oxygen - sensing mechanisms enable the cell to adapt to low oxygen environments and are also critical for normal development and apoptosis .
disruptions of oxygen - sensing pathways can lead to the development of certain forms of cancer [ 115 ] .
oxygen sensing is mediated partly via the egln hydroxylases ( egln1/egln2/egln3 ) that are dependent upon molecular oxygen , iron ( ii ) and -ketoglutarate to perform proline hydroxylation on their target [ 1620 ] .
therefore these enzymes are considered oxygen and also metabolic sensors . the availability of molecular oxygen is absolutely required for the hydroxylation reaction , because it donates the oxygen atom to the hydroxyl group .
but equally important for the hydroxylation reaction is the electron donor -ketoglutarate , a metabolite from the krebs cycle . since egln is dependent upon metabolites that take part in the krebs cycle , they are directly tied into the cellular metabolic network ( fig .
egln prolyl hydroxylases ( also referred to as phd ) belong to the 2-oxoglutarate - dependent oxygenase superfamily .
the reaction proceeds via the incorporation of dioxygen into the prolyl residue forming hydroxyproline at the substrate . as electron donor for this reaction serves -ketoglutarate ( = 2-oxoglutarate ) which is oxidized to succinate and co2 .
the dependency of the metabolite -ketoglutarate for catalytic activity makes these enzymes sensitive to -ketoglutarate levels and ties them directly into the metabolic network .
the first identified substrate for the egln prolyl hydroxylases is the transcription factor hypoxia inducible factor  ( hif- ) [ 16 , 17 ] .
the identification of hif- as a direct hydroxylation substrate provided the first and direct link between tumour suppressor function and oxygen sensing .
the tumour suppressor von hippel lindau ( vhl ) acts as an ubiquitin ligase by targeting hydroxylated hif- for degradation when oxygen is available , as where under low oxygen environments , hif- escapes hydroxylation and subsequently escapes vhl recognition [ 35 , 16 , 2227 ] .
the escape from vhl recognition allows hif- to accumulate and to transactivate its target genes that are important for adaptation to low oxygen environment including energy metabolism and angiogenesis [ 2830 ] .
both hif - dependent and hif - independent vhl functions contribute to vhl - defective tumorgenesis [ 3134 ] .
the vhl disease is caused by inactivating germline mutations of the vhl gene and predisposes to a variety of tumours including haemangioblastoma of the retina and nervous system , clear cell renal carcinomas ( rcc ; the most common form of kidney cancer ) and phaeochromocytomas / paragangliomas , tumours of the sympathoadrenal nervous system .
hif- deregulation appears to have a causal role in vhl - defective clear cell rcc and in vhl - defective blood vessel tumours haemangioblastomas ( hb ) .
although hif- regulation has been a major focus of vhl research , the genotype  phenotype correlation in the vhl disease gave insight into hif - independent vhl function ( table 1 ) .
specific vhl germline mutations corresponding to a specific subset of tumour phenotypes have a different relationship to hif- deregulation ( categorized in type i / iia / iib / iic disease ) [ 3537 ] .
the type i disease associates with rcc and hb , and their tumours reflect deregulated high hif- expression , whereas a very different clinical outcome is observed in the type 2c - vhl disease .
more importantly , hif- is not deregulated and maintains in low levels in the type 2c tumours [ 31 , 32 ] .
these findings suggest that hif- deregulation is not necessary for phaeochromocytma development in vhl disease .
phenotype correlation in the vhl disease interestingly , a second hydroxylation target of the prolyl hydroxylases egln has recently been identified .
the 2-adrenergic receptor is a prototypic g protein - coupled receptor , which is hydroxylated by egln3 and also oxygen dependent degraded by pvhl .
the discovery of another hydroxylation substrate by the egln enzymes not only broadens the functionality of prolyl hydroxylation , but also expands our understanding of cellular response to oxygen and its relationship to disease .
therefore , the identification of other egln substrates and the clues from the genotype  phenotype correlation that emerged in the vhl disease clearly indicates hif - independent vhl functions . of great insights
are studies from egln3-mediated neuronal apoptosis during sympathetic neuronal development , which shed light onto the genesis of phaeochromocytoma by presumably hif - independent pathways .
phaeochromocytomas are rare , with only five to eight cases diagnosed per million people a year .
extra - adrenal phaeochromocytomas are sometimes referred to as paragangliomas . in short , these tumours are sympathetic nervous system tumours .
