tumors undergo sustained growth in a dynamic environment where oxygen and nutrients are often scarce ( denko , 2008 ; hanahan and weinberg , 2011 ) . to cope with the energetic requirements of rapid proliferation in these challenging conditions ( fritz and fajas , 2010 )
, tumor cells profoundly reorganize their core metabolism ( cairns et  al . , 2011 ;
glucose utilization , which provides atp , essential anabolic intermediates , and antioxidative defenses ( hsu and sabatini , 2008 ; vander heiden et  al . , 2009 ) , is boosted and dissociated from oxygen availability ( the warburg effect ; warburg , 1956 ; warburg et  al . , 1927 ) .
key to the warburg effect is the decrease of mitochondrial respiration ( frezza and gottlieb , 2009 ) , which allows cancer cells to grow in the hypoxic conditions found in the interior of the tumor mass ( hsu and sabatini , 2008 ) .
the molecular mechanisms that inhibit oxidative phosphorylation ( oxphos ) in tumors are understood only partially .
the transcription factor hif1 ( hypoxia - inducible factor 1 ) decreases the flux of pyruvate into the krebs cycle and , hence , the flow of reducing equivalents needed to power the electron transport chain ( etc ) and stimulates glycolysis by inducing glucose transporters and glycolytic enzymes ( denko , 2008 ; semenza , 2010b ) .
hif is activated by hypoxia as well as by the accumulation of the krebs cycle metabolites succinate and fumarate that inhibit the prolyl hydroxylases ( phds ) responsible for proteasomal degradation of the hif1 subunit ( selak et  al . , 2005 ) .
succinate accumulation can originate from loss - of - function mutations in any of the genes encoding for succinate dehydrogenase ( sdh ) subunits ( or their assembly factor sdhaf2 ) , which cause hereditary paraganglioma - pheochromocytoma syndrome and are associated to a number of other neoplasms ( bardella et  al . , 2011 ) .
within this conceptual framework , we have analyzed the activity of trap1 , an evolutionarily conserved chaperone of the hsp90 family mainly located in the mitochondrial matrix and overexpressed in a variety of tumor cell types , where it exerts antiapoptotic functions through mechanisms that are only partially understood ( altieri et  al . , 2012 ; kang et  al .
our results indicate that trap1 supports tumor progression by downmodulating mitochondrial respiration through a decrease in the activity of sdh , which leads to hif1 stabilization even in the absence of hypoxic conditions , by increasing succinate levels .
we found that trap1 is localized in mitochondria of cancer cell models ( figures s1a and s1b available online ) , as expected ( altieri et  al . , 2012 ) , and that downregulation of trap1 expression by rnai abrogated any transforming potential .
in fact , knockdown of trap1 expression made saos-2 osteosarcoma cells , hct116 colon carcinoma cells , and hela cervix carcinoma cells ( dubbed shtrap1 cells ; figures s1c 
s1e ) unable to both form foci ( figure  1a ) and grow in soft agar ( figure  1b ) without affecting the rate of cell growth ( figure  1c ) .
notably , shtrap1 tumor cells lost the ability to develop tumor masses when injected into nude mice ( figure  1d ) .
conversely , when the trap1 complementary dna ( cdna ) was expressed in either rwpe-1 prostate epithelial cells or fibroblasts , these nontransformed cells acquired the capacity to form colonies in soft agar ( figures 2a and 2d ) , and downregulation of trap1 expression in rwpe-2 prostate cells , which are transformed by expression of v - ki - ras in rwpe-1 cells ( rasola et  al . , 2010a ) , abolished their tumorigenic features ( figure  2b ) .
moreover , stable transfection of a murine trap1 cdna , which is insensitive to human - directed small hairpin rna ( shrna ) constructs , reinstalled the tumorigenic capability of shtrap1 cells ( figure  2c ) .
mitochondrial localization of trap1 was essential for its proneoplastic activity , as expression of a trap1 cdna devoid of its mitochondrial targeting sequence was not tumorigenic in either cancer or nontransformed cells ( figures 2d and 2e ) .
we then asked whether trap1 promotes transformation by acting on mitochondrial metabolism , thus contributing to the warburg phenotype .
