..............................................................

Chemokine receptor CXCR4downregulated by von Hippel–Lindautumour suppressor pVHLPeter Staller1*, Jitka Sulitkova1, Joanna Lisztwan1, Holger Moch2,Edward J. Oakeley1 & Wilhelm Krek1*

1Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66,CH-4058 Basel, Switzerland2Institute of Pathology, University of Basel, Schonbeinstrasse 40, CH-4031 Basel,Switzerland

* Present address: Institute of Cell Biology, ETH Honggerberg, HPM F 42, CH-8093 Zurich, Switzerland

.............................................................................................................................................................................

Organ-specific metastasis is governed, in part, by interactionsbetween chemokine receptors on cancer cells and matchingchemokines in target organs. For example, malignant breastcancer cells express the chemokine receptor CXCR4 and com-monly metastasize to organs that are an abundant source of theCXCR4-specific ligand stromal cell-derived factor-1a (ref. 1). Itis still uncertain how an evolving tumour cell is reprogrammed toexpress CXCR4, thus implementing the tendency to metastasizeto specific organs. Here we show that the von Hippel–Lindautumour suppressor protein pVHL negatively regulates CXCR4expression owing to its capacity to target hypoxia-induciblefactor (HIF) for degradation under normoxic conditions. Thisprocess is suppressed under hypoxic conditions, resulting inHIF-dependent CXCR4 activation. An analysis of clear cellrenal carcinoma that manifests mutation of the VHL gene inmost cases revealed an association of strong CXCR4 expressionwith poor tumour-specific survival. These results suggest amechanism for CXCR4 activation during tumour cell evolutionand imply that VHL inactivation acquired by incipient tumourcells early in tumorigenesis confers not only a selective survivaladvantage but also the tendency to home to selected organs.

Inactivation of the VHL tumour suppressor gene is linked to thedevelopment of several different tumour types in humans, includ-ing hereditary and sporadic clear cell carcinoma of the kidney2. Thebest characterized function of pVHL is as a recognition subunit ofan E3 ubiquitin protein ligase complex that targets the a-subunits ofthe DNA-binding transcription factor HIF (ref. 3) for ubiquitin-mediated degradation in the presence of oxygen4. Tumour-derivedpVHL mutants are defective in this regard and manifest constitutiveactivation of HIF target genes5,6. Indeed, HIF activation is an earlyevent in the evolution of neoplastic kidney lesions in VHL disease7.To identify novel pVHL-regulated genes, we compared the geneexpression profile of pVHL-deficient renal cell carcinoma (RCC)cells previously stably transfected with an empty vector (designatedA498(neo)) with those engineered to stably produce haemaggluti-nin (HA)-tagged wild-type pVHL30 (designated A498(HA–pVHL30)) using Affymetrix gene chip technology. This approachidentified 101 genes that were significantly upregulated in pVHL-expressing A498 cells relative to vector control cells, and 64 that weresignificantly downregulated (Supplementary Fig. S1).

We were particularly intrigued by the fact that, among the genesmost strongly suppressed by the reintroduction of functionalpVHL30 in A498 cells is the gene encoding the G-protein-coupledchemokine receptor CXCR4 (Supplementary Fig. S1), the receptorfor the chemokine stromal cell-derived factor-1a (SDF-1a; alsoreferred to as CXCL12)8,9. Chemokine receptor–ligand interactionshave been implicated in the homing of various subsets of haema-topoietic cells to specific anatomical sites10,11 and to determine themetastatic destination of malignant breast cancer cells1. The latterfinding provided molecular support for the ‘chemoattractiontheory’ of organ-specific metastasis12,13. A key unanswered question

concerns the molecular mechanism through which cells acquireCXCR4 during the evolution of tumour cells. The fact that the geneon top of the ‘list’ of pVHL-suppressed genes was CXCR4 providedus with an unexpected avenue down which to pursue this question.

