Abstract
It has long been recognised that cancer cells critically depend on reprogrammed patterns of metabolism that can enable robust and abnormally high levels of cell proliferation. As mitochondria form hubs of cellular metabolic activity, it is reasonable to propose that pathways within these organelles can form targets that can be manipulated to compromise the ability of cancer cells to cause disease. However, mitochondria are highly multi-functional, and the full range of mechanistic inter-connections are still being unravelled to enable the full potential of targeting mitochondria in cancer therapeutics. Here, we aim to highlight the potential of modulating mitochondrial dynamics to target key metabolic or apoptotic pathways in cancer cells. Distinct roles have been demonstrated for mitochondrial fission and fusion in different cancer contexts. Targeting of factors mediating mitochondrial dynamics may be directly related to impairment of oxidative phosphorylation, which is essential to sustain cancer cell growth and can also alter sensitivity to chemotherapeutic compounds. This area is still lacking a unified model, although further investigation will more comprehensively map the underlying molecular mechanisms to enable better rational therapeutic strategies based on these pathways.
Introduction
Mitochondria can be considered the central hubs of cellular metabolism. These organelles are primarily appreciated for their ability to generate ATP with maximal bioenergetic efficiency via oxidative phosphorylation (OXPHOS). Interrelated to this primary role, mitochondria coordinate a range of additional pathways that ultimately control the growth and survival of the host cell (Spinelli & Haigis 2018). There is flux through the electron transport chain (ETC) with oxygen as the final electron acceptor, and through this process, reactive oxygen species (ROS) can be created, partly as a pathogenic effect as integrity of the system deteriorates, or alternatively as a normal physiologic signalling mechanism (Sena & Chandel 2012). The ETC transfers electrons from NADH and FADH2 formed from the tricarboxylic acid (TCA) cycle, which is compartmentalised in the mitochondrial matrix. Due to this, the ETC is inextricably tied to the balance of cellular redox status (e.g. the ratio of NAD+ to NADH) and to the flux of carbon atoms through TCA cycle intermediates. The flow of carbon and nitrogen atoms derived from extracellular nutrients ultimately channels through the mitochondria, which houses key portions of the urea cycle, nucleotide and lipid biosynthetic cascades. Last, another key role of mitochondria is to control the apoptosome and initiation of the intrinsic apoptosis pathway (Danial & Korsmeyer 2004). For all these roles, it has been exciting to speculate on the potential of targeting mitochondrial biology to reveal liabilities within the altered metabolism of cancer cells that can be exploited for therapeutics (Vyas et al. 2016). One particularly emerging area revolves around mitochondrial dynamics and the constant membrane remodelling displayed by these organelles. Since structure generally reflects function, it has been of great interest to understand how these remodelling events control bioenergetic and apoptotic mechanisms and further how these pathways contribute to the resilience of cancer cells.
Regulation and roles of mitochondrial dynamics
Perhaps reflecting their evolutionary origin from ancient proteobacteria (Roger et al. 2017), mitochondria need to be appreciated for their ability to reprogram their size, shape and localisation under different specific contexts. Mitochondrial dynamics primarily involves constant cycling between two opposing, but essential, pathways. New mitochondria are formed by the growth and expansion of existing organelles followed by fission. Mitochondrial fission is primarily promoted by the GTP-binding protein dynamin-related protein 1 (which in human is encoded by the gene DNM1L) (Fig. 1A) (Bleazard et al. 1999, Cribbs & Strack 2007, Taguchi et al. 2007). For simplicity, we will herein refer to DNM1L by its more common alias dynamin-related protein 1 (DRP1). DRP1 localises onto the mitochondrial outer membrane via the concerted action of a family of adaptor proteins including mitochondrial fission factor and mitochondrial elongation factor 1 and 2 (MIEF1/MIEF2) (Kraus et al. 2021). Once localised on mitochondria, DRP1 forms ring-like oligomeric constrictions that drive organelle pinching and scission, a process that was initially characterised in yeast experimental systems (Lackner et al. 2009, Kamerkar et al. 2018). To note, the entire process of fission may involve the concerted action of yet additional dynamin members (Lee et al. 2016) and coordinately interact with endoplasmic reticulum (ER) membranes. Interestingly, in mammalian cells, DRP1 activity can be coordinated by multiple regulatory mechanisms including phosphorylation at serine 637 by cAMP-dependent protein kinase (aka PKA), which inhibits the fission activity of DRP1 (Cribbs & Strack 2007, Cereghetti et al. 2008). In contrast, phosphorylation of serine 616 by cyclin-dependent kinase1 or mitogen-activated protein kinase (MAPK) family members (aka extracellular signal-regulated kinase (ERK)) can activate DRP1 activity and thus forms a pro-fission regulatory site (Taguchi et al. 2007, Kashatus et al. 2015, Serasinghe et al. 2015). Furthermore, DRP1 can be modified by SUMOylation via the MAPL/MUL1 E3 ligase, which regulates DRP1 function at mitochondria–ER contact sites (Braschi et al. 2009, Prudent et al. 2015). As such, DRP1 serves as a nexus point to integrate a number of upstream post-translational signals.
