von hippel - lindau ( vhl ) disease is a rare inherited syndrome which predisposes individuals to a variety of malignant and benign tumors including renal cell carcinoma and pheochromocytoma ( pcc ) .
renal cell carcinomas are cancers of the kidney that account for approximately 102,000 deaths worldwide each year [ 2 , 3 ] .
renal clear cell carcinoma ( rcc ) , arising in the proximal convoluted tubules of the kidney transport system , is the most common subtype of renal cell carcinomas ( comprising about 88% of tumors ) and is tightly associated with inactivating mutations of the vhl gene [ 4 , 5 ] .
pcc , the other principal vhl - related cancer , is a rare catecholamine - secreting cancer originating in chromaffin cells of the adrenal gland [ 68 ] .
although these tumors can be benign , the malignancy rate ranges from 10 to 15% .
malignant pcc is identified histologically by the presence of metastasis ( commonly to lymph nodes , liver , lungs , and bone ) .
therefore , a greater understanding of the biology underlying pcc is needed in order to advance diagnostic testing and prognosis . in approximately one - third of cases
, pcc arises in patients with germ - line mutations in predisposing genes such as vhl , nf-1 , men2/ret , and sdh subunits , tmem127 , max , or hif2a , among others [ 1214 ] .
studies indicate that vhl is among the most frequently targeted genes in pcc , mostly affected by genetic mechanisms such as mutations and loss of heterozygosity ( loh ) [ 1517 ] . in keeping with knudson 's two - hit hypothesis , tumors from patients who have a germ - line mutation in one vhl allele are susceptible to somatic inactivation of the remaining allele .
indeed , studies show that a somatic  second hit ,  which can arise through epigenetic or genetic mechanisms , results in a loss of vhl gene expression , abnormal vhl protein function , and consequent tumorigenesis [ 1921 ] .
vhl is a component of the vhl elongin bc complex  composed of the proteins vhl , cul2 or cul5 , rbx1 , and elongin b / elongin c ( elongins b and c are encoded by tceb1 and tceb2 , resp . ) .
this complex acts as an e3 ubiquitin - ligase and drives the proteasomal degradation of targeted proteins [ 22 , 23 ] .
the hypoxia - inducible factor 1 ( hif1- , encoded by hif1a ) , the primary target of this complex , regulates over 80 genes associated with tumor progression , glycolysis , angiogenesis , and metastasis and is negatively regulated by the vhl elongin bc complex [ 24 , 25 ] .
hypoxia inducing factor 1 ( hif1 ) is composed of an alpha subunit , which is negatively regulated by the vhl elongin bc complex and a beta subunit , which is constitutively expressed . under normoxic conditions ,
the hydroxylation of hif1- at two prolyl residues ( p402 and p564 ) by phd - containing proteins creates a binding site for vhl and results in proteasomal degradation of hif1- ( figure 1(a ) ) [ 2729 ] . in hypoxic conditions ,
phd - containing proteins no longer hydroxylate hif1- and vhl can not add destabilizing ubiquitin polymers to hif1-. hif1- can then heterodimerize with hif1- and translocate into the nucleus where it binds to hypoxia response elements ( hre ) and promotes the expression of genes , such as pdk1 , pfkl , glut1 , and vegf , that mediate the cellular hypoxic response .
genetic alterations affecting vhl or other complex components can lead to abnormal stabilization of hif1- , resulting in aberrant translocation of hif1 to the nucleus and ectopic activation of target genes , such as vegf , pdk , and epo , to elicit a hypoxic response , even in normoxic conditions ( figure 1(c ) ) [ 3032 ] .
previous studies of pcc have reported that disruption of the vhl elongin bc protein complex occurs through gene copy number loss , mutation , or epigenetic silencing of the vhl gene and that this disruption leads to tumorigenesis through activation of hif1 targets [ 16 , 20 , 33 , 34 ] .
the role of the other components of the vhl elongin bc complex is largely uncharacterized . in the present study , we investigated dna - level alterations  gene copy number losses ( cnl ) and promoter hypermethylation  affecting other components of the vhl elongin bc protein complex in pcc .
we assessed the effects of these alterations at the gene expression level and the impact of complex component disruption on enrichment of hif1-target expression .