To validate the oligonucleotide microarray results, we performedNorthern blot analysis. pVHL-negative A498(neo) cells express highlevels of CXCR4 messenger RNA (Fig. 1a, lane 1), whereas theirpVHL-expressing counterparts, A498(HA–pVHL30), do not (lane2). The disappearance of CXCR4 mRNA from pVHL-expressingcells was correlated with a downregulation of HIF2a proteinexpression. A498(neo) cells also exhibited strong surface expressionof the CXCR4 receptor as demonstrated by immunofluorescencemicroscopy (Fig. 1b, upper panel). Thus, pVHL30 negatively regu-lates, directly or indirectly, CXCR4 mRNA and protein productionin RCC cells.

The above experiments were performed with an A498 cloneexpressing pVHL30. To test whether pVHL19 behaves similarly andto assess whether certain naturally occurring tumour-derivedmutants of pVHL are defective in CXCR4 regulation, we infectedA498 cells with empty control retrovirus or with retrovirusesencoding untagged pVHL30, pVHL19 or the HIF degradation-defective tumour mutant pVHL30(N78S)14. Expression of pVHL30

or pVHL19 suppressed CXCR4 mRNA production (Fig. 1c, lanes 2and 3), whereas expression of pVHL30(N78S) did not (Fig. 1c, lane4). pVHL30 mutants that exerted residual degradation activitytowards HIF2a such as pVHL30(Y98H) or pVHL30(R167W)14 hadintermediate suppressive effects on CXCR4 mRNA abundance (datanot shown). These results suggest that pVHL is a negative regulatorof CXCR4 gene expression in RCC cells and indicate that thisfunction is affected in tumour-derived mutants of pVHL that failto regulate the HIF system. Moreover, they imply a link between lossof function of pVHL, the ensuing expression of HIFa and theoverproduction of CXCR4 mRNA.

In keeping with the above, CXCR4 mRNA was strongly inducedin pVHL-positive human embryonic kidney (HEK-293) cells andprimary human proximal renal tubular epithelia cells (RPTECs)when they were subjected to low oxygen concentrations (1%)(Fig. 1d, e, respectively). In addition, the hypoxia-mimetic sub-stance cobalt chloride induced CXCR4 mRNA in RPTECs (Fig. 1e).The kinetics of CXCR4 mRNA accumulation in response to hypoxiaclosely followed that of GLUT3, an established HIF target15 (Fig. 1e).Taken together, these results suggest that CXCR4 is a hypoxia-inducible gene.

To substantiate this conclusion we cloned the human CXCR4promoter including the first intronic region. Sequence analysis ofthis region revealed four potential hypoxia-response elements(HREs) located within 2.6 kilobases upstream of the transcriptionalstart site and one at position þ1 kb within the intron (Fig. 2a). Aluciferase reporter containing this CXCR4 promoter fragment,when introduced into VHL-positive HEK-293 cells, was activatedabout twofold by hypoxia (data not shown). Co-transfection ofwild-type HIF1a enhanced hypoxia-inducible reporter activity(about 10-fold; Fig. 2a) and a prolyl-hydroxylation-defectivemutant of HIF1a, HIF1a(P564A), which escapes pVHL control,activated the CXCR4 promoter also under normoxic conditions(about 7.5-fold; Fig. 2a). A progressive deletion analysis of theCXCR4 promoter revealed that the HRE located at 21.3 kbupstream of the transcriptional start is critical for hypoxia/HIF1a-inducible reporter activity (Fig. 2a), and mutation of thissequence in the context of the full-length CXCR4 promoter ren-dered the promoter insensitive to hypoxia and HIF1a (Fig. 2a).Finally, an oligonucleotide comprising this sequence (referred tohereafter as CXCR4-HRE (CHRE)) bound baculovirus-producedHIF1a–ARNT (Aryl hydrocarbon receptor nuclear translocator)heterodimers in electrophoretic mobility-shift assays (Fig. 2b,lane 3). Oligonucleotide binding of HIF1a–ARNT complexeswas specific, as shown by competition and supershift experiments

letters to nature

NATURE | VOL 425 | 18 SEPTEMBER 2003 | www.nature.com/nature 307© 2003 Nature Publishing Group

(Fig. 2b, lanes 4–12 and 13–15, respectively). Similarly, whole cellextracts of A498 cells gave rise to a prominent gel-shift complex(Fig. 2c, lane 2), which competed specifically with wild-type but notwith a mutant CHRE oligonucleotide (Fig. 2c, lanes 6–8 and 9–11,respectively). In addition, anti-HIF2a antibodies abolished (Fig. 2c,lane 12) or supershifted (lanes 13 and 14) this complex, suggestingthat it contains HIF2a. Anti-HIF1a antibodies had no effect (Fig.2c, lane 15). These results strongly suggest that CXCR4 is a noveltarget gene of the DNA-binding transcriptional activator HIF.

