tumor hypoxia tumor hypoxia tumor hypoxia is the situation where tumor cells have been deprived of oxygen. as a tumor grows, it rapidly outgrows its blood supply, leaving portions of the tumor with regions where the oxygen concentration is significantly lower than in healthy tissues. hypoxic microenvironements in solid tumors are a result of available oxygen being consumed within 70 to 150 μm of tumour vasculature by rapidly proliferating tumor cells thus limiting the amount of oxygen available to diffuse further into the tumor tissue. in order to support continuous growth and proliferation in challenging hypoxic environments, cancer cells are found to alter their metabolism. furthermore, hypoxia is known to change cell behavior and is associated with extracellular matrix remodeling and increased migratory and metastatic behavior. tumour stroma and extracellular matrix in hypoxia changes in the glycolytic pathway a particular change in metabolism, historically known as the warburg effect results in high rates of glycolysis in both normoxic and hypoxic cancer cells. expression of genes responsible for glycolytic enzymes and glucose transporters are enhanced by numerous oncogenes including ras, src, and myc. hif-1 induced changes in gene expression hif regulates interactions of cancer cells with ecm and ecm biosynthesis during cancer progression, tumor cells acquire comprehensive metabolic reprogramming, and tissue hypoxia is a prominent feature of solid tumors leading to cell metabolism adaptive changes. hypoxia inducible factor-1α (hif-1α) is a key oxygen-regulated transcriptional activator, playing a fundamental role in the adaptation of tumor cells to hypoxia by upregulating the transcription of target genes related to multiple biological processes, including cell survival, proliferation, angiogenesis and anti-apoptosis. significant hif1a expression has been noted in most solid tumors studied, which include cancers of the gastric and colon. these genes include: solute carrier family 2 (glut1), hexokinase (hk), phosphoglucose isomerase (pgi), phosphofructokinase (pfkl), fructose-bisphosphate aldolase (aldo), glyceraldehyde-3-phosphate dehydrogenase (gapdh), phosphoglycerate kinase (pgk), phosphoglycerate mutase (pgm), enolase 1 (enoa), pyruvate kinase (pk), pyruvate dehydrogenase kinase, isozyme 1 (pdk1) and lactate dehydrogenase a (ldh-a). in addition to alterations in oxygen concentration associated with hypoxic microenvironments, glucose concentration gradients found in tumors also influence the rate of aerobic and anaerobic glycolysis. a carbohydrate-response element (chore) is responsible for regulating glycolytic enzyme gene expression in response to changing glucose concentrations through a binding interaction at the same consensus sequence as hif-1. interactions of hif-1 and chore with the dna sequence 5’-rcgtg-3’ leads to increased expression of genes listed above. glut1 transporter expression glut1 glucose transporter glut1 is a member of the glut transporter family of 14 hexose transporters responsible for facilitating the transport of hexose sugars along the concentration gradient. glut1 is the most abundantly expressed of the family thought to maintain basal glucose transport in almost all cell types. glut1 levels, in response to hypoxic conditions, have been shown to increase with changes at both the mrna and protein levels. moreover, transport of glut1 has been shown to increase under these hypoxic conditions. with the role of transporting sugars from the extracellular to the intracellular environment, glut1, along with other members of the glut family, can be rate-controlling for cellular glycolytic metabolism. having an increased level of glut1, in the case of hypoxic tumors, increases the flux of glucose into the cells allowing for a higher rate of glycolysis and thus greater risks of metastasis (as elaborated upon below). hexokinase 2 expression hexokinase (hk) is the first enzyme in the glycolytic pathway converting glucose to glucose-6-phosphate through an atp-dependent phosphorylation event. important for glycolysis to proceed, the hexokinase reaction activates glucose for subsequent steps. in hypoxic tumors, hexokinase mrna abundance is significantly increased as well as protein levels. increased expression of hexokinase 2, in some cases nearly 10-fold, allows for an increased flux of glucose through the glycolytic pathway subsequent to the increased uptake by glut1.