CANCER CELL IS CHARACTERISED BY HIGH RATE OF GLYCOLYSIS

Introduction

Glycolysis is an anaerobic, sequential, enzymatic and catabolic multi-step process that converts a single glucose molecule into two pyruvates in the cytoplasm coupled with the production of two NADH molecules and a net yield of two molecules of ATP. This ten step pathway was elucidated in the 1940s (Li et al., 2015). Pyruvate, the end-product of glycolysis has several fates within the cell, depending on the energy need of the body. First, in the presence of enough cellular oxygen (or under aerobic conditions), pyruvate is oxidised with loss of carboxyl group as CO₂ and the remaining two carbon unit becomes the acetyl group of acetyl coenzyme A (AcetylCoA). This acetyl group is further metabolised in the tricarboxylic acid (TCA) cycle and fully oxidized to yield CO₂. This ultimately results in the production of 36 molecules of ATP (Annibaldi and Widmann, 2010).  Second, under condition of low cellular oxygen (as is the case in contracting muscle), otherwise known as anaerobic condition, pyruvate can be reduced to lactate through oxidation of NADH to NAD⁺ – a process referred to as lactic acid fermentation. Lastly, in microorganisms such as brewer’s yeasts and in certain plant tissues, pyruvate is reduced to ethanol, again with oxidation of NADH to NAD⁺ (Garrett and Grisham, 2017)

Cancer, on the other hand, can be defined as a heterogeneous group of genetic diseases characterised by unregulated clonal expansion of somatic cells brought about by multiple genetic and epigenetic changes (Evan and Vousden, 2001).  Cancer development and progression in humans involves multi-step processes that usually take place over many decades.  During these processes, the cancer cells acquire multiple allelic mutations in genes such as proto-oncogenes, tumour suppressor (TS) genes and other genes that control cell proliferation (Hahn and Weinberg, 2002).  These allelic mutations lead to the production of dysregulated proteins leading to the activation of oncogenes or the inactivation of TS genes; promoting abnormal regulation of signalling pathways involved in cell cycle regulation, genetic stability, apoptosis and cell differentiation.  This imbalance in cellular regulations drives the process of oncogenesis (Hahn and Weinberg, 2002).

In addition to genetic changes, it is also known that the tumour microenvironment plays key part in the transition from benign to malignant cancer. This is achieved by conferring an adaptive pressure that selects cells for their clonal expansion (Annibaldi and Widmann, 2010). In 2000, Hanahan and Weinberg published a seminal review tagged ‘’the hallmarks of cancer’’ (Hanahan and Weinberg, 2000) which aimed at summarising the promoting features of cancer cells into 6 major hallmarks. These include evading apoptosis, sustained angiogenesis, tissue invasion and metastasis, self-sufficiency in growth signals, insensitivity to anti-growth signals and lastly, limitless replicative potential. A decade later, the authors added two emerging hallmarks: reprogramming energy metabolism and evading immune response, and two enabling traits: genomic instability and mutation, and tumour promoting inflammation (Hanahan and Weinberg, 2011; Yousef and Carmen, 2017). The impact of Hanahan and Weinberg hallmarks of cancer I & II have been overwhelming as they serve as blueprints for understanding core traits of cancer.

This piece of work seeks to understand the relationship between cancer and high rate of glycolysis observed in tumours. It will elucidate on the additional hallmarks of cancer mentioned above, i.e. reprogramming energy metabolism in cancer cell by pointing out to key experiments pertaining, and that serve as proof, to this phenomenon.  It also aims at understanding the mechanism or proffer answers to the question of why there is a high rate of glycolysis in cancer cells.

The Warburg effect (Aerobic glycolysis)

Earlier work on cancer metabolism was pioneered by the biochemist and Nobel Award winner Otto Warburg (Justus et al., 2015). He proposed a direct association between mitochondrial impairment and cancer development by hypothesising that a defect in mitochondria respiration was responsible for the development of cancer (Warburg, 1956; Weinhouse et al., 1956). This postulation was motivated by his observation that cancer cells showed an increased rate of glycolysis when compared to normal cells, even in the presence of oxygen, which would normally inhibit glycolysis – a phenomenon known as Pasteur effect (inhibition of glycolysis in the presence of oxygen) (Warburg, 1956). As pointed out in the introduction above, one would expect pyruvate to be diverted to the Tricarboxylic Acid – TCA – cycle and then oxidative phosphorylation, but this is not the case in cancer cell metabolism.  Cancer cells would rather by-pass this cellular norm and instead, increase the rate of breakdown of sugar via glycolysis.  Recall that the process of oxidative phosphorylation occurs in the mitochondrial matrix and that pyruvate must be transported into the matrix via specific pyruvate transporters (example being the mitochondrial pyruvate carrier – MPC – complex) after pyruvate must have been delivered to the intermembrane compartment from the outer mitochondrial membrane via the action of a voltage-gated porin complex (Michael, 2017).  However, this is not the case in cancer cells. It preferentially adopts the process to glycolysis to process its energy. The increased rate of glycolysis in the cytoplasm would have led Warburg to conclude that the mitochondria was defective in cancer cells, and this defect could, in effect, contribute to cancer development. A defective mitochondrion would mean that cancer cells are unable to carry out oxidative phosphorylation to produce more energy in the form of ATP, and consequently, cells must increase the rate of glycolysis to get enough energy to meet up the increasing biomass observed in cancer.