Next we asked whether the presence of CXCR4 on the surface ofA498 cells would confer chemoattraction. As shown in Fig. 3a (leftpanel), stimulation of A498 cells with increasing concentrations ofSDF-1a resulted in directed migration of these cells in a trans-wellassay. This response was abrogated by the restoration of wild-typepVHL (Fig. 3a, right panel). Because directional cell migration andtissue invasion require changes in the dynamics of the actincytoskeleton, we next tested whether CXCR4 signalling in RCCcells results in the activation of downstream effectors such as LIMkinase 1 (LIMK-1)16. The latter is important in controlling actindynamics through inactivation of the actin depolymerization factorcofilin17. Stimulation of A498 cells with SDF-1a led to a rapidactivation of LIMK-1 (Fig. 3b, left panel) and extracellular signal-related kinases (ERKs), which regulate cell proliferation and thecell’s motility machinery (Fig. 3c, upper panel). Restoration ofpVHL function abrogated these responses (Fig. 3b, c). The loss ofpVHL tumour suppressor function in RCC cells is thereforeintimately linked to the acquisition of a novel property, namelythe enhanced responsiveness of such cells to chemotactic signals.

Next we asked whether CXCR4 is overexpressed in human renaltumours and, if so, whether its upregulation is correlated with theinactivation of VHL. As a surrogate of the latter, we determined theexpression of the HIF target genes encoding carbonic anhydrase(CA9) and glucose transporter 1 (GLUT1) because upregulation ofthese genes is correlated with VHL status in clear cell RCC.Expression levels of CXCR4, CA9 and GLUT1 were assessed in 29clear cell RCC and 5 papillary RCC using real-time quantitativepolymerase chain reaction (PCR) and compared with those innormal renal tissue (n ¼ 7). As illustrated in Fig. 4a, mRNA levels

of CXCR4, CA9 and GLUT1 were significantly higher in clear cellthan in papillary RCC (P , 0.05; Supplementary Table S3) ornormal renal tissue. Because papillary RCCs are not characterizedby VHL inactivation, these findings argue that CXCR4 expressionfollows VHL inactivation and HIF target gene activation in clear cellRCC.

To evaluate the expression of CXCR4 in a wide range of RCC, wescreened a renal cancer tissue microarray (TMA) containing 532different elements for CXCR4 protein expression by immunohis-tochemistry. The antibody used in this study detects high levels ofCXCR4 expression in breast cancer tissue (Fig. 4b), consistent withearlier results. Glomeruli and tubules in normal kidney tissuestained weakly, whereas intensive staining of lymphocellular infil-trates was observed (Fig. 4c). With this antibody we observed apattern of CXCR4 expression on the TMA ranging from absent/weak to strong (Fig. 4d–g, respectively). To determine whetherCXCR4 expression is correlated with clinical aggressiveness in clearcell RCC, we focused our analysis on 195 of 407 clear cell carcinomasamples on the TMA for which tumour-specific survival data wereavailable. Within this cohort, strong CXCR4 expression wasdetected in 93 samples (47.6%). In the remainder of the TMAelements, staining was either weak or absent. Statistical analysis ofthese data revealed no significant correlation of high levels ofCXCR4 expression with tumour stage and/or differentiation grade(Supplementary Table S4). However, we found a striking positivecorrelation between strong CXCR4 expression and poor tumour-specific survival (P ¼ 0.001) in a univariate analysis (Fig. 4 h).Importantly, multivariate Cox proportional hazards analysissuggests that this correlation was independent of tumour stageand differentiation grade (Table 1). No correlation with tumour-specific survival was observed for CA9: it displayed strong staining

Figure 1 pVHL and hypoxia regulate CXCR4 expression. a, Panels from the top: CXCR4

mRNA expression in A498(neo) and A498(HA–pVHL30) cells, and immunoblots of HIF2a,

pVHL and CDK2. b, A498(neo) and A498(HA–pVHL30) cells stained with anti-CXCR4

monoclonal antibody (green) and propidium iodide (red). c, Retrovirally infected A498 cell

pools stably expressing pVHL30, pVHL19 or pVHL30(N78S) were analysed for the

expression of CXCR4 mRNA and for HIF2a, pVHL and CDK2 proteins. d, e, CXCR4 and

GLUT3 mRNA expression in HEK-293 cells (d) and RPTECs (e) treated with hypoxia or

cobalt chloride (100 mM) as shown. WB, western blot.

