1):15–22. 120. Galluzzi L, Kepp O, Vander Heiden MG, Kroemer G. Metabolic targets for cancer therapy. Nat Rev Drug Discov. 2013;12(11):829–46. 121. Chen CL, Uthaya Kumar DB, Punj V, Xu J, Sher L, Tahara SM, et al. NANOG metabolically reprograms tumor-initiating stem-like cells through tumorigenic changes in oxidative phosphorylation and fatty acid metabolism. Cell Metab. 2016;23(1):206–19. 122. Cardwell CR, Hicks BM, Hughes C, Murray LJ. Statin use after diagnosis of breast cancer and survival: a population-based cohort study. Epidemiology. 2015;26(1):68–78. 123. Fiorillo M, Peiris-Pages M, Sanchez-Alvarez R, Bartella L, Di Donna L, Dolce V, et al. Bergamot natural products eradicate cancer stem cells (CSCs) by targeting mevalonate, rho-GDI-signalling and mitochondrial metabolism. Biochim Biophys Acta Bioenerg. 2018;1859(9):984–96. 124. Altman BJ, Stine ZE, Dang CV. From Krebs to clinic: glutamine metabolism to cancer therapy. Nat Rev Cancer. 2016;16(12):773. 125. Krall AS, Christofk HR. Rethinking glutamine addiction. Nat Cell Biol. 2015; 17(12):1515–7. 126. Pavlides S, Whitaker-Menezes D, Castello-Cros R, Flomenberg N, Witkiewicz AK, Frank PG, et al. The reverse Warburg effect: aerobic glycolysis in cancer associated fibroblasts and the tumor stroma. Cell Cycle. 2009;8(23):3984–4001.

Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.