Physiologically, normal cells have a low rate of glycolysis with most energy efficiently generated by oxidative phosphorylation in the mitochondria. In general cancer cells mainly use glycolysis to generate adenosine triphosphate (ATP).
Glycolysis takes place in the cytoplasm, where glucose is broken down to pyruvate, producing 2 ATP molecules per glucose molecule (Figure 1.7). Pyruvate is then converted into acetyl-CoA by pyruvate dehydrogenase in the mitochondria (Figure 1.8). Acetyl-CoA enters the tri-carboxylic acid (TCA) cycle (also known as the citric acid cycle or the Krebs cycle) where it is broken down via nine further enzymatic steps to oxaloacetate168 (Figure 1.8). Cells can also metabolise glutamine using the TCA cycle, which can be another useful source of energy for cancer cells169 (Figure 1.8). During the TCA cycle nicotinamide adenine dinucleotide (NADH) is produced which then serves as an electron donor in the electron transport chain (ETC), where 30-36 molecules of ATP are produced for each glucose molecule via oxidative phosphorylation (OXPHOS) in the inter-membrane space of mitochondria168 (Figure 1.9). Oxygen is used as an electron acceptor for OXPHOS. In the absence of oxygen (hypoxia) or functioning mitochondria, non-malignant cells rely on glycolysis for ATP generation. It has also been recently shown that cancer cells can use intracellular glycogen as a means of maintaining cell viability and proliferation170 and in response to acute hypoxia, cancer cells can increase glycogen storage171. This is consistent with the previously described glycogen shunt in a number of other cell types172.
Interestingly, even when oxygen is plentiful, rapidly growing cancer cells have glycolytic rates of 200 times that of a normal cell173. Cancer cells in general are metabolically adapted to grow and proliferative rapidly under conditions of low pH and oxygen tension with limited nutrients174 where non-transformed cells would struggle to grow175. An important study by Sonveaux et al. in 2008176 showed that normoxic tumour areas can oxidise lactate as a significant carbon source, sparing glucose and allowing it to diffuse further away from the tumour vasculature into hypoxic areas where anaerobic metabolism was used to metabolise the glucose to lactate, which was then used by the normoxic areas.
Figure 1.7. Glycolysis. The breakdown of glucose by enzymes into pyruvate, ATP and NADH in the cytosol173. ATP, adenosine triphosphate; ADP, adenosine diphosphate; NAD, nicotinamide adenosine dinucleotide.
Figure 1.8. The tri-carboxylic acid cycle. Acetyl-CoA enters the TCA cycle in the
mitochondria where it is broken down via nine further enzymatic steps to oxaloacetate168. Glutamine can also enter the TCA cycle. TCA, tricarboxylic acid cycle; PHD, prolyl hydroxylase, NAD, nicotinamide adenosine dinucleotide; GTP, guanosine triphosphate; FAD, flavin adenosine dinucleotide.
Figure 1.9. The electron transport chain. Electrons from the reduced NADH and succinate
generated by the TCA cycle are transferred through a chain of protein complexes embedded in the inner mitochondrial membrane177. The oxidation steps lead to protons moving from the inner matrix to the intermembrane space, creating a hydrogen concentration gradient which produces both an electrical potential and a pH potential. This leads to the conversion of ADP to ATP by ATP synthase. These coupled reactions are also referred to as OXPHOS. ETC, electron transport chain; NADH, nicotinamide adenine dinucleotide; TCA, tricarboxylic acid cycle; ADP, adenine di-phosphate; ATP, adenine tri-
The exact reason for the switch in glucose metabolism pathways in cancer cells is not known, although there are a number of theories: 1) an inherent feature of the malignant phenotype, called the Warburg effect178,179; 2) as a consequence of a hypoxic tumour microenvironment167, or 3) the existence of a pseudo-hypoxic tumour metabolic profile173.
Otto Warburg was the scientist who discovered the ‘oxidative glycolytic’ phenotype of cancer cells. In these cells, glycolysis is uncoupled from the mitochondrial TCA cycle and OXPHOS, and is consequently characterised by excess production of lactate leading to an acidic extracellular environment178. Warburg hypothesised that this metabolic reprogramming confers a survival advantage to cancer cells that will inevitably reside in a hypoxic environment if the tumour outgrows the vascularisation. The acidic extracellular environment does favour the survival of cancer cells, especially those that have lost wild type TP53 function, over normal cells and may promote invasion, as well as preventing apoptosis upon detachment (anoikis) facilitating metastasis180. This is highly relevant for the colonic epithelium as over 75% of human colonic tumours have lost functional wild type TP53 activity181. Analysis of CRC patient samples has identified high levels of GLUT-1, monocarboxylate transporter (MCT)-1 and HIF-1a which indicates increased glycolytic metabolism and lactate production182. In CRC, LDHA5, which converts pyruvate to lactate, is linked to activation of the HIF pathway as well as an aggressive phenotype183–185. Elevated levels of lactate also correlate with poor patient prognosis and overall survival in cervical and ovarian cancers186,187. Warburg also hypothesised that these metabolic changes occurred due to mitochondrial defects, however this topic remains controversial. Evidence is mounting to suggest that damaged mitochondria are not the cause of the increased aerobic glycolysis exhibited by most tumour cells, but that the primary functions of activated oncogenes and inactivated tumour suppressors are to reprogram cellular metabolism (reviewed in188). Carbon labelling metabolic studies have identified that in solid tumours, where glucose supply can be low189, glutaminolysis, the TCA cycle, the pentose phosphate pathway and nucleotide biosynthesis as well as glycolysis are all enhanced in tumour cells190. Nevertheless, the mitochondrial genome is susceptible to mutations because of the large
There is some evidence to suggest that mitochondria are damaged in CRC. One study showed that mitochondrial microsatellite instability is an early and independent event in colorectal precancerous lesions which occurs in 30% of CRC patients and is linked to poor prognosis192. Patients with lower mitochondrial DNA (mtDNA) copy number show higher TNM stages and poorer differentiation193. CRC cell lines including DLD1, HCT116, SW837 and HT29 have been shown to have mutations in mtDNA leading to a subtle elevation in ROS191; low levels of ROS are highly mitogenic, whereas high levels of ROS are toxic194. It has also been shown that HCT116 CRC cells treated with 1% oxygen for at least 20 passages, have larger mitochondrial mass, but decreased mtDNA and reduced sensitivity to 5-FU193.