Table 1 Cox analysis for clear cell renal carcinoma

Variable Relative risk P.............................................................................................................................................................................

CXCR4 expression 1.8 ,0.01Differentiation grade 1.9 ,0.001Tumour stage 3.0 ,0.0001.............................................................................................................................................................................

letters to nature

NATURE | VOL 425 | 18 SEPTEMBER 2003 | www.nature.com/nature308 © 2003 Nature Publishing Group

in 169 (87%) of the 195 samples (data not shown). CA9 expressionwas also not correlated with tumour stage and grade (data notshown). These results imply that a high level of CXCR4 expression isa predictor of poor tumour-specific survival, which in turn suggeststhat monitoring CXCR4 expression in patients with RCC mayprovide additional prognostic information.

The data presented in this report suggest a potential mechanismof how and when evolving clear cell RCC cells may acquire CXCR4and thus the tendency to metastasize to specific secondary sites. It isknown that the survival of patients with clear cell RCC is limited bythe development of metastasis. The association of strong CXCR4expression with poor tumour-specific survival implies that CXCR4expression levels influence the metastatic behaviour of clear cellRCC. The acquisition of CXCR4 expression is likely to occur duringthe initial phases of tumour progression, set off by the functionalinactivation of pVHL. Whether CXCR4 has additional roles, such asthe promotion of cell proliferation, that could confer a selectiveadvantage for the tumour cell at the primary site remains to beinvestigated. Finally, activation of HIF is also the result of intratu-moral hypoxia and non-VHL oncogenic pathways18–20 (for example,

in glioblastoma the expression of CXCR4 is localized to regions ofnecrosis and angiogenesis21). Hence, the mechanism underlyingtumour cell-specific production of CXCR4 proposed here mayapply to solid tumours in general.

One of the most remarkable features of metastatic tumours is thedegree to which they differ genotypically and phenotypically fromtheir primary tumour. A key question that follows is whether thesechanges have already taken place in the primary tumour, allowing itto spread to a specific secondary site, or whether primary tumourcells that are carried to secondary organs undergo these changesafter they have been exposed to the new environment. The resultspresented here suggest that the expression of a receptor important

Figure 2 CXCR4 is a target gene of HIF. a, Luciferase activity after co-transfection of

indicated CXCR4 reporter plasmids and HIF1a alleles in HEK-293 cells. Numbers indicate

fold activation relative to b-galactosidase (b-gal) standard and control. Black squares

indicate potential HREs. Lower panel: schematic representation of the human CXCR4

locus. b, Gel-shift assay using a probe spanning the CHRE and cell extracts from

baculovirus-infected Sf9 cells coexpressing human HIF1a and ARNT. Competitor

oligonucleotides (amounts in nanograms) and antibodies used are indicated. c, Extracts of

A498 cells were assayed for CHRE DNA binding. Competition and supershift experiments

are indicated as described for b. Arrows in b and c indicate specific DNA binding

complexes consisting of HIF1a/ARNT (b) and HIF2a/ARNT (c). WT, wild type.

Figure 3 pVHL suppresses SDF-1a-mediated chemotaxis of RCC cells. a, Left:

chemotactic response of A498 cells to different concentrations of SDF-1a. Results are

expressed as the ratio between cells migrating towards the chemokine gradient and cells

migrating in the negative control. Right: chemotaxis of A498 cells expressing the indicated

pVHL species after exposure to 10 nM SDF-1a. b, In vitro LIM kinase-1 assay after

treatment of A498, A498(neo) and A498(HA–pVHL30) cells with SDF-1a. c, Cells were

treated for indicated durations with 10 nM SDF-1a and processed for immunoblotting

with anti-phospho-ERK1/ERK2 specific antibodies (upper panels) or anti-ERK1/2 total

protein antibodies (lower panels).