the most frequent causes of phaeochromocytoma susceptibility are vhl missense mutations , activating mutations in c - ret , mutations in neurofibromatosis type 1 ( nf1 ) and mutations in succinate dehydrogenase subunits of mitochondrial complex ii ( sdh b / c / d ) .
loss of nf1 has been reported to promote survival of embryonic sympathetic neurons in the absence of the nerve growth factor ngf .
further , mutations in subunits of mitochondrial complex ii suggest impairment of mitochondria - mediated apoptosis and have led to the early hypothesis that failure of apoptosis in neuroendocrine precursor cells could result in the development of phaeochromocytoma and paraganglioma . given the evidence that nf1 promotes sympathetic neuronal survival and further that these neoplasias originate from the sympathetic nervous system , a closer look into the sympathetic neuronal development reveals important clues to the genesis of these tumours . since the discovery by rita levi - montalcini and viktor hamburger of the neurotrophic factor ngf , our understanding of developmental apoptosis in the sympathetic nervous system has greatly increased . during neuronal development
competition for ngf is an important developmental process for matching the size of a neuronal population , especially in the peripheral nervous system .
as much as 50% of neurons produced during embryogenesis die by apoptosis during neuronal development .
abnormal ngf signalling has been linked to paediatric nervous system tumours such as neuroblastoma [ 44 , 45 ] and disease - associated mutations such as nf1 have been shown to enhance signalling by ngf receptors and promote neuronal survival in the absence of ngf . in the last decades it became evident that jnk / c - jun signalling is required for apoptosis when ngf is limiting in the developing nervous system [ 4650 ] .
egln3 is induced in sympathetic neurons deprived of ngf and further has pro - apoptotic activity when overexpressed .
given that egln3 is known to hydroxylate hif- and has been implicated in developmental apoptosis in sympathetic neurons , the following questions arise : ( i ) does egln3-mediated apoptosis depend upon its hydroxylation activity ?
( ii ) does it depend upon hif hydroxylation or does it involve hydroxylation of unknown substrates ? ( iii ) is failure of egln3-mediated apoptosis implicated in the genesis of phaeochromocytomas and other tumours arising from the neural crest origin ?
the ability of egln3 to induce neuronal apoptosis appears to be unique among the egln family members .
induction of apoptosis is dependent upon egln3 hydroxylation activity , because catalytic impaired egln3 fails to induce apoptosis [ 9 , 52 ] .
importantly , egln3-induced apoptosis is not diminished in the presence of stable hif1 or hif2 variants that can not be hydroxylated on their prolines .
this suggests that hydroxylation targets of egln3 other than hif- are crucial for apoptosis function .
egln2 and egln3 are induced by hypoxia and dampen the hif- response under chronic hypoxia [ 5357 ]
. however , egln1 appears to be the primary hif prolyl hydroxylase under normal conditions .
consistent with this , mice lacking egln2 or egln3 are viable and grossly normal whereas mice lacking egln1 are not viable .
conditional inactivation of egln1 in mice leads to polycythemia due to hif- stabilization and increased transcription of hif target genes including erythropoietin [ 60 , 61 ] .
further , patients carrying egln1 mutations have been reported to develop polycythemia concluding that egln1 couples hif- stability in vivo[13 , 62 , 63 ] .
consistent with the unique role of egln3 in neuronal apoptosis are studies obtained from the egln3-deficient mice that clearly demonstrates the requirement for egln3-mediated apoptosis during the sympathetic neuronal development .
egln3 deficient sympathetic neurons are resistant to apoptosis after ngf withdrawal , as well as certain neurotoxins .
consistently , egln3 mice have an increased number of cells in the super cervical ganglia and in the adrenal medulla and show abnormalities in the sympathoadrenal system including systemic hypotension .
in fact , it appears that some of the adrenal abnormalities are caused through deregulation of the 2-adrenic receptor ( 2-ar ) .
loss of egln3 results in 2-ar up - regulation and accordingly , hypoxia stabilizes the 2-ar .
this is consistent with the observations from the type 2c vhl patients that develop phaeochromocytoma .
type 2c patients often show excessive secretion of catecholamines ( endogenous 2-ar ligands ) and increased sympathonervous system activity . in summary
, this points towards distinct and unique functions within the family of egln prolyl hydroxylases .
identification of novel egln hydroxylation targets will open new oxygen sensing pathways independent of what we have learned from hif-.
given the evidence that ( i ) phaeochromocytomas are sympathetic nervous system tumours and that ( ii ) egln3 is critical for apoptosis during sympathetic neural development , an attractive hypothesis emerged in which failure of egln3-mediated apoptosis during sympathetic development predisposes to the genesis of phaeochromocytomas and perhaps other neoplasia arising from neural crest origin .
interestingly , apparently unlinked phaeochromocytoma lesions ( vhl , nf1 , c - ret and sdh ) all appear to act on a single common pathway by decreasing egln3-mediated apoptosis at the time during development when levels of ngf become limiting ( fig .
egln3 appears to act downstream of the c - jun in the ngf signalling pathway .
therefore , a single pathway was established in which the genetic phaeochromocytoma defects act either directly on egln3 or upstream of egln3 to impair apoptosis ( fig .
2 ) . for instance , the genetic defect in sdh acts directly on egln3-mediated apoptosis function .
sdh inhibition results in accumulation of intracellular succinate , which in turn can product - inhibit the egln prolyl hydroxylases [ 9 , 71 ] .
this succinate - mediated inhibition not only results in hif stabilization , but also importantly inhibits egln3-mediated apoptosis .
the succinate - mediated product - inhibition of egln3 was overcome by re - addition of -ketoglutarate restoring ngf - mediated apoptosis .
this suggests that sdh inhibition acts on the prolyl hydroxylases via succinate and not as alternatively suggested through the generation of reactive oxygen species .
, ngf is required for neuronal survival but is also limiting . neurons that successfully compete for ngf survive whereas unsuccessful neurons undergo c - jun - dependent apoptosis .
c - jun transcriptionally activates prolyl hydroxylase egln3 . as ngf levels become limiting , familial phaeochromocytoma mutations ( vhl , nf1 , c - ret , sdh and kif1b ) all decrease apoptosis mediated by egln3 .
in addition to the predisposing sdh genetic lesion , other phaeochromocytoma lesions have been implicated upstream of egln3 to promote neuronal survival in the ngf signalling pathway . in the case of vhl predisposing lesion ,
pvhl suppresses junb and all vhl mutants tested ( including type iic vhl disease mutant ) abrogate its ability to do so .
junb acts as an antagonist of c - jun and increased junb levels attenuate the induction of c - jun - mediated apoptosis in sympathetic neurons deprived from ngf .
finally , previous evidence indicated that ret ( the receptor for glial - derived neurotrophic factor ) and nf1 could act through this pathway by modulating the action of the ngf receptor trka .
activation of c - ret , like loss of pvhl , leads to the induction of junb and attenuates apoptosis when ngf becomes limiting .
thus , when mutated , all the genetic phaeochromocytoma lesions ( sdh , vhl , c - ret and nf1 ) impair ngf - mediated apoptosis in neuroendocrine precursors during development .
these findings provide an explanation as to why the mutations in tumour suppressors that are found in familial phaeochromocytoma are rare in sporadic phaeochromocytoma .
this is because the pathway is no longer critical once development is completed . in summary ,
all the genetic lesions associated with phaeochromocytoma act on a single common pathway that impinges upon egln3 apoptotic activity during sympathetic neuronal development .
mutations in sdh , vhl , c - ret and nf1 would allow sympathetic neuronal precursors to escape from developmental apoptosis and set the stage for their neoplastic transformation
. it will be interesting to determine not only if egln3 is mutated in these neoplasias , but also , if failure of egln3 developmental apoptosis plays a broader role in paediatric cancers and other forms of hereditary cancer .
an important challenge is to identify the link between this enzyme and apoptosis , which presumably involves hydroxylation of a protein other than hif-. early studies indicated that egln3 mrna and protein expression ( at that time referred as sm-20 ) increases shortly after removal of ngf in primary sympathetic neurons and peaks between 10 and 15 hrs at a time when cells undergo apoptosis .
overexpression of egln3 is sufficient to promote cell death in ngf - maintained sympathetic neurons [ 51 , 73 ] .
cell death is caspase dependent and accompanied by an increase of cytochrome c in cytosolic and mitochrondria - enriched subcellular fractions .
the mechanism underlying egln3-induced cytochrome c is not known although it appears to involve an increase in cytochrome c mrna .
other studies have identified egln3 as a growth factor inducible gene in vascular smooth muscle cells   and later proposed , that it might function in growth arrest , differentiation and cell death during muscle differentiation [ 76 , 77 ] .