we used a blue native ( bn)-page approach ( figure  3a ) , which allows the separation and characterization of protein complexes under nondenaturing conditions ( wittig and schgger , 2008 ) , to investigate a possible interaction between trap1 and etc complexes . by cutting bn - page bands and running them on an sds - page
, we could observe the association between trap1 and both complex iv ( cytochrome oxidase , cox ) and complex ii ( succinate dehydrogenase , sdh ) ( figure  3a ) .
moreover , by performing an immunoblot directly on the bn - page , we found trap1 to be in correspondence with both complex iv and complex ii bands ; notably , these bands were diffused , and trap1 colocalized with their upper portion , suggesting that trap1 contributes to form a multimeric complex of higher molecular weight than the etc complex per se ( figure  3b ) .
we confirmed the interaction between trap1 and complex ii / sdh through further approaches , including ( 1 ) immunoprecipitation , finding coimmunoprecipitation ( coip ) of trap1 with sdh and vice versa ( figure  3c ) , and ( 2 ) mitochondrial protein crosslinking with dimethyl 3,3-dithiobis - propionimidate ( dtbp ) , a homobifunctional compound that reacts with the primary amines of two interacting proteins at an average distance of about 8   ( giorgio et  al
. , 2009 ) , followed by trap1 immunoprecipitation in order to determine whether trap1 and sdh are closely associated .
we found that two trap1/sdh complexes are formed in mitochondria ( figure  3d ) .
complex ii couples the krebs cycle to oxphos by oxidizing succinate to fumarate and then transferring electrons to coenzyme q ; hence , the enzyme is called either sdh or succinate : coenzyme q reductase ( sqr ; cecchini , 2003 ; lemarie and grimm , 2011 )
. sqr activity can be assessed spectrophotometrically in permeabilized mitochondria after inhibition of the other etc complexes by recording the reduction of 2,6-dichlorophenolindophenol ( dcpip ) in the presence of succinate as an electron donor and coenzyme q1 as an intermediate electron acceptor .
the slope of the absorbance decline of dcpip is directly proportional to sqr activity ( see figure  s2a ) .
we found that sqr enzymatic activity was increased in mitochondria from shtrap1 cells relative to those derived from control cells ( figures 4a , s2a , and s2b ) .
trap1 did not affect either the cytochrome oxidase enzymatic activity of complex iv ( figure  s2c ) or complex ii protein levels ( figure  s2d ) or mitochondrial mass ( figure  s2e ) .
specificity of trap1 inhibition on sdh was assessed by ( 1 ) expression of the mouse trap1 ( in shtrap1 cells ) that is insensitive to human - directed shtrap1 constructs , which resulted in inhibition of sqr activity to values similar to those of mock cells ( figure  4a ) , and ( 2 ) using an inhibitor of trap1/hsp90 atpase activity ( felts et  al . , 2000 ) , 17-allylamino-17-demethoxygeldanamycin ( 17-aag ) , whose availability to mitochondria was recently shown in  situ ( xie et  al . , 2011 ) ; 17-aag specifically increased sqr activity in control mitochondria , whereas shtrap1 mitochondria were insensitive to the drug ( figures 4a , 4b , and s2b ) .
notably , the effect of 17-aag was unrelated to hsp90 , as hsp90 protein levels were the same in mock and shtrap1 cells ( figure  s2f ) .
the sqr activity of etc complex ii was further inhibited in mitochondria from control cells that progressed through the focus - forming assay compared to mitochondria from the same cells kept in standard culture conditions , whereas no change in sqr activity could be appreciated in mitochondria from shtrap1 cells during the focus - forming experiments ( figure  4b ) .