letters to nature

NATURE | VOL 425 | 18 SEPTEMBER 2003 | www.nature.com/nature 309© 2003 Nature Publishing Group

for homing to distant organs is directly regulated by the hypoxiapathway and that its upregulation is already seen in early tumourstages. The propensity to metastasize, at least for certain cancers,may therefore be determined by the identities of the mutant allelesacquired relatively early during multistep tumorigenesis22. A

MethodsCell culture, retroviral infections and reporter-gene assaysCell culture conditions and procedures for retroviral infections as well as plasmidconstructions are available from the authors on request. A 2.7-kb fragment of the humanCXCR4 promoter and the intronic region was amplified by PCR from A498 genomic DNAand subcloned into pGL2b and pGL2-promoter vector (Promega), respectively. HEK-293cells were subjected to 12 h of hypoxia in a three-gas incubator (Nabco) 24 h aftertransfection. Luciferase activity was corrected for b-galactosidase activity and at least threeindependent experiments were performed.

Gene microarray analysisAnalysis was performed with HG_U95A GeneChips (Affymetrix). Scanning was done inan Affymetrix GeneChip scanner. Analysis was performed using Affymetrix MicroarraySuite v5 and GeneSpring 4.2.1 (Silicon Genetics). Changes in gene expression wereassessed by looking for concordant changes between replicates by using a signed Wilcoxonrank test. The ‘change’ threshold P was less than 0.003 for increase and more than 0.997 fordecrease. After concordance analysis these values become less than 9 £ 1026 and morethan 0.999991 respectively. Any gene whose detection P was more than 0.05 in allexperimental conditions was discarded from the analysis. Cluster analysis was performedwith dCHIP software (www.biostat.harvard.edu/complab/dchip).

Immunoblotting and northern analysisImmunoblot analysis for HIF2a (EPAS1-ab199; Novus Biologicals), pVHL23, CDK2 (M2;Santa Cruz), ERK (Cell Signaling Technology catalogue no. 9102) and phospho-ERK (Cell

Signaling Technology catalogue no. 9101) were performed with standard techniques. TotalRNA was prepared with TRIzol reagent (Life Technologies). Blots were hybridized inExpressHyb (Clontech) to 32P-labelled probes.

DNA binding assaysPreparation of cell lysates and binding reactions were performed as described24,25.Oligonucleotide sequences for vascular endothelial growth factor and CXCR4 are availablefrom the authors on request. Antibodies used were: H1alpha67 (Novus Biologicals),affinity-purified anti-human HIF1a chicken polyclonal antibody (gift fromM. Gassmann), anti-HIF2a (EPAS1-ab199, HIF2alpha-ab8365, Novus Biologicals; C-16,Santa Cruz) and anti-E2F-1 (KH-95, Santa Cruz).

Chemotaxis assaysCell migration was assayed with trans-well inserts with 8-mm pore membranes. HumanSDF-1a was obtained from R&D Systems. Membranes were precoated with fibronectin(0.5 mg ml21). After starvation for 24 h, cells were detached with PBS containing 2.5 mMEDTA, centrifuged and resuspended in DMEM, 0.1% BSA, 12 mM HEPES pH 7.4 at2 £ 105 cells per 100 ml. After 4 h, cells on the lower surface were fixed and stained withcrystal violet. Five different fields of the membrane were counted to assess the number ofcells that had migrated.

In vitro kinase reactionsPolyclonal rabbit serum to human LIMK-1 (anti-LIMK-1) was raised against a fusionprotein of full-length LIMK-1 and maltose-binding protein (J.L., unpublished work) andaffinity-purified23. A498 cells were serum-starved for 24 h, treated with 1 nM SDF-1a forthe indicated times, subjected to immunoprecipitation as described26 and processed forkinase reactions in the presence of glutathione S–transferase–cofilin16.

Quantitative PCRTotal RNA was extracted from frozen tissue, treated with DNase I using the RNeasy system(Qiagen) and reverse transcribed with random hexamer primers (Amersham) and AMVreverse transcriptase (Roche). Complementary DNA was analysed by the fluorogenic

Figure 4 CXCR4 expression in human renal cell carcinoma. a, Quantitative reverse

transcriptase PCR analysis of normal kidney tissues and renal cell carcinoma. Fold

expression levels are normalized to 18S rRNA and compared with the average values

in normal kidneys. Red bars, CXCR4; blue bars, GLUT1; green bars, CA9.

b–g, Immunohistochemical evaluation of CXCR4 expression. b, Invasive breast cancer.

c, Normal renal tissue; arrows indicate a glomerulum (left) and a tubulus (right).

Lymphocellular infiltrates stain intensively. Examples of weak (d) and strong (e–g) CXCR4

expression in clear cell RCCs. Original magnifications: b, c, £ 400; d, e, £ 50;

f, g, £ 400. h, Kaplan–Meier analysis of tumour-specific survival in patients with clear

cell RCC according to CXCR4 expression. P ¼ 0.001.

letters to nature

NATURE | VOL 425 | 18 SEPTEMBER 2003 | www.nature.com/nature310 © 2003 Nature Publishing Group

50-nuclease PCR assay. Primers and probes (Applied Biosystems): CXCR4 forward

50-TGGGTGGTTGTGTTCCAGTTT-3

0, reverse 5

0-ATGCAATAGCAGGACAGGATGA-3

0,

probe 5 0 -FAM-CATGGTTGGCCTTATCCTGCCTGGTA-TAMRA-3 0 (where TAMRArepresents carboxytetramethylrhodamine). GLUT1, CA9 and 18S rRNA were detectedwith Assays-on-Demand primer and probe sets Hs00197884_m1, Hs00154208_m1 andHs99999901_s1, respectively (Applied Biosystems). Gene-specific PCR products weremeasured continuously by an ABI PRISM 7000 Sequence Detection System (AppliedBiosystems) during 40 cycles. 18S rRNA was used for normalization. Expression values fortumour samples were compared with the expression in normal renal tissue. Student’s t-testwas applied to analyse the expression between different grades, tumour stages and tumourtypes using StatView 5.0 PPC (SAS Inst. Inc., Cary, North Carolina). P , 0.05 wasconsidered as significant.

Immunofluorescence and tissue microarray analysisIndirect immunofluorescence staining of CXCR4 was performed as described27 withMAB173 (R&D Systems). Confocal laser scanning microscopy was performed on a FV500/BX61 microscope (Olympus). Immunohistochemistry was performed on a tissuemicroarray containing 532 renal tumours and normal tissue28 obtained from the renalnephrectomy series at the University of Basel29. Approval to analyse the tissues wasobtained from the ethical commission in Basel. Upon antigen retrieval, standard avidin–biotin complex immunohistochemistry (ABC-Elite; Vectra Laboratories) was used toevaluate CXCR4 (MAB 172; R&D Systems; dilution 1:2000) and CA9 (M75 (ref. 30),dilution 1:160) expression. Colour was developed with 3-amino-9-ethylcarbazole andsections were counterstained with haematoxylin. CXCR4 and CA9 positivity was assessedsemiquantitatively by staining intensity as negative, weak or strongly positive by twoindependent investigators without prior knowledge of the clinical follow-up data. Caseswere accepted only as strongly positive if reviewers independently defined them thus.Contingency table analysis was used to analyse the relationship of CXCR4 and CA9expression to differentiation grade and tumour stage. Clear cell RCCs were analysed forpatient survival with the Kaplan–Meier method. Statistical differences between the groupswere determined with the log-rank test. A Cox proportional hazards analysis was used totest for independent prognostic information. Tumour-specific survival data were obtainedby reviewing the hospital records, by direct communication with the attending physicians,and from the Cancer Registry of Basel. Patients were evaluated from the time of biopsydiagnosis to the last known follow-up. Tumour-specific clinical follow-up data wereavailable from 195 patients.

Received 8 May; accepted 26 June 2003; doi:10.1038/nature01874.

1. Muller, A. et al. Involvement of chemokine receptors in breast cancer metastasis. Nature 410, 50–56

(2001).

2. Kaelin, W. G. Jr Molecular basis of the VHL hereditary cancer syndrome. Nature Rev. Cancer 2,

673–682 (2002).