finally , we explored whether similar disruptions were present in another vhl - inactivated cancer type , rcc .
our results indicate that , while vhl is disrupted in both pcc and rcc , other components of the vhl elongin bc complex , particularly rbx1 and cul5 , are significantly disrupted in pcc and their status might be an important clinical consideration in pcc .
information regarding dna copy number alterations affecting vhl tumor suppressor complex components ( vhl , tceb1 , tceb2 , rbx1 , cul2 , and cul5 ) was obtained from 171 pcc tumors available through the cancer genome atlas project ( tcga ) .
gene dosage alterations were assessed using the affymetrix snp6.0 platform at the broad tcga genome characterization center . processed level 3
briefly , raw copy number data was segmented using a circular binary segmentation algorithm   and mapped to hg18 genome assembly . in order to exclude polymorphic variations , a fixed set of common germ - line copy number variant probes
coordinates were converted to hg19 using a local repository of galaxy , running the liftover utility .
segmented data was loaded into the integrative genomics viewer ( igv ) [ 42 , 43 ] , and information regarding the six complex component genes was exported as a.tdm file .
dna copy number alterations were defined as follows : ( 1 ) dna copy number loss ( signal intensity log2 ratio <
0.3 ) , copy number neutral ( log2 ratio between 0.3 and 0.3 ) , or copy number gain ( log2 ratio > 0.3 ) .
gene - level transcription estimates , in the form of rsem normalized counts , were obtained for the six complex component genes analyzed and were obtained from processed rna sequencing data derived from 171 tumors and 4 adjacent nonmalignant tissues .
gene expression profiles were generated using the illumina hiseq 2000 rna sequencing platform by the university of north carolina and tcga genome characterization center .
individual expression profiles were loaded into igv , and expression information for each gene was exported .
methylation analyses using the illumina infinium humanmethylation450 platform were performed at johns hopkins university , university of southern california , and tcga genome characterization center .
probes mapping to the six complex component genes were extracted . only probes mapping to the promoter region , which are most likely to have an effect on gene expression , were selected for further analysis .
the ratio of the intensity of the methylated bead type to the combined locus intensity ( termed as beta values ( v ) ) was calculated using beadstudio software . to assess the difference in probe methylation between pcc and nonmalignant tissue , a delta beta value ( dv ) was calculated for each probe :
an average v was calculated for each probe in the nonmalignant cohort , and these values were subtracted from the pcc vs on a tumor - by - tumor basis .
data was derived from 171 pcc tumors samples that also contained expression and copy number information .
vhl complex component genes ( cul2 , cul5 , tceb1 , tceb2 , and rbx1 ) as well as 3 genes known to be frequently mutated in pcc ( ret , hras , and nf1 ) were classified as having an inactivating mutation if the result was a frame shift insertion , frame shift deletion , splice site mutation , missense mutation , or a nonsense mutation .
somatic mutation data was also analyzed in the same way in a cohort of 417 out of 522 rcc tumors where dna sequence data was available .
copy number data and mutation data for 522 rcc were downloaded from cbioportal for cancer genomics ( http://www.cbioportal.org ) [ 45 , 46 ] ; of these , 411 samples had concurrent copy number and mutation data .
the same criteria used for pcc were applied to define copy number loss and gain .
mutations with a neutral or low mutation assessor score were not considered in mutation frequency calculations . in order to assess the effect of dna - level alteration on gene expression of the vhl tumor suppressor complex components
, pcc was divided into up to three groups based on copy number alteration status ( copy number loss , copy number neutral , and copy number gain ) and expression was compared between groups using graphpad software v6 . for most genes ( vhl , cul5 , tceb1 , and tceb2 ) three group comparisons were performed using a kruskal - wallis test . since the majority of copy number alterations were cnl rather than gain , a two - group comparison was also performed for each of the six genes comparing cnl and neutral copy number using a mann - whitney u test .
rbx1 did not show copy number gain in any sample ; therefore only a mann - whitney u test was performed for this gene . in all comparisons ,
correlation between gene expression and promoter hypermethylation was assessed through spearman correlation analysis using graphpad software v6 .
each probe was correlated separately using a gene expression matrix of the 171 pcc samples .