< d-glucose hexokinase expression up-regulated by hif-1 α-d-glucose 6-phosphate atp adp po3-4 h2o glucose 6-phosphatase phosphoglucose isomerase expression phosphoglucose isomerase (pgi) is a housekeeping cytosolic enzyme with roles in both glycolysis and gluconeogenesis pathways. it is responsible for catalyzing the interconversion of glucose 6-phosphate and fructose 6-phosphate. extracellularly, pgi is known as an autocrine motility factor (amf) eliciting mitogenic, motogenic, differentiation functions as well as tumor progression and metastasis. activation of pgi through proposed hif-1 induced mechanisms results in increased conversion of glucose 6-phosphate to fructose 6-phosphate and also contributes to cell motility and invasion during cancer metastasis. α-d-glucose 6-phosphate phosphoglucose isomerase expression up-regulated by hif-1 β-d-fructose 6-phosphate phosphoglucose isomerase 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatases expression regulatory pathway of pfk-1 by fructose-2,6-bisphosphate 6-phosphofructo-2-kinases/fructose 2,6-bisphosphatases (pfkfbs) belong to a family of bifunctional atp-dependent enzymes responsible for controlling the level of glycolysis intermediate fructose-1,6-bisphosphate. hif-1-induced expression of these enzymes (pfk-2/fbpase-2) subsequently alters the balance of fructose-2,6-bisphosphate which plays an important role as an allosteric activator of phospho-fructokinase 1 (pfk-1). pfk-1 is an enzyme that controls one of the most critical steps of glycolysis. regulation of pfk-1 is also mediated by the cellular energy status in result of atp's inhibitory effect. greater quantities of fructose-2,6-bisphosphate in cancer cells, in result of hif-1 expression of pfk-2/fbpase-2, thus activates pfk-1allowing for an increased glycolytic flux converting fructose-6-phosphate to fructose-1,6-bisphosphate. allosteric regulation of glycolysis by fructose-2,6-bisphosphate allows cancer cells to maintain a glycolytic balance to match their bioenergetic and biosynthetic demands. β-d-fructose 6-phosphate (f6p) phosphofructokinase (pfk-1) expression up-regulated by hif-1 β-d-fructose 1,6-bisphosphate (f1,6bp) atp h+ + adp fructose-1,6-bisphosphate aldolase expression fructose-1,6-bisphosphate aldolase (aldo) belongs to a family include aldolase a, b and c. unique in glycolysis, aldolase enzymes cleave fructose-1,6-bisphosphate into two 3-c molecules including glyceraldehyde-3-phosphate (gap) and dihydroxyacetone phosphate (dhap). with the hif-1 mediated expression of aldolase a under hypoxic conditions, the catalysis of fructose-2,6-bisphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate is increased thus leading to increased glycolytic flux. β-d-fructose 1,6-bisphosphate (f1,6bp) fructose-1,6-bisphosphate aldolase expression up-regulated by hif-1 d-glyceraldehyde 3-phosphate (gadp) dihydroxyacetone phosphate (dhap) + glyceraldehyde-3-phosphate dehydrogenase expression the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (gapdh), is responsible for the oxidative conversion of glyceraldehyde-3-phosphate (gadp) to 1,3-bisphosphoglycerate (1,3bpg). up-regulation of glyceraldehyde-3-phosphate dehydrogenase expression is maximal (4-5 fold) following hypoxic conditions of ~24 hours in vascular endothelial cells. various models for the exact glyceraldehyde-3-phosphate dehydrogenase activation mechanisms have been proposed. glyceraldehyde 3-phosphate (gadp) glyceraldehyde phosphate dehydrogenase expression up-regulated by hif-1 d-1,3-bisphosphoglycerate (1,3bpg) nad+ + pi nadh + h+ phosphoglycerate kinase 1 expression hypoxia has been shown to induce a 10-fold accumulation of phosphoglycerate kinase 1 (pgk-1) mrna in mouse hepatoma (hepa 1c1c7) cells. phosphoglycerate kinase 1 is an enzyme involved in the conversion of 1,3-bisphosphoglycerate (1,3-bpg) to 3-phosphoglycerate (3-p-g) leading the production of atp from adp. induction of gene expression by hif-1 is thought to be dependent on the presence of aromatic hydrocarbon receptor nuclear translocator (arnt1). arnt's n-terminal region and hif-1 are thought to work together to induce transcription of phosphoglycerate kinase 1. 1,3-bisphosphoglycerate (1,3-bpg) phosphoglycerate kinase expression up-regulated by hif-1 3-phosphoglycerate (3-p-g) adp atp phosphoglycerate kinase phosphoglycerate mutase expression phosphoglycerate mutase b (pgm-b) is one of the latter glycolytic enzymes responsible for the conversion of 3-phosphoglycerate (3pg) to 2-phosphoglycerate (2pg). both protein and mrna levels were shown to increase 2-3-fold in research exposing fetal rat lung fibroblasts to hypoxic conditions. increased levels appeared to be regulated at the transcriptional level as per many of the other glycolytic enzymes. maximum up regulation was shown following 16 hours thus supporting its role in contributing to an increased glycolytic flux for adaption of cells to hypoxia. 3-phosphoglycerate (3pg) phosphoglycerate mutase expression up-regulated by hif-1 2-phosphoglycerate (2pg) enolase 1 expression enolase 1, also known as α-enolase, is encoded by the enoa gene and is responsible for converting 2-phosphoglycerate to phosphoenolpyruvate in the glycolytic pathway. both enolase 1 overexpression and its post-translational modifications could be of value for diagnostic and prognostic work in terms of cancer. although the exact roles of post-translational modifications have not been completely elucidated, patterns are shown between certain cancer cell types suggesting they may have important influence on function, localization and immunogenicity. aside from its role in promoting glycolytic flux and anaerobic energy production, it has been shown to induce a specific humoral and cellular immune response. on all levels, hypoxia-induced over-expression of enolase 1 may possess important roles in hypoxic tumors including the most straightforward increase in anaerobic respiration. 