3. Semenza, G. L. HIF-1 and tumor progression: Pathophysiology and therapeutics. Trends Mol. Med. 8,

S62–S67 (2002).

4. Pugh, C. W. & Ratcliffe, P. J. The von Hippel–Lindau tumor suppressor, hypoxia-inducible factor-1

(HIF-1) degradation, and cancer pathogenesis. Semin. Cancer Biol. 13, 83–89 (2003).

5. Iliopoulos, O., Levy, A. P., Jiang, C., Kaelin, W. G. Jr & Goldberg, M. A. Negative regulation of

hypoxia-inducible genes by the von Hippel–Lindau protein. Proc. Natl Acad. Sci. USA 93,

10595–10599 (1996).

6. Wiesener, M. S. et al. Constitutive activation of hypoxia-inducible genes related to overexpression of

hypoxia-inducible factor-1a in clear cell renal carcinomas. Cancer Res. 61, 5215–5222 (2001).

7. Mandriota, S. J. et al. HIF activation identifies early lesions in VHL kidneys: evidence for site-specific

tumor suppressor function in the nephron. Cancer Cell 1, 459–468 (2002).

8. Bleul, C. C. et al. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks

HIV-1 entry. Nature 382, 829–833 (1996).

9. Oberlin, E. et al. The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by

T-cell-line-adapted HIV-1. Nature 382, 833–835 (1996).

10. Forster, R. et al. CCR7 coordinates the primary immune response by establishing functional

microenvironments in secondary lymphoid organs. Cell 99, 23–33 (1999).

11. Peled, A. et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on

CXCR4. Science 283, 845–848 (1999).

12. Chambers, A. F., Groom, A. C. & MacDonald, I. C. Dissemination and growth of cancer cells in

metastatic sites. Nature Rev. Cancer 2, 563–572 (2002).

13. Liotta, L. A. An attractive force in metastasis. Nature 410, 24–25 (2001).

14. Clifford, S. C. et al. Contrasting effects on HIF-1a regulation by disease-causing pVHL mutations

correlate with patterns of tumorigenesis in von Hippel–Lindau disease. Hum. Mol. Genet. 10,

1029–1038 (2001).

15. Maxwell, P. H. et al. Hypoxia-inducible factor-1 modulates gene expression in solid tumors and

influences both angiogenesis and tumor growth. Proc. Natl Acad. Sci. USA 94, 8104–8109 (1997).

16. Nishita, M., Aizawa, H. & Mizuno, K. Stromal cell-derived factor 1a activates LIM kinase 1 and

induces cofilin phosphorylation for T-cell chemotaxis. Mol. Cell. Biol. 22, 774–783 (2002).

17. Arber, S. et al. Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase.

Nature 393, 805–809 (1998).

18. Aebersold, D. M. et al. Expression of hypoxia-inducible factor-1a: A novel predictive and prognostic

parameter in the radiotherapy of oropharyngeal cancer. Cancer Res. 61, 2911–2916 (2001).

19. Harris, A. L. Hypoxia—a key regulatory factor in tumour growth. Nature Rev. Cancer 2, 38–47 (2002).

20. Semenza, G. Signal transduction to hypoxia-inducible factor 1. Biochem. Pharmacol. 64, 993–998

(2002).

21. Rempel, S. A., Dudas, S., Ge, S. & Gutierrez, J. A. Identification and localization of the cytokine SDF1

and its receptor, CXC chemokine receptor 4, to regions of necrosis and angiogenesis in human

glioblastoma. Clin. Cancer Res. 6, 102–111 (2000).

22. Bernards, R. & Weinberg, R. A. A progression puzzle. Nature 418, 823 (2002).

23. Hergovich, A., Lisztwan, J., Barry, R., Ballschmieter, P. & Krek, W. Regulation of microtubule stability

by the von Hippel–Lindau tumour suppressor protein pVHL. Nature Cell Biol. 5, 64–70 (2003).

24. Takahashi, Y., Takahashi, S., Shiga, Y., Yoshimi, T. & Miura, T. Hypoxic induction of prolyl 4-

hydroxylase a (I) in cultured cells. J. Biol. Chem. 275, 14139–14146 (2000).