17-aag could still reactivate the sdh enzyme in mitochondria of trap1-expressing cells undergoing the focus - forming process ( figure  4b ) , indicating that even the enhanced inhibition of sqr activity occurring during the in  vitro transformation progression is mediated by trap1 and remains reversible .
in further accord with an inhibitory function of trap1 on etc complex ii , mitochondria from mef cells stably expressing trap1 showed a diminished sqr activity compared to controls , and this inhibition was increased during the focus - forming assay ; 17-aag reactivated sdh selectively in mitochondria from trap1-expressing mefs ( figure  4c ) .
trap1 expression was shown to be increased in a variety of tumor types ( kang et  al .
we analyzed the sqr activity of etc complex ii in a set of human colorectal cancer samples and compared it with that measured in the surrounding nontransformed mucosa for each patient . in all colorectal cancer samples at stage iv , characterized by metastases to lymph nodes and to distant sites , and in the majority of samples of stage i  iii , characterized by the absence of distant metastases , trap1 was upregulated relative to normal mucosa ( costantino et  al .
when we measured sqr activity in extracts from these samples , we found that trap1 upregulation was always paralleled by a decrease in sqr activity , and that this decrease could be partially rescued by adding 17-aag before starting recordings ( figure  4d ) . in a small subset of stage i  iii colorectal cancers , trap1 expression was not induced relative to surrounding nontumor tissues . in these samples , we could not detect any difference in sqr activity between samples from tumor and normal mucosa ( figure  4e ) , strengthening the link between trap1 and the regulation of complex ii activity .
the sqr assays described so far measure the maximal enzymatic activity of complex ii , as the complex is made accessible in permeabilized mitochondria and exposed to an excess of substrates .
we next analyzed whether trap1 also affects the oxygen consumption rate ( ocr ) of living cells .
downregulation of trap1 markedly increased mitochondrial - dependent respiration in all cancer cell models we tested ( figures 5a , s3a , and s3b ) ; in full accord with the effect of the drug on sqr activity ( see figure  4a ) , the trap1 inhibitor 17-aag increased ocr only in trap1-expressing cells ( figure  s4a ) .
in shtrap1 cells , the extra ocr was used to make atp , as it was inhibited by  the atp synthase blocker oligomycin ; moreover , addition of the uncoupler fccp increased respiration well above the basal level , indicating an increased respiratory capacity that remained fully sensitive to etc inhibition by rotenone ( figure  5a ) .
the comparison with control cells is striking because , unlike shtrap1 cells , they already utilize their maximal respiratory capacity under basal conditions , as shown by the lack of ocr increase with fccp ( figure  5a ) , an arrangement implying that any additional atp requirement must be provided by glycolysis .
consistently , we found that oxphos marginally contributes to atp synthesis in mock cells , whereas a high proportion of the intracellular atp content is provided by glycolysis , with a marked increase of glycolytic atp during the in  vitro tumorigenic process ; instead , in shtrap1 cells , most of the atp comes from oxphos ( figure  5b ) .
moreover , expression of the trap1 cdna in nontransformed fibroblasts markedly inhibited basal ocr and abolished any respiratory reserve ( figure  5c ) , mimicking the respiratory pattern of trap1-expressing tumor cells .
expression in shtrap1 cells of the murine trap1 insensitive to human - directed shtrap1 constructs determined an ocr pattern indistinguishable from that of mock cells ( figure  5d ) .
a low concentration of the etc complex ii inhibitors 3-nitropropionic acid ( 3-np ) , which inactivates sdh after covalent binding with an arg residue in the catalytic core of sdha ( huang et  al .
, 2006 ) , or thenoyltrifluoroacetone ( ttfa ) , which blocks electron transfer from succinate to coenzyme q at the quinone - binding site in subunits b and d ( huang et  al . , 2006 ) , inhibited ocr in shtrap1 cells but were inactive in the presence of trap1 ( figures s4b and s4c ) , paralleling the downmodulation of the sqr activity induced by 3-np only in trap1-expressing mitochondria ( figure  5e ) .
we also found that 3-np inhibits death in a dose - dependent fashion in shtrap1 , but not in mock saos-2 cells placed in conditions of long - term starvation that mimic the paucity of nutrients found in the inner tumor mass during the phases of its rapid accrual ( figure  5f ) .
moreover , treatment with 3-np partially restored the ability of shtrap1 cells to form colonies in soft agar , whereas it was ineffective on the colonies formed by control cells ( figure  5 g ) .
it was shown that succinate induces hif1 by inhibiting phds , the enzymes that hydroxylate hif1 , allowing its subsequent ubiquitin - dependent degradation ( selak et  al . , 2005 ) .
we observed that during the focus - forming assay , the intracellular level of succinate increased only in trap1-expressing cells ( figure  6a ) , matching the downmodulation of their sdh enzymatic activity ( figure  4b ) . in keeping with these results , hif1 was detectable exclusively in trap1-expressing cells during the focus - forming process ( figure  6b ) , whereas it was hydroxylated on pro residues ( i.e. , primed for proteasomal degradation ) both in culture conditions , independently of the presence of trap1 , and in shtrap1 cells exposed to focus - forming conditions ( figure  6c ) .
hif2 , which shares some redundant functions with hif1 and whose expression is increased in a broad spectrum of cancer cell types ( keith et  al . , 2012 ) , was not stabilized in our experimental conditions ( figure  6d ) .
we then used pimonidazole , a compound that is reductively activated under hypoxic conditions and forms protein adducts by reacting with cys residues ( arteel et  al . , 1998 ) , to understand whether hif1 stabilization could at least partially depend on hypoxic conditions occurring during the formation of foci .
remarkably , we could not detect any induction of pimonidazole - protein adducts in trap1-expressing cells during the process of in  vitro tumorigenesis , even when hif1 had already been stabilized ( figure  6e ) , which demonstrates that pseudohypoxic conditions elicited by the presence of trap1 are sufficient to promote hif1 stabilization .
tumor samples obtained from nude mice xenografted with trap1-expressing saos-2 cells ( see figure  1d ) were characterized by densely packed cells , amidst which fibrotic and necrotic areas could be observed ( figure  6f , marked as f and n , respectively ) ; hif1 was clearly detected in the majority of cells , the signal being particularly strong in the nuclei of cells where proliferation markers were also evident ( compare the mib / ki67 and the hif1 staining in figure  6f ) . in these samples
, cells displayed a punctate trap1 signal that fits well with its mitochondrial localization ( see the high - magnification trap1 staining in figure  6f ) .
the addition of dimethyl succinate , a membrane - permeable succinate analog , to the focus - forming culture medium both stabilized hif1 ( figure  7a ) and rescued the capability to form colonies ( figure  7b ) in shtrap1 cells , while it did not further increase the tumorigenicity of trap1-expressing cells ( figure  7b ) . moreover , hif1 inhibition either with a cell - permeable esterified form  of -ketoglutarate ( 1-trifluoromethylbenzyl--ketoglutarate , takg ) , which reverses hif1 stabilization by restoring phd enzymatic activity ( mackenzie et  al .
, 2007 ; tennant et  al . , 2009 ) , or with rnai on hif1 or hif1 , the latter being the stable subunit of the heterodimeric hif transcription factors ( keith et  al . , 2012 ) , fully abolished formation of foci in trap1-expressing tumor cells and in mefs transfected with a trap1 cdna ( figures 7c7f ) . taken together , these data indicate that trap1 prompts neoplastic growth by inducing a succinate - dependent stabilization of hif1.
tumor cells tend to increase their glycolytic activity without a matching increase of oxidative phosphorylation ( warburg , 1956 ; warburg et  al . , 1927 ) .
inhibition of the tumor suppressor p53 or activation of the transcription factor hif1 curtails oxphos by inducing the autophagic degradation of respiratory complexes and by abrogating the synthesis of some of their subunits ( such as sdhb ) or assembly factors ( denko , 2008 ; semenza , 2010a ; vousden and ryan , 2009 ) .
conversely , oxphos inhibition can play a causal role in tumorigenesis . inactivating mutations in mitochondrial dna ( mtdna ) genes encoding for subunits of etc complexes
i and iii were found associated with renal oncocytomas ( gasparre et  al . ,
2008 ) as well as thyroid and prostate cancers ( abu - amero et  al . , 2005 ;
however , these mutations are confined to a small set of neoplasms , and the lack of clear - cut molecular mechanisms hampers the definition of whether oxphos inhibition as such can play a general tumorigenic role .
key findings of the  present work demonstrate that the mitochondrial chaperone  trap1 , which is widely expressed in most tumors , but not  in  highly proliferating , nontransformed cells ( http://www.proteinatlas.org/ ) , is a component of the molecular machinery that decreases mitochondrial respiration and that this event is crucial for neoplastic progression .