Roles of mitochondrial dynamics regulating quality control of the mitochondrial network. (A) Mitochondrial fission results in the scission of a mitochondrion into daughter mitochondria which is driven by the GTPase DRP1 in a phosphorylation- and ER-dependent manner. (i) Fission allows organelle division to sustain mitosis. (ii) Fission facilitates the isolation of damaged mitochondria to be tagged by autophagy cargo receptors (e.g. LC3) for degradation by autophagosomes/autolysosomes via mitophagy. (iii) DRP1 becomes SUMOylated in a BAX/BAK-dependent manner to trigger cytochrome c release from the mitochondrial outer membrane, resulting in apoptosis initiation (for simplicity, roles of OPA1 in regulating cytochrome c from cristae are not shown). (B) Mitochondrial fusion results in the merging of mitochondria together facilitated by the GTPases MFN1/2 which function on the outer membrane and OPA1 which is located on mitochondrial inner membranes. (i) OPA1 plays key roles in organising the proper formation of cristae junctions, thus promoting overall assembly of the ETC for oxidative phosphorylation function. (ii) MFN2 can be phosphorylated by PINK1 to recruit Parkin to signal for mitophagy. (iii) In normal cells, OPA1 oligomers at cristae junctions serve to restrict cytochrome c within cristae folds. Upon apoptotic stimuli, OPA1 oligomers are proposed to disassemble to release cytochrome c into the intermembrane space before outer membrane permeablisation via DRP1- and BAX/BAK-dependent mechanisms.
Citation: Endocrine-Related Cancer 30, 1; 10.1530/ERC-22-0229
Roles of mitochondrial dynamics regulating quality control of the mitochondrial network. (A) Mitochondrial fission results in the scission of a mitochondrion into daughter mitochondria which is driven by the GTPase DRP1 in a phosphorylation- and ER-dependent manner. (i) Fission allows organelle division to sustain mitosis. (ii) Fission facilitates the isolation of damaged mitochondria to be tagged by autophagy cargo receptors (e.g. LC3) for degradation by autophagosomes/autolysosomes via mitophagy. (iii) DRP1 becomes SUMOylated in a BAX/BAK-dependent manner to trigger cytochrome c release from the mitochondrial outer membrane, resulting in apoptosis initiation (for simplicity, roles of OPA1 in regulating cytochrome c from cristae are not shown). (B) Mitochondrial fusion results in the merging of mitochondria together facilitated by the GTPases MFN1/2 which function on the outer membrane and OPA1 which is located on mitochondrial inner membranes. (i) OPA1 plays key roles in organising the proper formation of cristae junctions, thus promoting overall assembly of the ETC for oxidative phosphorylation function. (ii) MFN2 can be phosphorylated by PINK1 to recruit Parkin to signal for mitophagy. (iii) In normal cells, OPA1 oligomers at cristae junctions serve to restrict cytochrome c within cristae folds. Upon apoptotic stimuli, OPA1 oligomers are proposed to disassemble to release cytochrome c into the intermembrane space before outer membrane permeablisation via DRP1- and BAX/BAK-dependent mechanisms.
Citation: Endocrine-Related Cancer 30, 1; 10.1530/ERC-22-0229
Roles of mitochondrial dynamics regulating quality control of the mitochondrial network. (A) Mitochondrial fission results in the scission of a mitochondrion into daughter mitochondria which is driven by the GTPase DRP1 in a phosphorylation- and ER-dependent manner. (i) Fission allows organelle division to sustain mitosis. (ii) Fission facilitates the isolation of damaged mitochondria to be tagged by autophagy cargo receptors (e.g. LC3) for degradation by autophagosomes/autolysosomes via mitophagy. (iii) DRP1 becomes SUMOylated in a BAX/BAK-dependent manner to trigger cytochrome c release from the mitochondrial outer membrane, resulting in apoptosis initiation (for simplicity, roles of OPA1 in regulating cytochrome c from cristae are not shown). (B) Mitochondrial fusion results in the merging of mitochondria together facilitated by the GTPases MFN1/2 which function on the outer membrane and OPA1 which is located on mitochondrial inner membranes. (i) OPA1 plays key roles in organising the proper formation of cristae junctions, thus promoting overall assembly of the ETC for oxidative phosphorylation function. (ii) MFN2 can be phosphorylated by PINK1 to recruit Parkin to signal for mitophagy. (iii) In normal cells, OPA1 oligomers at cristae junctions serve to restrict cytochrome c within cristae folds. Upon apoptotic stimuli, OPA1 oligomers are proposed to disassemble to release cytochrome c into the intermembrane space before outer membrane permeablisation via DRP1- and BAX/BAK-dependent mechanisms.
Citation: Endocrine-Related Cancer 30, 1; 10.1530/ERC-22-0229
In terms of cellular function, DRP1-mediated fission to produce smaller mitochondria has been understood to be critical for organelle propagation during mitosis (Taguchi et al. 2007). DRP1 SUMOylation is also well established to enable proper cytochrome c release in a BAK/BAX-dependent manner to activate apoptosis (Frank et al. 2001, Wasiak et al. 2007, Prudent et al. 2015). For housekeeping, DRP1-mediated fission is critical for destruction of stressed mitochondria via mitophagy (Twig et al. 2008), potentially by directing specific capture of damaged mitochondrial regions by autophagy cargo receptors such as p62/sequestosome1 and LC3 (Burman et al. 2017). The control of mitochondrial fate following fission appears to involve DRP1 cross-talk with the actin cytoskeleton and lysosomes, which can coordinate towards beneficial vs destructive fission events, respectively (Kleele et al. 2021). So far, Syntaxin 17 is the best understood SNARE (SNAP-REceptor protein family member) that promotes DRP1-mediated fission at mitochondria–ER contact sites (Arasaki et al. 2015).
Counteracting fission, mitochondria undergo fusion via the stepwise action of three GTP-binding regulatory factors (Fig. 1B). Fusion is initially driven by mitofusin 1 (MFN1), a transmembrane protein which resides in the mitochondrial outer membrane (Schrepfer & Scorrano 2016). MFN1 functions in concert with its related family member MFN2 (which is also implicated in mitochondria–ER tethering) (Chen et al. 2003, 2005, Naon et al. 2016). MFN1 and MFN2 may play partially redundant roles, but these proteins are better understood to form active heterocomplexes in a nucleotide-dependent manner to tether two opposing mitochondria as a first initiating step towards organelle fusion (Engelhart & Hoppins 2019). Subsequently, the formation of higher order MFN1/2 complexes has been proposed to help directly promote outer membrane fusion. Interestingly, MFN2 has also been shown to be phosphorylated by phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) to then directly bind and recruit the ubiquitin E3 ligase Parkin (PRKN) to damaged mitochondria (Chen & Dorn 2013).
