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Hallmarks of Cancer Stem Cell Metabolism
Patricia Sancho*, David Barneda and Christopher Heeschen*
Centre for Stem Cells in Cancer & Ageing, Barts Cancer Institute, Queen Mary University of London,
UK, EC1M 6BQ
* Correspondence: Dr. Patricia Sancho, PhD, [email protected] or Dr. Christopher Heeschen,
MD, PhD, [email protected]; Centre for Stem Cells in Cancer & Ageing, Barts Cancer
Institute, Queen Mary University of London, UK.
Keywords: Cancer stem cells, Tumour-initiating cells, Oxidative phosphorylation, Glycolysis,
Metastasis, Metformin, Myc.
Acknowledgements: This work was supported by the ERC Advanced Investigator Grant (Pa-CSC
233460 to C.H.), the European Community's Seventh Framework Programme (FP7/2007-2013) under
grant agreement n° 256974 (EPC-TM-NET to C.H.) and n° 602783 (CAM-PaC to C.H.), the 2015
SU2C Lustgarten CRUK Pancreatic Cancer Dream Team Award (to C.H.) and the Pancreatic Cancer
Research Fund (to P.S.).
ABSTRACT
To cope with their high proliferation rate, cancer cells adapt their cellular metabolism using
glycolysis instead of from oxidative phosphorylation (OXPHOS) for production of ATP and building
blocks (Warburg effect). However, not all cancer cells behave equally due to substantial (epi-)genetic
heterogeneity. Despite having an identical genetic background, a subset of cells bears stemness
features, thus termed cancer stem cells (CSCs), resulting in a hierarchical organisation of the tumour
reminiscent of normal tissues. As opposed to differentiated cancer cells representing the bulk of the
tumour, CSCs in various, but not all human cancer types preferentially rely on mitochondrial
OXPHOS, thus rendering CSCs less dependent on the sparse supply with nutrients in the tumour
microenvironment. Moreover, the metabolic plasticity as the ability to switch between OXPHOS and
glycolysis depending on circumstances, is also limited in CSCs of various cancers. This apparent
fixation of CSCs on mitochondrial function for energy supply, but also for maintenance of their
stemness properties represents a previously unrecognised Achilles heel amendable for therapeutic
intervention. Elimination of highly chemoresistant CSCs as the root of many cancers via inhibition of
mitochondrial function may prevent relapse from disease and thus improve patients’ long-term
outcome.
I. INTRODUCTION
Cellular Metabolism
In non-transformed, mostly slowly proliferating or even quiescent somatic cells, mitochondria
are the main source of energy production through the tricarboxylic acid (TCA) cycle coupled to
oxidative phosphorylation (OXPHOS), which takes place in the mitochondrial matrix. Several carbon
fuels such as pyruvate, glutamine and fatty acids can feed the cycle to produce reducing equivalents
(nicotinamide adenine dinucleotide phosphate, NADH; Flavin adenine dinucleotide, FADH2) that are
subsequently used as electron donors for the electron transport chain (ETC). The transport of
electrons across the different complexes of the ETC is coupled to the generation of a proton motive
force, used by the ATP synthase (complex V) to generate ATP1.
Cancer cells, however, are characterised by a high proliferation rate and thus need to adapt
their cellular metabolism in order to provide support for increased division rates: rapid ATP
generation to maintain energy status, increased biosynthesis of macromolecules and tight regulation
of the cellular redox status2. Moreover, tumour cells must evade the checkpoint controls that under
physiological conditions inhibit proliferation in challenging metabolic conditions as found in the
tumour microenvironment. Levels of glucose, glutamine and oxygen are spatially and temporally
heterogeneous and frequently sparser as compared to conditions in well-perfused organs.
Accordingly, tumour cells reprogram their metabolic pathways to meet their needs during the process
of tumour growth, but also during metastasis.
For this purpose, cancer cells shift from ATP generation via OXPHOS to ATP generation via
glycolysis, despite still sufficient oxygen concentrations in the tumour microenvironment (Warburg
effect). As a result, many transformed cells derive a substantial amount of their energy via aerobic
glycolysis, which is more rapid than OXPHOS, but also far less efficient in terms of ATP generated
per unit of glucose consumed, resulting in an abnormally high rate of glucose uptake. Under these
circumstances, glucose is also metabolised through the pentose phosphate pathway (PPP) and other
alternative pathways2, which produce large quantities of reduced NADPH and other macromolecules
to generate the necessary building blocks required for sustaining high rates of cellular division.
Cancer Stem Cells
It is important to note that not all cancer cells behave equally, both at the functional and
metabolic level. Convincing evidence demonstrated that substantial (epi-)genetic heterogeneity exists
within each individual tumour. First, multiple subclonal populations of cancer cells are assumed to
foster tumour adaptation and therapeutic failure through Darwinian selection. Secondly, cancer
heterogeneity also exists within each of these subclones, despite their identical genetic background,
via the acquisition of stemness features in a subset of cells, thus resulting in a hierarchical
organisation of the tumour that is vaguely reminiscent of that found in many normal tissues (Fig. 1)3.
At the apex of this hierarchy are populations of cancer stem cells (CSCs) capable of self-renewal,
bearing long-term in vivo tumourigenicity as well as generating more differentiated progenies
constituting epigenetically defined intraclonal bulk4.
This new view of intraclonal functional heterogeneity bears the potential to fundamentally
change the way we analyse and treat cancer. This will require a thorough understanding of the
mechanisms underlying this close relationship between stem cells and their malignant counterparts as
well as their metabolic features. Although CSCs do not necessarily arise from tissue stem cells, these
cells have acquired stemness features allowing them to indefinitely self-renew and give rise to their
respective differentiated progenies. Epigenetic regulation mimicking, at least in part, normal
differentiation contributes to the generation of these hierarchically organised clones that, although
sharing common mutation profiles, bear diverse gene expression patterns and functions5.
Accumulating evidence also suggests striking parallels between mechanisms orchestrating normal
embryogenesis and those that invoke tumourigenesis and CSCs in particular6. Thus, it is of utmost
importance to conclusively define the potentially distinct metabolic features of CSCs.
II. METABOLIC PHENOTYPE OF CANCER STEM CELLS
Originally, it was hypothesised that CSCs bear a metabolic phenotype in analogy to normal
tissue hierarchy where multipotent stem cells are fundamentally glycolytic, while differentiated
somatic cells rely on OXPHOS7. Similar patterns have been reported for induced pluripotent stem
cells (iPSCs), where the reprogramming process is associated with a switch from OXPHOS to a
glycolytic programme, which indeed is essential for effective acquisition of a pluripotent state. These
findings suggested that not only are metabolic phenotype and stemness intrinsically linked, but rather
cellular metabolism actually controls stemness properties. Thus, it was postulated that activation of
the glycolytic programme favours stemness via different mechanism including enhanced antioxidant
capacity via the PPP as the most relevant one8.
Subsequently, a number of investigations aimed to validate this concept of glycolysis-driven
stemness to the cancer field. Experimental evidence obtained for CSCs in breast cancer seemed to
support such hypothesis. For example, Dong et al. demonstrated that the metabolic switch from
OXPHOS to aerobic glycolysis was essential for maintaining CSC functionality, due to decreased
ROS levels 9. Moreover, glycolysis was also found to represent the favoured metabolic programme of
CSCs in nasopharyngeal carcinoma10 and hepatocellular carcinoma11. Interestingly, elevated
expression of the oncogene MYC was defined as the main driver of stemness for these three cancer
types12, which is well in line with findings for iPS cells as discussed above. While the MYC levels did
not determine the metabolic wiring of iPS cells, their tumorigenic potential as evidenced by teratoma
formation was intrinsically linked to a MYC-driven glycolytic program13. Therefore, MYC seems to
be a likely candidate determining the connection between glycolysis and stemness, intimately
associated to the tumorigenic potential of iPS cells, but also for certain cancer types as listed above.
Importantly, however, accumulating evidence now demonstrates that CSCs in other cancer
types may actually rely on mitochondrial OXPHOS as the preferred energy production. To date, this
has been shown convincingly for glioblastoma14, lung cancer15, pancreatic cancer16 and leukaemia17.