Although it remains true, in most cases, that cancer cells exhibit a higher rate of glycolysis, the idea that mitochondrial defect was responsible for cancer development no longer holds true (Koppenol et al., 2011). It has now been proven that cancer cells have active and functional mitochondrial, contrary to Warburg’s theory (Ju et al., 2014; Xu et al., 2015)

Aerobic glycolysis in cancer

Increase in biomass and replication of the genome prior to cell division to create two daughter cells are key features of proliferating cells. The cell must therefore, generate enough energy and synthesise biomolecules at a sufficient rate to meet the demands for proliferation. Proliferation in cancer cells is always on the high rate and unregulated.  For developing tumours to survive, it needs to alter energy metabolism and nutrient uptake to favour its malignant growth. This alteration in energy metabolism and nutrient uptake led to the observation of Otto Warburg that cancer cells preferentially use glycolysis over mitochondrial oxidative phosphorylation for glucose-dependent ATP production even in the presence of ample oxygen, a phenomenon known as the ‘’Warburg effect’’ or aerobic glycolysis (Jones and Thompson, 2009; Warburg, 1956)

Cancer development has been previously linked to an increase in lactate production when compared to normal cells, even in the presence of ample oxygen which should ideally inhibit glycolysis (a concept known as ‘’pasteur effect’’). This peculiar feature of cancer cells has been confirmed by many subsequent studies (Warburg et al., 1926). Interestingly, this effect is not peculiar to cancer cells alone, as normal cells such as proliferating lymphocytes also show enhanced glycolysis (Greiner et al., 1994). The massive lactate production as a survival feature of cancer cells is necessary to regenerate, via lactate dehydrogenase, NAD⁺ needed to sustain glycolysis.

The paradigm of a purely ‘’glycolytic’’ cancer cells has been consistently challenged. Recent research shows that some glioma, hepatoma and breast cancer cell lines possess functional mitochondria and equally source their ATP mainly from oxidative phosphorylation (Kashiwaya et al., 1994). Interestingly, some cancer cells can switch between fermentation and oxidative metabolism, depending on the absence or presence of glucose and the environmental conditions. It is therefore, imperative to understand the mechanism by which cancer cells can reversibly regulate their energy metabolism. Such a situation is the glucose-induced suppression of respiration and oxidative phosphorylation (Crabtree, 1929; Diaz-Ruiz et al., 2009). This is referred to as the ‘’crabtree effect’’ and is usually short-term and reversible.

Molecular mechanisms that support the high rate of glycolysis in cancer cells.

This section will concentrate on some of the processes and/or mechanisms that contribute or support the fact that there is a high rate of glycolysis in cancer cells. This will be discussed in the following sections.

 The need to generate sufficient biosynthetic precursors

Cancer cells face the challenges of how to provide the requisite bioenergy and biosynthetic precursors to meet up with the ever-increasing genome and biomass. It is therefore, imperative for cancer cells to devise strategies to ensure the constant supply of these precursors, and this it achieves through aerobic glycolysis. It can be inferred here that glycolysis provides precursors for several biosynthetic pathways, and may provide a range of precursors required for RNA and DNA synthesis, via the Pentose Phosphate Pathway (PPP); and for certain amino acids and glycerol for lipids. This could support the need for the Warburg effect observed in cancer cells. This could also explain why cancer cells employ glycolysis for energy uptake, despite the low ATP production involved when compared to oxidative phosphorylation that yields tremendous amount of ATP. To buttress the hypothesis of a possible upregulation of the PPP mentioned above, Deberardinis and Tong together with their teams have reported that the non-oxidative branch of the PPP appears to be the main source of ribose-5-phosphate in tumour cells (Deberardinis et al., 2008; Tong et al., 2009). This, in effect, supports the need for increased glycolysis as ribose-5-phosphate is a key precursor for nucleotide biosynthesis.