Fusion of the mitochondrial inner membrane has been long understood to depend on the GTPase, optic atrophy 1 (OPA1) (Cipolat et al. 2004). Due to identification of OPA1 mutations linked to autosomal dominant optic atrophy (Ban et al. 2010), the underlying regulation of this protein has been characterised at genetic, biochemical and structural levels. Multiple splice isoforms undergo proteolytic processing to yield final long and short mature forms of OPA1, which are proposed to form a complex on cristae junctions close to the mitochondrial outer membrane (Ishihara et al. 2006). OPA1 complexes associated with the mitochondrial inner membrane have been proposed to directly interact with bilayer lipid in a manner sufficient to drive GTP-dependent membrane tubulation (Yan et al. 2020, Yu et al. 2020, Zhang et al. 2020). By controlling membrane architecture, OPA1 is thus understood to fine-tune cristae organisation to thereby regulate ETC complexes and overall OXPHOS capacity (Cogliati et al. 2013, Patten et al. 2014). These OPA1-specific observations are interrelated with roles proposed for MFN1/2-dependent mitochondrial fusion to sustain OXPHOS function by preserving mtDNA content or quality (Nakada et al. 2001, Ono et al. 2001, Chen et al. 2007, 2010). As such, multiple mechanisms link mitochondrial fusion to the overall maintenance of OXPHOS. Interestingly, in addition to bioenergetic roles, complexes formed following OPA1 proteolytic processing are also proposed to critically suppress unwanted release of cytochrome c from cristae folds into the mitochondrial intermembrane space (Cipolat et al. 2006, Frezza et al. 2006). In this model, upon apoptosis induction, the OPA1-mediated brake is relaxed, allowing cytochrome c to leave the cristae folds as a priming step before full DRP1- and BAX/BAK-mediated mitochondrial outer membrane permeabilisation. Given these multiple ties with cell metabolism and cell death, it is reasonable to surmise that mitochondrial dynamics would play roles controlling cancer cell biology.
Mitochondrial fission as a driver of cell transformation
Strategies for targeting mitochondria are based on the notion that cancer cells are critically reliant on altered cell metabolism. This concept is in large part based on seminal arguments made by Otto Warburg, reflecting how pure samples of cancer cells consumed high amounts of glucose and channelled this largely towards glycolysis releasing high amounts of lactate into the culture medium (Warburg 1956). The choice of glycolysis rather than the bioenergetically more efficient TCA cycle was unexpected, given the sufficient oxygen available for the cancer cells, thus characterising the Warburg effect and its key feature, aerobic glycolysis. Indeed, it was concluded that the reprogrammed metabolic patterns in cancer cells may represent an injured state of respiration and OXPHOS capacity, leading to overall lower bioenergetic efficiency. Insights gathered over the last two decades now lead to an appreciation that a shift towards the more embryonic like glycolytic state can offer metabolic benefits for tumour cells once they enter high levels of proliferation (DeBerardinis & Chandel 2020). Glucose consumption flux can function at higher rates than OXPHOS, which may provide a means for ATP production to more rapidly respond to different circumstances or function at dysregulated elevated levels (Epstein et al. 2017). High consumption of glucose can also overall lead to increased levels of carbon that eventually refuel metabolic intermediates to critically drive the biosynthesis of nucleotides and lipids required for cell growth (Vander Heiden et al. 2009). Last, it is clear that another key benefit of elevated glucose flux is enhanced activity of the pentose phosphate pathway, leading to increased cellular reducing capacity and generation of NADPH to further support biosynthetic pathways (Patra & Hay 2014).
As roles for mitochondrial dynamics in cancer were beginning to be explored, initial results undoubtedly suggested that increased mitochondrial fission was playing a causal role in promoting cancer cell transformation (Fig. 2). For example, it was noted in breast and lung cancer cell lines that mitochondrial sizes can be heterogeneous and cell-type specific, but cancer cells tended to contain smaller, fragmented mitochondria (Rehman et al. 2012, Zhao et al. 2013). Moreover, increased levels of fission factor DRP1 could be detected in these cancer cell models and also in lung tumour or breast metastasis patient samples. Further analyses seemed to suggest that increased fission (or decreased fusion) is necessary to promote breast cancer cell migratory properties and lung cancer tumour formation in xenografts. Other independent studies could further show that oncogenic RAS and BRAF may be promoting mitochondrial fission via a two-pronged mechanism that involved elevation of DRP1 levels and MAPK/ERK-mediated phosphorylation of DRP1 at its pro-fission S616 site (Kashatus et al. 2015, Serasinghe et al. 2015). This direct mechanism of DRP1 activation could be demonstrated using experimental systems involving normal non-oncogenic fibroblasts that were transformed via ectopic expression of oncogenic RAS-V12G. Additional supporting results included evidence for elevated phospho-DRP1 levels in melanoma and pancreatic adenocarcinoma patient samples. Mechanistically, silencing of DRP1 also reduced xenograft tumour sizes using pancreatic cancer models with activated MAPK/ERK signalling (Kashatus et al. 2015). Interestingly, elevated levels of DRP1-mediated mitochondrial fission were associated with lower mitochondrial membrane potential, decreased OXPHOS capacity, but increased mitochondrial ROS (Serasinghe et al. 2015). In agreement, vemurafenib, the gold standard targeted therapy for activated BRAF, was shown to trigger death in melanoma cell models, block MAPK/ERK-mediated DRP1 phosphorylation and shift towards mitochondrial fusion with a reactivation of OXPHOS (Ferraz et al. 2020). These findings thus outline a model in which cell transformation critically relies on increased mitochondrial fission and overall lower bioenergetic output.
