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Translational studies in urologic oncology

Clinical implications of hypoxia inducible factor in renal cell carcinoma

Marc C. Smaldone, M.D., Jodi K. Maranchie, M.D., FACS*Department of Urology, University of Pittsburgh Medical Center and University of Pittsburgh Cancer Institute, Pittsburgh, PA 15232, USA

bstract

Management of renal cell carcinoma (RCC) has made considerable strides in the past decade, due in large part to identification of the vonippel Lindau (VHL) tumor suppressor as a negative regulator of hypoxia inducible factor � (HIF-�) protein expression. Stabilization ofIF-� appears to be critical for renal tumorigenesis, and is observed even in VHL-independent RCC. Thus, an understanding of theathways that regulate expression and activation of the different HIF-� isoforms is key to delineating the mechanism of renal transformationnd for the development of novel therapeutics. A number of agents targeting HIF-� or its transcriptionally-regulated genes have shownromise in treatment of RCC. However, more effective treatment strategies are still needed. This report provides a directed review of recentiscoveries defining the role of HIF in renal tumorigenesis and their relevance to the clinical advances in targeted therapy for advancedCC. © 2009 Elsevier Inc. All rights reserved.

Urologic Oncology: Seminars and Original Investigations 27 (2009) 238–245

eywords: Renal cell carcinoma; HIF; VHL; VEGF; Therapy

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The incidence of renal cell carcinoma (RCC) has in-reased steadily over the past several decades. An estimated8,000 new cases of RCC were diagnosed in 2006, withreater than 12,000 expected deaths from the disease [1].his rise has been attributed to the widespread use of non-

nvasive abdominal imaging procedures for improved de-ection [2]. However, despite a higher proportion of patientsith localized disease at diagnosis, mortality has also risen

teadily over the same time period [3], suggesting a funda-ental shift in cancer biology. Although tumors localized to

he kidney are potentially cured by surgical resection, one-hird of patients present with advanced disease, and half ofhose remaining will ultimately relapse. These lesions areoth radio- and chemo-resistant, and standard immunother-pies lead to complete response in fewer 15% of patients4]. Clear cell RCC is well known for its intense vascularitynd high expression of angiogenic factors. Insight into thisiology came with the 2000 discovery that the von Hippelindau tumor suppressor gene (VHL), lost in greater than5% of clear cell RCCs, functions as a negative regulator ofypoxia inducible factor-� (HIF-�). However, HIF-� in-

* Corresponding author. Tel.: �1-412-605-3019; fax: �1-412-605-030.

qE-mail address: [email protected] (J.K. Maranchie).

078-1439/09/$ – see front matter © 2009 Elsevier Inc. All rights reserved.oi:10.1016/j.urolonc.2007.12.001

uction is not limited to the subset of clear cell RCC withHL loss, suggesting that it plays a fundamental role in

enal transformation. Novel therapeutic agents targetingIF-� or its transcription targets have demonstrated prom-

sing antitumor activity in clinical trials.

ypoxia inducible factor 1

The heterodimer transcription factor HIF-1 was firstdentified in 1991 as a regulator of renal production ofrythropoietin (Epo), the glycoprotein hormone that con-rols RBC production and maintains physiologic oxygenomeostasis. Deletion analysis of the 3= flanking region ofpo revealed the minimal essential sequence of 5=-TACGTGCT-3= [5] required for oxygen-dependent regu-

ation. HIF-1 was subsequently purified from this hypoxia-esponse element (HRE), yielding two subunits, HIF-1� andIF-1� [6]. The latter proved to be the aryl hydrocarbon

eceptor nuclear translocator (ARNT), which is constitu-ively expressed in all cell types [7]. In contrast, HIF-1� isightly regulated at the protein level by oxygen-dependentbiquitination followed by proteasomal degradation, nownown to be mediated by VHL. Under physiologic oxygenonditions, HIF-1� protein is virtually undetectable. Hyp-xia leads to abundant protein levels, nuclear translocation,nd transactivation of target genes harboring the HRE se-
uence [8,9]. More than 100 HIF transcription targets have
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239M.C. Smaldone, J.K. Maranchie / Urologic Oncology: Seminars and Original Investigations 27 (2009) 238–245

een described to date, involving a myriad of diverse path-ays required for response to hypoxia or environmental

tress, including erythropoiesis, angiogenesis, proliferation,poptosis [10], and anaerobic metabolism (Fig. 1).

The N-terminus of HIF-1� contains the sequences re-uired for dimerization and DNA binding. The C-terminusarbors two distinct activation domains: the oxygen depen-ent degradation domain (ODD), and the carboxy-terminalctivation domain (CAD) (Fig. 2). As its name implies, theDD contains the residues required for oxygen-mediatedegradation and can confer this property to other proteinshen expressed as a fusion [11]. Stabilized protein, how-

ver, is not transcriptionally active until a second oxygen-ependent event occurs at the CAD to permit binding ofo-factor CBP/p300, which promotes dimerization and nu-lear translocation. Under normal oxygen conditions, ansparagine within the CAD is hydroxylated by factor inhib-ting HIF (FIH-1), a 2-oxoglutarate-dependent oxygenasehat requires oxygen, iron (Fe(II)) and 2-oxoglutarate asubstrates. Hydroxylation prevents tertiary structural

ig. 1. Pathways and representative genes transcriptionally regulated byypoxia inducible factors HIF1 and HIF2. Although significant overlapxists, array studies indicate differential induction by the two isoforms withpoptosis and gluconeogenesis preferentially induced by HIF-1 and angio-enesis preferentially induced by HIF-2. Single asterisks indicate genespecifically regulated by HIF-2 whereas double asterisks indicate thosepecifically regulated by HIF-1.

ig. 2. Schematic of HIF-1� and HIF-2� indicating conserved activation
xygen dependent degradation domain. CAD: Carboxy-terminal activation doma
hanges required for CBP/p300 binding [12,13]. FIH-1 isnhibited by low oxygen levels, iron sequestration, or heavyetals (cobalt), conditions known to promote HIF activa-

ion. Binding of CBP/p300 was also recently shown to beegulated by the mammalian target of rapamycin (mTOR)ia binding of an mTOR-signaling motif (TOS) located inhe N-terminus of HIF-1� [14].