Interestingly, metabolic rewiring to OXPHOS rendered CSCs derived from these tumours resistant to
inhibition of glycolysis, which may confer these cells with a higher degree of independency from
microenviromental nutrient supply, as discussed in more detail below. Although the mechanisms of
action for the observed OXPHOS phenotype have not been well characterised for all cited tumour
types, regulatory proteins of mitochondrial biogenesis and structure seem to play a crucial role in
maintaining stem-related properties and functionality14,
16
. Indeed, findings for pancreatic cancer
clearly demonstrate that expression of the transcription factor PPARGC1A (PGC-1α), a master
regulator of mitochondrial biogenesis, was essential for the OXPHOS functionality in pancreatic
CSCs and, most importantly, self-renewal and maximal in vivo tumorigenic capacity16. Intriguingly, a
MYC-driven glycolytic programme was only found in more differentiated tumour cells and
overexpression of MYC actually counteracted stemness via negatively controlling PGC-1α
expression. These data seem to challenge the concept for MYC favouring stemness via activation of
glycolysis, as demonstrated for iPSCs and some other cancer types. However, those apparently
contradictory findings may be reconciled by a concept where MYC serves as a general modulator of
the differentiation state, promoting either stemness or differentiation in a context and cell typedependent manner.
Still, the apparent inconsistency in the predominant metabolic phenotype of CSCs isolated
from various cancer types may actually, at least in part, be related to the utilised model systems. Most
early studies in breast cancer demonstrating a highly glycolytic phenotype for the contained putative
CSCs were performed in established for cell lines, for which the existence of a true CSC
subpopulation remains at least questionable. Moreover, contradictory results regarding the CSC
metabolic phenotype for individual cancer types can be found, e.g. for ovarian cancer, where CSCs
have been reported to be either primarily glycolytic18 and OXPHOS-dependent19. The latter was
reported for primary cultures, which were derived from tumours obtained either directly from patients
or following in vivo expansion in immunocompromised mice (PDX models); an approach also used
for studies in glioblastoma14 and pancreatic cancer16. Thus, it remains to be determined if the
glycolytic phenotype originally reported for some cancer types will eventually be validated in
clinically more relevant models.
This matter is further complicated by recent reports that the metabolic plasticity, the ability to
switch between OXPHOS and glycolysis depending on circumstances, also appears to vary
considerably between CSCs derived from different tumour types. For example, while ovarian CSCs
are characterised by marked metabolic flexibility rendering them highly resistant to metabolic
targeting18, limited metabolic plasticity of CSCs has been reported for other tumour entities 14, 16.
Again, it appears that a more restricted metabolic phenotype was mainly found in primary cultures or
cell isolated from freshly resected tumours, where CSCs were shown to mostly rely on OXPHOS.
However, irrespective of the underlying primary metabolic phenotype, limited metabolic plasticity
represents an important feature with significant implications for designing new metabolism-centred
therapeutic strategies. Specifically, a strict dependency on mitochondrial OXPHOS with low
plasticity should render CSCs highly vulnerable to mitochondrial targeting, thus eliminating the
source of relapse and metastasis.
Addiction to oxidative mitochondrial metabolism could also be considered an adaptation of
CSCs to their respective microenvironment. Indeed, OXPHOS equips CSCs with increased resistance
to nutrient deprivation and, in general, to the metabolic austerity characterising many solid tumours.
Although OXPHOS operates at a significantly lower rate, it constitutes a far more efficient source for
energy generation. Thus, CSCs may generate a selective advantage in the context of specific tumour
microenvironments, as they use more efficiently limited nutrients. In addition, lactate excreted by the
more differentiated cancer cells running on glycolysis may serve as fuel for oxidative respiration in
cellular subsets dependent on mitochondrial metabolism, such as CSCs, constituting a metabolic
symbiosis system20. In line with this hypothesis, it has been demonstrated that certain glycolysis endproducts such as high-energy lactanes and ketones promote expression of stemness-associated genes
and shifting cells towards OXPHOS21.