an example of the correlation of the probe , cg07288693 , located in the promoter region of rbx1 , is shown in supplementary figure 1 supplementary material available online at http://dx.doi.org/10.1155/2014/546347 . in order to assess possible effects of hif1-target genes due to the disruption of the vhl elongin bc tumor suppressor complex
, we evaluated a gene - set enrichment analysis ( gsea ) for every sample using the single sample gene - set enrichment analysis ( ssgsea ) .
briefly , ssgsea calculates separate enrichment scores ( es ) for each pairing of a sample and gene set .
each ssgsea es represents the degree to which the genes in a particular gene set are coordinately up- or downregulated within a sample .
a rank normalized expression matrix for 171 pcc samples and 20,533 genes was used as input on the ssgsea implementation in genepattern public server .
ssgsea was performed using default parameters using the semenza_hif1_targets gene set available from the molecular signatures database v4.0 ( broad institute ) .
this gene set contains 36 genes that are transcriptionally regulated by hypoxia - inducible factor 1 ( hif1 ) .
we first examined the mutation status of the vhl gene in 241pcc tumors from the catalogue of somatic mutations in cancer ( cosmic ) .
consistent with literature reports , 24 out of 241 ( 10% ) cases harbored vhl gene mutation .
the data from tcga also showed a very low frequency of vhl mutation , at 2% .
we next analyzed the copy number status of component genes : vhl , rbx1 , cul2/cul5 , tceb1 , and tceb2 . in a cohort of 171 pcc tumors from the cancer genome atlas ( tcga ) , the frequency of gene disruption for three of these complex components was remarkably high ( rbx1 , 30.4% ; vhl , 26.9% ; cul5 , 21.6% ) , while two remaining complex components exhibited the modest disruption frequencies in pcc tumors : tceb1 ( 6.4% ) and tceb2 ( 2.9% ) .
interestingly , copy number gains were infrequent in all complex component genes ; no gene displayed gain in more than 5% of cases ( figure 2(a ) ) .
strikingly , when complex gene disruption was considered cumulatively , 59.6% of pcc harbored genetic loss of at least one of the complex components , while 24.3% harbored disruption of 2 or more complex components ( figure 2(c ) ) .
gene cnl events were not mutually exclusive : 17.5% of pcc had genetic loss of both vhl and any other complex component ; 33.3% harbored loss of a complex component other than vhl , and 8.8% harbored loss of vhl alone ( figure 2(b ) ) .
these findings highlight the importance of vhl elongin bc protein complex disruption at the genetic level in pcc .
we next investigated whether gene silencing by aberrant dna methylation affected any of the complex component genes .
gene methylation data was available for 171 pcc tumors and 4 adjacent solid nonmalignant tissues .
we found that all but one probe ( cg03160045 ) had a v of less than 0.14 in both pcc and nonmalignant samples , indicating that complex component genes were not highly methylated .
further , the dv (  pcc v  and  nonmalignant v  ) were close to zero in all cases , for all probes , suggesting that methylation of complex component genes did not differ between tumor and nonmalignant samples .
interestingly , we found that methylation was significantly negatively correlated with rbx1 expression ( supplementary figure 1 ) ; however , given the low dv for all probes , we could not confidently attribute the effects of methylation to expression levels of rbx1 .
we next examined whether genetic disruption of other complex component genes might lead to overactivity of hif1 and aberrant expression of hif1-target genes .
we performed single sample gene - set enrichment analysis ( ssgsea ) on the panel of 171 pcc and found that positive hif1-target gene - set enrichment ( supplementary table 1 ) occurred to some degree in all cases ( median es = 6132.4 ) ( figure 2(c ) , supplementary table 2 ) .
gene cnl of at least one complex component could explain hif1-target gene enrichment in 59.6% of cases .
since 33.1% of cases harbored genetic loss of vhl components other than vhl , and tgca mutation data indicated a vhl mutation rate of only 2% , inactivation of other vhl elongin bc complex components is likely involved in vhl elongin bc complex dysfunction in pcc and this could impact hif1-target expression ( figures 2(b ) and 2(c ) ) .
we next evaluated whether gene copy number alterations to the vhl elongin bc complex components were correlated with expression of these genes .