indeed , we find that trap1 behaves as an oncogene since ( i ) without trap1 , tumorigenesis is blunted both in  vitro and in  vivo , and ( ii ) trap1 expression confers tumorigenic potential to nontransformed cells .
we observe that trap1-mediated inhibition of sdh limits the maximal rate of respiration and leads to succinate accumulation followed by hif1 , but not hif2 , stabilization .
remarkably , the membrane - permeable succinate analog dimethyl succinate could both elicit hif1 stabilization and rescue the tumorigenic phenotype of shtrap1 cells , highlighting the mechanistic connection  between trap1-dependent succinate accumulation and hif1-dependent tumor formation .
once activated by inhibition of the proteasomal degradation of its  subunit ( either hif1 or hif2 ) , and by the ensuing association with the stable  subunit ( hif1 ) , hif1 can boost evolution of neoplasms by promoting angiogenesis , epithelial - mesenchymal transition , and the glycolytic switch ( brahimi - horn et  al . , 2011 ; semenza , 2010a ) . in multiple tumor models ,
both hif1 and hif2 promote neoplastic progression by regulating sets of genes that are only partially shared , and independent roles for hif1 and hif2 can depend on the cancer type or on its growth and progression stages ( keith et  al . , 2012 ) .
however , both  subunits can play a tumor suppressor function in specific tumor types , such as renal cell carcinoma ( hif1 ) or lung adenocarcinoma ( hif2 ) , through poorly defined mechanisms . in our model , hif1 stabilization , and the ensuing hif activation ,
have a crucial proneoplastic role , as promoting hif1 degradation or knocking down either hif1 or hif1 abolishes the neoplastic potential of trap1-expressing cells .
instead , we could not observe any hif2 stabilization , ruling out its role in the trap1-dependent tumorigenic process .
it is interesting that hif2 accumulates at higher o2 concentrations than hif1 ( keith et  al . , 2012 ) , and the possibility exists that , at variance with what we observe for hif1 , pseudohypoxic conditions are insufficient or unable to stabilize hif2. hif1 stabilization emerges after several days of in  vitro transformation .
this is not at all surprising because excess succinate can be utilized in multiple pathways , including increased heme synthesis ( frezza et  al . , 2011 ) , and because sdh becomes more strongly inhibited by trap1 during the focus - forming assay .
the latter observation also suggests that a threshold sdh inhibition must be reached to allow for succinate accumulation . despite a partial respiratory inhibition , trap1-expressing cells fully utilize their residual respiratory capacity to produce atp , as shown by ocr experiments , but reorient their metabolism toward glycolysis to meet any energy demand that exceeds respiratory capacity , in complete accord with warburg s observations .
trap1 is likely to begin a feedforward loop , as it inhibits sdh and respiration ( hence oxphos ) and induces hif1 , which in turn further inhibits oxphos ( denko , 2008 ; semenza , 2010b ) and directly downregulates sdh by induction of mir-210 ( puissgur et  al . , 2011 ) .
notably , we have determined that trap1-dependent stabilization of hif1 occurs in a pseudohypoxic way ( i.e. , in the absence of any hypoxic stress ) .
this observation has potential implications for the kinetics of tumor development , as trap1 could induce hif1 transcriptional activity even before the dysregulated accrual of the tumor mass creates hypoxic areas in its inner core . at variance with the complete block of the sdh enzyme , which is an extreme case caused by loss - of - function mutations only seen in specific subsets of tumors ( bardella et  al . , 2011 ) , the partial and reversible sdh inhibition caused by trap1 and its increased expression levels would mediate a more general proneoplastic function , which fits with trap1 identification as a bona fide inducible target of the proto - oncogene c - myc ( coller et  al . , 2000 ) .
tumor cells could be endowed with a multichaperone mitochondrial complex , as trap1 interacts with cyclophilin d , hsp90 , and hsp60 ( ghosh et  al . , 2010 ; kang and altieri , 2009 ) .
given the multiplicity of chaperone client proteins , we can not exclude further interactions of trap1 with other mitochondrial components .
for instance , trap1 is involved in the inhibition of the mitochondrial permeability transition pore ( kang et  al . , 2007 )
, whose opening irreversibly commits cells to death ( rasola and bernardi , 2007 ; rasola et  al . , 2010b ) , and we have observed that keeping the pore locked can be used by tumor cells to evade apoptosis ( rasola et  al . , 2010a ) .