In addition to constituting a major source of ATP for cancer cells, mitochondria participate in
controlling multiple signalling pathways, including the release of Cytochrome C to initiate apoptosis,
the release of bioactive ROS and the production of metabolites such as acetyl-CoA regulating protein
acetylation1. As such mitochondria also appear to regulate stemness properties, irrespective of the
underlying metabolic phenotype in individual cells 16, 22, 23. Indeed, enhanced mitochondrial biogenesis
appears to represent a key factor for CSC functionality in both glycolytic and OXPHOS-dependent
CSC14, 16. Increased mitochondrial mass, a surrogate marker for elevated biogenesis, can be easily
tracked and may identify cells with increased self-renewal capacity and chemoresistance16
independently of the cancer type. Specifically, the use of this metabolic biomarker allowed to identify
a subpopulation of CSCs with low mitochondrial mass but increased metabolic plasticity driven by
MYC within the pancreatic CSC compartment (CD133+/Mitolow)16. Consequently, these cells showed
increased resistance to mitochondrial targeting with respective inhibitors. However, this metabolic
plasticity came at the expense of a reduced self-renewal capacity and in vivo tumorigenic potential,
suggesting a delicate balance between stemness and metabolic plasticity, thus adding another level of
complexity to the metabolic features of CSCs. Whether the state of these two different CSC
subpopulations with differential plasticity and tumorigenic potential is dynamic and cells are able to
transition between states, or is hard wired due to distinct genetic backgrounds remains to be
elucidated.
III. TARGETING CELLULAR METABOLISM
The apparent OXPHOS dependence of CSCs in various tumours and the recently identified
role of mitochondria in the regulation of stemness properties22, suggest that targeting mitochondrial
metabolism could be an effective pharmacological strategy for the elimination of CSCs. Moreover, as
mitochondria in cancer cells are often altered by mutations in their vulnerable DNA, the
pharmacological disruption of certain mitochondrial processes could damage CSCs without affecting
healthy tissues relying on OXPHOS. Pharmacological agents targeting OXPHOS at various levels are
currently explored in preclinical and clinical studies for cancer treatment (Fig. 2)
Targeting mitochondrial OXPHOS could be an effective strategy to eliminate cancer cells
which cannot fully meet their energetic demands by glycolysis, either due to limited availability of
glucose in poorly vascularised tumours, glycolysis inhibition by current therapies such as PI3K
inhibitors, or restricted metabolic plasticity as observed in CSCs14, 16. Inhibition of mitochondrial
respiration by agents blocking electron transport chain (ETC) complexes selectively induces apoptosis
in CSCs versus non-CSCs16. The fact that such agents are also effective in eliminating primarily
glycolytic CSCs in breast cancer or nasopharyngeal carcinoma24,
25
, highlights the importance of
mitochondria for CSCs beyond energy production. In fact, tumour cells displaying mutations
impairing TCA cycle or ETC, thus predominantly relying on glycolysis for ATP production, still
require active mitochondria for the generation of metabolites from glutamine via reductive
carboxylation26.
Drug screens aiming for the identification of compounds that selectively eliminate CSCs
resulted in the selection of several FDA-approved compounds that inhibit mitochondrial activity. For
example, the antibiotic salinomycin, which inhibits OXPHOS, was identified in a screen targeting
breast CSCs and eliminated the CSC gene expression signature in subsequent in vivo studies27.
Remarkably, salinomycin was also selected in an independent screen aimed at targeting colorectal
cancer cells in glucose-deprived multicellular tumour spheroids with inner hypoxia28, which could
reflect the microenvironment of CSCs in solid tumours. Beside salinomycin, four other compounds
were identified (nitazoxanide, niclosamide, closantel and pyrvinium pamoate), all of them inhibiting
mitochondrial respiration. Niclosamide, an anti-helmitic drug which uncouples mitochondrial
OXPHOS, was also among the selected compound in two independent drug screens in breast and
ovarian CSCs29, 30. Apart from the direct inhibition of mitochondrial complexes, OXPHOS can also be
suppressed by inhibitors of mitochondrial translation, e.g. the antibiotic tigecycline, which was
selected in a drug screen because of its selective toxicity against leukemic cells, which depend on
OXPHOS for energy production31. As mitochondria originally evolved from bacteria, it is not
surprising that multiple antibiotics can disrupt mitochondrial function. Indeed, a recent study
suggested that CSCs from multiple tumour types could be eradicated by treatment with certain widely
prescribed antibiotics via disrupting mitochondrial respiration, either by inhibition of mitochondrial
ribosomes or direct targeting of OXPHOS23.
The anti-diabetic agent metformin has also emerged as a promising candidate for targeting
OXPHOS in CSCs16. Interest in metformin rose when it was shown in retrospective studies that it is
associated with a lower incidence of cancer in diabetic patients and improved survival when cancer
has been diagnosed, respectively. Although the regulation of glucose and insulin levels may also
contribute to the reported effects, compelling evidence demonstrated that the anti-tumoural activity of
metformin involved the impairment of OXPHOS via direct inhibition of mitochondrial Complex I32.
Remarkably, metformin selectively induced apoptosis in pancreatic CSCs as a result of their inability
to switch to glycolysis and subsequent energy crisis16. Still, a minor subset of CSCs displaying low
mitochondrial mass and a predominantly glycolytic metabolism were inherently resistant to
metformin. The presence of these cells in pancreatic tumours appeared to account for the observed
univocal relapse of tumours in mice treated with metformin and, among other factors including
insufficient dosing in many patients, may also explain the negative outcome of the first clinical trials
testing the effects of metformin in pancreatic cancer patients33.
These data suggest that metformin and other drugs impairing mitochondrial ATP production
need to be combined with agents targeting the mechanism of resistance allowing some CSCs to
overcome OXPHOS inhibition, e.g. modulation of the PGC1-/MYC ratio. Intriguingly, metformin
resistance in pancreatic CSCs was prevented/reversed by knockdown of MYC expression using RNAi
or indirectly via BRD4 inhibition, suggesting the therapeutic potential of MYC inhibitors in
combination with mitochondrial targeting. Alternatively, agents interfering with mitochondrial
function at various levels may be more effective in targeting all CSCs. In fact, while drug resistance
was observed with other OXPHOS inhibitors, such as rotenone or resveratrol, resistance was not seen
for menadione, which acts via dual mechanism – inhibition of Complex I and induction of
mitochondrial ROS. On the other hand, the efficacy of metformin may also be limited by its
requirement of organic cation transporters (OCTs) for cellular uptake, which restricts its effects in
healthy tissues, but also limits its potential use to tumour cells expressing OCTs. Phenformin, another
biguadine formerly used in diabetes, could overcome these limitations as it is more hydrophobic and
can be delivered to mitochondria more efficiently than metformin. Phenformin also promotes cancer
cell death by inhibiting Complex I and has offered promising preclinical results in certain cancers
such as non-small-cell lung carcinoma34.
Thus, an important factor concerning the design of novel pharmacological strategies targeting
mitochondria is to ensure the efficient delivery of the drug to the mitochondria of cancer cells. CSCs
relying on OXPHOS will present an elevated Mitochondrial Membrane Potential (Δψm), which can
be exploited for selectively increasing drug delivery to the mitochondria of these cells. Delocalised
lipophilic cations (DLCs) such as triphenylphosphonium (TPP) accumulate in the mitochondrial
matrix and can be conjugated to small compounds for selective drug delivery to mitochondria35. The
mitochondrial accumulation of Mito-Chromanol, a vitamin E analogue conjugated to TPP induced
cell death by inhibiting OXPHOS in breast cancer cells without affecting non-tumour cells36.
Conjugation with TPP has also been utilised to selectively deliver the chaperone inhibitor Gamitrinib
to active mitochondria and disrupt energy production in tumour cells by impairing protein folding in
mitochondria37. However, mitochondria-penetrating peptides might be preferred for the treatment of
certain tumours, as they can deliver cargo molecules irrespective of Δψm. Their conjugation to
chemotherapeutic agents such as doxorubicin directs their activity towards mtDNA, promoting drug
selectivity for cancer cells with reduced mtDNA integrity while their stable mitochondrial localisation
prevents the acquisition of resistance by drug efflux38.