rna sequencing data for the 171 pcc tumors was downloaded from tcga , and samples were grouped according to their copy number status .
intriguingly , gene dosage in four of the five vhl complex components that were altered at the copy number level 
rbx1 , cul5 , vhl , and tceb1was significantly positively correlated with expression ( p < 0.0001 ) ( figure 3 ) .
tceb2 , which had a gene cnl frequency of 2.9% , was not significantly correlated with expression ; cul2 was not altered at the copy number level .
these findings suggest that underexpression of rbx1 , cul5 , vhl , and tceb1 is a selected event in pcc .
we evaluated if gene cnl affects components of the vhl elongin bc protein complex in another cancer type characterized by inactivation of vhl : rcc .
we queried gene cnl and mutation frequencies of the six vhl elongin bc complex genes in rcc using resources available at the cbioportal for cancer genomics .
in rcc , negligible gene cnl and mutation frequencies were observed for all of the vhl complex genes except for vhl itself ( figure 4 ) . across 522 rcc tumors ,
vhl was mutated in 179 ( 34.3% ) samples and lost at the copy number level in 419 ( 71.3% ) samples , while 39.7% of samples exhibited concurrent mutation and cnl , rbx1 , cul2 , cul5 , tceb2 , and tceb2 did not appear to be significantly altered in rcc at dna level , with no complex components exceeding a cnl frequency of 4% and no mutation frequency reaching 1% . by contrast ,
vhl mutation frequency in pcc was only 2% according to tcga data and , of the 171 pcc tumors from tcga , only 26.3% of samples exhibited cnl of vhl , with rbx1 and cul5 also undergoing frequent cnl . from these data , we observe markedly different genetic profiles when comparing genes coding for the vhl elongin bc complex between rcc and pcc . in rcc , it appears that vhl is the sole contributor to disruption of vhl elongin bc complex , whereas , in pcc , vhl elongin bc complex loss of function may occur frequently through cnl of vhl , rbx1 , and cul5 and through mutation of vhl .
we evaluated the mutation status of three other genes known to be frequently mutated in pcc ( hras , ret , and nf1 ) to assess their relationship with alterations in vhl complex component genes [ 13 , 50 ] .
of the 171 pcc tumors analyzed , 39 had mutations in either hras , ret , or nf1 ( supplementary figure 2 ) .
twelve of the 39 cases had no disruption in the vhl complex , while 27 displayed vhl elongin bc protein complex disruption .
the frequency of cases harboring mutations in ret , hras , or nf1 did not significantly differ between cases with or without vhl complex disruption ( p = 0.1942 , chi - square test ) . in rcc , nf1 , ret , and hras were not frequently mutated ( 2% , 0% , and < 1% , resp . ) , and these mutations were not mutually exclusive with vhl mutations .
oxygen - sensing pathways are paramount for cell survival and normal cellular function , while they also play a key role in tumor progression and aggressiveness .
hif1 pathways allow cells to survive in conditions of temporary oxygen deprivation ( e.g. , hif is essential in embryonic development ) . since abnormal accumulation of hif1- subunits
can induce hif1 pathways to promote tumor progression and aggressiveness , its levels need to be tightly regulated . this function
is mainly achieved by the vhl elongin bc complex . in vhl - related tumors , such as pcc and rcc [ 20 , 34 ] ,
hif1 activity is aberrantly and constitutively high , mimicking a hypoxic environment , irrespective of oxygen levels [ 31 , 51 , 52 ] .
genetic lesions affecting the vhl gene are usually considered the cause of hif1- accumulation [ 25 , 26 ] . since genetic mechanisms disrupting the vhl gene
are only present in a fraction of pcc , we have tested the hypothesis that deregulation of other protein components of the vhl elongin bc complex might also result in activation of hif1 pathways . indeed , gene cnl affecting rbx1 and cul5 significantly impacted gene expression .
we also noticed a negative correlation between hypermethylation and expression for rbx1 ; however , the low methylation levels of probes across all samples , including nonmalignant samples , imply that hypermethylation is not a major mechanism of rbx1 downregulation .
it has been shown that underexpression of rbx1 , due to gene cnl , might interfere with the keap1/cul3/rbx1 complex , which also displays a e3 ubiquitin - ligase activity in thyroid and ovarian cancer .