thus , trap1 could take part in several mitochondrial changes that crucially contribute to the neoplastic phenotype .
targeting its chaperone activity and molecular interactors could dismantle the metabolic and survival adaptations of neoplastic cells , paving the way to the development of highly selective , mitochondriotropic antineoplastic drugs .
experiments were performed on different cell models : human saos-2 osteosarcoma cells , human hct116 colorectal carcinoma cells , rwpe1/rwpe2 prostate epithelial cells , hela cervix carcinoma cells , and mefs .
trap1 , hif1 , and hif1 expression was knocked down by stable transfections with shrnas .
stable transfection of a trap1 cdna was performed in mef cells that show negligible levels of the endogenous protein .
tissue samples from both tumor and normal , noninfiltrated peritumoral mucosa were obtained from patients with colorectal carcinoma during surgical cancer removal after express written informed consent was obtained from all patients .
three different tumorigenesis assays were performed : ( 1 ) focus - forming assays , in which cells were grown to confluence and kept in culture for the subsequent 25  days ; tumor cells lose contact inhibition and overgrow to form foci ; ( 2 ) soft agar assays , in which cells are embedded in an agar matrix ; only transformed cells , which escape anoikis death signals , can grow to form colonies ; ( 3 ) cell injection in nude mice , in order to follow the growth of primary tumors .
samples obtained from these assays were also exploited for investigating changes in complex ii enzymatic activity in succinate levels and in hif1 stabilization and distribution .
cytofluorimetric analyses were utilized to analyze cell death induction with the use of annexin v - fitc and propidium iodide probes as well as mitochondrial mass with the use of n - acridine orange .
mitochondria were isolated through sequential centrifugations after mechanical cell disruption . in order to establish submitochondrial protein localization ,
bn - page experiments were carried out on isolated mitochondria in order to identify etc complexes .
after a first electrophoresis  in nondenaturing conditions , bands were visualized with coomassie blue staining , cut , and run on sds - page for the identification  of  protein  components by western immunoblot .
crosslinking assays  were  performed on isolated mitochondria incubated with the membrane - permeable , homobifunctional reagent dtbp prior to trap1 immunoprecipitation . complex ii enzymatic activity was investigated , measuring the sqr activity with classical spectrophotometric approaches on cell or tumor lysates .
complex iv enzymatic activity was investigated , measuring the oxidation of reduced cytochrome c. each measurement of the respiratory chain ( rc ) complex activity was normalized for protein amount and for citrate synthase activity . in  vivo respiration
was followed in a kinetic mode by measuring the oxygen consumption rate ( ocr ) of cell monolayers with an extracellular flux analyzer .
immunohistochemical inspections were performed on serial sections of paraffin - embedded tumor samples obtained from xenografted nude mice following standard procedures .
immunoelectron microscopy ( immuno - em ) inspections were performed on antibody - labeled fixed cells using the gold - enhanced protocol .
intracellular succinate level was analyzed on lysates obtained by scraping cells  placed in a cold methanol / acetonitrile solution . after spinning down the  insoluble material , the supernatant
was collected , and metabolites were  separated using a liquid chromatography system coupled online to an ltq orbitrap mass spectrometer equipped with an electrospray ionization source .
student s t test was used to compare pairs of data groups . in all figures ,
bar graphs report mean   sd values ( n  3 ) ; p  < 0.05 .
 summarywe report that the mitochondrial chaperone trap1 , which is induced in most tumor types , is required for neoplastic growth and confers transforming potential to noncancerous cells . 
 trap1 binds to and inhibits succinate dehydrogenase ( sdh ) , the complex ii of the respiratory chain . 
 the respiratory downregulation elicited by trap1 interaction with sdh promotes tumorigenesis by priming the succinate - dependent stabilization of the proneoplastic transcription factor hif1 independently of hypoxic conditions . 
 these findings provide a mechanistic clue to explain the switch to aerobic glycolysis of tumors and identify trap1 as a promising antineoplastic target . 
Introduction
Results
Discussion
Experimental Procedures