IV. CONCLUSIONS
In various cancers, CSCs have now been shown to bear a distinct metabolic phenotype and are highly
dependent on OXPHOS with very limited metabolic plasticity. The reason for this may be
multifactorial – beside gaining a greater independency from the sparse nutritional support in the
tumour microenvironment, increasing evidence now also suggests that the tight control of
mitochondrial ROS production in CSCs is a prerequisite for mainlining their stemness and high
fidelity. This apparent metabolic vulnerability provides a vast set of new therapeutic opportunities to
more efficiently eliminate these highly tumourigenic cells, even though resistance may arise in some
instances. The latter could be related either to the acquisition of metabolic plasticity in a subset of
CSCs or to the pre-existence of a small subset of CSCs with advanced metabolic features. As such
CSCs display reduced stemness properties, they do not significantly participate in cancer progression
in treatment naïve tumours, but metabolic targeting provides these cells with an advantage and they
eventually take and drive relapse of the disease. Importantly, these resistant CSCs can be tracked by
using tissue biopsies or by analysing circulating CSCs (liquid biopsies; Fig. 1) in order to monitor
treatment response as well as arising resistance. A more effective strategy could be to combine
distinct targeting strategies or to use mitochondria-targeting agents with dual mechanism of action,
which may help to improve the still miserable outcome of many cancer patients.
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Figure 1
Cancer stem cells in pancreatic cancer progression and metastasis. Intraclonal heterogeneity is formed by CSCs and
their differentiated progenies (Left). CSCs are capable of undergoing unlimited cell division while retaining their stem cell
identity (self-renewal) and giving rise to non-CSCs with limited proliferative capacity (differentiation). CSCs evolve as the
tumour progresses via (epi-)genetic alterations, but also in response to interactions with their niche, leading to diverse CSC
subclones with distinct functionality4. Both CSCs and non-CSCs can acquire mobility, which may be driven by EMT
processes, but only arising metastatic CSCs can initiate secondary lesions4 and are tractable as circulating CSCs in the blood.
These cells must survive the hostile environment of the blood stream, evade immune surveillance and extravasate at a distant
location to form metastatic lesions. (Centre) Importantly, circulating CSCs may also evolve, after a period of dormancy,
from disseminated non-CSCs through still poorly understood processes. CSCs can also (re)colonize their tumours of origin,
or other sites, in a process called “tumour reseeding”. Thus, CSCs circulating in the blood represent metastatic CSCs from
various sources (Right).
Figure 2
Figure 2. Targeting CSCs through mitochondrial disruption. OXPHOS-dependent CSCs could be eliminated via
different strategies aiming to impair their mitochondrial energy metabolism. (1) Direct inhibition of OXPHOS using the antidiabetic agents metformin and phenformin, which inhibit the ETC Complex I. Subsequent energy crisis results in the
specific induction of CSC death. (2) Conjugation of pharmacologic agents to mitochondrial carriers such as TPP or
mitochondria penetrating peptides to selectively deliver and accumulate them in mitochondria. (3) Chemotherapeutic agents
conjugated to mitochondrial carriers to selectively disrupt mtDNA integrity and indirectly block OXPHOS by impairing
ETC proteins coded in mtDNA genes. (4) Blockade of mitochondrial protein biosynthesis via inhibition of mitochondrial
ribosomes using Tigecycline and other FDA-approved antibiotics. These compounds impair OXPHOS and demonstrated
toxicity against CSCs. (5) Similarly, the functionality of ETC components can also be targeted by the mitochondrial delivery
of the chaperone inhibitor Gamitrinib. (6) Cell signalling by OXPHOS-generated mitochondrial ROS is crucial for cancer
cell proliferation and can be targeted by the mitochondrial accumulation of anti-oxidants such as Mito-Chromanol.
Conversely, CSCs can be eliminated by inducing toxic ROS levels in mitochondria using the ROS generator Menadione.
(7) Finally, OXPHOS can also be impaired at the level of mitochondrial carbon metabolism, either by altering the enzymes
involved in the TCA cycle or fatty acid oxidation (FAO) or by interfering with the supply of mitochondrial fuels.