HL regulation of HIF-1�

Greater than 75% of clear cell RCCs harbor biallelic lossf the von Hippel Lindau (VHL) tumor suppressor gene15]. These tumors are uniquely vascular and characterizedy elevated expression of VEGF and glucose transporter-1Glut-1). By 1999, the multi-subunit von Hippel Lindauumor suppressor complex, comprised of elongin B, elongin, Cul2, and RBx1, had been characterized. Due to strikingomology to the SCF (Skp1-Cdc53/Cul-1-F-box protein)omplex in yeast, it was identified as an E3 ubiquitin ligaseith VHL as the substrate recognition component [16]. Theiscovery of HIF-1� and HIF-2� as the first two confirmedargets of VHL-mediated degradation greatly advanced ournderstanding of renal tumorigenesis, and opened the dooro novel molecularly targeted therapies [17]. VHL binds tohe ODD under normal oxygen conditions, leading to poly-biquitination and subsequent degradation of HIF-�. VHLinding requires hydroxylation of one of two proline resi-ues within the ODD by a family of prolyl hydroxylasesPHD 1–3) [18]. Analogous to FIH-1, PHDs are dioxygen-ses, dependent upon molecular oxygen, 2-oxoglutarate,nd iron and inhibited by hypoxia, iron chelation, or cobalt19,20]. When oxygen levels are low, hydroxylation doesot occur and VHL cannot bind HIF-�, leading to stabili-ation and protein accumulation.

lternate HIF-� isoforms

A homologous HIF-2� subunit (the product of thePAS-1 gene) with 48% sequence identity with HIF-1� was

solated from endothelial cells in 1997 [21]. Like HIF-1�,IF-2� is stabilized and activated by hypoxia and dimerizes

ding domains. Shaded boxes represent critical hydroxylation sites. ODD:
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240 M.C. Smaldone, J.K. Maranchie / Urologic Oncology: Seminars and Original Investigations 27 (2009) 238–245

ith ARNT to form HIF-2. Further, HIF-2 activates tran-cription of target genes by binding the same HRE asIF-1�. However, HIF-1� and HIF-2� are not functionally

edundant. Whereas HIF-1� homozygous inactivation ismbryonic lethal due to lack of vascular formation andlobal hypoxia [22], embryos of HIF-2� double knockoutice demonstrate normal systemic vasculature but impaired

ung maturation and catecholamine production [23]. Fur-her, HIF-2� expression is more tightly controlled and un-etected in most cells. It promotes an undifferentiated phe-otype in pluripotent stem cells emphasizing the need forersistent silencing in many tissues after development [24].IF-2� is the predominant isoform, however, in developingidney, small vessels of the CNS, adrenal medulla, lung andntestine. Interestingly, although mature normal kidney andarly cystic lesions predominantly express the HIF-1� iso-orm [25] clear cell RCC demonstrates a shift toward ex-ression of HIF-2� [26,27]. Conversely, expression of HIF-�, described in 1998, is down-regulated with progressiono cancer [28]. This isoform is highly abundant in heart,lacenta, and skeletal muscle. Six different splice variantsave been described, three of which are targeted by VHL-ediated degradation [29]. HIF-3� resembles HIF-1� and

2� in the ODD, but lacks the CAD transactivation domain.t appears to function as a dominant negative regulator ofypoxia-inducible gene expression in the human kidney30].

IF-� as a renal oncogene

Recent studies have shown that HIF-�, particularly HIF-�, plays an important role in the development in VHL-eficient RCC and is not solely a marker for disruption ofhe VHL pathway. Immunohistochemical examination ofarly kidney lesions from VHL patients show a coordinatedoss of VHL and increase in HIF-1�. Intriguingly, there isn apparent switch from HIF-1� accumulation to HIF-2�ccumulation in such lesions coincident with increasingysplasia [26]. While the relative contributions of HIF-1�nd HIF-2� to the pathogenesis of the VHL phenotype haveet to be defined, these findings suggest that HIF-2� is morencogenic than HIF-1� in the setting of VHL-defectiveCC. The evidence supporting this theory was nicely sum-arized by Kim et al. in a recent review [31]. Briefly,

uman RCC lines express either both HIF-1� and HIF-2�r HIF-2� alone, suggesting that there may be a selectionressure to maintain HIF-2� expression or lose HIF-1�xpression [17]. Second, HIF-2� variants lacking prolylydroxylation sites prevent tumor inhibition by VHL innimal models, whereas analogous HIF-1� mutants do not32,33]. Third, down-regulation of HIF-2� with viral vectorairpin RNAs in human VHL–/– RCC cells is sufficient torevent tumor formation in nude mice [34,35]. Finally,athologic changes observed in mice engineered to lack
HL can be prevented by simultaneous deletion of HIF-1� a
36]. Bias toward HIF-2� in renal transformation is perhapsn part due to the fact that HIF-1� preferentially inducesro-apoptotic pathways not targeted by HIF-2� [10]. De-pite the fact that both target the same HRE, array studiesemonstrate that expression of genes involved in the gly-olytic pathway are driven preferentially by HIF-1� [37],hereas HIF-2� preferentially promotes growth and angio-enesis [38]. Consistent with this, although both HIFsqually activate exogenous reporter constructs containinghe Epo HRE, HIF-2 preferentially binds the endogenouspo promoter [39] suggesting the involvement of isoform-pecific nuclear co-factors.

onhypoxic regulation of HIF

There is a growing body of evidence that HIF-� subunitsre alternatively activated by reactive oxygen species (ROS)nder normal oxygen conditions [40]. Transcriptional in-uction and activation of HIF-� are seen in response to aide array of inflammatory cytokines and growth factor

timuli, including TNF-� [41,42], angiotensin II [43], IL-1�44], and thrombin [45]. We showed that in VHL-deficientCC cells, where HIF-2� protein levels are abundant, nor-oxic transcriptional activity is critically dependent upon

xpression of the Nox4 NADPH oxidase, an endogenousenerator of ROS found in greatest abundance in the distalenal tubules [46,47]. A role in tumorigenesis was con-rmed by Nox inhibition in xenografts, demonstrating thatADPH oxidases promote tumor growth [48]. These stud-

es suggest a role for Nox4 as a future target for treatmentf RCC.

HIF-� protein is also stabilized by binding to heat shockrotein 90 (hsp90) via a mechanism that is independent ofoth oxygen and VHL. The specific hsp90 inhibitoreldanamycin leads to HIF-� degradation [49]. ARNT com-etes with hsp90 for binding of HIF-� and protects therotein from geldanamycin-mediated degradation [50]. Thisathway is thought to play a role in adaptation to hypoxia byttenuating HIF-� activation.

IF in non-clear cell RCC

Although the HIF pathway was originally linked toHL-deficient clear cell RCC, over-expression of HIF isocumented in all renal histologic subtypes, including nas-ent renal tumors expected to have limited artifact fromumor ischemia. Increased expression of HIF-1� or HIF-2�as seen in 50% and 100% of chromophobe tumors and in5% and 50% of hereditary type I papillary tumors (HPRC),espectively [27]. Loss of fumarate hydrogenase in heredi-ary leiomyomatosis and papillary RCC (HLPRC) leads tolevated fumarate, which directly stabilizes HIF-� by com-etitive inhibition of HIF prolyl hydroxylases [51]. HIF is
lso elevated in clear cell RCC of tuberous sclerosis where

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he VHL pathway is intact [52]. Although the impact of HIFn non-VHL tumorigenesis is less well defined, these studiesint at a fundamental role in renal transformation.

irect targeting of HIF

Over 40 inhibitors of HIF-1 activity have been identified,ncluding inhibitors of mRNA expression, protein expres-ion, DNA-binding activity, or HIF-1 mediated gene tran-cription [53], with varying degrees of overlap with HIF-2.IF-� mRNA translation requires expression of mamma-

ian target of rapamycin (mTOR) via a PI3 kinase-depen-ent pathway [54]. Inhibitors of mTOR (rapamycin, tem-irolimus, everolimus) bind FK506 binding protein 12FKBP12). The resulting FKBP12/drug complex then bindsTOR, inducing G1 growth arrest. This prevents transla-

ional initiation of proteins including HIF-� [55]. Preclini-al studies show that VHL loss sensitizes cells to mTORnhibition in a HIF-dependent manner [56]. A Phase II studyf single-agent temsirolimus (CCI-779) in cytokine refrac-ory advanced RCC yielded a 7% objective response rate,nd a 51% stable disease rate at 24 weeks [57]. In combi-ation with interferon-�, temsirolimus resulted in 11% par-ial response with median time to progression of 9 months58]. A large (n � 626) Phase III, randomized study ofrst-line temsirolimus vs. IFN vs. temsirolimus plus IFN inoor-risk RCC revealed a survival benefit for temsirolimuslone (median survival 10.9 months) compared with IFN7.3 months) or the combination (8.4 months) [59]. Theurvival benefit in this trial was greatest in patients withoorest risk features and with non-clear cell histology.verolimus (RAD-001) is an orally administered mTOR

nhibitor that has demonstrated antitumor activity as well asrolonged time to progression in a Phase II trial of heavilyretreated patients [60]. A randomized Phase III trial ofverolimus vs. placebo is ongoing.

IF-responsive growth factors and angiogenesis

Many of the HIF-responsive genes described to datencode growth factor receptors or their ligands, includingascular endothelial growth factor (VEGF), platelet derivedrowth factor (PDGF), and transforming growth factor-�TGF-�) [61]. Disruption of these pathways with neutraliz-ng antibodies or small molecule inhibitors has shownromise in clinical trials.

VEGF stimulates endothelial cell proliferation and sur-ival and suppresses the antitumor immune response62,63]. Furthermore, the VEGF tyrosine kinase receptor isxpressed by RCC cells, suggesting the possibility of anutocrine feedback loop in addition to paracrine effects ofEGF on endothelial cells [64]. Multiple VEGF isoformsave been described. VEGF-A is involved in angiogenesis,
hile VEGF-C and VEGF-D have been linked to lym- s
hangiogenesis [65]. Bevacizumab (Avastin) is a human-zed monoclonal antibody that binds and neutralizes all theajor isoforms of VEGF. In a randomized Phase II trial,

evacizumab led to a significant delay in time-to-progres-ion relative to placebo (4.8 vs. 2.5 months) in patients withdvanced RCC who had failed prior high dose IL-2 [66].wo randomized, Phase III trials comparing interferon vs.

nterferon plus bevacizumab have been completed [67] andill help determine the role of bevacizumab as a primary

reatment modality. Early analysis of one study shows aesponse rate of 31% for the combination relative to 13%or interferon alone, with a corresponding benefit in pro-ression free survival of 10.2 vs. 5.4 months [68]. Neutral-zation of serum VEGF with a fusion of the VEGF receptornd human immunoglobulin (the VEGF-trap) showed min-mal response in an early Phase I trial of 15 patients withdvanced solid malignancies. However, one subject withetastatic RCC maintained stable disease for greater than 6onths [69], and Phase II trials for advanced RCC are

lanned.Tyrosine kinase inhibitors also demonstrate single-agent

ctivity in advanced RCC. Sunitinib (SU11248) inhibits theeceptor tyrosine kinases VEGFR-2, PDGFR, FMS-like ty-osine kinase 3 (FLT-3), and c-KIT. Initial trials demon-trated sunitinib’s utility as a second-line agent for patientsith metastatic RCC who had failed initial cytokine therapy

70,71]. A recent randomized multicenter Phase III clinicalrial of sunitinib vs. interferon as first-line treatment foretastatic RCC revealed a response rate of 37% vs. 9% with

rogression free survival of 11 vs. 5 months and improveduality of life [72]. Initially developed as an RAF-1 inhib-tor, sorafenib (BAY43-9006), also inhibits VEGFR-2,EGFR-3, FLT-3, c-KIT, and PDGFR, and has shownromising activity and a good safety profile in RCC [73]. Ahase II “randomized discontinuation” trial of sorafenib vs.lacebo showed a progression free survival of 24 weeks vs.weeks [74]. In a subsequent Phase III trial, sorafenib

roduced definitive responses in 10% of patients, and sta-ilized disease in an additional 74%. There was a significantmprovement in progression-free survival vs. placebo (me-ian 5.5 vs. 2.8 months) [75]. These encouraging earlyesults and acceptable side effect profiles formed the basisor recent FDA approval of both Sunitinib and sorafenib.

Other VEGFR inhibitors are currently under investigation.xitinib (AG013736) inhibits VEGFR-1, VEGFR-2, PDGFR,

nd c-KIT. Early results have shown promising activity and aood tolerance [76]. Pazopanib (GW786034) targetsEGFR-1, VEGFR-2, VEGFR-3, PDGFR�, PDGFR�, and

-kit [77]. It recently completed Phase I testing and is currentlyeing evaluated in both European and U.S. trials.

Platelet-derived growth factor (PDGF)-B promotes vas-ular pericytes and maintenance of established vasculature78]. Preclinical data suggest that involution of blood ves-els may require dual inhibition of VEGF and PDGF79,80]. Many of the TKIs described above also demon-
trate activity against the PDGF receptor (PDGFR), re-

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ecting their structural similarities. Imatinib mesylateGleevec), an inhibitor of c-Abl and c-Kit used to treathronic myeloid leukemia and gastrointestinal stromal tu-ors, is also a potent inhibitor of PDGFR. Although initial

tudies reported minimal single-agent activity in advancedCC, combination trials of imatinib with VEGFR inhibitorsre underway.

TGF-� is another HIF-responsive growth factor, whichromotes proliferation by activating epidermal growth fac-or receptor (EGFR) expressed by RCC [81]. EGFR inhibi-ion abrogates RCC tumor growth in xenografts [82,83].mall molecule inhibitors (erlotinib and gefitinib), andonoclonal antibodies (cetuximab) targeting EGFR are

ow FDA-approved for other indications. Unfortunately,hey have thus far displayed limited single-agent activitygainst advanced RCC [84,85]. Lapatinib, an EGFR/Erb1yrosine kinase inhibitor, showed no overall survival benefits second-line therapy following cytokine failure, but didenefit a subset of patients with tumor overexpression ofGFR [86].

ombination therapy

Combining agents that target different points in theHL-HIF pathway may enhance therapeutic efficacy. Ahase II trial combining bevacizumab and erlotinib in pa-

ients with metastatic RCC reported a 25% overall responseate in 59 patients who had failed prior cytokine therapy87]. This led to a randomized Phase II trial comparingevacizumab plus erlotinib vs. bevacizumab plus placebo asrst line therapy for metastatic RCC that unfortunatelyemonstrated no discernible difference in response rate orrogression free survival [88]. Studies to evaluate the safetyf combination of small molecule inhibitors with monoclo-al antibodies or inhibitors targeting different pathways ofngiogenesis or proliferation are underway. Until clinicalrials demonstrate conclusive evidence that combinationherapy is both safe and effective, combinations should bevoided.

uture targets

Many other HIF target genes are currently under inves-igation as potential therapy targets. Cyclin D1 and its cat-lytic partner cyclin-dependent kinase 4 (cdk4) promote cellroliferation via phosphorylation of RB1 [38]. Flavopiridol,cdk inhibitor, showed an overall response rate of 12% and

table disease rate of 41% in a Phase II study of 34 patientsith advanced RCC. Unfortunately, this was associatedith significant drug-related toxicity [89]. Carbonic anhy-rase IX (CAIX) is another HIF transcription target up-egulated in RCC [90]. In conjunction with low dose IL-2,irect targeting of CAIX with G250, a chimeric anti-CAIX
onoclonal antibody, has shown promising results in early
linical trials [91]. There is evidence that the tyrosine kinaseeceptor c-Met and its ligand, hepatocyte growth factor, arenduced by HIF [92,93]. Activating mutations of the c-METroto-oncogene, characteristic of HPRC, have also beeneported in a subset of sporadic clear cell tumors [94,95].nhibitors of c-MET are in development for both clear cellnd papillary RCC. The small molecule, chemotin, disruptsinding of p300 to the CAD of HIF-�. It has been shown tobrogate tumor growth in RCC xenografts [96]. Inhibitorsf HSP90 including geldanamycin reduce HIF-� proteinxpression and activity [97]. As noted, suppression of theox4 NADPH oxidase decreases HIF-� mRNA expression

nd activity and abrogates tumor growth in xenografts.hese and other targets that disrupt HIF�/HIF� or HIF-oactivator interactions will provide new therapeutic targetsn the treatment of RCC.

onclusions

Recent advances in the understanding of the role of HIFn RCC biology have had a dramatic impact on the devel-pment of novel targeted therapies in patients with ad-anced disease. Small molecule inhibitors (temsirolimus,unitinib, and sorafenib), and monoclonal antibodies (bev-cizumab) have demonstrated promising results in Phase IInd Phase III clinical trials as both adjunct and primaryherapies. The tyrosine kinase inhibitors sunitinib and sor-fenib have been approved by the FDA and are currentlyeing implemented in clinical practice. Combination ther-py with and without adjuvant cytokine therapy is stillnder early investigation. Further definition of the VHL-IF pathway will continue to provide insight into RCC

umorigenesis and novel therapeutic targets for this complexatient population.

eferences

[1] Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2006. CA CancerJ Clin 2006;56(2):106–30.

[2] Pantuck AJ, Zisman A, Belldegrun AS. The changing natural historyof renal cell carcinoma. J Urol 2001;166(5):1611–23.

[3] Hock LM, Lynch J, Balaji KC. Increasing incidence of all stages ofkidney cancer in the last two decades in the United States: Ananalysis of surveillance, epidemiology, and end results program data.J Urol 2002;167(1):57–60.

[4] Bukowski RM. Cytokine therapy for metastatic renal cell carcinoma.Semin Urol Oncol 2001;19(2):148–54.

[5] Semenza GL, Wang GL. A nuclear factor induced by hypoxia via denovo protein synthesis binds to the human erythropoietin gene en-hancer at a site required for transcriptional activation. Mol Cell Biol1992;12(12):5447–54.

[6] Wang GL, Jiang BH, Rue EA, et al. Hypoxia-inducible factor 1 is abasic-helix-loop-helix-PAS heterodimer regulated by cellular O2 ten-sion. Proc Natl Acad Sci USA 1995;92(12):5510–4.

[7] Jain S, Maltepe E, Lu MM, et al. Expression of ARNT, ARNT2,HIF1 �, HIF2 � and Ah receptor mRNAs in the developing mouse
Mech Dev 1998;73(1):117–23.

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[8] Salceda S, Caro J. Hypoxia-inducible factor 1 � (HIF-1�) protein israpidly degraded by the ubiquitin-proteasome system under normoxicconditions. Its stabilization by hypoxia depends on redox-inducedchanges. J Biol Chem 1997;272(36):22642–7.

[9] Kallio PJ, Wilson WJ, O’Brien S, et al. Regulation of the hypoxia-inducible transcription factor 1� by the ubiquitin-proteasome path-way. J Biol Chem 1999;274(10):6519–25.

10] Sowter HM, Ratcliffe PJ, Watson P, et al. HIF-1-dependent regula-tion of hypoxic induction of the cell death factors BNIP3 and NIX inhuman tumors. Cancer Res 2001;61(18):6669–73.

11] Srinivas V, Zhang LP, Zhu XH, et al. Characterization of an oxygen/redox-dependent degradation domain of hypoxia-inducible factor �

(HIF-�) proteins. Biochem Biophys Res Commun 1999;260(2):557–61.

12] McNeill LA, Hewitson KS, Claridge TD, et al. Hypoxia-induciblefactor asparaginyl hydroxylase (FIH-1) catalyses hydroxylation at the�-carbon of Asparagine-803. Biochem J 2002;367:571–5.

13] Freedman SJ, Sun ZY, Poy F, et al. Structural basis for recruitment ofCBP/p300 by hypoxia-inducible factor-1 �. Proc Natl Acad Sci USA2002;99(8):5367–72.

14] Land SC, Tee AR. Hypoxia inducible factor 1� is regulated by themammalian target of rapamycin (mTOR) via an mTOR-signalingmotif. J Biol Chem 2007;282:20534–43.

15] Gnarra JR, Tory K, Weng Y, et al. Mutations of the VHL tumorsuppressor gene in renal carcinoma. Nat Genet 1994;7(1):85–90.

16] Lisztwan J, Imbert G, Wirbelauer C, et al. The von Hippel-Lindautumor suppressor protein is a component of an E3 ubiquitin-proteinligase activity. Genes Dev 1999;13(14):1822–33.

17] Maxwell PH, Wiesener MS, Chang GW, et al. The tumor suppressorprotein VHL targets hypoxia-inducible factors for oxygen-dependentproteolysis. Nature 1999;399(6733):271–5.

18] Aprelikova O, Chandramouli GV, Wood M, et al. Regulation of HIFprolyl hydroxylases by hypoxia-inducible factors. J Cell Biochem2004;92(3):491–501.

19] Ivan M, Kondo K, Yang H, et al. HIF� targeted for VHL-mediateddestruction by proline hydroxylation: Implications for O2 sensing.Science 2001;292(5516):464–8.

20] Jaakkola P, Mole DR, Tian YM, et al. Targeting of HIF-� to the vonHippel-Lindau ubiquitination complex by O2-regulated prolyl hy-droxylation. Science 2001;292(5516):468–72.

21] Tian H, McKnight SL, Russell DW. Endothelial PAS domain protein1 (EPAS1), a transcription factor selectively expressed in endothelialcells. Genes Dev 1997;11(1):72–82.

22] Ryan HE, Lo J, Johnson RS. HIF-1 � is required for solid tumorformation and embryonic vascularization. EMBO J 1998;17(11):3005–15.

23] Tian H, Hammer RE, Matsumoto AM, et al. The hypoxia-responsivetranscription factor EPAS1 is essential for catecholamine homeostasisand protection against heart failure during embryonic development.Genes Dev 1998;12(21):3320–4.

24] Holmquist L, Jogi A, Pahlman S. Phenotypic persistence after reoxy-genation of hypoxic neuroblastoma cells. Int J Cancer 2005;116(2):218–25.

25] Wiesener MS, Jurgensen JS, Rosenberger C, et al. Widespread hy-poxia-inducible expression of HIF-2� in distinct cell populations ofdifferent organs. FASEB J 2003;17(2):271–3.

26] Mandriota SJ, Turner KJ, Davies DR, et al. HIF activation identifiesearly lesions in VHL kidneys. Evidence for site-specific tumor sup-pressor function in the nephron. Cancer Cell 2002;1(5):459–68.

27] Kim CM, Vocke C, Torres-Cabala C, et al. Expression of hypoxiainducible factor-1� and 2� in genetically distinct early renal corticaltumors. J Urol 2006;175(5):1908–14.

28] Gu YZ, Moran SM, Hogenesch JB, et al. Molecular characterizationand chromosomal localization of a third �-class hypoxia inducible
factor subunit, HIF3�, Gene Expr 1998;7(3):205–13.
29] Maynard MA, Qi H, Chung J, et al. Multiple splice variants of thehuman HIF-3 � locus are targets of the von Hippel-Lindau E3ubiquitin ligase complex. J Biol Chem 2003;278(13):11032–40.

30] Maynard MA, Evans AJ, Hosomi T, et al. Human HIF-3�4 is adominant-negative regulator of HIF-1 and is down-regulated in renalcell carcinoma. FASEB J 2005;19(11):1396–406.

31] Kim WY, Kaelin WG Jr. Molecular pathways in renal cell carci-noma—rationale for targeted treatment. Semin Oncol 2006;33(5):588–95.

32] Maranchie JK, Vasselli JR, Riss J, et al. The contribution of VHLsubstrate binding and HIF1-� to the phenotype of VHL loss in renalcell carcinoma. Cancer Cell 2002;1(3):247–55.

33] Kondo K, Klco J, Nakamura E, et al. Inhibition of HIF is necessaryfor tumor suppression by the von Hippel-Lindau protein. Cancer Cell2002;1(3):237–46.

34] Zimmer M, Doucette D, Siddiqui N, et al. Inhibition of hypoxia-inducible factor is sufficient for growth suppression of VHL–/– tu-mors. Mol Cancer Res 2004;2(2):89–95.

35] Kondo K, Kim WY, Lechpammer M, et al. Inhibition of HIF2� issufficient to suppress pVHL-defective tumor growth. PLoS Biol2003;1(3):E83.

36] Rankin EB, Higgins DF, Walisser JA, et al. Inactivation of thearylhydrocarbon receptor nuclear translocator (Arnt) suppresses vonHippel-Lindau disease-associated vascular tumors in mice. Mol CellBiol 2005;25(8):3163–72.

37] Hu CJ, Wang LY, Chodosh LA, et al. Differential roles of hypoxia-inducible factor 1� (HIF-1�) and HIF-2� in hypoxic gene regulation.Mol Cell Biol 2003;23(24):9361–74.

38] Raval RR, Lau KW, Tran MG, et al. Contrasting properties ofhypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma. Mol Cell Biol 2005;25(13):5675–86.

39] Rankin EB, Biju MP, Liu Q, et al. Hypoxia-inducible factor-2(HIF-2) regulates hepatic erythropoietin in vivo. J Clin Invest 2007;117(4):1068–77.

40] Pouyssegur J, Mechta-Grigoriou F. Redox regulation of the hypoxia-inducible factor. Biol Chem 2006;387(10–11):1337–46.

41] Haddad JJ, Olver RE, Land SC. Antioxidant/pro-oxidant equilibriumregulates HIF-1� and NF-� B redox sensitivity. Evidence for inhibi-tion by glutathione oxidation in alveolar epithelial cells. J Biol Chem2000;275(28):21130–9.

42] Haddad JJ, Land SC. A non-hypoxic, ROS-sensitive pathway medi-ates TNF-�-dependent regulation of HIF-1�. FEBS Lett 2001;505(2):269–74.

43] Richard DE, Berra E, Pouyssegur J. Nonhypoxic pathway mediatesthe induction of hypoxia-inducible factor 1� in vascular smoothmuscle cells. J Biol Chem 2000;275(35):26765–71.

44] Jung YJ, Isaacs JS, Lee S, et al. IL-1�-mediated up-regulation ofHIF-1� via an NF�B/COX-2 pathway identifies HIF-1 as a criticallink between inflammation and oncogenesis. FASEB J 2003;17(14):2115–7.

45] BelAiba RS, Djordjevic T, Bonello S, et al. Redox-sensitive regula-tion of the HIF pathway under nonhypoxic conditions in pulmonaryartery smooth muscle cells. Biol Chem 2004;385(3/4):249–57.

46] Shiose A, Kuroda J, Tsuruya K, et al. A novel superoxide-producingNAD(P)H oxidase in kidney. J Biol Chem 2001;276(2):1417–23.

47] Maranchie JK, Zhan Y. Nox4 is critical for hypoxia-inducible factor2-� transcriptional activity in von Hippel-Lindau-deficient renal cellcarcinoma. Cancer Res 2005;65(20):9190–3.

48] Block K, Gorin Y, Hoover P, et al. NAD(P)H oxidases regulateHIF-2� protein expression. J Biol Chem 2007;282(11):8019–26.

49] Isaacs JS, Jung YJ, Mimnaugh EG, et al. Hsp90 Regulates a vonHippel Lindau-independent hypoxia-inducible factor-1�-degradativepathway. J Biol Chem 2002;277(33):29936–44.

50] Isaacs JS, Jung YJ, Neckers L. Aryl hydrocarbon nuclear translocator(ARNT) promotes oxygen-independent stabilization of hypoxia-in-ducible factor-1� by modulating an Hsp90-dependent regulatory
pathway. J Biol Chem 2004;279(16):16128–35.

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51] Isaacs JS, Jung YJ, Mole DR, et al. HIF overexpression correlateswith biallelic loss of fumarate hydratase in renal cancer: Novel role offumarate in regulation of HIF stability. Cancer Cell 2005;8(2):143–53.

52] Liu MY, Poellinger L, Walker CL. Up-regulation of hypoxia-induc-ible factor 2� in renal cell carcinoma associated with loss of Tsc-2tumor suppressor gene. Cancer Res 2003;63(10):2675–80.

53] Semenza GL. Development of novel therapeutic strategies that targetHIF-1. Expert opinion on therapeutic targets 2006;10(2):267–80.

54] Hay N. The Akt-mTOR tango and its relevance to cancer. Cancer Cell2005;8(3):179–83.

55] Hudson CC, Liu M, Chiang GG, et al. Regulation of hypoxia-induc-ible factor 1� expression and function by the mammalian target ofrapamycin. Mol Cell Biol 2002;22(20):7004–14.

56] Thomas GV, Tran C, Mellinghoff IK, et al. Hypoxia-inducible factordetermines sensitivity to inhibitors of mTOR in kidney cancer. NatMed 2006;12(1):122–7.

57] Atkins MB, Hidalgo M, Stadler WM, et al. Randomized Phase IIstudy of multiple dose levels of CCI-779, a novel mammalian targetof rapamycin kinase inhibitor, in patients with advanced refractoryrenal cell carcinoma. J Clin Oncol 2004;22(5):909–18.

58] Smith J, Ko Y-J, Dutcher J, et al. Update of a Phase I study ofintravenous CCI-779 given in combination with interferon-� to pa-tients with advanced renal cell carcinoma. J Clin Oncol 2004;22:385S[Abstract].

59] Hudes G, Carducci M, Tomczak P, et al. A Phase 3, randomized3-arm study of temsirolimus (TEMSR) or interferon-� (IFN) or thecombination of TEMSR�IFN in the treatment of first-line, poor riskpatients with advanced renal cell carcinoma (adv RCC). J Clin Oncol2004;22(3):454–63 [abstract].

60] Amato R, Misellati A, Khan M, et al. A Phase II trial of RAD001 inpatients (Pts) with metastatic renal cell carcinoma (MRCC) J ClinOncol 2006;24:224s [Abstract].

61] Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer2003;3(10):721–32.

62] Gabrilovich DI, Ishida T, Nadaf S, et al. Antibodies to vascularendothelial growth factor enhance the efficacy of cancer immunother-apy by improving endogenous dendritic cell function. Clin CancerRes 1999;5(10):2963–70.

63] Ohm JE, Gabrilovich DI, Sempowski GD, et al. VEGF inhibits T-celldevelopment and may contribute to tumor-induced immune suppres-sion. Blood 2003;101(12):4878–86.

64] Fox SB, Turley H, Cheale M, et al. Phosphorylated KDR is expressedin the neoplastic and stromal elements of human renal tumors andshuttles from cell membrane to nucleus. J Pathol 2004;202(3):313–20.

65] Shibuya M, Claesson-Welsh L. Signal transduction by VEGF recep-tors in regulation of angiogenesis and lymphangiogenesis. Exp CellRes 2006;312(5):549–60.

66] Yang JC, Haworth L, Sherry RM, et al. A randomized trial ofbevacizumab, an antivascular endothelial growth factor antibody, formetastatic renal cancer. N Engl J Med 2003;349(5):427–34.

67] Rini BI, Halabi S, Taylor J, et al. Cancer and Leukemia Group B90206: A randomized Phase III trial of interferon-� or interferon-�plus antivascular endothelial growth factor antibody (bevacizumab)in metastatic renal cell carcinoma. Clin Cancer Res 2004;10(8):2584–6.

68] Escudier B, Pluzanska A, Koralewski P, et al. Bevacizumab plusinterferon alfa-2a for treatment of metastatic renal cell carcinoma: arandomised, double-blind phase III trial. Lancet 2007;370:2103–11.

69] Dupont J, Camastra D, Gordon M, et al. Phase I study of VEGF Trapin patients with solid tumors and lymphoma. Proceedings of theAmerican Society for Clinical Oncology 2003;22:194 [Abstract].

70] Motzer RJ, Michaelson MD, Redman BG, et al. Activity of SU11248,a multitargeted inhibitor of vascular endothelial growth factor recep-tor and platelet-derived growth factor receptor, in patients with met-
astatic renal cell carcinoma. J Clin Oncol 2006;24(1):16–24.
71] Motzer RJ, Rini BI, Bukowski RM, et al. Sunitinib in patients withmetastatic renal cell carcinoma. JAMA 2006;295(21):2516–24.

72] Motzer RJ, Hutson TE, Tomczak P, et al. Sunitinib vs. interferon � inmetastatic renal-cell carcinoma. N Engl J Med 2007;356(2):115–24.

73] Escudier B, Lassau N, Angevin E, et al. Phase I trial of sorafenib incombination with IFN �-2a in patients with unresectable and/ormetastatic renal cell carcinoma or malignant melanoma. Clin CancerRes 2007;13(6):1801–9.

74] Ratain MJ, Eisen T, Stadler WM, et al. Phase II placebo-controlledrandomized discontinuation trial of sorafenib in patients with meta-static renal cell carcinoma. J Clin Oncol 2006;24(16):2505–12.

75] Escudier B, Eisen T, Stalder WM, et al. Randomized Phase III trial ofthe RAF kinase and VEGFR inhibitor sorafenib (BAY 43–9006) inpatients with advanced renal cell carcinoma (RCC). Proceedings ofthe American Society of Clinical Oncology Annual Meeting 2005;Orlando, FL.

76] Rini B, Rixe O, Bukowski R, et al. AG-013736, a multi-targettyrosine kinase inhibitor, demonstrates antitumor activity in a Phase2 study of cytokine refractory, metastatic renal cell cancer (RCC).J Clin Oncol 2004;23:380 [Abstract].

77] Kumar R, Knick VB, Rudolph SK, et al. Pharmacokinetic-pharma-codynamic correlation from mouse to human with pazopanib, a mul-tikinase angiogenesis inhibitor with potent antitumor and antiangio-genic activity. Mol Cancer Ther 2007;6(7):2012–21.

78] Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch.Nat Rev Cancer 2003;3(6):401–10.

79] Bergers G, Song S, Meyer-Morse N, et al. Benefits of targeting bothpericytes and endothelial cells in the tumor vasculature with kinaseinhibitors. J Clin Invest 2003;111(9):1287–95.

80] Benjamin LE, Golijanin D, Itin A, et al. Selective ablation of imma-ture blood vessels in established human tumors follows vascularendothelial growth factor withdrawal. J Clin Invest 1999;103(2):159–65.

81] Knebelmann B, Ananth S, Cohen HT, et al. Transforming growthfactor � is a target for the von Hippel-Lindau tumor suppressor.Cancer Res 1998;58(2):226–31.

82] Prewett M, Rothman M, Waksal H, et al. Mouse-human chimericanti-epidermal growth factor receptor antibody C225 inhibits thegrowth of human renal cell carcinoma xenografts in nude mice. ClinCancer Res 1998;4(12):2957–66.

83] Smith K, Gunaratnam L, Morley M, et al. Silencing of epidermalgrowth factor receptor suppresses hypoxia-inducible factor-2-drivenVHL–/– renal cancer. Cancer Res 2005;65(12):5221–30.

84] Motzer RJ, Amato R, Todd M, et al. Phase II trial of anti-epidermalgrowth factor receptor antibody C225 in patients with advanced renalcell carcinoma. Invest New Drugs 2003;21(1):99–101.

85] Rowinsky EK, Schwartz GH, Gollob JA, et al. Safety, pharmacoki-netics, and activity of ABX-EGF, a fully human anti-epidermalgrowth factor receptor monoclonal antibody in patients with meta-static renal cell cancer. J Clin Oncol 2004;22(15):3003–15.

86] Ravaud A, Gardner J, Hawkins R, et al. Efficacy of lapatinib inpatients with high tumor EGFR expression: Results of a Phase III trialin advanced renal cell carcinoma (RCC). J Clin Oncol 2006;24:217s[Abstract].

87] Hainsworth JD, Sosman JA, Spigel DR, et al. Treatment of metastaticrenal cell carcinoma with a combination of bevacizumab and erlo-tinib. J Clin Oncol 2005;23(31):7889–96.

88] Bukowski R, Kabbinavar F, Figlin R, et al. Results of a randomizedPhase II trial of bevacizumab �/– erlotinib in mRCC. J Clin Oncol2006;24:18S [Abstract].

89] Van Veldhuizen PJ, Faulkner JR, Lara PN Jr., et al. A Phase II studyof flavopiridol in patients with advanced renal cell carcinoma: Resultsof Southwest Oncology Group Trial 0109. Cancer Chemother Phar-macol 2005;56(1):39–45.

90] Grabmaier K, MC AdW, Verhaegh GW, et al. Strict regulation ofCAIX(G250/MN) by HIF-1� in clear cell renal cell carcinoma. On-
cogene 2004;23(33):5624–31.

[

[

[

[

[

[

[


91] Bleumer I, Oosterwijk E, Oosterwijk-Wakka JC, et al. A clinical trialwith chimeric monoclonal antibody WX-G250 and low dose inter-leukin-2 pulsing scheme for advanced renal cell carcinoma. J Urol2006;175(1):57–62.

92] Pennacchietti S, Michieli P, Galluzzo M, et al. Hypoxia promotesinvasive growth by transcriptional activation of the met protoonco-gene. Cancer Cell 2003;3(4):347–61.

93] Hayashi M, Sakata M, Takeda T, et al. Up-regulation of c-metprotooncogene product expression through hypoxia-inducible fac-tor-1� is involved in trophoblast invasion under low-oxygen tension.
Endocrinology 2005;146(11):4682–9.
94] Natali PG, Prat M, Nicotra MR, et al. Overexpression of the met/HGFreceptor in renal cell carcinomas. Int J Cancer 1996;69(3):212–7.

95] Schmidt L, Duh FM, Chen F, et al. Germline and somatic mutationsin the tyrosine kinase domain of the MET proto-oncogene in papillaryrenal carcinomas. Nat Genet 1997;16(1):68–73.

96] Kung AL, Wang S, Klco JM, et al. Suppression of tumor growththrough disruption of hypoxia-inducible transcription. Nat Med 2000;6(12):1335–40.

97] Kong X, Lin Z, Liang D, et al. Histone deacetylase inhibitors induceVHL and ubiquitin-independent proteasomal degradation of hypoxia-
inducible factor 1�. Mol Cell Biol 2006;26(6):2019–28.

clinical implications of hypoxia inducible factor in renal cell carcinoma

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