Model for mitochondrial fission as a critical driver of cell transformation. Activated oncogenes can increase mitochondrial fission via modulation of dynamin-related protein 1 (DRP1) expression and phosphorylation status. Increased mitochondrial fission shifts the bioenergetic state from oxidative phosphorylation (OXPHOS) towards glycolysis, while reducing mitochondrial membrane potential and increasing ROS generation, which contributes to cell transformation and tumour growth potential. Strategies to enhance mitochondrial fusion have been explored as a therapeutic via activation of mitofusin 2 (MFN2) or inhibition of DRP1 which can reduce tumour growth and further inhibit mitochondrial function or homeostasis.
Citation: Endocrine-Related Cancer 30, 1; 10.1530/ERC-22-0229
Model for mitochondrial fission as a critical driver of cell transformation. Activated oncogenes can increase mitochondrial fission via modulation of dynamin-related protein 1 (DRP1) expression and phosphorylation status. Increased mitochondrial fission shifts the bioenergetic state from oxidative phosphorylation (OXPHOS) towards glycolysis, while reducing mitochondrial membrane potential and increasing ROS generation, which contributes to cell transformation and tumour growth potential. Strategies to enhance mitochondrial fusion have been explored as a therapeutic via activation of mitofusin 2 (MFN2) or inhibition of DRP1 which can reduce tumour growth and further inhibit mitochondrial function or homeostasis.
Citation: Endocrine-Related Cancer 30, 1; 10.1530/ERC-22-0229
Model for mitochondrial fission as a critical driver of cell transformation. Activated oncogenes can increase mitochondrial fission via modulation of dynamin-related protein 1 (DRP1) expression and phosphorylation status. Increased mitochondrial fission shifts the bioenergetic state from oxidative phosphorylation (OXPHOS) towards glycolysis, while reducing mitochondrial membrane potential and increasing ROS generation, which contributes to cell transformation and tumour growth potential. Strategies to enhance mitochondrial fusion have been explored as a therapeutic via activation of mitofusin 2 (MFN2) or inhibition of DRP1 which can reduce tumour growth and further inhibit mitochondrial function or homeostasis.
Citation: Endocrine-Related Cancer 30, 1; 10.1530/ERC-22-0229
Additional insights into the key roles of mitochondrial fission in controlling metabolic states were revealed from studies in genetic models for pancreatic cancer (Nagdas et al. 2019). In this model, Cre-lox-mediated inactivation of DRP1 was shown to improve survival outcomes of tumour-bearing mice and decrease the levels of severe subtypes of pancreatic intraepithelial neoplasia. Importantly, DRP1-targeted cell models, when propagated in culture, showed a clear loss of glucose consumption. These observations are in line with the association of mitochondrial fragmentation and a shift from OXPHOS towards glycolysis. Surprisingly, parallel analysis of mouse tumour-derived cells indeed indicated patterns of metabolic reprogramming. However, analyses of tumours originating from the Cre-lox DRP1-targeted cells suggested additional in vivo selection pressures which caused reactivation of glycolytic activity. Therefore, these data support a model in which DRP1-mediated mitochondrial fission is key for promoting tumour growth through modulation of metabolic programmes in the tumour cell to favour glycolysis at the expense of OXPHOS.
The concept of targeting mitochondrial fission as a therapeutic has been explored (Fig. 2), for example initially in lung cancer xenografts (Rehman et al. 2012). Since the prototypical DRP1 inhibitor Mdivi 1 has off-target effects on the ETC (Bordt et al. 2017), more specific small molecules such as the ellipticine analogue Drpitor1 have since been identified with modelled interactions at the DRP1 GTP-binding pocket (Wu et al. 2020). It is promising that Drpitor1 or its derivative could be shown to effectively inhibit growth in a range of cancer cell lines and reduce tumour growth in a lung cancer xenograft model. Considering the reciprocal nature of mitochondrial dynamics, increased mitochondrial fusion has also been proposed to be functionally equivalent as a block of fission. This concept included pioneering work showing tumour inhibitor effects from MFN2 overexpression in lung cancer models (Rehman et al. 2012). A more recent advancement came from a drug screen aimed to identify FDA-approved compounds that could activate MFN2 gene expression (Miret-Casals et al. 2018). The top candidate in this screen was leflunomide, a drug that was first introduced in the clinic over 20 years ago with anti-inflammatory properties for treatment of arthritis. Mechanistically, leflunomide is understood to inhibit dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo pyrimidine biosynthesis pathway. In this way, a novel link was suggested between inhibition of pyrimidine biosynthesis and upregulation of MFN2, which was sufficient to drive abnormally high levels of mitochondrial fusion. The therapeutic potential of manipulating mitochondrial fusion was further explored using genetic models of KRAS-dependent pancreatic cancer. In these experimental systems, CRISPR-Cas9-mediated gene targeting of DRP1 indeed impaired xenograft tumour formation (Yu et al. 2019), consistent with a key role for DRP1 in cancer cell growth. It was further noted that DRP1-deficient cells, which had higher levels of mitochondrial fusion, also showed impaired OXPHOS, which contrasted with other findings reporting elevated OXPHOS correlating with increased fusion (Serasinghe et al. 2015, Nagdas et al. 2019). In addition, other strategies to drive mitochondrial fusion via MFN2 overexpression or leflunomide treatment all suppressed tumour growth and lowered OXPHOS.