Overexpression of glucose transporters

Commonly, substrate supply is the controlling step of a metabolic pathway for some cancer cell lines (Kashiwaya et al., 1994; Rodríguez-Enríquez et al., 2009). Accordingly, the overexpression of glucose transporters has been reported as one of the main determinants of the Warburg effect (Yamamoto et al., 1990). Glucose derivatives such as ¹⁸Fluoro-deoxyglucose (FDG) is used as a tracer in positron emission tomography (FDG-PET) scan for imaging uptake of glucose in tissues in vivo (Czernin and Phelps, 2002). Enhanced glucose uptake visualised by FDG-PET correlates with poor prognosis and higher metabolic potential in many tumour types.

The high affinity glucose transporters (Glut 1 and 3) have been shown to be overexpressed in cancer cell lines (Macheda et al., 2005) and their inhibition in vitro has been shown to impair growth of tumours in cells (Cao et al., 2007). This is a clear evidence of their direct involvement in tumour growth. Overexpression of glucose transporters in tumour cells indicates that there is an increased uptake of glucose into the cell and hence, an increase in the rate of glycolysis to metabolise the excess glucose. As a result, glucose transport could be a suitable target for pharmacological anti-tumour agents.

Increased activity (or downregulation) of glycolytic pathway enzymes

The high rate of glycolysis may also be explained by the increased activity of glycolytic pathway enzyme. To prove this, Pelicano et al. has shown that each of the enzymes involved in the glycolytic pathway is overexpressed or downregulated in several cancer cell lines (Pelicano et al., 2006). Other reports have supported the fact above, pointing out that each of the enzymes in the pathway shows a several-fold increase in activity compared to their normal counterparts (Marín-Hernández et al., 2006). It is important to note here that only the rate limiting enzymes in the pathway would have significant impact for the quantitative increase in glycolysis. As a result, only hexokinase (HK, especially HKII isoform), phosphofructokinase (PFK) and pyruvate kinase (PK) may be implicated, and their regulation and expression pattern change in some tumours – see figure 1 below (Diaz-Ruiz et al., 2011).  For example, the activity of PFK is tightly regulated according to the energy state in a cell – inhibited when the cell no longer requires ATP. However, in cancer cells like leukemia and lymphoma, the L and P isoforms of PFK are predominant. Surprisingly, the allosteric properties of these isoforms allow the maximal activity of the enzyme even in low energy demand condition (they respond less effectively to their inhibitors – citrate and ATP) while they are highly activated by lower concentrations of fructose-2, 6-bisphosphate (Vora et al., 1985; Vora et al., 1980). The above points also support the observation of a high rate of glycolysis in cancer cells as the rate-limiting enzymes are found to be overexpressed and/or upregulated as the case may be. Fig. 1 summarises some of the enzymes that are overexpressed, upregulated and/or downregulated in tumours. It also highlights the role of the enzymes in either the glycolytic pathway or the TCA cycle.

The pyruvate crossroad

Pyruvate is located at the intersection between two of the main catabolic pathways of the cell: glycolysis and Krebs cycle (see figure 1 above). Pyruvate can be transported into the mitochondrial matrix and be converted to acetyl-coA (via the action of pyruvate dehygrogenase) which subsequently enters the Krebs cycle to yield reducing equivalents to be later used by respiratory chains to drive oxidative phosphorylation (Diaz-Ruiz et al., 2011). On the other hand, the metabolite can reside within the cytosol and be reduced by lactate dehydrogenase to lactate.

Under aerobic glycolysis which predominates in cancer cells, pyruvate is said to be inefficiently metabolised by mitochondrion and subsequently deviating the metabolic flux into lactate production (Diaz-Ruiz et al., 2011). Three events have been suggested to be responsible for this:

• The restriction of pyruvate transport into mitochondrial matrix
• The inhibition of the pyruvate dehydrogenase complex
• The over-activation of lactate dehydrogenase

All three events promote aerobic glycolysis and therefore, favour the development of tumours. The last two events will be elaborated further here.  First, using pyruvate dehydrogenase (PDH) complex as a case study, PDH catalyses the oxidative decarboxylation of pyruvate to produce acetyl-coA, a key intermediate in Krebs cycle (see figure 1 above). Its activity is tightly controlled according to the energy need of the cell. It is inhibited when the cell’s energy store is high, and vice versa (Strumiło, 2005). Two key enzymes covalently and reversibly regulate the activity of PDH – PDH kinases (pdhk) phosphorylate and thus inactivate it whereas PDH phosphatases (PDP) revert this inactivation (Strumiło, 2005).  The activity of pdhk1, an isozyme of pdhk has been shown to be upregulated by c-Myc and hypoxia-inducible factor-1a (HIF-1a) in cancer cells, which lead to inhibition of mitochondrial respiration in cancer cells (Saunier et al., 2016). Again, the transcription of pdhk3, another isozyme of pdhk has been reported to be induced by HIF-1a (Saunier et al., 2016). Taken together, these data suggest that the phosphorylation of PDH is an important factor for the formation and/or progression of tumour.

Lastly, using lactate dehydrogenase (LDH) as a case study.  Pyruvate is reduced in the cell cytosol by LDH in order to keep a constant supply of NAD⁺. The latter is required to drive glycolysis. See figure 2 below for the chemical reaction catalysed by LDH. This enzyme has been found to be overexpressed in a variety of cancer cell lines (Goldman et al., 1964) and the disruption of its expression stimulates respiration and decreases tumour cell viability in hypoxic conditions (Fantin et al., 2006; Le et al., 2010). Over-expression of the M-subunit of LDH homo- or hetero-tetramer has been detected in several human tumours (Fantin et al., 2006).

Krebs Cyle and Oxidative phosphorylation defects or weakening:

Defects in the Krebs cycle and oxidative phosphorylation pathways could also be responsible for the increased glycolysis rate observed in tumour cells. Research has confirmed high citrate efflux in mitochondria isolated from a hepatoma cell line (Parlo and Coleman, 1984, 1986). The leaked citrate is oxidised by isocitrate dehydrogenase to produce NADPH which is required for lipid synthesis – as a consequence of an impaired Krebs cycle. However, a study contrary to this report has been published. The only difference between the two research results is that a different cell line was used in the latter. The synthesised lipids are key to building up the membranes of the fast-growing cancer cells. Again, mutations in succinate dehydrogenase, which participates in the mitochondrial respiratory chain as complex II commonly occur in phaeochromocytomas and paragangliomas; suggesting that this TCA enzyme may contribute to tumour growth (Gottlieb and Tomlinson, 2005). Another example is the mutation observed in isocitrate dehydrogenase, which is known to be implicated in adult cases of glioblastoma and seems to have a major role in the development of the tumour by a gain-of-function effect (Dang et al., 2009; Yan et al., 2009). The above findings suggest that a defect in the mitochondrial Krebs cycle enzymes could also be responsible for the switch to glycolysis as characterised by cancer cells. Furthermore, there is also the influence of various cancer-associated mutations, with many having considerable impacts on metabolism (Potter et al., 2016).

Both a decrease in ADP translocation to the mitochondrial matrix as well as the inhibition of the ATP synthase have been reported as highlighted in figure 1 above (Lee and Yoon, 2015; Sonveaux et al., 2008).  Both scenarios would restrict ATP production in mitochondria, thereby forcing the cell to rely mostly on glycolysis-derived ATP. It is worth noting that several studies have shown that other cancer cell lines possess fully functional mitochondria which is contrary to the Warburg effect (Guppy et al., 2002; Pasdois et al., 2003; Rodríguez-Enríquez et al., 2006)

Another interesting feature of cancer cell energy metabolism is their extensive consumption of glutamine, which is the most abundant amino acid in mammals (Kovacević and Morris, 1972). Glutaminolysis has been reported to increase in cancer cell lines (Matsuno and Hirai, 1989). Glutamine is involved in numerous anabolic pathways (such as nucleic acid) and can be degraded in the Krebs cycle thereby generating ATP through both substrate level and oxidative phosphorylation (Kovacević and Morris, 1972; Reitzer et al., 1979).

Although pyruvate, via the action of pyruvate carboxylase, could have also supplied oxaloacetate to Krebs cycle, this enzyme is reported to be suppressed in certain cancers (liver, brain and breast) (Deberardinis et al., 2008). Again, there is also the influence of various cancer-associated mutations with many having considerable impacts on metabolism (Potter et al., 2016)

Conclusion

The aim of cancer cells is to ensure they by-pass the body’s defence or regulatory mechanisms to promote their own survival.  To survive and grow, and in addition to the hallmarks of cancers discussed, altered energy metabolism is one strategy cancer cells utilise to acquire enough energy and precursors to duplicate their genome and biomass.

Cancer cells utilise the concept of aerobic glycolysis to ensure that they have sufficient energy and biosynthetic precursors to ensure they develop, progress and survive the cell’s surveillance mechanism.  They achieve this by employing a variety of survival mechanisms, among which include the overexpression of glucose transporters, up- or downregulation or overexpression of key enzymes in glycolysis, Krebs cycle and/or oxidative phosphorylation pathways, in addition to activating proto-oncogenes and inactivating tumour-suppressor genes.

This paper has highlighted the relationship between cancer and high rate of glycolysis (observed in tumours) and discussed some of the various mechanisms cancer cells employ to achieve this. Some of the key pathways or mechanisms discussed above, including increased expression of glucose transporters, over-activation of lactate dehydrogenase, key enzymes implicated in the Krebs cycle could serve as targets for drug design.

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