25. Wang, G. L. & Semenza, G. L. Purification and characterization of hypoxia-inducible factor 1. J. Biol.

Chem. 270, 1230–1237 (1995).

26. Lisztwan, J. et al. Association of human CUL-1 and ubiquitin-conjugating enzyme CDC34 with the

F-box protein p45(SKP2): Evidence for evolutionary conservation in the subunit composition of the

CDC34-SCF pathway. EMBO J. 17, 368–383 (1998).

27. Amara, A. et al. HIV coreceptor downregulation as antiviral principle: SDF-1a-dependent

internalization of the chemokine receptor CXCR4 contributes to inhibition of HIV replication. J. Exp.

Med. 186, 139–146 (1997).

28. Moch, H. et al. High-throughput tissue microarray analysis to evaluate genes uncovered by cDNA

microarray screening in renal cell carcinoma. Am. J. Pathol. 154, 981–986 (1999).

29. Moch, H. et al. Prognostic utility of the recently recommended histologic classification and revised

TNM staging system of renal cell carcinoma: A Swiss experience with 588 tumors. Cancer 89, 604–614

(2000).

30. Pastorekova, S., Zavadova, Z., Kostal, M., Babusikova, O. & Zavada, J. A novel quasi-viral agent,

MaTu, is a two-component system. Virology 187, 620–626 (1992).

Supplementary Information accompanies the paper on www.nature.com/nature.

Acknowledgements We thank all members of the laboratory for discussions; K. Struckmann for

providing the tumour RNA samples; G. Keller and B. Mohr for human GLUT3 and CXCR4

cDNAs, respectively; and R. Bernards, U. Muller, N. Hynes, G. Thomas and members of our

laboratory for critically reading the manuscript. This work was supported by the Robert Wenner

Award, the Dr Josef Steiner Foundation and the Novartis Research Foundation. H.M. is

supported by a grant from the Swiss National Science Foundation. P.S. is supported by an EMBO

long–term fellowship.

Competing interests statement The authors declare that they have no competing financial

interests.

Correspondence and requests for materials should be addressed to W.K.

(wilhelm.krek@cell.biol.ethz.ch). The data for the microarray experiment are deposited in

ArrayExpress under accession number E-MEXP-13.

..............................................................

The BTB protein MEL-26 is asubstrate-specific adaptorof the CUL-3 ubiquitin-ligaseLionel Pintard1*, John H. Willis2*, Andrew Willems3,Jacque-Lynne F. Johnson4, Martin Srayko4†, Thimo Kurz2, Sarah Glaser1,Paul E. Mains4, Mike Tyers3, Bruce Bowerman2 & Matthias Peter1

1Institute of Biochemistry ETH, Honggerberg 8093, Zurich, Switzerland2Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA3Department of Medical Genetics and Microbiology, Samuel Lunenfeld ResearchInstitute, Mt Sinai Hosp., 600 University Ave, Univ. Toronto, Toronto, Ontario,M5G 1X5, Canada4Genes and Development Research Group and Department of Biochemistry andMolecular Biology, University of Calgary, Calgary, Alberta, T2N 4N1, Canada

* These authors contributed equally to this work

† Present address: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108,

01307 Dresden, Germany

.............................................................................................................................................................................

Many biological processes, such as development and cell cycleprogression are tightly controlled by selective ubiquitin-depen-dent degradation of key substrates. In this pathway, the E3-ligaserecognizes the substrate and targets it for degradation by the 26Sproteasome. The SCF (Skp1–Cul1–F-box) and ECS (Elongin C–Cul2–SOCS box) complexes are two well-defined cullin-based E3-ligases1–3. The cullin subunits serve a scaffolding function andinteract through their C terminus with the RING-finger-contain-ing protein Hrt1/Roc1/Rbx1, and through their N terminus withSkp1 or Elongin C, respectively. In Caenorhabditis elegans, theubiquitin-ligase activity of the CUL-3 complex is required fordegradation of the microtubule-severing protein MEI-1/kataninat the meiosis-to-mitosis transition4. However, the molecularcomposition of this cullin-based E3-ligase is not known. Here

letters to nature

NATURE | VOL 425 | 18 SEPTEMBER 2003 | www.nature.com/nature 311© 2003 Nature Publishing Group