expression of egln3 was also reported in fibroblasts upon activation of a temperature - sensitive form of p53
. however , egln3-induced apoptosis appears not to be impaired in cells lacking p53 or expressing a dominant negative p53 protein , indicating that p53 might not function downstream of egln3 to induce apoptosis . in addition , a recent study indicated that egln3 s ability to induce apoptosis correlates with the formation of aggresome - like structures .
recently , an unbiased genome - wide approach has been undertaken to understand how egln3 causes neuronal cell death .
this led to the identification of the kinesin kif1b as a downstream effector of egln3 .
kif1b , a member the kinesin 3 family , consists of two major splice variants  and . kif1b and kif1b are motor proteins implicated in anterograde transport of mitochondria and synaptic vesicle precursors , respectively [ 79 , 80 ] . the recent study , which identified kif1b as an egln3 downstream target showed how kif1b is both necessary and sufficient for neuronal apoptosis when ngf becomes limiting , but it remained unclear how egln3 regulates kif1b. interestingly , kif1b maps on to the chromosomal region 1p36.2 , a region of the genome that is frequently deleted in neural crest - derived tumours including neuroblastomas .
the existence of one or more human tumour suppressor genes on chromosome 1p has been suspected for decades [ 8284 ] .
further suggestion that kif1b acts as a 1p36 tumour suppressor comes from the current model for phaeochromocytoma development .
phaeochromocytomas develop when sympathetic neuronal precursors escape from egln3-mediated developmental apoptosis , suggesting that mutations in kif1b may be relevant to phaeochromocytoma and other tumours of neuronal origin .
indeed , inherited loss - of - function kif1b missense mutations have been identified in phaeochromocytomas and neuroblastomas and an acquired loss - of function mutation in a medullablastoma , arguing that kif1b is a pathogenic target of these deletions .
nonetheless , it has been further reported that the remaining kif1b allele in 1p deleted tumours and cell lines is often wild - type , contrary to the knudson twohit scenario [ 64 , 8588 ] .
also , the existence of multiple neuroblastoma and phaeochromocytoma suppressor genes on 1p has been suggested [ 89 , 90 ] .
additional studies are needed to address how often it is deregulated , epigenetically or genetically , in cancer .
a spotlight is now on understanding the mechanistic basis of how egln3 regulates kif1b and how this translates into cell death .
kif1b appears not be a direct egln3 hydroxylation target , but egln3 hydroxylation activity is required for kif1b regulation .
therefore egln3 presumably involves hydroxylation of a protein that in turn might regulate kif1b to induce apoptosis .
in 1924 , otto warburg observed that cancer cells are highly glycolytic in the presence of oxygen and have reduced rates of oxidative phosphorylation .
recent studies have argued that cancer cells might benefit from this persistence of high lactate production in the presence of oxygen , referred to as aerobic glycolysis or pseudo - hypoxia [ 92 , 93 ] . from a bioenergetic perspective
, it remains a conundrum how highly metabolic proliferative cancer cells undergo a pathway that results in 10 times less atp production compared to their normal counterparts that oxidize their glycolysis endproduct within the mitochondria via the krebs cycle ( pyruvate conversion into acetyl - coa , fig .
interestingly , three major enzymes of the krebs cycle ( sdh , fh , idh ) have been recently identified as bonafide tumour suppressors , but more importantly , inactivation in either of them directly affects egln activity [ 9 , 10 , 14 , 71 ] .
this is because the egln activity is not only dependent upon molecular oxygen to perform their hydroxylation reaction , but also require the krebs cycle metabolite -ketoglutarate as electron donor , which ties them directly into this metabolic network .
therefore an attractive hypothesis arises : cancer cells favour aerobic glycolysis to inhibit egln activity in order to escape either certain oncogenic apoptotic signals and/or activate oncogenic hif-. about 70% or more of low - grade gliomas bear loss of function mutation in idh1 and idh2 [ 94 , 95 ] .
subsequently egln prolyl hydroxylases are inhibited in their hydroxylase activity with the consequences of hif- stabilization .
presumably egln3-mediated apoptosis might be impaired in these settings as well , but this has not been investigated yet .
further , these studies follow the discoveries that germline mutation in sdh are linked to phaeochromocytoma and that fh mutation lead to leiomyosarcoma and renal cell carcinoma , both of which affect also the egln prolyl hydroxylase activity [ 9 , 10 , 71 ] .