Hypoxia is a feature of solid tumours (Chapter 1, Section 1.8) and can lead to expression of many genes, via HIF-1a, that have a role in metabolism such as GLUT-1, HK2 and LDHA, all which have been shown to be relevant in CRC tumorigenesis and linked to poor prognosis182,195 (Figure 1.10). HIF-1 also promotes the expression of pyruvate dehydrogenase kinase (PDK)-1 expression162 which acts to block conversion of pyruvate to acetyl-CoA, effectively blocking the TCA cycle and OXPHOS, forcing the cell to use glycolysis for ATP generation. Upregulation of PDK1 also protects cells from ROS damage. There is evidence to suggest that HIF-1 down regulates mitochondrial biogenesis162 by inducing microRNA (miRNA)-210 transcription resulting in a reduction of iron-sulphur cluster assembly enzyme (ISCU) and COX10, two important elements of the mitochondria electron transport chain and the TCA cycle196. HIF transcription factors have also been shown to affect the function and stability of other genes which influence cellular metabolism; the best characterised examples are v-myc avian myelocytomatosis viral oncogene homolog (MYC) and p53. MYC binds E-boxes in the promoter of target genes when associated with MYC-associated protein X (MAX) as a heterodimer197. MAX is itself regulated by binding of MAX dimerisation protein (MXD1/MAD) and MAX interactor 1 (MX11/MAD2)198. MX11 binding to MAX inhibits binding of MYC:MAX to E-boxes. HIF-1α can also interfere with the MYC:MAX heterodimer; through upregulation of MX11, increasing competition for MAX199 and by direct binding of MAX to displace MYC. Conversely, HIF-2α binds and stabilises the MYC:MAX heterodimer to promote MYC associated transcriptional changes200. HIFα can
be outcompeted in tumours with high expression levels of MYC201, therefore, HIF-2α interaction with the MYC:MAX heterodimer is more likely to influence the hypoxia- induced metabolic transformation in non-MYC-amplified tumours. There are no known mutations of MYC in CRC. However, MYC is frequently amplified in CRC; Soga et al. recently implicated amplification of MYC, which is commonly seen in CRC, leads a change in expression of 121 metabolic genes and 39 transporter genes in CRC promoting metabolic reprogramming202.
The relationship between TP53 and hypoxia is controversial; hypoxia has been shown to induce TP53 stability in some conditions, but not in others203,204. It appears that lower oxygen tensions elicit strong stabilisation of TP53, through DNA damage-response mechanisms205. Stabilisation of TP53 increases the entry of glycolytic intermediates into the pentose phosphate and folate pathway by modulation of key enzymes206,207. In this way, TP53 can increase antioxidant production, although at the expense of ATP production. Expression of TP53 is also important for the assembly and function of COX in the ETC. Loss of TP53 in normoxia results in a similar phenotype observed in hypoxia as the malate-aspartate shuttle ceases to function effectively208.
Pseudo-hypoxia is the activation of the hypoxia response pathway under non-hypoxic conditions which is commonly seen in cancers209, although this has not been reported specifically in CRC to date. Defects in some TCA cycle enzymes lead to decreased hydroxylation of HIFα subunits and trigger the pseudo-hypoxia response210. Isocitrate dehydrogenase (IDH) is mutated in glioma and acute myeloid leukaemia (AML); succinate dehydrogenase (SDH) and fumarate hydratase (FH) are mutated in pheochromocytoma, para-ganglioma, leiomyoma, and renal carcinoma188. There are no known mutations in these enzymes in CRC211.
Figure 1.10. Cellular effects of change in O2 tension. As a result of changes in O2 tension there are; (A) Changes in pathways as a result of changes in O2 tension, (B) changes in specific proteins as a result of changes in O2 tension and (C) changes in transcript expression152. ROS, reactive oxygen species; PHD, prolyl hydroxylase domain-containing protein; OXPHOS, oxidative phosphorylation; HIF, hypoxia inducible factor; LDHA, lactose dehydrogenase A; MCT4, mono-carboxylate transporter 4; HK2, hexokinase 2; GLUT1, glucose transporter 1; PGAM, phosphoglycerate mutase; TIGAR, TP53 induced glycolysis regulatory phosphatase; COX, cytochrome C oxidase; ALDH4, aldehyde dehydrogenase 4; PDK1, pyruvate dehydrogenase kinase 1.