Taken together, it is clear that loss of DRP1 and mitochondrial fission can impair cancer cell growth, as seen in a number of different tumour types. Alternatively, forcing mitochondrial dynamics the other direction towards increased fusion can also impair cancer cell growth. There is a complex interrelationship between mitochondrial dynamics and the metabolic reprogramming that takes place in a transformed cell. Elevated levels of fission may be accompanied by increased reliance on glycolysis and a suppression of OXPHOS. However, manipulations to ectopically promote fusion also can impair OXPHOS, which is likely sensitive to a variety of perturbations.
Mitochondrial OXPHOS as a critical component of cell transformation
Based on roles in normal physiologic contexts, a coordinated balance of mitochondrial dynamics is essential to maintain critical metabolic and bioenergetic functions. Genetic deletion of either mitochondrial fission or fusion factors leads to embryonic lethality in mouse models (Chen et al. 2003, Ishihara et al. 2009). Since both sides of mitochondrial dynamics are required to sustain cell homeostasis in the non-transformed context, it remained perplexing that cancer cells would be preferentially more addicted to mitochondrial fission. Indeed, a major overriding function of mitochondria fusion is to maintain OXPHOS and sustain metabolic fitness (Fig. 3A). As such, before directly considering roles of mitochondrial fusion in cancer, it is worthwhile to discuss the other accumulating evidence that demonstrates more generally how mitochondrial metabolism is critical for driving cancer cell growth.
Critical roles of the electron transport chain in maintaining cancer cell biosynthetic pathways. (A) Mitochondrial dynamics can maintain overall mitochondrial fitness and function of the ETC to thereby maintain the generation of ROS, NAD+ or ubiquinone. (B) ETC function is critical to maintain biosynthesis of aspartate, asparagine, pyrimidines and purines. NAD+ or ubiquinone electron acceptors are required at multiple reactions and can halt biosynthesis when limiting (highlighted in green). ETC supports the generation of aspartate (highlighted in yellow) which is required to maintain biosynthesis of asparagine, pyrimidines and purines.
Citation: Endocrine-Related Cancer 30, 1; 10.1530/ERC-22-0229
Critical roles of the electron transport chain in maintaining cancer cell biosynthetic pathways. (A) Mitochondrial dynamics can maintain overall mitochondrial fitness and function of the ETC to thereby maintain the generation of ROS, NAD+ or ubiquinone. (B) ETC function is critical to maintain biosynthesis of aspartate, asparagine, pyrimidines and purines. NAD+ or ubiquinone electron acceptors are required at multiple reactions and can halt biosynthesis when limiting (highlighted in green). ETC supports the generation of aspartate (highlighted in yellow) which is required to maintain biosynthesis of asparagine, pyrimidines and purines.
Citation: Endocrine-Related Cancer 30, 1; 10.1530/ERC-22-0229
Critical roles of the electron transport chain in maintaining cancer cell biosynthetic pathways. (A) Mitochondrial dynamics can maintain overall mitochondrial fitness and function of the ETC to thereby maintain the generation of ROS, NAD+ or ubiquinone. (B) ETC function is critical to maintain biosynthesis of aspartate, asparagine, pyrimidines and purines. NAD+ or ubiquinone electron acceptors are required at multiple reactions and can halt biosynthesis when limiting (highlighted in green). ETC supports the generation of aspartate (highlighted in yellow) which is required to maintain biosynthesis of asparagine, pyrimidines and purines.
Citation: Endocrine-Related Cancer 30, 1; 10.1530/ERC-22-0229
Indeed, there is a large body of evidence to support key roles for mitochondrial function in different cancer cell models. For example, it was earlier noted that breast cancer or glioblastoma cell lines that could be depleted of mtDNA (Rho zero-status) had impaired anchorage-independent growth and tumorigenic phenotypes and increased sensitivity to alkylating or platinum-based chemotherapy agents (Cavalli et al. 1997). This dependence was confirmed in independent studies using Rho zero osteosarcoma, melanoma and breast cancer cell models, which more firmly demonstrates the critical role of mtDNA-dependent mitochondrial function (such as OXPHOS) for tumorigenic growth (Weinberg et al. 2010, Tan et al. 2015). Further studies using mitochondria with an inactive cytochrome b suggested that cancer cells, for example an osteosarcoma cell model, were also reliant on ROS generated from the ETC complex 3 (Fig. 3A) (Weinberg et al. 2010). Here, cell hybrids were generated that carried mitochondria with a mutant cytochrome c allele capable of partial ETC function to allow ROS production (but not OXPHOS), and this was sufficient to rescue anchorage-independent growth. Therefore, outputs besides ATP generation can be important downstream of the ETC to support cancer cell phenotypes. Consistent with important functional roles, the overall reliance on mitochondria for cancer progression could be demonstrated using wider genetic strategies. For example, targeting of mitochondrial transcription factor A (TFAM), which regulates the expression of mitochondrial genes and is further required to maintain mtDNA integrity (Larsson et al. 1998), suppresses tumour formation in mutant KRAS-dependent mouse models (Weinberg et al. 2010). Similarly, targeting of peroxisome proliferator-activated receptor-gamma, coactivator 1 alpha (PGC-1 alpha, encoded by PPARGC1A), which drives mitochondrial biogenesis, reduces metastatic properties in mouse models of breast cancer (LeBleu et al. 2014).
It was interesting that there were also pharmacological strategies for targeting mitochondria. Treatment of colon cancer cells with the biguanides metformin or phenformin could inhibit complex 1 of the ETC and furthermore slow down xenograft tumour growth in mice (Wheaton et al. 2014). Further mechanistic insights were provided from a chemical biology study aimed to find more specific inhibitors of OXPHOS, leading to identification of IACS-010759 (Molina et al. 2018). This compound was characterised to inhibit ETC function by interacting with the ND1 subunit of complex 1, and impressively, IACS-010759 was able to inhibit OXPHOS and cell growth with an IC50 of 1.4 nM. Moreover, IACS-010759 was shown to effectively inhibit proliferation in a large range of breast, pancreatic and ovarian cancer cell lines, with relatively minimal effects on non-transformed cells. For in vivo efficacy, IACS-010759 was able to inhibit tumorigenesis of neuroblastoma and glioblastoma xenograft models that were glycolysis impaired and thus strongly reliant on OXPHOS. Therefore, inhibition of the ETC complex 1 using IACS-010759 has strong potential as a pre-clinical cancer therapeutic, since this molecule could be further confirmed to have good pharmacokinetic properties in several mammalian models.
Mechanistic insights came from metabolomic profiling of acute myelogenous leukaemia (AML) cell models treated with IACS-010759 (Molina et al. 2018). Many IACS-010759-induced changes were consistent with OXPHOS inhibition, including accumulation of complex 1 substrate, NADH, increased levels of NMPs, but decreased levels of NTPs. In addition, it was clear that blocking OXPHOS with IACS-010759 led to compensatory higher glucose consumption, which bypassed entry into the TCA cycle to be released as lactate. TCA intermediates, as a result, received more replenishment of carbon derived from glutamine. As such, a blockade at complex 1 led to the expected lower bioenergetic charge and altered metabolic fuel choices. The further clue was that, of all amino acids, IACS-010759 led primarily to depletion of aspartate (Fig. 3B). This effect was intriguing considering other results highlighting aspartate as a critical metabolic intermediate, which is formed from the TCA cycle intermediate, oxaloacetate.
A CRISPR screen for genes that interacted with phenformin treatment led to the finding that ETC inhibition critically impaired aspartate production to limit cell growth (Birsoy et al. 2015). It was also revealed that a key role of the ETC (via the electron transfer step at complex 1) was to recycle NADH back to regenerate a pool of NAD+ (Sullivan et al. 2015). As NAD+ serves as a key electron acceptor in multiple biosynthetic reactions, decreased NAD+/NADH ratios effectively suppress a range of reactions needed for cell growth. For example, conversion of alpha-ketoglutarate to oxaloacetate in the oxidative arm of the TCA cycle requires NAD+ at several steps (Fig. 3B, left panel). Reductive generation of oxaloacetate involves conversion of glutamate to alpha-ketoglutarate via glutamate dehydrogenase, and this also requires an electron acceptor like NAD+. Therefore, both biosynthesis routes for aspartate critically require NAD+. Nucleotide biosynthesis is notably highly NAD+ dependent. De novo biosynthesis of pyrimidines starts with the addition of aspartate to carbamoyl-phosphate (and later requires further electron acceptors at the reaction catalysed by DHODH) (Fig. 3B, middle panel). De novo biosynthesis of purines requires aspartate to generate the intermediate (N-succinylo-5-aminoimidazole-4-carboxyamide ribonucleotide and incorporates a second molecule of aspartate to form adenosine monophosphate (AMP) (Fig. 3B, right panel). Transformation of inosine monophosphate (IMP) to form guanosine monophosphate (GMP) further requires NAD+ at the reaction catalysed by inosine monophosphate dehydrogenase. Experimentally, it could be shown in osteosarcoma, glioblastoma and lung carcinoma cell lines that supplementation of alpha-ketobutyrate can forcibly drive back NAD+ recycling and rescue growth of cells with blocked ETC function (Sullivan et al. 2015). As such, the ETC supports nucleotide synthesis by maintaining redox balance and the regeneration of electron acceptors such as NAD+.
Consistent with the primacy of redox balance, further critical interrelated pathways could be identified. When a subunit of ubiquinol–cytochrome c reductase was inactivated in a KRAS-dependent lung cancer mouse model, activity of complex 3 was blocked, leading to a suppression of tumorigenesis in xenograft experiments (Martínez-Reyes et al. 2020). Similar effects could be confirmed in an acute lymphoblastic leukaemia model. Dissection of this block revealed that a key role of complex 3 was also to regenerate the electron acceptor ubiquinone (aka coenzyme Q) from its reduced form, ubiquinol. Interestingly, the expression of a Ciona (invertebrate) alternate oxidase enzyme to regenerate ubiquinone could rescue multiple ETC functions such as NAD+ recycling, aspartate synthesis and OXPHOS and thereby rescued tumour growth. Ubiquinone was thus proposed as a pinnacle electron acceptor which enables NAD+ generation at complex 1 and succinate dehydrogenase activity at complex 2 and serves as the electron acceptor for the enzyme DHODH for pyrimidine biosynthesis.
Overall, the importance of ETC-dependent redox balance has been confirmed across a range of cancer contexts. To note, it has been proposed that the main role of the ETC was to generate aspartate for further conversion to asparagine via the action of asparagine synthetase (ASNS) (Krall et al. 2021) (Fig. 3B, left panel). In this way, asparagine biosynthesis was proposed to be a key metabolic pathway downstream of OXPHOS that was required for tumorigenesis via activation of MTORC1 cell growth and biosynthetic signalling pathways. In line with this model, asparagine supplementation could bypass the effects of different ETC inhibitors and rescue cell growth. These observations thus link OXPHOS function with asparagine biosynthesis as a key signalling mechanism in cancer cells, which is intriguing in light of other findings highlighting how asparagine can drive aggressive disease in a number of tumour models. ASNS was identified using in vivo screens to select for genes preferentially expressed in highly metastatic breast cancer subclones (Knott et al. 2018). Higher ASNS expression can correlate with poorer patient survival in breast, bladder and squamous head and neck cancers as well as renal clear cell carcinoma patients. In this way, gene targeting of ASNS, dietary restriction of asparagine and reduction of bioavailable asparagine (by treatment with l-asparaginase) are being explored therapeutically in pre-clinical breast and lung cancer mouse models (Hettmer et al. 2015, Knott et al. 2018, Krall et al. 2021). In this regard, asparagine limitation has been a long-established strategy to treat certain types of leukaemia that are highly dependent upon asparagine availability (Richards & Kilberg 2006).
In sum, the ETC provides critical maintenance of the redox balance, in addition to ATP generation, to support cancer cell growth. From all evidence, key molecules of this redox mechanism include NAD+, ubiquinone, aspartate and asparagine. A large part of this dependence may be to form nucleotides, as best demonstrated for pyrimidine biogenesis and the activity of DHODH, which can be critical determinants of tumorigenesis (Bajzikova et al. 2019). The coordination of electron flow with OXPHOS appears to be integrated into the core set of metabolic pathways required for cancer cell survival and has been shown to be highly adaptable to various perturbations, and this flexibility likely contributes to the overall resiliency of cancer cells.
Activation of mitochondria in aggressive or resilient cancer cells
As mitochondrial function can positively regulate cancer cell growth, a key issue is whether cell transformation can reprogram mitochondria. We summarised above how some RAS-dependent cancers activate DRP1 to promote mitochondrial fission and suppress OXPHOS (Kashatus et al. 2015, Serasinghe et al. 2015). However, mitochondrial activation in cancer is also widely reported. For example, two independent studies have highlighted mitochondrial biogenesis and upregulation of PGC-1 alpha selectively in circulating tumour cells using experimental models of aggressive breast cancer (LeBleu et al. 2014, Andrzejewski et al. 2017). One set of findings indicated that metastatic circulating cells are more reliant upon increased OXPHOS and its resulting increased ATP supply (LeBleu et al. 2014). High PGC-1 alpha levels were indeed found to correlate in breast cancer patient samples with higher rates of metastases and poorer prognosis. In a different system, it was shown in an inducible pancreatic ductal adenocarcinoma model that upon shut-off of KRAS expression, there is in vivo selection resulting in a dormant group of resilient cancer-initiating cells. Notably, these dormant cells featured reprogrammed gene expression including upregulation of PGC-1 alpha and downstream OXPHOS genes, with an accompanying increase in mitochondrial oxygen consumption activity. Therefore, sub-populations of aggressive tumour cells appear to upregulate mitochondrial function to support their cancer stem-cell like phenotype. In accordance, others have observed in mammary epithelial cells that transformation into a stem-cell like invasive epithelial–mesenchymal transition-associated state involves an increase in mitochondrial fusion. These higher levels of fusion were found to be driven through the action of miR200c and upregulation of PGC-1 alpha and MFN1 (Wu et al. 2019).
Higher levels of mitochondrial function are associated with sub-populations of aggressive or resilient cancer cells. Cancer cell resistance following a chemotherapeutic treatment continues to be a hurdle for the design of effective strategies in the clinic. As one example, it was earlier observed that treatment of glioblastoma cells with phosphoinositide-3-kinase inhibitors leads to a more invasive resistant cell state associated with AKT and MTORC1 activation (Caino et al. 2015). Another feature of this resistant cell state included a repositioning of active mitochondria to the cell periphery, potentially to provide more localised ATP production to fuel cell invasion. It was also noted that metastatic sub-population of breast cancer cells with elevated PGC-1 may also be especially resistant to treatment with ETC complex 1 inhibitors (Andrzejewski et al. 2017), suggesting that OXPHOS was not necessarily essential in these cases or more likely that mitochondrial biogenesis increases global bioenergetic fitness or flexibility to support resilience in the face of metabolism-targeting compounds. Other pre-clinical work has shown with triple-negative breast cancer patient-derived xenografts (PDX) in mice that treatment using standard front-line neoadjuvant chemotherapy (doxorubicin + cyclophosphamide) leads to in vivo selection of residual remnant tumours (Echeverria et al. 2019). Moreover, transcriptomic analysis of resulting residual tumour material indicated a prominent upregulation of mitochondrial gene expression, which was further reflected in increased OXPHOS function in isolated cells. Interestingly, residual PDX tumour cells remained highly sensitive to the complex 1 inhibitor IACS-010759, indicating that OXPHOS function was still essential in this chemoresistant state.
Increased mitochondrial function can also be associated with specific molecular sub-categories of cancer. A search of mutational signatures in breast and ovarian cancer patient data sets found that mutation in BRCA1/2 or DNA homologous recombination pathways were linked with alterations in OXPHOS or mitochondrial metabolism genes (Lahiguera et al. 2020). It was further dissected using high-grade serous ovarian cancer cells that the loss of homologous recombination shifted the burden towards PARP-dependent DNA repair pathways, which are highly dependent upon supplies of NAD+ and ATP. In this way, tumour growth of BRCA2 mutant ovarian cancer cells were highly sensitive to OXPHOS inhibition by metformin. To sum up, despite the vast heterogeneity that exists across and within each cancer type, a recurring observation is the increased reliance on mitochondrial capacity, which could be supporting cell resiliency via bioenergetic, biosynthetic or redox-related mechanisms.
Targeting mitochondrial fusion in cancer
While altered mitochondrial functional capacity in cancer is widely observed, specific changes in mitochondrial fission or fusion factors are less clear. One informative report surveyed the status of six mitochondria dynamics genes across the Cancer Genome Atlas (TCGA) (Anderson et al. 2018). In this search, four of these genes (OPA1, MFN1, DNM1L (DRP1) and FIS1) were notably altered across the overall range of data sets. Furthermore, OPA1 and MFN1 showed the highest rates of amplification, particularly in high-grade serous ovarian cancer, head and neck squamous carcinoma and lung cancer sets. DNM1L was also detected to be amplified, although at lower rates, in these cancer sets. Pancreatic adenocarcinoma showed the highest levels of DNM1L amplification although it did not show alterations in the other mitochondrial genes. Other analyses of RNAseq data have highlighted that OPA1 showed significant mRNA overexpression in a large range of tumour types, including AML, breast, oesophageal, renal and stomach cancer (Zamberlan et al. 2022).