similarly , underexpression of cul5 linked to genetic loss events has been documented in breast tumors .
together , these results suggest that gene cnl has an impact on decreased gene expression of rbx1 and cul5 , in addition to vhl , and may subsequently contribute to dysfunction of the vhl elongin bc complex in pcc .
interestingly , other frequent somatic mutations in pcc , such as those affecting hras , ret , and nf1 , seem to occur independently of vhl elongin bc protein complex disruption .
it has been well documented that loss of function alterations to vhl , components of the succinate dehydrogenase ( sdh ) complex , and hif2a , as well as pseudohypoxia characterize cluster 1 pcc tumors and correlate with increased hif1 signaling [ 25 , 26 , 55 , 56 ] .
we propose the fact that tumors involving dna - level alterations of the vhl complex component genes should be considered as part of this cluster .
an enrichment analysis ( gsea ) of the panel of 171 pcc tumors revealed that hif1 gene targets were positively enriched in all cases ( figure 2(c ) ) .
the high frequency of samples showing alterations in at least one of the complex components ( 59.6% ) at least partially explains the positive enrichment of hif1 targets . in cases that did not have clear dna - level alterations to members of the vhl elongin bc complex , upregulation of hif1-target genes
may simply be due to the presence of hypoxic cells in the biopsy of samples used to generate the data . from these observations
, we suggest that gene cnl of other vhl complex components , namely , rbx1 and cul5 , along with vhl , cnl , and mutation , facilitates dysfunction of this complex and the consequent accumulation of hif1-. 
 vhl is also frequently disrupted in rcc .
previous studies have shown that the vhl gene is affected by somatic mutations in 50% of cases , while hypermethylation is observed in 1020% of sporadic rcc .
we analyzed disruption of vhl and other complex components in rcc and compared these results to their disruption in pcc .
interestingly , vhl seems to be the only gene significantly disrupted in rcc , with 71.3% of cases undergoing cnl and other 34.3% with vhl mutation .
these results indicate that rcc tumors are likely dependent on elimination of vhl rather than other complex components in order to generate conditions of pseudohypoxia .
the genetic landscape of the vhl elongin bc complex genes in pcc , however , showed vhl gene to be less frequently inactivated at the dna level , with the burden of genetic inactivation of the vhl elongin bc complex seeming to fall somewhat equally on rbx1 , cul5 , and vhl . the data presented here provide a rationale for a more comprehensive interrogation of the role of other vhl complex components ( namely , rbx1 and cul5 ) in the hif1-mediated oxygen - sensing pathway in pcc . in summary , we present compelling evidence that hif1-mediated pseudohypoxic conditions are genetically selected in pcc via the disruption of multiple vhl complex components and we provide further rationale for exploring this pathway as a therapeutic target in pcc with potential application to rcc and other vhl - related diseases .
 pheochromocytomas ( pcc ) are rare tumors that arise in chromaffin tissue of the adrenal gland . 
 pcc are frequently inherited through predisposing mutations in genes such as the von hippel - lindau ( vhl ) tumor suppressor . 
 vhl is part of the vhl elongin bc protein complex that also includes cul2/5 , tceb1 , tceb2 , and rbx1 ; in normoxic conditions this complex targets hypoxia - inducible factor 1 alpha ( hif1a ) for degradation , thus preventing a hypoxic response . vhl inactivation by genetic mechanisms , such as mutation and loss of heterozygosity , inhibits hif1a degradation , even in the presence of oxygen , and induces a pseudohypoxic response . 
 however , the described < 10% vhl mutation rate can not account for the high frequency of hypoxic response observed . 
 indeed , little is known about genetic mechanisms disrupting other complex component genes . here 
 , we show that , in a panel of 171 pcc tumors , 59.6% harbored gene copy number loss ( cnl ) of at least one complex component . 
 cnl significantly reduced gene expression and was associated with enrichment of gene targets controlled by hif1 . 
 interestingly , we show that vhl - related renal clear cell carcinoma harbored disruption of vhl alone . 
 our results indicate that vhl elongin bc protein complex components other than vhl could be important for pcc tumorigenesis and merit further investigation . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion