ESPACIO PÚBLICO Y EQUIPAMIENTO COLECTIVO

Early work by Gray identified the importance of tissue oxygenation for sensitivity to radiation damage. Histological assessment of lung adenocarcinoma suggested that due to unrestrained growth, tumour cells are forced away from their supplying vessels resulting in a large diffusion distance for oxygen in respiring tissue.(153) The importance of hypoxia in solid tumours is increasingly recognised in resistance to radiotherapy and chemotherapy.(154, 155)

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Figure 1.2.2-1: Diagram of glycolysis pathway.(156)

Enlarging tumours may result in an exponential reduction in tissue oxygenation. In experimental mouse tumours, bulky tumours greater than 400mm3 had median pO2 of less than 10mmHg. At pO2 below 10mmHg, intracellular acidosis developed and coincided with a reduction in ATP levels, rise in inorganic phosphate and a drop in energy charge.(157) ATP is necessary for normal cell proliferation and survival and comes primarily from either glycolysis, the conversion of glucose to pyruvate in the cytoplasm resulting in a net 2 ATP for each glucose or, from the TCA cycle which uses pyruvate in a series of reactions donating electrons via NADH and FADH2 to respiratory chain complexes within mitochondria (Figure 1.2.2-1). This process, of which oxygen is the final electron acceptor, generates 36 ATP per glucose molecule. In low oxygen conditions, pyruvate does not enter the TCA cycle and is instead metabolised to lactic acid by lactate dehydrogenase, a process called anaerobic

43 glycolysis. Cancer cells consume glucose at a high rate and produce lactic acid rather than using the TCA cycle, even in the presence of oxygen.

A normal cellular response to hypoxia is increased glucose utilisation as a result of elevated hypoxia-inducible factor 1α (HIF-1α). This in turn induces increased levels of glucose-transporter 1, a transmembrane protein overexpressed in many tumours but undetectable in normal tissues, leading to increased glucose uptake.(158) Under normoxic conditions, HIF-1α is continuously synthesised and degraded. Hypoxia stimulates anaerobic glycolysis by stabilization of HIF-1α and its transcription of glycolytic enzyme genes causing increased glucose uptake and lactate production. HIF-1α binds to the DNA sequence 5’-RCGTG-3’ and increases expression of genes encoding glucose transporters and glycolytic enzymes including aldoseA, enolase1, lactate dehydrogenase A, phosphofroctokinase L, phosphoglycerate kinase 1 and pyruvate kinase M, as well as angiogenic growth factors (eg VEGF), hexokinase II, and haemopoitic factor.(156) Even in the absence of hypoxia, a variety of oncogenic induced proteins can lead to stabilization of HIF-1 or inhibition of its degradation.(159) Any proliferative advantage conveyed to tumours by aerobic glycolysis is not immediately apparent. In terms of ATP production, anaerobic metabolism of glucose is very inefficient, producing only 2 ATP compared with 38 ATP per glucose with complete oxidation. In fact, this evolutionary adaptation is one of the earliest steps in carcinogenesis and allows tumour cells to survive beyond normal tissue constraints. In the 1960’s, Thomlinson and Gray observed that viable tumour cells were not observed greater than 160µm from blood vessels. As tumour cells proliferate and expand, the population inevitably moves further away from its blood supply, leading to reduced substrate availability.(156) Tumour angiogenesis is

44 required to permit further growth in addition to the molecular mechanisms already described. Angiogenic factors, such as VEGF promote increased vascularity of tumours. As new microvessels tend to be highly irregular and tortuous, blood flow and oxygen delivery tends to remain poor and hypoxia is likely to remain a strong selective force.(160) The ability to tolerate transient hypoxia due to the glycolytic adaptation contributes to metastatic potential. The increased presence of [H+] in the tumour microenvironment is toxic to adjacent normal cells that lack the mechanisms to adapt to extracellular acidosis. This allows tumours to continue to proliferate and may enhance invasive potential and ability to metastasize by causing degradation of the extracellular matrix and promoting angiogenesis.(161)

Anaerobic glycolysis, activated by HIF1 is not sufficient for hypoxic adaptation alone as hypoxic stress results in the production of reactive oxygen species (ROS) by mitochondria that would be toxic were it not for further adaptation. Pyruvate dehydrogenase kinase 1 (PDK1) is a direct HIF1 target gene that inhibits conversion of pyruvate to acetyl-CoA, attenuating mitochondrial respiration and reducing production of ROS. In hypoxic conditions, HIF1α null mouse embryo fibroblasts fail to activate PDK-1 and undergo apoptosis with a significant rise in ROS. Forced expression of PDK1 by independent retroviral infection increased ATP, prevented hypoxia-induced ROS generation and apoptosis.(162)

The serine/threonine kinase Akt is also associated with increased glucose uptake and aerobic glycolysis independent to HIF1. Akt promotes increased glucose utilization without increasing oxygen consumption. Akt mobilises glucose transporters and activates hexokinase 2 (HK2) to phosphorylate and trap intracellular glucose. Activation of the Atk oncogene is sufficient to cause the switch to aerobic glycolysis

45 characteristic of cancer cells, but does not increase proliferation of cancer cells in vivo.(163)

In addition to the Atk oncogene, Myc can upregulate the activation of a number of glycolytic enzymes including Glut1,(164) PDK1,(165) LDH-A.(166, 167) Although the Myc transcription factor would appear to enhance the Warburg effect via increasing glycolysis, it has also been shown to encourage respiration in mitochondria leading to increased ROS which would be toxic to tumour cells.(168) A further adaptation of tumour cells can compensate for this however. One of the most frequently mutated genes, p53, can promote glycolysis by interfering with mitochondrial respiration. Inactivation of p53 in tumour cells can reduce mitochondrial respiration and this effect is mediated by synthesis of Cytochrome C Oxidase 2 (SCO2). SCO2 is necessary for construction of the mitochondrial cytochrome c oxidase (COX) complex, the major site of oxygen consumption in eukaryote cells.(169) Mutant p53 has also been demonstrated to promote glucose influx to cancer cells via increased transcription of type II hexokinase, an enzyme that converts glucose to glucose-6-phosphate, the initial step in glycolysis.(170) The ability of cancer cells to adapt to the tumour microenvironment by upregulating glycolysis confers an evolutionary survival advantage but overcoming problems with energy production alone is not the only adaptation required by malignant cells.