It may be prudent to mention that both OPA1 (3q29) and MFN1 (3q26) lie close to a known oncogene, PIK3CA (3q26), while DNM1L (12p11) lies close to KRAS (12p12). Therefore, it needs to be better established if alterations in mitochondria dynamics genes represent driver, disease-modifying or passenger effects. There is ample evidence that silencing of DRP1 or MFN1/2 proteins can impair cancer cell growth, so these functions appear to be essential under various contexts (Caino et al. 2015, Kashatus et al. 2015, Serasinghe et al. 2015, Nagdas et al. 2019, Wu et al. 2019, Yu et al. 2019). Other results further demonstrate that OPA1-dependent maintenance of the inner mitochondrial membrane can be essential for cancer cells. Silencing of OPA1 could inhibit in vitro migratory phenotypes and in vivo tumour growth in breast cancer experimental models (Zamberlan et al. 2022). Towards an eventual therapeutic, it may be promising that small-molecule MLYS22 has been developed as an OPA1 inhibitor and has been shown to effectively phenocopy genetic knockdown in cancer cell biology and tumorigenesis experiments. Interestingly, the full scope of OPA1 function at the inner membrane remains to be clarified. Indeed, the study first characterising MLYS22 highlighted a role for OPA1 in controlling angiogenesis and endothelial cell biology, largely attributed to nuclear factor kappa B (NF-KB)-dependent gene expression changes (Herkenne et al. 2020). The effects of OPA1 targeting on migration and breast cancer tumour formation were largely attributed to a mechanism involving altered levels of miRNAs in the 148/152 family, which could be leading to mis-regulation of cyclin, integrin or MAPK/ERK-dependent pathways (Zamberlan et al. 2022). These observations may thereby highlight wider mechanisms linking OPA1 or mitochondrial metabolism to gene expression regulation.
Pleiotropic nature of mitochondrial dynamics in cancer cells
Overall, the consideration of mitochondrial dynamics in cancer remains controversial, particularly if one aims to place, for example, fission as pro- or anti-cancer. Looking across various models, evidence can be found to implicate both mitochondrial fission and fusion, depending on the context, to be critical for maintaining the growth or survival of cancer cells. The lack of simple relationships between mitochondrial fission and fusion in cancer mechanisms might be unsatisfying. The sheer complexity of this puzzle was highlighted in work where roles for both sides of mitochondrial dynamics were dissected using the same high-grade serous ovarian cancer model (Anderson et al. 2018). In these cells, DRP1 or OPA1 could be inactivated using CRISPR/Cas9 approaches. These derivatives were then screened in a high-throughput manner to determine how loss of fission vs fusion altered growth inhibitory sensitivity across approximately 2100 anti-cancer drugs. Across the 2100 tests, OPA1 targeting more frequently led to drug resistance as compared to DRP1 targetting. Therefore, OPA1 may be playing more pro-cell death roles across a range of all possible cancer drug interactions. Despite this generalisation, simple models do not appear possible since OPA1 targeting can also sensitise to certain drug treatments (i.e. OPA1 as an anti-apoptotic factor). Similar double-edged sword effects could be observed following DRP1 targeting with increased resistance or sensitivity, depending on the specific drug. The screen data could be further dissected into effects depending on type of drug (e.g. apoptosis, cytoskeletal and DNA damage), but even with this separation, clear associations between gene and drug category did not emerge. Regardless, further mechanistic progress was possible from this screen by filtering and focussing on the observations that both OPA1 or DRP1 targeting can lead to hypersensitivity to a range of drugs known as Smac-mimetics that induce apoptosis by suppressing inhibitor of apoptosis proteins (IAPs) such as XIAP and cIAP1/2. Ultimately, attention to the details is still very much required as different underlying pathways appear to explain these effects. DRP1 targeting appears to push towards apoptosis by inducing cytochrome c leakiness, while OPA1 targeting is distinct and may activate ATF4 and an integrated stress response. Clearly, further work will be required to deconvolute the other exciting mechanisms of mitochondrial fission and fusion that likely lie in these screen data.
Conclusions and unresolved questions
Here, we aimed to summarise the potential roles of mitochondrial dynamics in controlling cancer cell biology. We discussed how initial data postulated that mitochondrial fission and a suppression of respiration may be a prominent feature of transformed cells. However, other compelling studies indicate that mitochondrial respiration may be especially required to support redox balance and nucleotide biosynthesis. In fact, activation of mitochondrial function is featured in a number of different aggressive, stem cell-like or chemotherapeutic resistant cancer cell states. The concept of targeting mitochondrial function or mitochondrial dynamics to reveal further drug dependencies has been explored, and evidence supports the potential of targeting fission and fusion pathways, but details are unclear.
To develop these findings towards new therapeutics, it will now be imperative to better understand how mitochondrial fission and fusion can play different roles in modulating cell metabolism and apoptosis. In many cases, we have observed how mitochondrial fission (or fusion) can be pleiotropic and have different functions depending on cell context. This heterogeneity likely reflects sets of complex interactions with other pathways specific to each host cell. These co-dependencies need to be clarified so that we can better predict how different cancer cell types may be reliant on particular sections of the mitochondrial dynamics cycle. These fundamental insights will be ultimately essential in order to rationally design drug treatments that can be combined with mitochondrial targeting to offer more effective therapeutic windows in the clinic.
Declaration of interest
The authors have no conflict of interests to declare.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Acknowledgements
KBP received studentship support from Queen’s University Department of Biomedical Sciences. CC is supported by a studentship from Natural Sciences and Engineering Research Council (NSERC), Canada. EYWC is supported by NSERC (operating grant RGPIN-2018-05162) and Cancer Research Society/Société de Recherche sur le Cancer, Canada.
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