2-phosphoglycerate (2pg) enolase 1 expression up-regulated by hif-1 phosphoenolpyruvate (pep) h2o enolase 1 pyruvate kinase expression hif-1 activated pyruvate kinase m comes in multiple isoforms known as pkm1 and pkm2. pyruvate kinase is shown to convert phosphoenolpyruvate to pyruvate forming atp from adp. along with phospho-fructokinase 1, pyruvate kinase is also allosterically activated by fructose-2,6-bisphosphate. in cancer cells pyruvate kinase m2 has been shown to interact directly with hif-1α enhancing hif-1 binding and p300 recruitment to hypoxia response elements. this positive feedback loop leads to hif-1 transactivation and an amplified effect on glucose metabolism. pyruvate kinase m2 is often considered the main regulator of cancer metabolism with roles in various parallel, feed-forward, positive and negative feedback mechanisms. the genetic difference between pyruvate kinase m1 and pyruvate kinase m2 is only 22 out of 531 amino acids which makes an immense difference. pyruvate kinase m2 has metabolic activity regulated by post-translational modifications including acetylation, oxidation, phosphorylation, hydroxylation and sumoylation. these different modifications can cause the shift from the metabolically active tetrameric form to the in-active monomeric form. the well-known egfr-activated extracellular signal-regulated kinase 2 (erk2) and death-associated protein kinase are both shown to bind and directly phosphorylate pyruvate kinase m2 leading to increased activity in the glycolysis pathway. in hypoxic conditions found in a solid tumor, pyruvate kinase m2 plays a large role in promoting anaerobic energy production. phosphoenolpyruvate (pep) pyruvate kinase expression up-regulated by hif-1 pyruvate (pyr) adp + h+ atp pyruvate dehydrogenase kinase expression the areas surrounding the phosphorylation sites on pyruvate dehydrogenase are shown in red. pyruvate dehydrogenase kinase phosphorylation of these sites leads to decreased dehydrogenase activity pyruvate dehydrogenase directly follows the glycolytic pathway and is responsible for the conversion of pyruvate to acetyl-coa which enters into the tca cycle. the tca cycle, although not directly requiring oxygen, requires the cycling of nadh to nad+ as performed by the electron transport chain under aerobic conditions. under anaerobic conditions, such as those found in hypoxic tumors, the tca cycle provides little atp yield due to the lack of electron transport chain function. in order to direct the glycolytically produced pyruvate away from the tca cycle, pyruvate dehydrogenase kinase is over-expressed in response to hypoxic conditions. pyruvate dehydrogenase kinase is not a glycolytic enzymes but more of a glycolytic regulator. pyruvate dehydrogenase kinases, transcriptionally activated by hif-1 in hypoxic conditions, are responsible for phosphorylating the e1 subunit of pyruvate dehydrogenase ultimately suppressing its function. by inhibiting this specific pathway, the glycolytic products are directed away from the mitochondrial tca cycle and towards lactate dehydrogenase. lactate dehydrogenase expression activated expression of lactate dehydrogenase a (ldh-a), parallels with deactivation of pyruvate dehydrogenase mediated by pyruvate dehydrogenase kinase. subsequent inactivation of pyruvate dehydrogenase following phosphorylation and increased expression of lactate dehydrogenase a shunts pyruvate away from the mitochondrial tca cycle. in many different tumor types lactate dehydrogenase a is found at elevated levels and has even been linked to poor prognosis and a greater metastatic potential the high levels of lactate production surface the question of whether lactate has some influence on the aggressive behaviour shown in hypoxic tumors. pyruvate lactate dehydrogenase expression up-regulated by hif-1 lactate nadh nad+ lactate dehydrogenase overview of glycolytic changes and consequences overview of the hif-1 effect on the expression of glycolytic enzymes increased expression of almost every glycolytic enzyme is seen in hypoxic tumor conditions. the over-expression of these proteins is mediated by hif-1 and completely alters normal cellular metabolism. with decreases in the rate of mitochondrial oxidation, lactate and protons begin to accumulate. high levels of glycolysis and the production of lactate, as shown in hypoxic tumor cells, is hallmark of cancer cells even in the presence of oxygen. to relieve tumors cells of acidosis, carbonic anhydrases appear to be highly expressed once again downstream of hif-1 activation. these enzymes catalyze the reversible hydration of carbon dioxide into bicarbonate and protons. they also assist in acidifying the extracellular environment and maintaining a slightly alkaline intracellular compartments contributing to tumor cell survival. lactate from the hypoxic tumor cells is excreted to the surrounding environment by carbonic anhydrase 9 and sodium-hydrogen exchanger 1 mct4. local aerobic cancer