this is because loss of function mutation in sdh and fh increases the accumulation of succinate and fumarate respectively .
the excess of these metabolites inhibits the proline hydroxylases with the consequences in either accumulation of oncogenic hif- and/or blunting egln3-mediated apoptosis [ 9 , 10 , 71 , 96 ] .
the krebs cycle enzymes ( sdh , fh and idh ) are tumour suppressors and linked to egln activity .
the egln prolyl hydroxylases are dependent upon the krebs cycle metabolite -ketoglutarate as electron donor for hydroxylation activity .
loss of function mutation in idh decreases -ketoglutarate levels and subsequently impairs egln function . on the other hand , mutation in sdh or fh
leads to accumulation of succinate or fumarate respectively , which in turn product - inhibits egln activity .
loss of function of sdh , fh or idh would lead to impaired krebs cycle activity and impaired oxidative phosphorylation with the consequence of enhanced glycolysis ( embden  meyerhof pathway ) to generate atp .
the generated pyruvate during glycolysis is redirected away from the krebs cycle by conversion into lactate .
cancer cells tend to convert most glucose to lactate regardless of whether oxygen is present ( aerobic glycolysis ) , an observation that was first made by otto warburg in 1924 . despite the striking difference in atp production
, cancer cells might favour aerobic glycolysis to escape from egln hydroxylation , resulting in the accumulation of oncogenic hif. and/or resistant to egln3-mediated apoptosis .
the recently identified mutations in the metabolic / mitochondrial enzymes ( fh , sdhb / c / d , idh1/2 ) provide convincing evidence that alteration in cellular metabolism contributes to the pathogenesis of cancer .
hence , 43 years later we are beginning to learn the depth of otto warburg s foresight that ( in his words )  cancer cells should be interpreted as a mitochondrial dysfunction and that  the prime cause of cancer is the replacement of the respiration of oxygen in normal body cells by a fermentation of sugar[97 , 98 ] .
even if this is the prime cause for some cancers , the underlying mechanistic basis remains controversial .
however , recently it has become more evident that the inactivation of the egln prolyl hydroxylases is a downstream effector of these mitochondrial alterations .
the identification of sdh and fh mutations has already led to the development of cell permeable -ketoglutarate derivates with the potential to suppress the transforming effects of these mutations through reactivation of the eglns .
the exciting part of identifying metabolic - enzyme mutations in specific cancers is that they are 
druggable. they might provide new opportunities as therapeutic targets that would be more susceptible and more effective than existing cancer therapies .
future work is needed to further elucidate the direct impact of cancer metabolism in prolyl hydroxylase functioning .
 introductionlessons from a rare diseasespecificity of function within the egln prolyl hydroxylasesfailure of egln3-mediated apoptosis in the genesis of phaeochromocytomaunderstanding the mechanistic basis of egln3 killingconnecting egln activity to the warburg conundrumfuture directionsoxygen - sensing mechanisms are often dysfunctional in tumours . 
 oxygen sensing is mediated partly via prolyl hydroxylation . 
 the egln prolyl hydroxylases are well characterized in regulating the hypoxia inducible factor  ( hif- ) hypoxic response , but also are implicated in hif - independent processes . 
 egln3 executes apoptosis in neural precursors during development and failure of egln3 developmental apoptosis can lead to certain forms of sympathetic nervous system tumours . 
 mutations in metabolic / mitochondrial enzymes ( sdh , fh , idh ) impair egln activity and predisposes to certain cancers . 
 this is because the eglns not only require molecular oxygen to execute hydroxylation , but also equally require the electron donor -ketoglutarate , a metabolite from the krebs cycle . 
 therefore egln enzymes are considered oxygen , and also , metabolic sensors . 
 -ketoglutarate is crucial for egln hydroxylation activity , whereas the metabolites succinate and fumarate are inhibitors of the egln enzymes . 
 since egln activity is dependent upon metabolites that take part in the krebs cycle , these enzymes are directly tied into the cellular metabolic network . 
 cancer cells tend to convert most glucose to lactate regardless of whether oxygen is present ( aerobic glycolysis ) , an observation that was first made by otto warburg in 1924 . despite the striking difference in atp production 
 , cancer cells might favour aerobic glycolysis to escape from egln hydroxylation , resulting in the accumulation of oncogenic hif and/or resistance to egln3-mediated apoptosis . 